Update on Wnt signaling in bone cell biology and bone disease

Review

Update on Wnt signaling in bone cell biology and bone disease

David G.Monroe a ,Meghan E.McGee-Lawrence b ,Merry Jo Oursler a ,c ,Jennifer J.Westendorf b ,c ,?

a Department of Medicine/Endocrine Research Unit,Mayo Clinic,200First Street SW,Rochester,MN 55905,USA

b Department of Orthopedi

c Surgery/Division of Orthopedic Research,Mayo Clinic,200First Street SW,Rochester,MN 55905,USA c

Department of Biochemistry and Molecular Biology,Mayo Clinic,200First Street SW,Rochester,MN 55905,USA

a b s t r a c t

a r t i c l e i n f o Article history:

Accepted 20October 2011

Available online 3November 2011Received by Meghan Jendrysik Keywords:Lrp5Lrp6

Sclerostin β-catenin R-spondin

Bone mineral density Polymorphisms

For more than a decade,Wnt signaling pathways have been the focus of intense research activity in bone biology laboratories because of their importance in skeletal development,bone mass maintenance,and therapeutic potential for regenerative medicine.It is evident that even subtle alterations in the intensity,amplitude,location,and duration of Wnt signaling pathways affects skeletal development,as well as bone remodeling,regeneration,and repair during a lifespan.Here we review recent advances and discrepancies in how Wnt/Lrp5signaling regulates osteoblasts and osteocytes,introduce new players in Wnt signaling pathways that have important roles in bone development,discuss emerging areas such as the role of Wnt signaling in osteoclastogenesis,and summarize progress made in translating basic studies to clinical therapeutics and diagnostics centered around inhibiting Wnt pathway antagonists,such as sclerostin,Dkk1and Sfrp1.Emphasis is placed on the plethora of genetic studies in mouse models and genome wide association studies that reveal the requirement for and crucial roles of Wnt pathway components during skeletal development and disease.

?2011Elsevier B.V.All rights reserved.

Contents 1.

Introduction .............................

..................................21.1.Wnt signaling pathways ..................

....................................21.1.1.Wnt –β-catenin signaling .................................................21.1.2.Non-β-catenin Wnt signaling pathways ......

....................................31.2.Osteoblast and osteoclast lineages:differentiation,maturation and coupling .............................52.

Wnts and Wntless ............................................................52.1.Wnts ...........................

....................................52.1.1.Wnt3a ..........................................................62.1.2.Wnt5a ..........................................................62.1.3.Wnt10b .........................................................62.1.4.Wnt14......................

....................................62.2.Wntless (Wls,Evi,Gpr177)....................................................72.3.R-spondins ............................................................73.

Wnt receptors ..............................................................73.1.LDL receptor-related proteins ...............

....................................73.1.1.Lrp5...........................................................73.1.2.Lrp6...........................................................83.1.3.Lrp4.......................

..

.....

.............................

8

Gene 492(2012)1–18

Abbreviations:AER,apical ectodermal ridge;Apc,adenomatous polyposis coli;Bmp,bone morphogenic protein;Ck1,casein kinase 1;CKI,conditional knock-in;CKO,conditional knock-out;Ctnnb1,catenin (cadherin-associated protein),beta 1;Daam1,disheveled-associated activator of morphogenesis 1;Dkk,Dickkopf;Dmp1,Dentin matrix acidic phospho-protein 1;Dvl,Disheveled;Fgf,?broblast growth factor;Fzd,Frizzled;Gsk,glycogen synthase kinase;GWAS,genome-wide association studies;HBM,high bone mass;Jnk,c-Jun N-terminal kinase;KO,knockout;Krm,Kremen;Lef1,Lymphoid enhancer binding factor;LiCl,lithium chloride;Lrg,leucine-rich repeat containing G protein-coupled receptor;Lrp,low-density lipoprotein receptor-related protein;Ocn,Osteocalcin;Opg,osteoprotegerin;OPPG,osteoporosis-pseudoglioma syndrome;Osx,Osterix;OSCS,osteopathia striata with cranial sclerosis;PCP,planar cell polarity;PKA,protein kinase A;PKC,protein kinase C;PTH,parathyroid hormone;Rankl,receptor activator of NF-κB ligand;Scl,sclerostin;Sfrp,secreted Frizzled-related protein;Shh,sonic hedgehog;SNP,single nucleotide polymorphism;Tcf,T cell factor;Tg,transgenic;Wif,Wnt inhibitory factor;Wise,Wnt modulator in surface ectoderm;Wls,Wntless;Wnt,Wingless-type MMTV integration site;Wtx,Wilm's tumor genes on chromosome X.

?Corresponding author at:Mayo Clinic,200First Street SW,Rochester,MN 55905,USA.Tel.:+15075385651;fax:+15072845075.E-mail address:westendorf.jennifer@https://www.doczj.com/doc/0210348478.html, (J.J.

Westendorf).0378-1119/$–see front matter ?2011Elsevier B.V.All rights reserved.doi:

10.1016/j.gene.2011.10.044

Contents lists available at SciVerse ScienceDirect

Gene

j o ur n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /g e n e

3.2.Frizzleds (8)

3.2.1.Fzd9 (8)

4.Wnt antagonists (9)

4.1.Secreted Wnt antagonists:Dkks,Sfrps,Wif1,Sost,and Sost-dc1 (9)

4.1.1.Dickkopfs(Dkk) (9)

4.1.2.Secreted frizzled-related proteins(Sfrps) (9)

4.1.3.Wnt inhibitory factor(Wif)-1 (10)

4.1.4.Sclerostin/SOST (10)

4.1.5.Sost-dc1(Ectodin,Wise,Usag1) (10)

4.2.Transmembrane modulators of Wnt signaling (11)

4.2.1.Kremen1/2 (11)

4.2.2.Ror2 (11)

4.2.3.Ryk (11)

5.β-catenin and associated intracellular proteins (11)

5.1.Ctnnb1(β-catenin) (11)

5.2.Adenomatous polyposis coli(Apc) (12)

5.3.Axin1/2 (12)

5.4.Gsk3β (12)

5.5.Tcf7and Lef1transcription factors (13)

5.5.1.Tcf7(Tcf1) (13)

5.5.2.Lef1 (13)

5.6.Wtx(FAM123B) (13)

6.Emerging areas for Wnts in bone biology (14)

6.1.Wnt signaling and osteoclasts (14)

6.2.Aging (14)

6.3.Cancer (14)

7.Summary and conclusions (14)

Acknowledgments (14)

References (14)

1.Introduction

Wnts are a large family of19secreted glycoproteins that trigger multiple signaling cascades essential for embryonic development and tissue regeneration.Proteins involved in the ampli?cation and transduction of Wnt signals are often altered in cancer or lineage progenitor cells,leading to abnormal cell cycle control and/or altered cell fate decisions(MacDonald et al.,2009;Polakis,2000).Mutations in several Wnt pathway components also contribute to human skeletal dysplasias.Most notably,mutations in the Wnt co-receptor LRP5 cause low or high bone mass depending on the nature of the alteration (Boyden et al.,2002;Gong et al.,2001;Little et al.,2002)and inactiva-tion of the secreted Wnt antagonist Sclerostin produces high bone mass,sclerosteosis and van Buchem's disease(Balemans et al.,2001; Brunkow et al.,2001).A loss-of-function mutation in LRP6,another Wnt co-receptor,is linked to an inherited disorder characterized by osteoporosis,coronary artery disease,and metabolic syndrome (Mani et al.,2007).Less well known is that inactivating mutations in WTX,an intracellular regulator ofβ-catenin stability,cause osteopathia striata with cranial sclerosis(OCTS)(Jenkins et al.,2009)and FZD9,a Wnt co-receptor,is deleted in patients with Williams–Beuren syndrome, which is partially characterized by low bone density(Francke,1999). During the last several years,polymorphisms in these and many more Wnt pathway components were linked to altered bone mineral density in genome wide association studies(Kiel et al.,2007b;Riancho et al., 2011;Rivadeneira et al.,2009;Sims et al.,2008;van Meurs et al., 2008).Thus,it has become clear that even subtle alterations in the intensity,amplitude,and duration of Wnt signaling pathways affects skeletal formation during development,as well as bone remodeling, regeneration,and repair during a lifespan.In this review,we provide an update to a2004review on Wnt signaling in osteoblasts and bone disease published in this journal(Westendorf et al.,2004).Emphasis is placed on new data from murine genetic studies assessing the requirement for and roles of Wnt pathway components during skeletal development and disease.These observations are discussed in context with current knowledge of molecular and physiological regulation of bone mass.Progress in translating these discoveries to treatments for altered bone mass conditions is also summarized.

1.1.Wnt signaling pathways

1.1.1.Wnt–β-catenin signaling

Wnts trigger several signaling cascades.The best known is the Wnt/β-catenin pathway(commonly called the canonical pathway), which features the stabilization and nuclear translocation of β-catenin as easily measurable outcomes.In the absence of Wnts,β-catenin associates with cadherins at the plasma membrane.Any excessβ-catenin is quickly sequestered by a protein complex containing Axin1/2,Apc,casein kinase(Ck)1,glycogen synthase kinase(Gsk)3β, and Wtx and degraded by ubiquitin-mediated proteolysis(Fig.1)(For more details see(Westendorf et al.,2004)).When certain Wnts(e.g., Wnt3a)are present,they crosslink cell surface molecules,Lrp5/6and a Frizzled(Fzd),which mobilizes Gsk3βand Ck1to the membrane where they phosphorylate serines on Lrp5/6,promote the formation of a signalosome,and recruit Disheveled(Dvl),Axin1/2,and caveolin (Bilic et al.,2007;MacDonald et al.,2009;Niehrs and Shen,2010;Zeng et al.,2005).This releasesβ-catenin from the destruction complex, increases its levels,and allows it to enter the nucleus where it can displace co-repressors from transcription factors(e.g.,Lef1,Tcf7) and regulate gene expression.Nuclear localization ofβ-catenin is often used as a metric of enhanced Wnt signaling.Expression levels of target genes(e.g.,Axin2,Lef1)are also commonly measured to study Wnt signaling.Althoughβ-catenin is activated by Wnts,it is important to remember that it is also mobilized by other signals(e.g.,Igf and Akt activation)and is not exclusive to the canonical Wnt signaling cascade. This point is especially important in bone,asβ-catenin deletion triggers bone loss via different mechanisms than Lrp5inactivation(subsequent sections).

The Wnt/β-catenin pathway stimulates cell proliferation and survival. Enhanced stimulation of the pathway is a feature of many cancers (Polakis,2000).Under normal physiological settings,multiple proteins keep this cascade in check.In addition to intracellular inhibitors

2 D.G.Monroe et al./Gene492(2012)1–18

(Axin2),the canonical pathway is neutralized by extracellular factors (Fig.1).Secreted frizzled related proteins (Sfrps)and Wnt inhibitory fac-tors (Wifs)directly bind Wnts and prevent their interactions with receptors.Other secreted proteins including Dickkopfs (Dkk),Sclerostin (Scl),and Sostdc1(Wise)bind to Lrp5/6receptors,inducing receptor internalization and/or reducing their availability to Wnts.Thirdly,some Wnts (e.g.,Wnt5a)trigger alternative signaling pathways by co-opting receptor components and thus competing with Wnts (e.g.,Wnt3a)that induce β-catenin stabilization.For example,Wnt5a induces the formation of a complex consisting of Lrp5/6,Ror1/2,and Fzd2(Sato et al.,2010).

1.1.

2.Non-β-catenin Wnt signaling pathways

In some contexts,Wnts neither stabilize β-catenin nor interact with Lrp5/6.Rather,through Fzds and Dvl,Wnts can trigger alternative intracellular events (Fig.1and reviewed by (Gao and Chen,2010)).Non-β-catenin cascades include the planar cell polarity (PCP)pathway,trimeric G-protein coupled receptor pathways including calcium ion (Ca 2+)signaling,Rho family GTPase pathways,and the Jnk pathways.Dvl has multiple conserved domains that allow it to interact with many binding partners,which determines which downstream pathways are engaged (Gao and Chen,2010).Furthermore,membrane-spanning receptors such Ror2and Ryk can activate Dvl-independent signaling (Angers and Moon,2009).

1.1.

2.1.Planar cell polarity (PCP)pathway.The most extensively studied non-β-catenin Wnt signaling pathways is the PCP pathway,which enables cells to orient relative to an axis along the plane of a tissue (Henderson and Chaudhry,2011).PCP signaling governs cell movement in the embryo via convergent extension (Sokol,1996)and determines cell fates,enabling the creation of asymmetric and highly aligned struc-tures such as hair follicles as well as orchestrating the polarized beating of motile cilia in numerous tissues (Devenport and Fuchs,2008;Jones et al.,2008).The establishment of polarity in the plane of the epithelium provides directional information during development.Wnt binding to

Fzd leads to Dvl-driven sorting of cellular components to either the proximal or distal regions of the cell and orients it within the tissue (Veeman et al.,2003).Thus far,little is known about PCP activation during bone remodeling.

1.1.

2.2.Wnt and Rho/Rac GTPases.Dvl activation of the Rho GTPase family member Rac1leads to Jnk activation and stimulation of the transcription factors c-Jun and ATF2(Li et al.,1999;Ohkawara and Niehrs,2011;Sato et al.,2010).Wnt3a causes chondrocyte de-differentiation by activating c-Jun/AP-1and suppressing Sox-9expression,supporting a role for a non-β-catenin/Wnt3a pathway in bone development (Hwang et al.,2005).Wnt binding to Fzd can also promote Dvl interactions with the adaptor protein disheveled-associated activator of morphogenesis (Daam)1,which activates the Rho guanine nuclear exchange factor WGEF (Wu and Herman,2006).WGEF induces RhoA/ROCK pathway activation,which promotes cytoskeletal reorganization to control cell shape and adhesion (Gao and Chen,2010).Dvl/Daam1interactions can also cause cytoskeletal reorganization by in ?uencing Pro ?lin independent of RhoA activation (Gao and Chen,2010).

1.1.

2.

3.Wnt and G-protein coupled receptor signaling.Evidence is mounting that Wnt activates trimeric G-protein signaling to control a number of downstream signaling pathways.G proteins are required for Wnt activity,but whether there are direct interactions between Fzd and G proteins remain unresolved (Katanaev and Tomlinson,2006;Katanaev et al.,2005;Liu et al.,1999,2005;Purvanov et al.,2010).Phys-ical interactions between Fzd and G proteins were observed under physiological conditions (Koval and Katanaev,2011).Thus,Wnt3a stimulated G αs and G αi/o ,but not G αq11association with Fzd receptors in brain tissue.Wnt/Fzd induced cAMP accumulation and PKA activa-tion though G αs protein (Witze et al.,2008).In contrast,G αi/o stimulated phospholipase C,intracellular Ca +2release and direct PKC activation.G protein signaling,speci ?cally,G αq11activation,was also required for nuclear localization of β-catenin following Wnt3a treatment (Tu et

al.,

Fig.1.Wnt signaling pathways.The “canonical ”Wnt –β-catenin signaling pathway is illustrated in the center of the diagram.Secreted and intracellular inhibitors of β-catenin are shown on the right side.Wnt signaling pathways that do not involve β-catenin are summarized on the left side.

3

D.G.Monroe et al./Gene 492(2012)1–18

Table1

Summary of bone phenotypes in mouse models of altered Wnt signaling in osteoblast lineage cells and the germline.

Gene KO/CKO/Tg/

CKI

Cre line Bone phenotype(s)References

APC CKO OCN-Cre Early postnatal lethality,osteopetrosis,increased trabecular bone density,

few osteoclasts,elevated Opg

Holmen et al.,2005

CKO Col2a1-Cre Early postnatal lethality,reduced mineralization at E14.5,increased

mineralization at E16.5and in ribs

Miclea et al.,2009 Axin2KO Germline Craniosynostosis,increased BMD Yu et al.,2005

Ctnnb1

(β-Catenin)CKO Wnt1-Cre Embryonic lethal,block in prechondrocyte condensation and craniofacial

development

Brault et al.,2001

CKO Prx1-Cre Lack of mineralization in head and distal skeletal elements,enhanced

chondrogenesis,lower osteoblastogenesis

Hill et al.,2005 CKO Dermo1-Cre Shortened limbs,twisted body axis,diminished intramembranous and

endochondral bone formation,ectopic cartilage

Day et al.,2005;

Hu et al.,2005 CKO Osx1-Cre Lack of cranial ossi?cation,increased chondrogenesis Rodda et al.,2005 CKO Col2a1-Cre Ectopic cartilage in long bones,normal intramembranous bone Day et al.,2005 CKO 2.3Col1a1-Cre Reduced bone mass,increased osteoclast numbers,decreased Opg Glass et al.,2005 CKO Ocn-Cre Early postnatal lethality,reduced cortical and trabecular bone density,

increased osteoclast numbers

Holmen et al.,2005

CKO Dmp1-Cre Premature postnatal lethality,impaired cortical and trabecular bone mass,

increased osteoclast number and activity,decreased Opg levels

Kramer et al.,2010a CKI/GOF Prx1-Cre:exon3Early postnatal lethality,no bone formation Hill et al.,2005

CKI/GOF Osx1-Cre:exon3Embryonic lethality,excessive premature ossi?cation Rodda et al.,2005 CKI/GOF 2.3Col1a1-Cre:exon3Premature postnatal lethality,failed tooth eruption,increased ossi?cation,

decreased osteoclast numbers and function,normal osteoblast numbers,

rib osteomata

Glass et al.,2005

Dkk1Het Germline High bone mass inversely proportional to Dkk1concentration in

hypomorphic animals Morvan et al.,2006; MacDonald et al.,2004,2007

Tg 2.3and3.6Col1a1Low bone mass,decreased osteoblast number,reduced serum osteocalcin

levels,lower matrix mineralization

Li et al.,2006

Tg 2.3Col1a1Osteopenia,reduced bone formation,normal PTH responsiveness Fleming et al.,2008;

Guo et al.,2010;

Yao et al.,2011 Dkk1d Hypomorphic mutation Increased bone mass that is inversely proportional to Dkk1expression,

distal forelimb postaxial polysyndactyly

MacDonald et al.,2007

Dkk2KO Germline Low bone mass Li et al.,2005

Fzd9KO Germline Osteopenia,decreased bone formation Albers et al.,2008

Gsk3βHet KO Germline Increased trabecular bone mass Kugimiya et al.,2008;

Noh et al.,2009

Krm1/2DKO Germline Increased BMD Ellwanger et al.,2008 Krm2KO Germline High bone mass at24weeks,increased bone formation Schulze et al.,2010 Krm2Tg 2.3Col1a1Osteoporosis,decreased bone formation,reduced cortical strength,

reduced Opg expression,and increased bone resorption

Schulze et al.,2010 Lef1Het KO Germline Reduced bone formation in females only Noh et al.,2009 KO Germline Reduced bone mass in all KO and Het mice JJW et al.,unpublished Lef1ΔN Tg 2.3Col1Increased trabecular bone mass Hoeppner et al.,2010 Lrp4Lrp4ECD Hypomorph Reduced BMD,increased bone turnover Choi et al.,2009

Lrp5KO Germline Decreased bone mass Fujino et al.,2003;

Kato et al.,2002 CKO 2.3Col1-Cre Normal vertebral bone mass Yadav et al.,2008

CKO Dermo1-Cre Normal vertebral bone mass Yadav et al.,2010

CKO Dmp1-Cre Decreased trabecular bone mass and cortical strength Cui et al.,2011

Tg Rat3.6Col1-HBM G171V Increased bone mass and strength Akhter et al.,2004;

Babij et al.,2003 CKI/GOF 2.3Col1-Cre:HBM G171V

cDNA

Normal vertebral bone mass and bone formation rates Yadav et al.,2008

CKI/GOF Dmp1-Cre:HBM G171V or

A214V Increased trabecular bone mass,bone strength,and bone

formation rates in distal femur and L5

Cui et al.,2011

CKI/GOF Prx1-Cre:HBM G171V Increased bone mass in limbs,but not vertebrae Cui et al.,2011

CKI/GOF Villin-Cre:HBM G171V Increased vertebral bone mass and bone formation rates Yadav et al.,2008

CKO Villin-Cre Decreased vertebral bone mass,bone formation rates and

osteoblast numbers

Yadav et al.,2008

CKI/GOF Vil1-Cre:HBM G171V or

A214V

Normal bone mass Cui et al.,2011 CKO Vil1-Cre Normal bone mass Cui et al.,2011

Lrp6Het KO Germline Decreased bone Holmen et al.,2004 Rs Hypomorphic mutation Decreased bone mineral density,no chance in osteoblast number,

elevated Rankl expression,increased bone resorption

Kubota et al.,2009 Lrp5/6KO/Het Germline Decreased bone Holmen et al.,2004 Rspo2KO Germline Decreased ossi?cation in distal phalanges and stunted?bula Nam et al.,2007

Sfrp1KO Germline Increased bone mass Bodine et al.,2004 Tg Sfrp1Decreased bone mass Yao et al.,2010

Sfrp4Tg 2.3Col1a1Low bone mass,fewer osteoblasts;LiCl rescued these defects Nakanishi et al.,2008 Tg Serum amyloid P Decreased bone mass Cho et al.,2010

Sclerostin (Scl,Sost)KO Germline Increased bone mass Balemans et al.,2003;

Krause et al.,2010

Tg Ocn+APO E Osteopenia Winkler et al.,2003

4 D.G.Monroe et al./Gene492(2012)1–18

2007).The βγsubunits of the trimeric G protein complex interact with Dvl in vertebrate cells.Fzd7and G protein βγsubunits are required for Wnt11to stimulate axis organization,indicating the βγsubunits as well as the αsubunit are involved in non-β-catenin G protein-mediated signaling (Angers et al.,2006;Penzo-Mendez et al.,2003).1.2.Osteoblast and osteoclast lineages:differentiation,maturation and coupling

Osteoblasts,osteocytes,and osteoclasts directly regulate bone mass.Osteoblasts originate from mesenchymal progenitor cells and are responsible for producing proteins,such as type 1collagen,that form a mineralizable matrix.Runx2,Sp7(osterix),Wnts,Lrp5,and β-catenin are among the crucial factors required for their speci ?cation from mesenchymal precursors and osteo-chondoprogenitors.Wnts and β-catenin subsequently contribute to proliferation and survival of osteoblasts (Westendorf et al.,2004).β-catenin also regulates the communication or coupling of osteoblasts with osteoclast precursors,which originate from hematopoietic stem cells,by controlling expression of osteoprotegerin (Opg),a competitive inhibitor of Rankl and Rank interaction,to affect bone resorption (Glass et al.,2005).Osteocytes are terminally differentiated osteoblasts embedded within the mineralized matrix that communicate changes in mechanical loading and the extracellular environment to osteoblasts and osteoclasts on the bone surface to stimulate fracture repair and in ?uence bone remodeling (Bonewald,2011).

Wnts and Wnt pathway components are essential for many stages of osteoblast lineage development and maturation.Knowledge in this area has advanced in the last decade due to the availability and utilization of genetic approaches that test the requirement or role for certain molecules in bone development,biology,and disease.These models include germ-line knockout (KO),conditional knockout (CKO)or knock-in (CKI),and transgenic (Tg)expression.Table 1summarizes bone phenotypes that result when Wnt pathway components are genetically altered in oste-oblast lineage cells or the germline.Table 2lists bone phenotypes of mice where β-catenin levels are altered in osteoclast lineage cells and their precursors.The CKO,CKI,and Tg strategies allow for tissue-speci ?c and/or inducible expression.Several promoters drive expression of Cre recombinase (for CKO or CKI strategies)or transgenes in

osteoblast and osteoclast lineage cells at different stages of maturation (Fig.2)(Van Koevering and Williams,2008).In the following sections,we review studies that utilized these technologies to advance our un-derstanding of Wnt pathways in bone biology and disease.2.Wnts and Wntless 2.1.Wnts

Wnts are secreted,cysteine-rich glycoproteins involved in control-ling cell proliferation,cell-fate speci ?cation,gene expression,and cell survival.Cells recognize Wnts with 10Frizzled receptors (Fzd)and Lrp molecules (Lrp5/6and potentially Lrp4).The large number of ligands and receptors creates great combinatorial diversity and contributes to widely variable cellular responses depending on the molecules present.Wnts were historically classi ?ed as either “canonical ”or “non-canonical ”based on their ability to activate β-catenin;however,in reality the distinction is not so clear because some Wnts stimulate both pathways depending on the cellular context.Understanding how Wnt molecules contribute to osteoblast function and overall bone homeostasis is crucial in developing treatments for the clinical intervention for various bone diseases,such as osteoporosis.

Expression analyses of the Wnt family members in various osteo-blastic models provide insight into the possible function and physio-logical source of each Wnt.Witte and colleagues pro ?led all 19Wnts during mouse limb development and cartilage differentiation (Witte et al.,2009).Each Wnt displayed a unique expression pattern and localization.Interestingly Wnt1,Wnt3a,Wnt8a,and Wnt8b were not detected at any developmental timepoint.Mak and colleagues found that expression levels of Wnt2,Wnt2b,Wnt4,Wnt5a,Wnt10b,and Wnt11were higher in mature murine osteoblasts compared to their progenitors (Mak et al.,2009).These studies provide an important spatial and temporal context to begin to understand how the Wnt ligands affect bone biology.

Many Wnt ligands affect various aspects of bone biology in vitro ,but their true importance in bone physiology will ultimately come from experimental observations in vivo .Currently available Wnt mouse models suggest that Wnt3a,Wnt5a,and Wnt10b are capable of regulating osteoblast function (Baksh et al.,2007;Boland et

Table 1(continued )Gene

KO/CKO/Tg/CKI Cre line Bone phenotype(s)

References

Tg SOST Osteopenia

Loots et al.,2005Sostdc1(Wise)KO

Germline

Supernumerary teeth,bone phenotype not determined

Ahn et al.,2010;Kassai et al.,2005;

Murashima-Suginami et al.,2008Tcf7(Tcf1)KO Germline Lower bone mass (modest),increased bone resorption

Glass et al.,2005Wif1

KO Germline Normal skeletal development,accelerated radiation-induced osteosarcoma formation

Kansara et al.,2009Tg

2.3Col1a1Normal bone,depletion of hematopoietic stem cells

Schaniel et al.,2011Wls (Gpr177)CKO Wnt1-Cre Craniofacial defects,defective anterior –posterior axis formation Carpenter et al.,2010;Fu et al.,2011

Wnt3a Het KO Germline Reduced BMD

Takada et al.,2007Wnt5a Het KO Germline Reduced BMD,increased adipogenesis Takada et al.,2007Wnt7b KO Germline No defects in skeletal development Rodda et al.,2005

Wnt10b

KO Germline Reduced BMD,increased adipogenesis

Bennett et al.,2005,2007;Stevens et al.,2010Tg Fabp4Increased BMD Bennett et al.,2005Tg Ocn Increased BMD

Bennett et al.,2007Wnt14

Tg

Col2a1

High expression blocked endochondral bone formation,

lower transgene expression promoted chondrocyte maturation and enhanced endochondral bone formation

Day et al.,2005

Wtx

KO Germline Sclerosis,increased osteoblastogenesis,but delayed mineralization Moisan et al.,2011

CKO Prx1-Cre Bone overgrowth,reduced marrow adiposity

CKO Osx1-Cre Increased cortical and trabecular bone mineralization CKO Col2a1Normal skeleton CKO Ocn-Cre

Normal skeleton

BMD:bone mineral density;CKI:conditional knock-in;CKO:conditional knockout mouse;GOF,gain of function;HBM,high bone mass mutations;Het:heterozygous knockout mouse;KO:global knockout;DKO:double global knockout;Opg:osteoprotegerin;Rs:ringelschwanz hypomorphic Lrp6mutation;Tg:transgenic.

5

D.G.Monroe et al./Gene 492(2012)1–18

al.,2004;Etheridge et al.,2004;Hu et al.,2005),whereas Wnt14contributes to endochondral bone formation (Day et al.,2005).2.1.1.Wnt3a

Germline deletion of Wnt3a causes early embryonic lethality;however,heterozygotic Wnt3a males display bone loss,with decreases in bone mineral density and trabecular number (Takada et al.,2007).Recombinant Wnt3a is commercially available and is used in numerous in vitro assays to stimulate canonical Wnt signaling in osteoblasts where it induces cell proliferation and survival (Almeida et al.,2005).Wnt3a also induces the proliferation of mesenchymal precursor cells (Boland et al.,2004).

2.1.2.Wnt5a

Wnt5a heterozygote males also display bone loss,with decreases in bone mineral density and trabecular number and increases in adipocyte number (Takada et al.,2007).Recombinant Wnt5a is also commercially available and is often used to stimulate non-canonical (or non-β-catenin)signaling pathways.However,care needs to be used in inter-preting results with rWnt5a as it activates or represses β-catenin/Tcf sig-naling depending on the receptor context (Mikels and Nusse,2006).

Thus,Wnt5a stabilizes β-catenin in the presence of Fzd4but inhibits β-catenin if it binds to Ror2.

2.1.

3.Wnt10b

Wnt10b levels are directly correlated with bone mineral density and indirectly related to marrow adiposity.Thus,transgenic overexpression of Wnt10b in either mature osteoblasts or marrow adipocytes increases bone formation (Bennett et al.,2005,2007).Wnt10b also inhibits fat accumulation in genetically predisposed mouse models of obesity (Wright et al.,2007)and is important for the maintenance of mesen-chymal progenitor cells (Stevens et al.,2010).The maintenance of a progenitor pool of preosteoblastic cells in the bone marrow possibly may be through activation of auxiliary pathways,such as Notch (Modder et al.,2011).The capability of Wnt10b to control osteoblastic lineage allocation could offer novel therapeutic interventions to osteoporotic-and/or obesity-related diseases.

2.1.4.Wnt14

Wnt14is expressed in the tissue surrounding mesenchymal condensations and differentiating osteoblasts (Guo et al.,2004;Kato et al.,2002).It activates β-catenin and induces Lef1expression (Day

Table 2

Summary of bone phenotypes in mouse models of altered Wnt signaling in osteoclasts and precursors.Gene

CKO/CKI Cre line

Bone phenotype(s)

References Ctnnb1(β-Catenin)

CKO

PPAR γ-tTA:TRE-Cre:exon6

Het mice:Osteoporosis,increased bone resorption,no change in bone formation;

Wei et al.,2011

KO mice:Osteopetrosis,reduced bone resorption,reduced

osteoclast precursor proliferation,no change in bone formation CKO Tie2-Cre:exon6Het:Osteoporosis;KO:partial embryonic lethality,osteopetrosis CKO Lyz-Cre:exon6

KO:Osteoporosis,increased bone resorption;Het:intermediate bone loss CKO Ctsk-Cre:exon 6Ibid

CKI/GOF Tie2-Cre:exon3

Embryonic lethality

CKI/GOF PPAR γ-tTA:TRE-Cre:exon3Osteopetrosis:more immature proliferating osteoclasts but fewer

mature osteoclasts,reduced bone resorption,no change in bone formation CKI/GOF Lyz-Cre:exon3Ibid CKI/GOF

Ctsk-Cre:exon 3

Ibid

BMD:bone mineral density;CKI:conditional knock-in;CKO:conditional knockout mouse;Het:heterozygous knockout

mouse.

Fig.2.Cell lineages involved in bone development and homeostasis.Osteoblasts and chondrocytes are derived from mesenchymal progenitor cells,whereas osteoclasts are derived from hematopoietic precursors.Various promoters (indicated at the top and bottom of the diagram in gray boxes)drive transgene or Cre expression in these cells at various stages of their maturation.Mature osteoblasts and osteocytes stimulate osteoclast maturation through Rankl –Rank interactions,but also secrete the decoy receptor Opg to regulate the process.Osteocytes secrete Scl to inhibit Lrp5activities.PTH and mechanical loading suppress Scl production by osteocytes to increase bone formation.

6 D.G.Monroe et al./Gene 492(2012)1–18

et al.,2005).High Wnt14expression blocked endochondral bone formation;however,lower transgene levels promoted chondrocyte maturation and enhanced endochondral bone formation(Day et al., 2005).These data demonstrate that Wnt14can contribute to bone formation.

2.2.Wntless(Wls,Evi,Gpr177)

The ability of Wnts to activate signaling cascades in either an autocrine or paracrine fashion requires that they be secreted from cells.Wntless(Wls)is a seven-pass transmembrane protein responsible for the processing and secretion of all Wnts(Banziger et al.,2006; Bartscherer et al.,2006;Goodman et al.,2006).Wls is expressed ubiqui-tously in human cells and rodent tissues(Jin et al.,2010;Yu et al.,2010), suggesting that Wnts are important in virtually all cell types,both developmentally and postnatally.Germline deletion of Wls causes embryonic lethality and Wnt protein accumulation in the Golgi (Fu et al.,2009).Conditional Wls knockouts with the Wnt1-Cre mouse strain caused craniofacial defects as well as defective anterior–posterior axis formation(Carpenter et al.,2010;Fu et al.,2011).Interestingly, the Wls gene itself is activated byβ-catenin and Lef1/Tcf-dependent transcription,which then assists the cellular traf?cking of Wnt proteins in a positive feedback mechanism(Fu et al.,2009).Several genome-wide association studies identi?ed WLS as a gene linked to altered bone mineral density(Hsu et al.,2010;Kumar et al.,2011;Rivadeneira et al.,2009;Styrkarsdottir et al.,2010).

2.3.R-spondins

R-spondins(Rspo1–4)are secreted factors that synergize with Wnts(e.g.,Wnt1,3a and7a,11)to promoteβ-catenin stabilization (Kim et al.,2006).In this regard,Rspo2and Rspo3are more potent that Rspo1,whereas Rspo4is a relatively weak activator(Kim et al., 2008).R-spondins interfere with Dkk1binding to Krm2/Lrp6,thereby preventing Lrp6internalization(Binnerts et al.,2007;Kim et al., 2008).R-spondins also bind to the leucine-rich repeat containing G protein-coupled receptor(Lgr)-4and-5with high af?nity and enhance Lrp6phosphorylation(Carmon et al.,2011;de Lau et al.,2011). Lgr4-null mice exhibit delayed osteoblast differentiation and minerali-zation during embryogenesis(Luo et al.,2009).Virtually all indices of bone formation were suppressed in both trabecular and cortical bone with concomitant downregulation of osteocalcin,bone siaoloprotein, and collagen transcripts in these animals.

R-spondins are required for development and reproduction(Aoki et al.,2007;Bell et al.,2008;Blaydon et al.,2006;Ishii et al.,2008; Parma et al.,2006),but little is known about their individual roles in skeletal development.Rspo2is necessary for hind limb development, ossi?cation of the most distal phalanges,and proper?bular growth (Nam et al.,2007).All four R-spondins appear to share similar mech-anisms of action;therefore,it is possible that functional redundancy between R-spondins may account for the lack of a signi?cant bone https://www.doczj.com/doc/0210348478.html,pound R-spondin mouse models may be needed to uncover the importance of Rspo function in bone during postnatal life.

A few studies examined the role of R-spondins in osteoblastic cell culture systems.In C2C12and primary mouse calvarial cells,Rspo1 synergized with Wnt3a to induce osteoblast differentiation and Opg expression(Lu et al.,2008),suggesting suppression of osteoclastogenesis through upregulation of Opg may contribute to overall bone anabolism. Furthermore,in MC3T3-E1mouse preosteoblasts,Wnt11promoted osteoblast differentiation and mineralization through Rspo2(Friedman et al.,2009).Together,these data identify R-spondins and Lgr4/5as modulators of Wnt signaling.In accordance,Rspo1protected arthritic mice from cartilage and bone damage in vivo(Kronke et al.,2010). Thus,R-spondins may represent a novel class of therapeutic agents to combat speci?c bone and cartilage diseases,although more research into the mechanism of R-spondins in bone and in vivo is necessary before any conclusions into the ef?cacy of these potential treatments can be drawn.

3.Wnt receptors

3.1.LDL receptor-related proteins

Low-density lipoprotein receptor-related proteins(Lrp)are evolu-tionarily conserved plasma membrane receptors with a variety of functions including lipid metabolism,cargo transport,and cellular signaling.Lrp5/6are low af?nity co-receptors for Wnts and high af?nity receptors for soluble Wnt antagonists:Scl,Sost-dc1,and Dkk1. Lrp4is also emerging as a regulator of bone mass density.

3.1.1.Lrp5

Lrp5is one of the most interesting molecules in bone biology at the present time.Its story is one of remarkable achievements in translational research and like all intriguing tales is not without controversy.LRP5 was?rst implicated in bone biology by researchers interested in the genetic cause for osteoporosis pseudoglioma(OPPG)syndrome,a juvenile-onset autosomal recessive disease of low bone mass(Gong et al.,2001),and by physicians caring for patients with remarkably high bone mass(HBM)who appeared resistant to high impact fractures (such as from an automobile accident)and who anecdotally had trouble staying a?oat while swimming(Boyden et al.,2002;Little et al.,2002). Molecular determinants for both of these conditions were mapped to the same region of chromosome11,which was later identi?ed as the LRP5locus(Boyden et al.,2002;Gong et al.,1996;Little et al.,2002). Following the identi?cation of mutations in LRP5coding regions that led to loss-of-function(in OPPG patients)or gain-of-function(in HBM individuals),the conditions were reproduced in animal models. Thus,germline deletion of Lrp5in all mouse tissues recapitulated the low bone density in OPPG patients(Fujino et al.,2003;Kato et al., 2002),while transgenic overexpression of LRP5-G171V(a gain-of-function mutation)with a relatively osteoblast-speci?c rat collagen1 promoter produced high bone mass with increased mechanical strength (Akhter et al.,2004;Babij et al.,2003).Subsequent experiments indicated that Lrp5was required for ef?cient Wnt signaling andβ-catenin activa-tion in osteoblasts,while the Lrp5gain-of-function mutations prevented Lrp5internalization and binding to other ligands such as Dkk1and Scl (Boyden et al.,2002;Ellies et al.,2006;Zhang et al.,2004).The Lrp5 research path subsequently merged with several others focused on Dkk1and Scl and has led to promising new anabolic therapies for low bone mass in less than two decades,making it an exciting example of translational research at its best.

The Lrp5story is not yet complete though because the physiological mechanisms by which Lrp5alterations regulate bone mass are not fully understood.Given that Lrp5is expressed in osteoblast-lineage cells,it is possible that Lrp5mutations directly alter the activities of bone-forming cells.However,the genetic mutations in the aforementioned patients and the Lrp5-de?cient animal models are present in all cells and tissues,thus leaving the possibility that Lrp5alterations indirectly affect bone formation.To determine if Lrp5directly regulates osteoblast-lineage cells,two groups made Lrp5CKO mice as well as Lrp5conditional knock-in(CKI)mice containing a HBM gain-of-function mutation(e.g.,G171V or A214V).Crossing these mice with ones expressing Cre under the control of various tissue-restricted promoters produced confounding results.In the?rst study,neither conditional deletion of Lrp5in osteoblast progenitors(with Dermo1-Cre)or mature osteoblasts(with2.3Col1a1-Cre),nor condi-tional knock-in of the Lrp5-G171V cDNA into mature osteoblasts (2.3Col1a1-Cre)affected vertebral bone volume density,osteoblast number,or bone formation rates as measured by static and dynamic histomorphometry(Yadav et al.,2008,2010).Rather Lrp5activity was inversely associated with serotonin synthesis in intestinal stem cells of the duodenum(Villin-Cre),which signaled back to osteoblasts to

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in?uence bone formation and regulate bone mass in an endocrine/ hormonal fashion(Yadav et al.,2008).Elevated levels of circulating serotonin were also observed in OPPG patients(Yadav et al.,2010), whereas patients with high bone mass due to a gain-of-function LRP5 mutation had lower than normal serotonin plasma levels(Frost et al., 2010).

Using different animals strains,microcomputed tomography and DEXA scanning,another study showed that conditional Lrp5deletion in pre-osteocytes and osteocytes(with Dmp1-Cre)reduced trabecular bone density in the distal femurs and L5vertebra,and weakened cortical bone strength.However Lrp5deletion in the intestinal stem cells(with a different villin promoter,Vil1-Cre)had no effect on bone mass(Cui et al.,2011).Accordingly,conditional expression of HBM alleles G171V or A214V(created by knocking in mutated exons 3and4only,in contrast to the cDNA used in the other study(Yadav et al.,2008))in osteocytes(with Dmp1-Cre)or the limb bud mesen-chyme(with Prx1-Cre)increased bone mass and strength(Cui et al., 2011).No signi?cant changes in serotonin levels were detected as a result of altering Lrp5activity(Cui et al.,2011).

These seemingly contradictory results could be attributed to differences in the genetic constructs,mouse models,methods used to measure bone density,bones tested,mouse environments, and/or serotonin assay techniques.These possibilities are relatively easy to address and doing so may in fact reveal important insights into fundamental cellular and endocrine mechanisms of bone formation. Lrp5is necessary for bone formation after loading(Akhter et al., 2004;Saxon et al.,2011)and PTH-induced high bone mass(O'Brien et al.,2008);results that best align with the need for Lrp5signaling in osteocytes(Cui et al.,2011).The role of Lrp5in less mature osteo-blasts,which arise from multiple sources(e.g.,pericytes,bone marrow derived mesenchymal progenitor cells,neural crest cells)could be less important if related Wnt/Dkk1/Scl(co-)receptors(e.g.,Lrp6)or alter-native growth and differentiation pathways compensate for altered Lrp5activity.Thus,exactly how Lrp5regulates bone mass is still unclear, but both direct regulation of osteoblast-lineage cells and indirect regulation via endocrine or paracrine signaling are viable options.

Beyond the existing controversy with the animal models,there is accumulating evidence that LRP5polymorphisms affect bone mass and fracture risk in human populations(Kiel et al.,2007a,2007b; Koay et al.,2004;Mizuguchi et al.,2004;Riancho et al.,2011; Rivadeneira et al.,2009;Sims et al.,2008;Urano et al.,2004;van Meurs et al.,2006,2008).Several of these LRP5variants alter canonical Wnt signaling(Kiel et al.,2007b),and a recent study showed that a lumbar spine bone mineral density-associated polymorphism, rs312009,affects Runx2binding to the LRP5promoter and alters gene transcription(Agueda et al.,2011).

3.1.2.Lrp6

Lrp6is more than70%identical to Lrp5at the amino acid level and has many similar properties as it binds to Wnts,Scl,and Dkks.An inherited autosomal dominant LRP6mutation that impairs Wnt signaling was found in a family with osteoporosis,coronary artery disease,and metabolic syndrome(Mani et al.,2007).Polymorphisms in LRP6are associated with low bone mineral density and fracture risk in humans(Riancho et al.,2011;Sims et al.,2008;van Meurs et al.,2006, 2008).Lrp6appears to have an earlier and perhaps broader role in development than Lrp5as Lrp6KO mice are not viable and show defective limb development(Pinson et al.,2000).However,Lrp6 heterozygous(+/?)mice have reduced total and trabecular bone mineral density(Holmen et al.,2004).Compound Lrp5?/?: Lrp6+/?mice have even lower bone mineral density than either the single or double heterozygotes as measured by DEXA;indicating that Lrp6and Lrp5genetically interact in skeletal development and have at least partially redundant functions in postnatal mice (Holmen et al.,2004).Mice carrying an Lrp6hypomorphic mutation, ringelschwanz(rs),that prevents it from being chaperoned to the cell surface also have reduced bone mineral density(Kubota et al.,2008). Osteoblast number and mineralization were not impaired in these animals,but Rankl expression was elevated on osteoblasts and corre-lated with increased bone resorption.Lrp6CKO mice have not yet been reported but are essential to determining its roles in osteoblast-lineage cells and perhaps unraveling clues to Lrp5's distinct functions.

Interesting biochemical studies revealed that Lrp6contributes to optimal PTH signaling in osteoblasts.In response to PTH stimulation, Lrp6binds the PTH receptor,PTHR1,and is phosphorylated by PKA (Wan et al.,2008,2011).This recruits Axin,stabilizesβ-catenin,and increases Tcf/Lef1-dependent gene transcription.The effects of PTH in Lrp6-insuf?cient animals were not determined;however,Lrp6 siRNAs ef?ciently blocked PTH stimulation of Tcf/Lef1activity in rat osteosarcoma cells(Wan et al.,2008).Lrp5was not tested in these assays because PTH stimulated bone formation in Lrp5-de?cient mice to the same extent as it did in wildtype mice(Iwaniec et al.,2007; Sawakami et al.,2006);however,later studies showed that Lrp5is necessary for increased bone formation,but not bone remodeling,in mice expressing a constitutively active(ca)PTHR1in osteocytes (O'Brien et al.,2008).Decreased Scl expression in the caPTHR1trans-genic animals appeared to be was responsible for the anabolic effects in osteocytes.The speci?c roles of Lrp5and Lrp6in PTH responsiveness will certainly become clearer in the near future as tissue-speci?c Lrp6 CKO mice are studied.

3.1.3.Lrp4

Lrp4(also known as Megf7)is an emerging regulator of bone mass.Lrp4-de?cient mice have polysyndactyly due to defective limb development in the apical ectodermal ridge(AER)as early as embryonic day9(Johnson et al.,2005;Simon-Chazottes et al.,2006;Weatherbee et al.,2006).Mice containing a Lrp4hypomorphic mutation,Lrp4ECD, exhibit impaired skeletal growth,reduced trabecular bone volume, and increased bone turnover(Choi et al.,2009).Lrp4antagonizes canonical Wnt signaling and modulates several important development signaling pathways involving Wnts,Bmps,Fgfs,and Shh in skeletal and tooth development(Johnson et al.,2005).Lrp4is expressed on human and rat osteoblasts and osteocytes(Leupin et al.,2011).It directly binds to Wnt antagonists,including Scl(Leupin et al.,2011)and Sost-dc1 (also called Wise)(Ohazama et al.,2008).Lrp4suppression by RNA interference allowed for osteoblast mineralization in vitro, even in the presence of Scl(Leupin et al.,2011).

LRP4appears to control bone density in humans as well.Like its cousins LRP5and LRP6,LRP4polymorphisms are associated with altered bone mineral density and lower fracture incidence in genome-wide association studies(Kumar et al.,2011;Rivadeneira et al.,2009; Styrkarsdottir et al.,2009).In addition,two mutations(R1170W and W1186S)in the extracellular region of LRP4were found in patients exhibiting bone overgrowth(Leupin et al.,2011).These amino acid substitutions impaired LRP4association with Scl,an inhibitor of bone formation.

3.2.Frizzleds

Fzds are highly versatile seven-pass membrane proteins that contribute to activation of bothβ-catenin and non-β-catenin signaling pathways by virtue of their interactions with Dvl and the existence of potential phosphorylation sites for cAMP-dependent PKA,PKC,and Ck2in their intracellular domains.There is a paucity of information about the roles of speci?c Fzds in bone biology;however,data from Fzd9-de?cient mice demonstrate that it contributes to optimal bone formation.

3.2.1.Fzd9

Patients with Williams–Beuren syndrome have low bone density and hemizygous deletion of a region on chromosome7that includes

8 D.G.Monroe et al./Gene492(2012)1–18

FZD9(Francke,1999).Fzd9KO and heterozygote mice have reduced bone mineral density and low bone formation rates(Albers et al., 2011).β-catenin was not affected by Fzd9-de?ciency,but Stat1levels were reduced.This led to the reduction of interferon-stimulated genes,including Isg15,which encodes an ubiquitin-like molecule. Interestingly Isg15-de?cient mice also have low bone density.Isg15 overexpression restored the ability of Fzd9-de?cient osteoblasts to mineralize their extracellular matrix in vitro.Fzd9expression was upregulated during osteoblast maturation.Thus,Fzd9is a crucial regulator of late stages of bone mineralization.

4.Wnt antagonists

4.1.Secreted Wnt antagonists:Dkks,Sfrps,Wif1,Sost,and Sost-dc1

Secreted Wnt antagonists generally utilize two distinct mechanisms to inhibit Wnt signaling.Sfrps,Cerberus and Wif1bind to Wnts and/or Fzds to directly interfere with association of the ligand with its receptor (Fig.1).In contrast,Dkk,Sost and Sost-dc1(Wise)bind to the Lrp5/6 co-receptor and inhibit Wnts from associating with the Fzd/Lrp receptor complex.Existing data suggest that some of these inhibitors are viable targets for new anabolic therapeutics.

4.1.1.Dickkopfs(Dkk)

The Dickkopf factors(Dkk1–4)have differing expression patterns during embryonic and postnatal development(Nie et al.,2005;Witte et al.,2009).Dkks bind and sequester the Lrp5/6and Krm1/2membrane complex to inhibit Wnt activity.Recent studies highlight the impor-tance of Dkk1,Dkk2,and Dkk3in osteoblastic function.

4.1.1.1.Dkk1.Dkk1is a well-characterized secreted Wnt inhibitor that is active in many tissues(reviewed in(Pinzone et al.,2009)).Several lines of clinical evidence indicate that DKK1regulates bone mass in humans.The?rst is that the gain-of-function mutations in LRP5 responsible high bone mass inhibit the ability of LRP5to bind DKK1(Ai et al.,2005;Boyden et al.,2002).Second,high DKK1 production by malignant plasma cells leads to osteolytic bone lesions in patients with multiple myeloma and blocks osteoblast differentiation (Tian et al.,2003).Data from various animal models con?rm that Dkk1 suppresses Wnt signaling and inhibits bone formation.Dkk1?/?mice die shortly after birth with severe developmental abnormalities(del Barco Barrantes et al.,2003),but Dkk1+/?mice have increased bone formation and bone mass without a compensatory change in bone resorption(Morvan et al.,2006).Conversely,transgenic overexpression of Dkk1using the rat collagen1α1promoter speci?c to osteoblasts signi?cantly decreased osteoblast number,bone formation rate,and serum osteocalcin levels(Fleming et al.,2008;Guo et al.,2010;Li et al.,2006;Yao et al.,2011).Finally,mice harboring the hypomorphic Dkk1d(doubleridge)allele,which display forelimb postaxial poly-syndactyly,are informative models to study Dkk1activity in bones (MacDonald et al.,2004).The trabecular and cortical bone density parameters of hypomorphic progeny of Dkk1+/?and Dkk1+/d mice are inversely proportional to the level of Dkk1expression. (Macdonald et al.,2007).These studies demonstrate that Dkk1is a negative regulator of bone in vivo.

In recent years,signi?cant interest in DKK1suppression as a treat-ment modality for various bone diseases has led to the development of an array of DKK1-neutralizing antibodies.In several murine multiple myeloma models,DKK1antibodies signi?cantly increased osteoblast numbers,serum osteocalcin levels,and trabecular bone volume (Diarra et al.,2007;Fulciniti et al.,2009;Heath et al.,2009).A Dkk1 antibody also increased bone formation at endosteal bone surfaces in a mouse model of ovariectomy-induced osteopenia(Glantschnig et al.,2011).Dkk1is expressed in most tissues,thus using Dkk1 antibodies to treat a chronic and systemic disease like osteoporosis may produce unwanted effects.However,local delivery of Dkk1-neutralizing antibodies may be a treatment option for fractures non-union because Dkk1inhibits fracture repair(Chen et al., 2007).Indeed,in a murine fracture repair model,anti-Dkk1anti-bodies increased the callus area,bone mineral content/density,and biomechanical properties of the injured bone(Komatsu et al., 2010).Collectively,these reports suggest that Wnt pathway activation through suppression of Dkk1may offer therapeutic treatments for select bone diseases and orthopedic conditions.

4.1.1.2.Dkk2.The molecular functions of Dkk2vary with cellular context.Like Dkk1,Dkk2effectively blocks Wnt1-dependent activation of Lef1/Tcf target genes and inhibits both the Wnt and osteogenic differ-entiation pathways in osteoarthritic osteoblasts(Chan et al.,2011b). However,Dkk2also activatesβ-catenin in Xenopus embryos(Wu et al.,2000).These opposing effects may be modulated by Krm2,which converts Dkk2from an agonist to an antagonist of Lrp6(Mao and Niehrs,2003).Dkk2-null mice are osteopenic with suppressed bone formation parameters(Li et al.,2002).Osteoblast cultures derived from bone marrow and calvaria of Dkk2?/?mice mineralize at a slower rate than wildtype cells.These data suggest that Dkk2stimulates bone formation at least in early development,in stark contrast to the bone inhibitory functions of Dkk1.Interestingly,Wnt7b may facilitate Dkk2induction of osteogenesis.Dkk2inhibited bone formation in the absence of Wnt7b,but induced terminal osteoblast differentiation in the presence of high Wnt7b levels(Li et al.,2005a).Thus,the effects of Dkk2on osteoblasts is critically dependent on cellular context,partic-ularly Krm2and Wnt7b levels.

4.1.1.3.Dkk3.Little information exists regarding Dkk3's activity in bone,but it was temporally co-expressed with osteogenic genes in Bmp2-producing C3H10T1/2mesenchymal progenitor cells implanted into mice(Aslan et al.,2006).The Dkk3-expressing cell im-plants had decreased bone quality as measured byμCT and biolumi-nescence imaging.Further investigation is required to fully determine Dkk3's role in bone formation.

4.1.2.Secreted frizzled-related proteins(Sfrps)

Sfrp1-5are secreted,cysteine-rich glycoproteins that share high homology to the Fzd receptors.Sfrps antagonize Wnts though direct binding,thereby preventing their functional association with Fzds on the cell surface(Kawano and Kypta,2003).Several Sfrps are expressed in skeletal tissue and cells of the osteoblastic lineage. They have varying effects on bone development and osteoblast function.

4.1.2.1.Sfrp1.Sfrp1action in bone has been extensively explored.Tar-geted disruption of Sfrp1increased trabecular but not cortical bone mineral density to a similar extent as PTH(Bodine et al.,2004, 2007),whereas transgenic Sfrp1overexpression decreased bone den-sity and attenuated the bone anabolic effects of PTH(Yao et al.,2010). Sfrp1expression is increased by dexamethasone and may be involved in glucocorticoid-induced osteoporosis(Wang et al.,2005).Mecha-nistically,Sfrp1appears to affect cell viability and maturation as its deletion reduced osteoblast and osteocyte apoptosis in vivo,and cell proliferation and differentiation in vitro(Bodine et al.,2004).In an immortalized human osteoblast cell line,Sfrp1potently suppressed Wnt signaling(Bodine et al.,2005).Similarly,Sfrp1signi?cantly in-creased osteoblast apoptosis with concomitant decreases in bone mineral density,trabecular bone volume,and cortical bone area in rat femurs(Wang et al.,2005).Sfrp1also binds Rankl and blocks osteoblast-induced osteoclastogenesis(Hausler et al.,2004).Collec-tively,these data clearly demonstrate that Sfrp1inhibits osteoblast viability and coupling to osteoclasts.

As with other secreted Wnt inhibitors,there is signi?cant interest in isolating Sfrp1antagonists to treat low bone mass conditions.A high throughput screen of potential small molecule inhibitors

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revealed a class of piperidinyl diphenylsulfonyl sulfonamide com-pounds that bind Sfrp1and inhibit its activity(Bodine et al.,2009; Moore et al.,2010).These compounds blocked Sfrp1-mediated apo-ptosis of preosteoblasts and stimulated bone formation in vitro (Moore et al.,2009);however,the effectiveness of these compounds in vivo has not been reported.In a rodent model of periodontal bone loss,Sfrp1polyclonal antibodies suppressed bone resorption and de-creased pathogen-induced in?ammation(Li and Amar,2007).Like Dkk1antagonists,Sfrp1-based therapeutics might be best suited for such localized conditions because of its broad expression pattern in multiple tissues.

4.1.2.2.Sfrp4.Sfrp4is expressed in human mesenchymal stem cells and in areas of bone formation at E1

5.5in the developing mouse limb(Etheridge et al.,2004;Witte et al.,2009).Transgenic overex-pression of Sfrp4using the osteoblast-directed rat2.3kb Col1a1pro-moter suppressed osteoblast proliferation and decreased bone formation(Nakanishi et al.,2008).Moreover,transgenic mice overex-pressing Sfrp4under the control of serum amyloid P promoter,which drives postnatal secretion of Sfrp1from the liver and into serum, exhibited low bone mass(Cho et al.,2010).Recombinant Sfrp4also inhibited osteoblast proliferation and partially suppressed the activity of Wnt3a in vitro(Nakanishi et al.,2006).Collectively,these data demonstrate that Sfrp4negatively regulates bone formation and de-creases bone mineral density through the inhibition of Wnt signaling.

SFRP4polymorphisms were associated with altered hip and spine bone mineral density in numerous populations(Cho et al.,2009; Karasik et al.,2003;Styrkarsdottir et al.,2008).Furthermore,the Sfrp4locus was associated with lower bone mineral density in the senescence-accelerated mouse P6(Nakanishi et al.,2006).

4.1.3.Wnt inhibitory factor(Wif)-1

Wif1is a secreted factor that inhibits Wnt signaling through direct interaction with Wnts(e.g.,Wnt-3a,-4,-5a,-7a,-9a,-11) (Malinauskas et al.,2011;Surmann-Schmitt et al.,2009).Wif1is expressed during Bmp2-induced osteoblast differentiation of C2C12 and MC3T3-E1cells.Moreover,WIF1was elevated in calvarial sutures of craniosynostosis patients(Coussens et al.,2007).These results sug-gest that Wif1may be part of a negative feedback loop that controls osteoblast differentiation and maturation(Vaes et al.,2005).Wif1 KO mice have a normal skeleton but are more sensitive to radiation-induced osteosarcomas(Kansara et al.,2009).Similarly,mice with osteoblast-speci?c Wif1overexpression display no overt bone pheno-type,but have disrupted stem cell quiescence leading to a loss of self-renewal potential,suggesting an important role for Sfrp4in regener-ation of the progenitor cell niche(Schaniel et al.,2011).

4.1.4.Sclerostin/SOST

Inactivating mutations in the SOST gene,which encodes the pro-tein Sclerostin(Scl),cause two rare bone sclerosing disorders,scler-osteosis and van Buchem disease.These diseases are characterized by endosteal hyperostosis,progressive generalized osteosclerosis, and high bone mass associated with increased osteoblastic activity and elevated bone formation markers(Wergedal et al.,2003).The mutations introduce premature transcriptional stop codons,interfere with splicing,or delete regulatory elements in SOST,thereby prevent-ing osteocytes from secreting suf?cient levels of fully functional Scl (Balemans et al.,2001,2002;Brunkow et al.,2001;Staehling-Hampton et al.,2002).As is often true in medicine,identi?cation of the genetic and molecular origin of these rare diseases has revealed an important mechanism in normal physiological processes and unleashed a?urry of activity to translate the information into therapy for more common disorders.

Unlike Dkks,Sfrps,and Wise,Scl is produced primarily by bone cells and is abundant in the osteocytic canalicular system(van Bezooijen et al.,2004;Winkler et al.,2003)but has also been detected in cementocytes in teeth,mineralized hypertrophic chondrocytes in the growth plate,and osteoarthritic cartilage(Chan et al.,2011a; van Bezooijen et al.,2009).Scl binds Lrp5/6and inhibits their associ-ation with Fzd and Wnts(Li et al.,2005b;Semenov et al.,2005).Scl inhibits proliferation and differentiation and stimulates apoptosis of osteogenic cultures(Sutherland et al.,2004;van Bezooijen et al., 2004;Winkler et al.,2003).In support of the Wnt inhibitory function of Scl in vivo,canonical Wnt signaling,bone density,and bone me-chanical strength are elevated in Sost knockout mice(Krause et al., 2010;Li et al.,2008);whereas transgenic overexpression of Sost in-duced osteopenia(Loots et al.,2005;Winkler et al.,2003).Collective-ly,these data clearly demonstrate that Scl is an important negative regulator of bone formation.

By virtue of its relatively exclusive expression in bone and its role in repressing bone formation from the extracellular space,Scl is an at-tractive target for anabolic therapeutics.Scl neutralizing antibodies have shown ef?cacy in multiple pre-clinical models(e.g.,rodents, non-human primates)and more recently in clinical trials for osteopo-rosis(reviewed in(Rachner et al.,2011)).For example,Scl antibodies increased bone mass and prevented bone loss associated with estro-gen de?ciency in ovariectomized rats(Li et al.,2009).In phase-2clin-ical studies,a fully humanized Scl neutralizing antibody increased bone formation parameters in post-menopausal osteoporotic women(Padhi et al.,2011).These studies and others indicate that Scl inhibitors may provide skeletal bene?ts for patients with osteopo-rosis and other diseases of low bone mass.Scl antibodies may also im-prove outcomes of orthopedic stabilization and?xation procedures that are complicated by low bone volumes.

Scl has quickly emerged as an important modulator of anabolic signaling pathways in bone,particularly PTH stimulation and me-chanical loading.Intermittent PTH stimulates bone formation;how-ever,the molecular mechanisms underlying this response are not fully understood(reviewed in(Kramer et al.,2010b)).PTH suppresses Scl expression both in vitro and in vivo(Bellido et al.,2005;Keller and Kneissel,2005)by inhibiting myocyte enhancer factor2,which normally activates Sost transcription through a speci?c enhancer element(Leupin et al.,2007).PTH-dependent bone anabolism is suppressed in mice where Sost is overexpressed,indicating that Scl levels can modulate PTH-induced bone formation(Kramer et al., 2010c;O'Brien et al.,2008).Scl expression is also suppressed in osteocytes by mechanical loading in vivo(Robling et al.,2006).This may contribute to high Wnt/β-catenin signaling that occurs after mechanical loading(Robinson et al.,2006).

4.1.

5.Sost-dc1(Ectodin,Wise,Usag1)

Sost-dc1(sclerostin domain containing1)is a secreted factor that belongs to the Dan/Cerberus family of proteins.It binds to Bmps to neutralize their activity(Itasaki et al.,2003;Kassai et al.,2005; Laurikkala et al.,2003;Yanagita et al.,2004).It also blocks Wnt1, Wnt3a,and Wnt10b activities in various cellular models(Beaudoin et al.,2005;Blish et al.,2008;Lintern et al.,2009).Sost-dc1inhibits Wnt activity by binding Lrp6(Itasaki et al.,2003;Lintern et al., 2009)and possibly Lrp4(Ohazama et al.,2010).Sost-dc1-de?cient mice have extra teeth due to excessive Bmp signaling and reduced apoptosis of developing odontogenic mesenchymal cells(Ahn et al., 2010;Kassai et al.,2005;Munne et al.,2009;Murashima-Suginami et al.,2008).The bone phenotype of the Sost-dc1-null models has not been characterized;however,several lines of evidence suggest a role in the skeleton.SOST-DC1polymorphisms were associated with attainment and maintenance of peak bone mass in Chinese women (He et al.,2011).Furthermore,Wnt10b suppressed SOST-DC1expres-sion in a human osteosarcoma cell model(Modder et al.,2011).Since osteoblast function is critically dependent on both Bmp and Wnt signals,a potential role of Sost-dc1in osteoblasts is intriguing,although further research is needed to clarify its role in bone.

10 D.G.Monroe et al./Gene492(2012)1–18

4.2.Transmembrane modulators of Wnt signaling

Several transmembrane proteins modulate Wnt signaling pathways by binding to Wnts or the secreted antagonists discussed above.These molecules include Kremen(Krm)1,Krm2,and the receptor tyrosine kinases,Ror2and Ryk.

4.2.1.Kremen1/2

Krm1and Krm2are single-pass transmembrane co-receptors for Dkk1.Krms and Dkk1form a ternary complex with Lrp6,which is rapidly endocytosed within5min to reduce Wnt/β-catenin signaling (Mao et al.,2002).Krms are expressed in developing limb buds.Double mutant Krm1?/?:Krm2?/?mice have elevated Wnt signaling,expanded AERs and ectopic postaxial forelimb digits(Ellwanger et al.,2008). Ectopic growth of digits is enhanced in triple mutant Krm1?/?: Krm2?/?:Dkk1+/?mice,demonstrating a genetic interaction between Krms and Dkk1in limb development.Double mutant Krm1?/?: Krm2?/?mice had increased bone volume and bone formation rates at12weeks of age(Ellwanger et al.,2008).Single mutant Krm1?/?and Krm2?/?mice had normal bone volume and bone formation rates at this age,but the Krm2?/?mice developed high bone mass associated with increased bone formation12weeks later,at 24weeks of age(Schulze et al.,2010).Transgenic expression of Krm2in mature osteoblasts under control of the2.3Col1a1promoter suppressed osteoblast maturation and Opg production(Schulze et al., 2010).Cortical strength was reduced and osteoclast activity was elevated.Krm2is predominantly expressed in bones of6week-old mice,whereas as Krm1is expressed in bone as well as other tissues (Schulze et al.,2010).These data suggest that Krm2is a potential bone-speci?c target for future for anabolic agents.

4.2.2.Ror2

The Ror family of membrane-spanning tyrosine kinases bind certain Wnts either alone or as Fzd co-receptors to activate non-β-catenin signaling in mammalian tissues(Grumolato et al.,2010;Minami et al.,2010).Wnt5a,for example,induces the formation of a complex consisting of Lrp5/6,Ror1/2,and Fzd2(Sato et al.,2010).Via Ror2, Wnt5a blocks Wnt3a-mediatedβ-catenin activation(Mikels and Nusse,2006).ROR2mutations are linked to several skeletal disorders (e.g.,dominant brachydactyly type B and recessive Robinow syndrome), further supporting a role for this pathway in endochondral bone forma-tion(Afzal et al.,2000;Angers and Moon,2009;DeChiara et al.,2000; Oldridge et al.,2000).

4.2.3.Ryk

Ryk is an atypical tyrosine kinase receptor that is predicted to lack intrinsic enzymatic activity,but may associate with Src kinases (Hovens et al.,1992;Wouda et al.,2008).Ryk's?y homolog,Drl, binds Wnt5a in the absence of Fzd or Dvl to regulate growth cone guidance(Bonkowsky et al.,1999).In HEK293T cells,RYK can be co-immunoprecipated with Wnt1,Wnt3a,Fzd and Dvl,and is required for Wnt-mediatedβ-catenin activation(Lu et al.,2004).Ryk activities in bone cells have not been reported.

5.β-catenin and associated intracellular proteins

5.1.Ctnnb1(β-catenin)

β-catenin is a cytoplasmic and nuclear protein encoded by the Ctnnb1gene.It is a key link in numerous signaling cascades,including the“canonical Wnt pathway”,is essential for embryonic development, and is hyperactivated by mutations in many cancers.Wnt ligation of Lrp5/6and Frizzled receptors inactivates theβ-catenin destruction complex consisting of Apc,Axin,Ck1,Gsk3,Wtx,and the E2ubiquitin ligase,βTrCP.Asβ-catenin accumulates,some is transported to the nucleus where it interacts with Lef1/Tcf transcription factors to regulate numerous genes,including Axin2,which in turn can provide feedback inhibition(Jho et al.,2002).β-catenin proteolysis is triggered by phosphorylation of several serine residues in its N-terminus by Ck1 and Gsk3β.Deletion of exon3in Ctnnb1,removes these residues and produces a stable protein that acts as a gain-of-function mutation (Harada et al.,1999).Over the last decade,numerous genetic studies were performed using mice in which exon3(to activateβ-catenin)or exons6–10(to eliminateβ-catenin)of Ctnnb1is?anked by loxP se-quences to dissectβ-catenin's role(s)in skeletal development through gain-and loss-of function,respectively.

β-catenin is essential for controlling mesenchymal cell fate decisions and linking bone formation to bone resorption.Ctnnb1-deletion in mesenchymal progenitors(as early as E9.5)caused severe defects in skeletal formation,characterized by reduced mineralization,defective osteoblastogenesis,and ectopic chondrogenesis(Brault et al.,2001;Day et al.,2005;Hill et al.,2005;Hu et al.,2005;Rodda and McMahon, 2006).Targeted Ctnnb1-deletion in committed osteoblast-lineage cells at a later stage in development(E14.5)also reduced bone mass,but surprisingly osteoblast numbers and bone formation rates were normal(Glass et al.,2005;Holmen et al.,2005;Kramer et al.,2010a). These Ctnnb1-de?cient osteoblasts and osteocytes produced less Opg, which allowed for more interactions between Rankl-positive osteo-blasts and Rank-expressing osteoclasts and promoted bone resorption.

Increasingβ-catenin activities through deletion of exon3produced nearly opposite phenotypes as the knockout mutations and early lethality.Thus,Ctnnb1gain-of-function mutations in mature osteo-blasts and osteocytes caused premature and excessive ossi?cation by reducing osteoclast numbers without changing osteoblast numbers (Glass et al.,2005;Rodda and McMahon,2006).In animals where β-catenin was activated in premature limb bud and craniofacial mesen-chyme(with Prx1-Cre),appendicular and skull bone elements were absent,suggesting thatβ-catenin stabilization negatively impacts this early stage of differentiation(Hill et al.,2005).

Because of the different phenotypes of the Ctnnb1and Lrp5-de?cient mice,it is crucial to discussβ-catenin's roles outside of the Wnt signaling pathway.Notably,β-catenin associates with cadherins to regulate epithelial cell growth,cell adhesions and migration. N-cadherin overexpression inhibits osteoblast proliferation and survival by blocking Wnt3a,PI3K/Akt and Erk signaling(Hay et al., 2009).β-catenin also links the membrane to the actin-cytoskeleton,which may transmit signals responsible for contact-mediated inhibition of cell growth,or in the case of bone homeostasis, signals from mechanical strains.Several reports demonstrated that mechanical loading activated a Lef/Tcf reporter and promoted nuclear β-catenin localization in primary calvarial cells(Armstrong et al., 2007;Hens et al.,2005).In murine calvarial osteoblasts,mechanical loading by biaxial strain increased nuclearβ-catenin levels through the activation of Akt and consequent inactivation of Gsk3β(Case et al.,2008).Gsk3βinhibition by mechanical strain stimulated Nfatc1as well asβ-catenin signaling to induce osteogenesis and inhibit adipo-genesis of multipotent mesenchymal cells(Sen et al.,2008).In osteo-cytes,the mechanosensory cells of bone,?uid?ow sheer stress indirectly stabilized and stimulated nuclear translocation ofβ-catenin through prostaglandin E2(PGE2)and EP2/4synthesis,PI3K/Akt and cAMP/PKA signaling,and Gsk3βinactivation to protect osteocytes from glucocorticoid apoptosis and stimulate gap junctions(Bonewald and Johnson,2008;Kamel et al.,2010;Kitase et al.,2010;Xia et al., 2010).Together these data indicate that mechanical strain activates multiple pathways,many of which converge onβ-catenin to control cell fate and promote bone formation.

Recently,the effects of alteringβ-catenin levels in osteoclast lineage cells were reported(Wei et al.,2011).Using a variety of Cre drivers,it was determined thatβ-catenin regulates osteoclastogenesis in a dosage-dependent manner(Table2).A minimum amount of β-catenin was required to induce the proliferation of osteoclast pro-genitors as complete Cttnb1deletion caused osteopetrosis.In contrast,

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Cttnb1haploinsuf?ciency accelerated osteoclastogenesis and produced an osteoporotic phenotype.High levels of constitutively activeβ-catenin inhibited osteoclast maturation and bone resorption to cause osteopetrosis.Wnt3a and two Gsk3βinhibitors attenuated osteoclast differentiation(Wei et al.,2011).Moreover,Wnt3a stabilized β-catenin in human osteoclast precursor cells from multiple myeloma patients in vitro to suppress osteoclast differentiation(Qiang et al., 2010).Rankl treatment suppressedβ-catenin expression in osteoclasts to suppress proliferation and induce terminal differentiation programs (Wei et al.,2011).Further analysis showed thatβ-catenin promotes osteoclast precursor proliferation in response to M-CSF by inducing expression of Gata2and Evi1,but blocks Rankl-induced osteoclast maturation by impairing c-Jun activity.These data suggest that thera-peutic strategies designed to increase bone mass by activating the canonical Wnt pathway may confer both anabolic and anti-resorptive effects.

Alterations inβ-catenin sequence and/or activity contribute to numerous diseases in humans.More than half of human bone and soft tissue sarcomas have excessβ-catenin activity(Iwao et al., 1999;Vijayakumar et al.,2011).Tumors have not yet been reported in mice expressing gain-of-functionβ-catenin mutations;however, benign rib osteomata were found in80%of animals(Glass et al., 2005),suggesting that sustainedβ-catenin activation combined with other genetic or epigenetic events may promote carcinogenesis. CTNNB1polymorphisms were linked to altered bone mineral density in some human population studies(Rivadeneira et al.,2009),but the molecular consequences of these variants have not been elucidated.

5.2.Adenomatous polyposis coli(Apc)

Apc is a tumor suppressor andβ-catenin binding protein.Defects in APC cause familial adenomatous polyposis,an autosomal dominant pre-malignant condition that usually progresses to colon cancer. Apc's major function in the cell is to inhibitβ-catenin activity.Apc is a scaffold for other components of theβ-catenin destruction complex in the cytoplasm.Apc also associates withβ-catenin in the nucleus where it preventsβ-catenin from associating with Lef/Tcf transcription factors(Neufeld and White,1997;Neufeld et al.,2000a,2000b).Although not as thoroughly studied asβ-catenin or Lrp5at the genetic level,the phenotypes of Apc CKO mice are consistent with its role as a negative regulator ofβ-catenin activity,as well as withβ-catenin being a crucial regulator of bone resorption.In both studies,conditional deletion of Apc in either chondrocytes(with Col2a1-Cre)or osteoblasts(with OCN-Cre) elevatedβ-catenin levels and produced early postnatal lethality as all mice died within5weeks(Holmen et al.,2005;Miclea et al.,2009). Similar to the Cttnb1CKI models,Apc deletion in mature osteoblasts increased trabecular bone volumes,but its deletion in progenitors cells caused severe delays and skeletal malformations.In the OCN-Cre driven Apc CKO mice,no defects in osteoblast development were detected in vitro or in vivo;however,osteoclasts were not detected in histological sections.Opg levels were elevated in these Apc CKO mice, whereas Rankl mRNA levels were reduced in osteoblasts.Cttnb1CKO mice made by the same group with the same OCN-Cre driver had nearly the opposite phenotype.Moreover,mice carrying osteoblast-speci?c deletions of both Apc and Cttnb1had phenotypes resembling the Cttnb1CKO animals.Together,these data support the conclusion that Apc is a negative regulator ofβ-catenin in skeletal progenitors and mature osteoblasts.SNPs in APC were found associated with altered trabecular volumetric bone mineral density in several human population studies(Miclea et al.,2010;Yerges et al.,2009).

5.3.Axin1/2

Axin1and Axin2are functionally equivalent scaffolding proteins required for the assembly of theβ-catenin destruction complex that includes Gsk3β,Dvd,Apc,and Wtx(Chia and Costantini,2005).In the presence of Wnt ligands,Axin and other components of the destruction complex are recruited to Lrp5/6at the cell membrane where they facilitate downstreamβ-catenin signaling(Bilic et al., 2007;MacDonald et al.,2009;Niehrs and Shen,2010;Zeng et al.,2005).Axin1is widely expressed and Axin1-de?cient mice do not survive past E9.5due to forebrain and neural tube defects (Zeng et al.,1997).In contrast,Axin2exhibits a more restricted expression pattern,and is upregulated by Wnt/β-catenin/Tcf signaling. Thus,Axin2is a negative feedback inhibitor of the Wnt/β-catenin pathway(Jho et al.,2002).Axin2KO mice are born with no noticeable morphologic abnormalities;however,skull doming was evident by postnatal day28(Yu et al.,2005).Further analysis revealed that nuclear β-catenin expression is elevated in cranial bones by seven days of age, leading to increased osteoblast progenitor proliferation,increased osteoblast differentiation,and craniosynostosis characterized by pre-mature fusion of the frontal/metopic suture(Liu et al.,2007a).Bone mass and strength of the axial skeleton was increased at six months of age in Axin2KO mice due to increased osteoblast differentiation, enhanced osteoblast function,and decreased osteoclast formation (Yan et al.,2009).Introducing Ctnnb1-de?ciency onto the Axin2 KO background attenuated the increased osteoblast activity and craniosynostosis phenotype in these mice(Liu et al.,2007a;Yan et al.,2009),whereas conditional activation ofβ-catenin recapitulated many aspects of the Axin2KO skeletal phenotype(Mirando et al., 2010),con?rming the role ofβ-catenin signaling in this model.

Interestingly,Axin2?/?mice displayed a runted phenotype com-pared to wildtype littermates.This disrupted growth was compounded in double mutant Axin2?/?:Axin1+/?mice(Dao et al.,2010).The runted phenotype appears to be due to Axin2's role in chondrocyte maturation.Axin2?/?mice have shorter hypertrophic zones in the growth plate and enhanced expression of type10collagen,a marker of mature chondrocytes(Dao et al.,2010).Thus,under normal circumstances Axin2expression inhibits late chondrocyte differentia-tion,as it does in osteoblasts.Because of its key role in osteoblast and chondrocyte development,Axin2may also contribute to musculoskeletal repair.Indeed,Axin2-de?cient mice demonstrated rapid healing in fracture models(Minear et al.,2010).

5.4.Gsk3β

Glycogen synthase kinases(Gsk)3alpha and beta are highly conserved and ubiquitous serine/threonine enzymes that participate in multiple signaling pathways,including both canonical and non-canonical Wnt signaling.It has long been known that Gsk3phosphory-lates multiple components of Wnt pathways,includingβ-catenin,Axin, and Apc(reviewed previously in(Westendorf et al.,2004)).Gsk3 phosphorylation of the N-terminus ofβ-catenin promotes its degradation by the26S proteosome.Seemingly paradoxically,Gsk3also has a positive role in promoting Wnt signaling.In response to ligands,Gsk3and Axin move to the membrane where Gsk3phosphorylates Wnt receptors, Lrp5/6(Bilic et al.,2007;MacDonald et al.,2009;Niehrs and Shen, 2010;Zeng et al.,2005).Lrp5/6phosphorylation results in the formation of a large multi-protein signalosome,which consequently sequesters Gsk3and facilitatesβ-catenin accumulation and enhanced gene transcription(reviewed by(Niehrs and Shen,2010)).

There is much evidence that Gsk3inhibition promotes bone forma-tion in vivo.Gsk3βsuppression by genetic deletion or pharmacological inhibition enhances bone density.Gsk3βKO mice do not survive embryogenesis;however,Gsk3β+/?mice have higher trabecular bone volume density,more osteoblasts per bone surface and increased bone formation rates(Kugimiya et al.,2007;Noh et al.,2009).The numbers of osteoclasts per bone perimeter and eroded surface areas were also elevated,indicating that increased bone formation was coupled to increased resorption(Kugimiya et al.,2007).Osteoblasts from Gsk3β+/?mice had more Runx2activity because the

12 D.G.Monroe et al./Gene492(2012)1–18

phosphorylation of an inhibitory residue in Runx2was suppressed in the absence of adequate Gsk3βlevels.Interestingly,Gsk3βhaploinsuf?-ciency or lithium chloride treatment rescued the cleidocranial dysplasia in Runx2+/?animals(Kugimiya et al.,2007).Lithium chloride also rescued the low bone mass phenotypes of Lrp5?/?and SAMP6 mice,and increased bone mass in wildtype mice(Clement-Lacroix et al.,2005).Other small molecule Gsk3βinhibitors also increase bone mass in wildtype and ovariectimized animals and improve vertebral strength(Clement-Lacroix et al.,2005; Kulkarni et al.,2006).Reductions in bone marrow adiposity and enhancements in osteocytic responses to mechanical strain were also observed,suggesting that Gsk3βin?uences mesenchymal cell fate and osteocyte responses to loading(Case et al.,2008;Sen et al.,2008).These positive effects on early progenitors and terminal osteocytes may be the primary reasons why bone formation is enhanced as a result of Gsk3βinhibition,despite increased bone resorption.

Oral lithium chloride has been a treatment option for bipolar disease for more than a half-century.Some studies indicate that patients taking lithium have lower fracture rates and less bone turnover than normal individuals,while other surveys did not observe any differences(Vestergaard et al.,2005;Wilting et al.,2007).Thus, lithium and other Gsk3inhibitors may be safe for short-term anabolic uses in some patients;however,long-term use and effectiveness remains to be proven.

In summary,Gsk3is an important contributor to bone formation in vivo and osteoblast maturation in vitro.Existing in vivo models are insuf?cient to determine whether enhanced bone formation is solely due to osteoblastic responses.Future studies in which Gsk3βis conditionally deleted at different stages of osteoblast and osteoclast development may unravel some of the complexities observed in vitro, with the germline knockout/heterozygote mice,and with lithium chloride response in patients.Finally,Gsk3proteins participate in multiple signaling pathways besides Wnt,Lrp5/6,Ror1/2,andβ-catenin.Most notable are their roles in G protein coupled receptor, Akt,and Bmp2signaling pathways.Untangling these pathways will require information on molecular structures and posttranslational modi?cations of relevant proteins.

5.5.Tcf7and Lef1transcription factors

T cell factors7(Tcf7)and lymphoid enhancer binding factor(Lef1) are nuclear proteins that link Wnt signaling andβ-catenin to the genome.They bind to the DNA sequence YCTTTGWW via a C-terminal DNA binding domain and toβ-catenin via N-terminal sequences.Central regions of Tcf7s and Lef1interact weakly with β-catenin and strongly with co-repressors,including Hdacs and Tle proteins.Temporal and spatial expression patterns,alternative splicing,and differential promoter usage of Lef1and the three Tcf7genes,Tcf7(Tcf1),Tcf7L1(Tcf3),and Tcf7L2(Tcf4),affect their activities during skeletal development(Glass et al.,2005; Waterman,2004;Westendorf et al.,2004).In adults,their expression is typically restricted to regenerating tissues.Thus far,no polymor-phisms in TCF7or LEF1have been associated with altered BMD;however, Tcf7(Tcf1)or Lef1KO mice indicate that they play important roles in bone turnover.

5.5.1.Tcf7(Tcf1)

Tcf1?/?mice were originally described as being of normal size (Verbeek et al.,1995;Roose et al.,1999);however,more careful analysis of the skeleton revealed modest decreases in bone mineral density at one month of age(Glass et al.,2005).These bone mass reductions were attributed to increased bone resorption as osteoclast numbers and activity were elevated,Opg levels were reduced,and no changes were noted in osteoblast numbers or function.The results were consistent with the Ctnnb1CKO animals reported in the same study.Mice heterozygous for Ctnnb1and Tcf7(Tcf1)also had reduced bone density as a result of increased osteoclast activity,demonstrating a genetic link between the molecules(Glass et al.,2005).Tcf7(Tcf1), Tcf7L2(Tcf4),andβ-catenin associated with the Opg promoter and Lef1cooperated withβ-catenin to activate Opg promoter activity in vitro(Glass et al.,2005).Tcf1also binds to the Runx2promoter, which was activated by canonical Wnt signaling(Gaur et al.,2005). Together these data demonstrate that Tcf7is a crucial mediator of β-catenin signaling in mature osteoblasts and indirectly regulates osteoclast activation.

5.5.2.Lef1

Lef1-de?cient mice die within a few days of birth with multi-organ defects due to impaired epithelial and mesenchymal cell interactions (van Genderen et al.,1994).Bone mass was not measured in these mice because of the early postnatal lethality(Noh et al.,2009;van Genderen et al.,1994);however,young female heterozygotes in this Lef1strain had low trabecular bone mass with reduced osteoblast activity(Noh et al.,2009).This phenotype was temporal and normal-ized as the animals aged.Male Lef1+/?animals did not have low bone mass unless the androgen receptor was inactivated(Noh et al.,2009). Thus,Lef1may have an age-and gender-related role in bone homeo-stasis.A new Lef1KO model in which a5'-exon that encodes the β-catenin binding domain was targeted has a similar gross phenotype as the original Lef1KO mice(JJW,unpublished).Bone structures were measurable by microcomputed tomography in a few animals of this strain that lived to three weeks of age.These Lef1KO mice have dra-matically lower trabecular bone mass and fewer trabeculae;howev-er,no changes were observed in heterozygotes.

The different bone phenotypes of the heterozygotes in the two Lef1mutant strains of mice may provide important information and insight into the functions of Lef1isoforms.Lef1contains two pro-moters that drive expression of a full-length protein(Lef1)and an N-terminally truncated isoform(Lef1ΔN).The original Lef1-de?cient mouse(van Genderen et al.,1994)would eliminate both isoforms, whereas the newer Lef1-de?cient mouse only inactivates the product of the?rst promoter(JJW,unpublished).Lef1overexpression inhibited osteoblast maturation and Runx2activity on the osteo-calcin promoter in vitro(Kahler and Westendorf,2003;Kahler et al.,2006),but Lef1ΔN had the opposite effect(Hoeppner et al., 2009).Lef1ΔN is present in mature osteoblasts and is induced by Bmp2,but repressed by Wnt3a(Hoeppner et al.,2009).Despite the absence of the N-terminal high af?nityβ-catenin binding domain,Lef1ΔN retains the ability to weakly interact withβ-catenin via second domain(Hoeppner et al.,2011).Thus,Lef1ΔN can facilitateβ-catenin activity,although in other scenarios it acts as a competitive inhibitor of Wnt activity(Hovanes et al.,2001). Transgenic mice expressing Lef1ΔN in mature osteoblasts with the 2.3Col1a1promoter have a modestly higher trabecular bone mass, increased bone formation rates,and elevated serum osteocalcin levels(Hoeppner et al.,2011).Together these data demonstrate that alternative isoforms of Lef1temporally and spatially regulate osteoblast function and trabecular bone mass.Lef1or Tcf7CKO mice have not yet been made but would be useful for dissecting their speci?c roles in bone.

5.6.Wtx(FAM123B)

WTX(also called FAM123B,OSCS or AMER1)is a tumor suppressor encoded on the X chromosome that contains somatic mutations in approximately30%of Wilms tumors.Germline loss-of-function mutations in WTX cause osteopathia striata with cranial sclerosis (OSCS),a disorder characterized by osteosclerosis in females and high incidence of lethality in male fetuses(Jenkins et al.,2009). WTX directly binds toβ-catenin,Apc,and Axin1/2and is a compo-nent of theβ-catenin destruction complex(Major et al.,2007); thus,loss-of-function mutations in WTX enhanceβ-catenin stability.

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Wtx is expressed in the developing mouse skeleton and skull at E14.5and contributes to both intramembranous and endochondral bone formation(Jenkins et al.,2009).Germline deletion of Wtx caused somatic overgrowth and developmental defects in mesen-chymal tissues,including bone.Wtx de?cient animals died within one day of birth but had enlarged cranial vaults,bowed long bones, and increased cortical bone mineral densities(Moisan et al.,2011). Heterozygous animals also had higher bone mineral densities.In contrast,adipogenesis was inhibited and Wtx-de?cient mice had less white and brown fat.Targeted deletion of Wtx with a variety of Cre lines demonstrated that Wtx is active in early mesenchymal progenitors as conditional deletion in mice expressing Prx1-and Osx-Cre displayed bone overgrowth;whereas deletion in commit-ted chondrocytes(Col2a1-Cre)or osteoblasts(Ocn-Cre)did not alter the skeleton(Moisan et al.,2011).Runx2and Osterix levels were higher in Wtx-de?cient embryos andβ-catenin activity was increased in progenitor cells.Interestingly,older Wtx-de?cient mice had persistent defects in matrix mineralization.Together these data indicate that Wtx is a negative regulator of mesenchymal and osteoblastic progenitors,but a positive regulator of osteoblast maturation into osteocytes.These studies did not examine how Wtx-deletion affects osteoclastogenesis.

6.Emerging areas for Wnts in bone biology

The remarkable advancements in our understanding of the molec-ular underpinnings of rare bone diseases and in how Wnts control bone formation and osteoblast proliferation,differentiation,and sur-vival have quickly led to the development of multiple therapies for more common diseases of altered bone mass,such as osteoporosis (Rachner et al.,2011).To fully understand the effects of these drugs, it will be crucial to also study how Wnts and Wnt antagonists affect other cells in the bone marrow and to examine their effectiveness during aging.

6.1.Wnt signaling and osteoclasts

In contrast to the plethora of data about Wnt signaling in osteo-blast lineage cells,there is a paucity of information about the cell autonomous in?uences of Wnts on osteoclasts.As has been discussed,β-catenin indirectly regulates osteoclastogenesis by raising the Opg to Rankl ratio in osteoblasts(Glass et al.,2005)and there is a threshold tolerance forβ-catenin expression during osteoclastogenesis(Wei et al.,2011).Other Wnt pathway components,including Wnts,Fzds, Lrps,and Tcf family members,are also expressed in osteoclast lineage cells(Qiang et al.,2010).Thus,Wnts/β-catenin signaling appears to reduce bone resorption.This area is clearly in need of further investiga-tion to fully resolve the scope of Wnt in?uences on bone metabolism and to understand the effectiveness of Wnt-based therapies on bone structure and function.

6.2.Aging

Work in non-bone cells provides evidence for bene?cial effects of Wnts on aging but also raise some concerns about therapies to promote Wnt signaling to prevent age-related bone loss.For example,Wnt signaling is required for T cell development and protects thymic epithelial cells against senescence(Pongracz et al.,2003;Talaber et al.,2011).Wnt/Tcf signaling also keeps preadipocytes in an undifferentiated proliferative stage to inhibit adipogenesis (Bilkovski et al.,2011).In contrast,Wnts are activated in several models of accelerated aging and increase?brosis of aged muscle progenitor cells(Brack et al.,2007;Liu et al.,2007b).β-catenin-mediated Wnt signaling also induces mesenchymal stem cell aging and DNA damage through the p53/p21pathway and ROS generation,promotes cardiovascular cell aging,and induces mitochondrial biogenesis to cause cell senescence(Naito et al., 2010;Yoon et al.,2010;Zhang et al.,2011).

6.3.Cancer

It has been extensively documented that Wnt/β-catenin signaling effectively promotes tumor development and progression in a number of cancers including breast,multiple myeloma,endometrial,and lung (Polakis,2000).Benign osteomas developed in mice expressing constitutively activeβ-catenin in osteoblasts(Glass et al.,2005) and Wif1depletion made animals more susceptible to radiation-induced osteosarcomas(Kansara et al.,2009);however,there is no increased incidence in cancer in families carrying LRP5gain-of-function mutations,nor are there any reports of increased tumors in Sost-or Dkk1-de?cient animals.Interestingly,anti-Wnt therapy is under development to as means to treat a number of cancers. How these therapies will in?uence bone metabolism will need to be examined,particularly in cancers that cause osteolysis such as multiple myeloma and breast cancer.

7.Summary and conclusions

In conclusion,much has been learned about the roles of Wnt path-way components in bone development,remodeling,and repair during the last decade though the use of genetic animal models.These studies were fueled by the desire to understand the molecular underpinnings for rare bone diseases and have quickly led to the development of multiple therapies for common diseases of altered bone mass(e.g., postmenopausal osteoporosis)and for regenerative medicine.Despite these rapid and measurable accomplishments,much remains to be learned about the effects of Wnts and Wnt antagonists on skeletal physiology and regeneration.Undoubtedly,new components of Wnt pathways will be identi?ed,receptor–ligand speci?cities will be de?ned,and a deeper understanding of how Wnt pathways interact with other signaling cascades in a variety of cell types will be achieved in the next decade.Meanwhile,clinical trials will test the effectiveness of current Wnt pathway drugs on a variety of endocrine and orthopedic conditions and advanced genome sequencing technologies will point us in new directions.Thus,continuing the bedside-to-bench exchange of information that has made this story so successful and compelling. Acknowledgments

The authors are supported by several NIH grants(AG04875, AR48147,AR60140,DE020194,DK91145).

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18 D.G.Monroe et al./Gene492(2012)1–18

免疫荧光操作步骤及注意事项

免疫荧光技术是在免疫学、生物化学和显微镜技术的基础上建立起来的一项技术。它是根据抗原抗体反应的原理，先将已知的抗原或抗体标记上荧光基团，再用这种荧光抗体（或抗原）作为探针检查细胞或组织内的相应抗原（或抗体）。利用荧光显微镜可以看见荧光所在的细胞或组织，从而确定抗原或抗体的性质和定位，以及利用定量技术（比如流式细胞仪）测定含量。 紫外光激发荧光物质放射荧光示意图 免疫荧光实验的主要步骤包括细胞片制备、固定及通透（或称为透化）、封闭、抗体孵育及荧光检测等。细胞片制备（通俗的说法是细胞爬片）是免疫荧光实验的第一步，细胞片的质量对实验的成败至关重要，原因很简单，如果发生细胞掉片，一切都无从谈起。这一步关键的是玻片（Slides or Coverslips）的处理以及细胞的活力，有人根据成功经验总结出许多有益的细节或小窍门，非常值得借鉴。固定和通透步骤最重要的是根据所研究抗原的性质选择适当的固定方法，合适的固定剂和固定程序对于获得好的实验结果是非常重要的。免疫荧光中的封闭和抗体孵育与其它方法（如ELISA或Western Blot）中的相同步骤是类似的，最重要的区别在于免疫荧光实验中要用到荧光抗体，因此必须谨记避光操作，此外抗体浓度的选择可能更加关键。最后需要注意的是，标记好荧光的细胞片应尽早观察，或者用封片剂封片后在4℃或-20℃避光保存，以免因标记蛋白解离或荧光减弱而影响实验结果。 由于操作步骤比较多，同时在分析结果时无法像WB那样可以根据分子量的大小区分非特异性识别，所以要得到一个完美的免疫荧光实验结果，除了需要高质量的抗体，以及对实验条件进行反复优化外，还必须设立严谨的实验对照。总之，免疫荧光实验从细胞样品处理、固定、封闭、抗体孵育到最后的封片及观察拍照，每步都非常关键，需要严格控制实验流程中每个步骤的质量，才能最终达到你的实验目的。 基本实验步骤：

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集团有限公司员工安全知识手册

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实施过程中加强检查和督促。 （4）、组织安全生产检查，包括综合检查、专业性检查、季节检查和日常检查，发现问题及时报请有关部门解决，及早发现事故隐患。 （5）、搞好劳动保护，做好劳保用品的发放工作、劳保用品的管理使用及保护女工和职业病防治等工作。 （6）、进行安全生产教育和培训，主要是对新工人的安全教育、岗位培训教育和独立操作、特殊工种、某些危险岗位的专门安全教育。 （7）、采取相应的组织技术措施，进行安全技术考核，从技术上保障安全。（8）、建立伤亡事故及时报告制度。事故发生后，立即做好抢救等善后工作，要组织事故分析会，查清事故原因，提出预防措施，以防类似的事故再次发生。要做到“三不放过”，即：事故原因分析不清不放过：事故责任者和当事人未受到教育不放过：没有防范措施不放过。 5、安全管理的原则要求 由于施工生产的特点是流动性大、工作条件差、手工露天作业多、沟坑、高空立体交叉作业多，较容易产生不安全因素，所以安全管理显得十分重要。强化施工现场安全管理的原则要求是： （1）、进场教育、标志明确、防范周密、定期检查，重点是防范。要进行进场前和经常性的安全教育，反复宣传，警钟长鸣。现场要设置醒目的安全标志，安全防护措施要齐全，教育施工人员照章作业，设专业人员在工地上巡视检查，发现违章行为及时纠正和处理。各种安全防护法规要不折不扣的执行。 （2）、安全第一的原则。企业管理的职能部门、管理人员、现场指挥人员和参加施工生产的所有人员，都必须树立安全第一的思想，抓生产的同时

免疫荧光技术

免疫荧光技术（Immunofluorescence technique）又称荧光抗体技术，是标记免疫技术中发展最早的一种。它是在免疫学、生物化学和显微镜技术的基础上建立起来的一项技术。很早以来就有一些学者试图将抗体分子与一些示踪物质结合，利用抗原抗体反应进行组织或细胞内抗原物质的定位。它是根据抗原抗体反应的原理，先将已知的抗原或抗体标记上荧光基团，再用这种荧光抗体（或抗原）作为探针检查细胞或组织内的相应抗原（或抗体）。利用荧光显微镜可以看见荧光所在的细胞或组织，从而确定抗原或抗体的性质和定位。 一、基本原理： 免疫荧光技术是根据抗原抗体反应的原理，先将已知的抗原或抗体标记上荧光素，制成荧光抗体，再用这种荧光抗体（或抗原）作为探针检测组织或细胞内的相应抗原（或抗体）。在组织或细胞内形成的抗原抗体复合物上含有标记的荧光素，利用荧光显微镜观察标本，荧光素受外来激发光的照射而发生明亮的荧光（黄绿色或橘红色），可以看见荧光所在的组织细胞，从而确定抗原或抗体的性质、定位，以及利用定量技术测定含量。 二、应用范围： 其应用范围极其广泛，可以测定内分泌激素、蛋白质、多肽、核酸、神经递质、受体、细胞因子、细胞表面抗原、肿瘤标志物、血药浓度等各种生物活性物质。根据诊断类别，又可分为传染性疾病、内分泌、肿瘤、药物检测、免疫学、血型鉴定等。 三、基本实验步骤：

1、细胞准备。对单层生长细胞，在传代培养时，将细胞接种到预先放置有处理过的盖玻片的培养皿中，待细胞接近长成单层后取出盖玻片，PBS洗两次；对悬浮生长细胞，取对数生长细胞，用PBS离心洗涤（1000rpm,5min）2次，用细胞离心甩片机制备细胞片或直接制备细胞涂片。 2、固定。根据需要选择适当的固定剂固定细胞。固定完毕后的细胞可置于含叠氮纳的PBS中4℃保存3个月。PBS洗涤3×5min. 3、通透。使用交联剂（如多聚甲醛）固定后的细胞，一般需要在加入抗体孵育前，对细胞进行通透处理，以保证抗体能够到达抗原部位。选择通透剂应充分考虑抗原蛋白的性质。通透的时间一般在5-15min.通透后用PBS洗涤3×5min. 4、封闭。使用封闭液对细胞进行封闭，时间一般为30min. 5、一抗结合。室温孵育1h或者4℃过夜。PBST漂洗3次，每次冲洗5min. 6、二抗结合。间接免疫荧光需要使用二抗。室温避光孵育1h.PBST漂洗3次，每次冲洗5min后，再用蒸馏水漂洗一次。 7、封片及检测。滴加封片剂一滴，封片，荧光显微镜检查。 四、注意事项： 1、染完之后没有封片前直接照一些，因为有的时候可能封片会出现问题，再想照反而没有了，另外不要拖太长时间，荧光会崔灭的。 2、荧光的片子一定要避光保存，保存的好的话，过一段时间仍然能照出很好的片子。

经典信号通路之Wnt信号通路

经典信号通路之Wnt信号通路 1、Wnt信号通路简介 Wnt信号通路是一个复杂的蛋白质作用网络，其功能最常见于胚胎发育和癌症，但也参与成年动物的正常生理过程. 2、Wnt信号通路的发现 Wnt得名于Wg (wingless) 与Int.wingless 基因最早在果蝇中被发现并作用于胚胎发育，以及成年动物的肢体形成INT 基因最早在脊椎动物中发现，位于小鼠乳腺肿瘤病毒(MMTV)整合位点附近。Int-1 基因与wingless 基因具有同源性。 果蝇中wingless 基因突变可导致无翅畸形，而小鼠乳腺肿瘤中MMTV复制并整合入基因组可导致一种或几种Wnt基因合成增加。 3、Wnt信号通路的机制 Wnt信号通路包括许多可调控Wnt信号分子合成的蛋白质，它们与靶细胞上的受体相互作用，而靶细胞的生理反应则来源与细胞和胞外Wnt配体的相互作用。尽管发应的发生及强度因Wnt配体，细胞种类及机体自身而异，信号通路中某些成分，从线虫到人类都具

有很高的同源性。蛋白质的同源性提示多种各异的Wnt配体来源于各种生物的共同祖先。 经典Wnt通路描述当Wnt蛋白于细胞表面Frizzled受体家族结合后的一系列反应，包括Dishevelled受体家族蛋白质的激活及最终细胞核内β-catenin水平的变化。Dishevelled (DSH) 是细胞膜相关Wnt受体复合物的关键成分，它与Wnt结合后被激活，并抑制下游蛋白质复合物，包括axin、GSK-3、与APC蛋白。axin/GSK-3/APC 复合体可促进细胞内信号分子β-catenin的降解。当“β-catenin 降解复合物”被抑制后，胞浆内的β-catenin得以稳定存在，部分β-catenin进入细胞核与TCF/LEF转录因子家族作用并促进特定基因的表达。 4、Wnt介导的细胞反应 经典Wnt信号通路介导的重要细胞反应包括： 癌症发生。Wnts, APC, axin，与TCFs表达水平的变化均与癌症发生相关。 体轴发育。在蟾蜍卵内注射Wnt抑制剂可导致双头畸形。 形态发生。 （此文档部分内容来源于网络，如有侵权请告知删除，文档可自行编辑修改内容， 供参考，感谢您的配合和支持）

员工安全教育培训资料

三车间员工安全基础知识培训资料 一、安全生产基础知识 1、什么是安全生产？ （1）概念：安全生产是指在生产过程中，要努力改善劳动条件，克服不安全因素，防止伤亡事故的发生，使劳动生产在保证劳动者安全健康和国家财产安全的前提下顺利进行，安全生产的对象有人的不安全行为和物的不安全状态。所以消除危害人身安全健康的一切不得因素，保障员工的安全和健康，舒适地工作，称之为安全。（2）安全生产的重要性：化工企业的安全生产不同于其他行业，由于化工生产具有一定的特殊性，生产工艺复杂，操作要求严格，生产现场的设备密集、压力容器数量较多，易燃易爆物品多，工艺要求高等特点，具有潜在的危险性，一旦发生事故后果是严重的，所以说化工企业的安全管理非常重要。 2、我国安全生产的方针是什么？ 我国安全生产的方针是对安全生产工作的总要求，它是安全生产的工作方向。其内容是：“安全第一，预防为主、综合冶理”。在各行各业的管理过程中，必须要遵循这一方针，这是国家保护劳动者的安全健康的一项基本政策。所谓“安全第一”，是指安全生产是国家一切生产企业的头等大事。当生产任务与安全发生矛盾时，应先解决安全问题，使生产在确保安全的前提下顺利进行。 所谓“预防为主”，是指在实现“安全第一”的许许多多的工作中，做好预防工作是最重要的，它要求我们防微杜渐，防患于未然，把事故危害消灭在发生之前，也就是我们经常所说的把隐患消灭在萌芽状态之中。事故一旦发生往往很难挽回。到那时，如果不做好预防工作，安全第一也就成了一句空话。 3、员工遵章守纪的意义是什么？ 员工遵章守纪是安全生产工作的基础。因此，在生产过程中，不仅要有熟练的技艺，而且必须自觉遵守各项操作规程和劳动纪律，按应有的标准进行，形成每个环节、每道工序、每个人应有的职业范围。如没有这些元规范要求，劳动过程中可能会发生混乱，对生产造成损失，给人身安全带来危害。所以，只有严守规章制度，严守劳动纪律、杜绝违章，做到“四不伤害”即不伤害自己，不伤害别人、不被别人伤害，保护他人不受伤害，才能杜绝“三违”（即违章指挥、违章作业、违反劳动纪律）行为，最大限度地减少和消除种类伤亡事故的发生，从而达到克服安全生产的薄弱环节，切实解决企业安全生产工作中存在的各类问题，使“安全第一，预防为主”的方针真正落到实处。 二、危险化学品的概念 是指有易燃、易爆、易腐蚀、放射性等性质。在生产经营储运、使用和废弃物处置过程中，容易造成人身伤亡和财产损毁。 1、目前我车间涉及的原辅料有：、氨、尾气氯化氢、硫、三氯化磷、硫酸二甲酯、甲醇等均为《危险化学品名录》（2002版）中所列的危险化学品，在使用和储存过程中存在着火灾、爆炸、中毒（窒息）、触电、机械伤害、物体打击、高处坠落、灼烫、粉尘伤害、噪声、淹溺、自然灾害等危险有害因素。 本项目涉及的中间产品及回收套用的三氯硫磷（中间产品）、甲基二氯化物（中间产品）、甲基一氯化物（（中间产品）、二甲基硫化磷酰胺（中间产品）、副产品盐酸属危险化学品。 2、、剧毒化学品管理，设置防盗、监控报警装置；加设可燃气体报警装置，有毒气体报警装置，严格按照剧毒品五双管理落实监管工作。易燃易爆物料：甲醇储罐按要求拟安装液位报警装置、气体泄漏检测报警装置和高液位联锁紧急切断设施：罐区加设防盗、监控装置。 3、危险化学品使用场所的主要安全规定 1、严格管理明火 2、避免摩擦撞击 3、防止电气火花 4、防止泄漏 5、操作人员应具备相应的操作技能 4、危险化学品生产岗位安全操作要点 1、上岗前必须做好岗位检查，杜绝任何设备带病运行，尤其是安全附件。 2、必须严格执行工艺技术规程，遵守工艺纪律，做到平稳运行。 3、必须严格执行安全操作规程：安全操作规程是生产经验的总结，往往是通过血的教训换来的。 4、控制溢料和漏料，严防滴漏，是否存在泄料和溢料主要取决于岗位员工对待安全生产的态度和责任心；正确判断和处理异常情况。 5、严格按要求佩戴相关的劳动用品，穿戴使用个人防护用品是保护自身安全、健康的最后一道防线。

住院患者服药依从性管理及影响因素分析

住院患者服药依从性管理及影响因素分析 目的了解住院患者服药的依从性及相关影响因素。方法选择在我院住院治疗的患者作为研究对象，依照Morisky等推荐的用药依从性标准对患者用药依从性进行评定，采用χ2检验和Logistic回归分析筛选患者用药依从性的影响因素。结果157例患者中，服药依从者96例，依从率61.1%；服药不依从者61例，不依从率38.9%。多因素Logistic回归分析显示药费支付方式、用药时间、是否需要长期服药的知晓情况对住院患者服药依从性有影响。表现为：公费或医保患者依从性高于自费患者（OR=1.248）；服药时间在2年～和≥3年患者的依从性高于服药时间<3个月患者（OR=1.240和2.597）；需要长期服药患者的依从性比不需要长期服药者更高（OR=2.213）。结论应加强用药患者的依从性教育，努力提高患者的用药依从性。 标签：住院患者；药物治疗；依从性；影响因素 住院患者往往都要经历漫长的疾病恢复期，有很多患者需要长期服药甚至终身服药，患者服药依从性无论对于在家服药患者还是住院患者都是一个普遍存在的问题，对住院患者进行科学、系统的用药指导，培养其良好的服药习惯、提高患者的服药依从性，对于提高患者治疗效果、减低并发症等都有重要意义[1]。为了了解我院住院患者服药依从性程度及其影响因素，以便為患者设计更好的治疗方案，更好的指导患者用药，最终达到提高治疗效果的目的[2-3]。为此，我们以问卷调查的形式对四川省仁寿县中鑫医院的157例住院患者进行了服药依从性的临床调查研究。 1 资料与方法 1.1一般资料选择2014年4月～12月在四川省仁寿县中鑫医院住院治疗的住院患者160例，作为研究对象，发放调查问卷160份，最终收回有效问卷157份，有效回收率98.13%。 1.2方法服药依从性判定标准：依照Morisky等推荐的服药依从性标准判定患者的服药依从性，依从性量表：①你是否有忘记点药的经历；②你是否为了快速达到目的，而自己增加服药次数；③当你自觉症状改善时，是否会自行停药； ④当你觉得药效不明显时，是否会自行停药。当有≥3个条目回答为”否”则判定为依从，有≤2个条目回答为”否”则判定为不依从。同时对患者的基本情况、疾病情况、治疗情况等进行问卷调查。 1.3统计学处理数据资料通过Epidata3.10进行管理，采用SPSS16.0统计软件对数据资料进行统计学分析，检验水准α为0.05。单因素分析采用χ2检验对各调查因素的服药依从性进行分析，采用多因素Logistic回归分析筛选影响患者服药依从性的影响因素。 2 结果

一些医学用量表

一、简易精神状态检查表(Mini—Mental State ExaminatiOn MMSE) 1．介绍 1975年Folstein等设计了一个用于评定老年人认知功能障碍等级的量表，并且被用于检查Alzheimer病早老性痴呆和治疗的效果，但是对于治疗后的改变其敏感性差。此量表因为设计合理，应用广泛和简洁，是医生很好的选择。 2．内容 （1）今年是公元哪年? 1 0 现在是什么季节? 1 0 现在是几月份? 1 0 今天是几号? 1 0 今天是星期几? 1 0 （2）咱们现在是在哪个城市? 1 0 咱们现在是在哪个区? 1 0 咱们现在是在什么街(胡同)? 1 0 咱们现在是在哪个医院? 1 0 这里是第几层楼? 1 0 （3）我告诉您三种东西，在我说完之后，请您重复一遍，这三种东西都是什么？3 2 1 0 树，钟，汽车(各1分共3分)。 （4）100－7=? 连续5次(各1分共5分) 5 4 3 2 1 0 （5）现在请您说出刚才我让您记住的那三样东西（各1分共3分） 3 2 1 0 （6）(出示手表)这个东西叫什么? 1 0 (出示铅笔)这个东西叫什么? 1 0 （7）请您跟着我说“大家齐心协力拉紧绳” 1 0 （8）我给您一张纸，请按我说的去做，现在开始：“用右手拿着这张纸，用两只手将它对折起来，放在您的左腿上”(每项1分共3分) 3 2 1 0 （9）请您念一念这句话，并且按着上面的意思去做“闭上您的眼睛” 1 0 （10）请您给我写一个完整的句子。 1 0 （11）(出示图案)请您照着这个样子把它画下来 1 0 3．解释 （1）检查需要5~10分钟，仅需要一只手表，铅笔和一张纸。 （2）每个单词朗读时间1秒钟，根据第一次复述结果评分，但是重复朗读单词直至患者可以复述，最多6次，如果患者最终仍不能完全复述所有单词，则认为回忆测试结果没有意义。（3）计算：每步1分，如果患者不能进行这项检查，那么要求患者倒着拼写“world”，每拼写正确一个字母，给1分。 （4）书写：句子必须包括主语和动词，符合逻辑，不要求语法和拼写正确，必须是患者自己写的，不能是检查者口述的句子。 （5）要求患者画2个互相交错的五角形，边长1英尺，应象例图显示的一样，必须包括10个角，2个必须交错，可以忽略图形的颤抖和扭转。 （6）此量表的内部一致性高，测试和再测试之间、检查者之间的一致性好。 （7）引导语：“我将您问一些问题，检查您的记忆力和思考问题的能力，有些问题很简单，有些问题对于每个人来说都较难，尽您最大努力回答问题，如果不会，您不必紧张。”（8）评分标准：总分30分，划分痴呆的标准：文盲≤17，小学≤20，中学（包括中专）≤22，大学（包括大专）≤23 （9）对于痴呆早期其敏感性差，对血管性、多发硬化性，帕金森性的认知功能障碍敏感性

免疫荧光抗体检测技术

实验七免疫荧光抗体检测技术 一、目的要求 通过本实验，了解和掌握直接免疫荧光和间接免疫荧光染色的原理和方法，并能应用于疾病的诊断和抗原抗体的分析。 二、实验原理 用化学或物理的方法将荧光素标记到抗体或抗原上，这种标记荧光素的抗体或抗原仍然能与相应抗原或抗体发生特异性结合。通过已知的荧光抗体或抗原，检测样本中相应的抗原或抗体，从而进行疾病的诊断和对抗原抗体的分析。 间接免疫荧光试验 三、实验材料 感染鸡马立克病病毒（MDV）的细胞培养物，抗MDV特异性单克隆抗体BA4;丙酮-乙醇（6:4，V/V）固定液，盖玻片，FITC标记的抗小鼠IgG荧光抗体，0.01mol/L PBS(pH 7.2)，蒸馏水，载玻片（洁净无油）；1000ml 烧杯，磁棒，磁力搅拌器，200μL移液器，吸水纸，镊子等。 四、实验方法 1.细胞片的制备与固定将消毒并处理过的盖玻片放于细胞培养瓶（皿）中，按常规操作，制备鸡胚成纤维细胞，并培养于细胞培养瓶（皿）中，待细胞生长成单层后，接种CVI988/Rispens疫苗株。48—72h后，直接收获盖片，用PBS洗涤1次，再以-20℃预冷的丙酮-乙醇（6:4，V/V）固定液固定5min，空气干燥，置密封容器于-20℃保存备用。如用96孔或24孔细胞培养板培养的细胞，可弃去培养液，用PBS洗涤1次，然后固定，固定方法同上。 2.抗体染色 ⑴将接种并固定的MDV病毒的盖玻片，用PBS洗涤1次，置架上。取未接种MD病毒的盖玻片，同上用PBS洗涤1次，置架上，作为阴性对照。盖玻片上滴加10μL—20μL工作浓度的单克隆抗体，96孔细胞培养板加入50μL/孔工作浓度的单克隆抗体。37℃水浴孵育30—45min。 ⑵盖玻片在PBS（Ph7.4,0.01mol/L）中用磁力搅拌器洗涤5—10min，如用96孔细胞培养板检测，则加入PBS 200μL/孔，洗涤3—4次。 ⑶取出盖片置架上，滴加工作浓度的荧光抗体（FITC标记的抗小鼠IgG的抗体）。37℃孵

Wnt信号通路

Maturitas78(2014)233–237 Contents lists available at ScienceDirect Maturitas j o u r n a l h o m e p a g e:w w w.e l s e v i e r.c o m/l o c a t e/m a t u r i t a s Review Wnt signaling and osteoporosis Stavros C.Manolagas? Division of Endocrinology and Metabolism,Center for Osteoporosis and Metabolic Bone Diseases,University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System,Little Rock,AR,USA a r t i c l e i n f o Article history: Received8April2014 Accepted11April2014 Keywords: Osteoblasts Osteoclasts Osteocytes RANKL OPG Bone therapies a b s t r a c t Major advances in understanding basic bone biology and the cellular and molecular mechanisms responsible for the development of osteoporosis,over the last20years,have dramatically altered the management of this disease.The purpose of this mini-review is to highlight the seminal role of Wnt signaling in bone homeostasis and disease and the emergence of novel osteoporosis therapies by targeting Wnt signaling with drugs. Published by Elsevier Ireland Ltd Contents 1.Introduction (192) 2.Wnt signaling (193) 3.Wnt/?-catenin signaling in bone health and disease (193) 4.Wnt signaling,osteocytes,and the mechanical adaptation of the skeleton (193) 5.Wnt/?-catenin signaling,the FoxO transcription factors,and the pathogenesis of osteoporosis (194) 6.Targeting Wnt signaling for the development of a novel bone anabolic therapy for osteoporosis (194) 7.Summary (195) 8.Research agenda (195) Contributors (195) Competing interest (195) Provenance and peer review (195) Funding (195) Acknowledgements (195) References (195) 1.Introduction The mammalian skeleton regenerates throughout life by the removal(resorption)of old bone by osteoclasts and its replacement with new bone by osteoblasts,during a process called remodeling [1].Osteocytes–former osteoblasts which are entombed within the mineralized matrix–sense the need for regeneration in a ?Correspondence to:Distinguished Professor of Medicine,Division of Endocrinol-ogy and Metabolism,University of Arkansas for Medical Sciences,4301W.Markham St.,Slot587,Little Rock,AR72205,USA.Tel.:+15016865130;fax:+15016868148. E-mail address:manolagasstavros@https://www.doczj.com/doc/0210348478.html, particular anatomical site and orchestrate the process by directing the homing of osteoclasts and osteoblasts to the site that is in need of remodeling,by producing and secreting key factors that control osteoclast and osteoblast generation[2,3].Under physiologic con-ditions,bone resorption and formation are balanced with the exact same amount of bone added in the site from which it was previously resorbed.With advancing age,the balance between resorption and formation is disturbed and bone mass declines.In addition bone progressively loses mechanical strength to an extent that is greater than the decline of bone mass because of the deterioration of its microarchitecture and the quality of its matrix and mineral(by mechanisms that are not well understood)and an increase in the number of dead or dysfunctional osteocytes as well as increased https://www.doczj.com/doc/0210348478.html,/10.1016/j.maturitas.2014.04.013 0378-5122/Published by Elsevier Ireland Ltd

员工安全培训手册(完整版)

员工安全教育培训手册 XXXX XXXX建设集团养护科技有限责任公司 2018年7月

前言 安全保障着员工的生命，员工决定着企业的生命．XXXX建设集团养护科技有限责任公司将始终贯彻＂安全第一，预防为主,综合治理＂的方针，致力通过全员、全方位、分级进行的安全教育，从而不断提高员工的安全意识、安全知识和技能，努力实现安全零事故的目标。 为此，公司特组织编写此教材，以作为新员工入厂安全教育和部门级和岗位级安全教育的基础教材。 因经验水平有限，书中多有不完善之处，敬请指正为感！ XXXX建设集团养护科技有限责任公司 安全环保部 2018年7月

目录 第一章公司安全生产管理概述 (4) 第二章员工在安全方面的权利和义务 (5) 第三章安全事故的引发和防止要点 (7) 第四章员工基本安全常识 (9) 第五章工伤认定的基本条件 (14) 附件1 常见安全标识 (16)

第一章公司安全生产管理概述 一、安全生产方针 我们必须长期坚持“安全第一，预防为主，综合治理”的管理方针。 要把安全作为一切工作的前提，任何时候安全是最要的，第一位的，生产和其他工作要服从于安全，做到不安全不生产，隐患不处理不生产，安全措施不落实不生产。 安全管理以预防为主。通过不断完善制度，从而消除安全隐患，以提高员工安全认识水平和技能水平，从而消除安全隐患和防止事故发生。 二、安全生产责任制 公司安全生产责任制是依据“管生产必管安全”的原则落实的。贯彻“谁主管，谁负责”，各级各类人员必须担负起自己职责范围内相应的安全责任。 三、安全教育体系 公司安全教育分三级：公司级（厂级）、部门级（车间）、岗位级（班组）。公司级安全教育主要是针对公司安全生产方针、制度和基本安全生产知识。部门安全教育主要是针对部门的安全生产制度、生产现场安全状况等。岗位级安全教育主要结合本岗位的操作规程、安全操作技能、安全防护方法等。 项目安全教育分为三级：项目级、工作处级、班组级。项目级安全教育主要是针对项目的安全制度和基本安全生产知识。工作处级教育主要是针对工作处的生产现场安全状况等。班组级安全教育主要结合本施工作业岗位的操作规程、安全操作技能、安全防护方法等。 新员工入厂必须经过三级安全教育，培训合格后才可上岗操作。转岗工必须经过转岗培训。 特种岗位人员必须经过专门培训，通过考试，取得特种岗位上岗证才能上岗操作。 第二章员工在安全方面的权利和义务

免疫荧光技术常见问题汇总

免疫荧光技术常见问题汇总 1、问：用免疫荧光方法做组织切片和培养细胞内的蛋白，两者操作过程有哪些不同？ 参考见解：只是固定方法不同。细胞固定用甲醇，切片固定用多聚甲醛，而染色方法是一样的。 2、问：近期要做免疫荧光双标，不知哪位有免疫荧光双标技术的详细步骤及其注意事项？ 参考见解：经验是： （1）选取一抗时要来源于两种不同的动物，用来源于rabbit和rat的抗体，二抗则是不同荧光信号标记的，比如donkey anti-rabbit-FITC（绿）和donkey anti-rat-Tex-Red（红）。 （2）两种一抗同时孵育，然后两种二抗同时孵育。抗体浓度、孵育时间要仔细摸索，感觉一抗4度孵育过夜比较好，背景比较清晰。 （3）阳性对照用的是阳性组织切片，阴性对照则分别是家兔和大鼠的IgG，荧光标记物对照是PBS+荧光标记物。 （4）封闭血清与二抗来源动物一致，用的是10%的正常donkey血清。 （5）其余步骤同一般免疫荧光单标操作。 3、问：本人拟做Brdu标记神经干细胞免疫荧光，二抗为FITC标记，想请教各位大侠： （1）抗体分装和荧光显微镜观察时是否一定要在暗室中进行？ （2）封片时是否用甘油缓冲液即可，还是用甘油和0.5mmol/L pH9.0-9.5的碳酸氢盐缓冲液等量混合封片？ （3）免疫酶染色中的3%过氧化氢及2N盐酸是否还需要用？ 参考见解： （1）你的二抗是用FITC标记的，为避免荧光分解，分装及荧光显微镜观察时均要避光； （2）封片最好用甘油加0.5mmol/L pH9.0-9.5的碳酸氢盐缓冲液，后者能使玻片透明； （3）关于免疫酶染色，如果是用过氧化物酶做标记，就必须用3%H2O2以去除内源性过氧化物酶；2N盐酸需要使用，目的是使DNA变性，让Brdu抗体能够充分地与已经掺入的Brdu结合。