OPEN
ORIGINAL ARTICLE
Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation
P Daniel 1,G Filiz 1,DV Brown 1,F Hollande 1,M Gonzales 1,2,G D’Abaco 3,N Papalexis 4,WA Phillips 5,6,J Malaterre 6,7,RG Ramsay 6,7and T Mantamadiotis 1,4
The cyclic-AMP response element binding (CREB)protein has been shown to have a pivotal role in cell survival and cell proliferation.Transgenic rodent models have revealed a role for CREB in higher-order brain functions,such as memory and drug addiction behaviors.CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues,and aberrant CREB expression is associated with tumours.It is the central position of CREB,downstream from key developmental and growth
signalling pathways,which gives CREB this ability to in?uence a spectrum of cellular activities,such as cell survival,growth and differentiation,in both normal and cancer cells.We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade.The mechanism by which CREB regulates glioblastoma (GBM)tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and
phosphoinositide 3-kinase (PI3K)pathways that then modulate the expression of three key cell cycle factors,cyclin B,D and proliferating cell nuclear antigen (PCNA).Cyclin D1is highly CREB-dependent,whereas cyclin B1and PCNA are co-regulated by both CREB-dependent and -independent mechanisms.The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells,which in turn allows CREB to regulate the activity of the PI3K itself.Given that CREB sits at the hub of key cancer cell signalling pathways,understanding the role of glioma-speci?c CREB function may lead to improved novel combinatorial anti-tumour therapies,which can complement existing PI3K-speci?c drugs undergoing early phase clinical trials.
Oncogenesis (2014)3,e108;doi:10.1038/oncsis.2014.21;published online 30June 2014
INTRODUCTION
Patients diagnosed with malignant glioblastoma (GBM)show a median survival of 14months;a statistic largely unchanged over the past decades.For gliomas,the only drug used as part of the standard therapy is the DNA alkylating/methylating agent temozolomide,which has led to an improvement in median overall survival,ranging between 0and 7months,depending on the methylation status of the patient’s DNA repair gene,MGMT.1Attempts at targeting speci?c factors in GBM have so far been unsuccessful,with such attempts exempli?ed by clinical trials conducted recently:the use of the promising angiogenesis inhibitor bevacizumab (Avastin)2or a combination of bevacizumab and a phosphoinositide 3-kinase (PI3K)pathway inhibitor provided no bene?t to patients.3Therefore there is a need to develop better approaches for treating gliomas to improve patient survival.To progress the discovery and testing of novel drugs and combinations of drugs,the understanding of the molecular genetic mechanisms and factors driving GBM development,growth and drug resistance must be clari?ed.Among the factors and pathways implicated in glioma development and growth,the kinases PI3K and mitogen-activated protein kinase (MAPK)are among the most studied.Highlighting the critical role of these kinases in cancer,480%of GBM patients harbour alterations such as epidermal growth factor
receptor (EGFR)ampli?cation,EGFRvIII-activating mutation and/or downstream PIK3CA-activating mutations or phosphatase and tensin homolog (PTEN)deletions,4contributing to the hyperactivation of the downstream effectors such as extracellular signal–regulated kinase and AKT,key drivers of pathogenesis in GBM.5Although aspects of the immediate upstream and downstream components of these pathways are relatively well understood,the feedback loops and nuclear target networks controlling these pathways in GBM biology are not as well de?ned.
As many cancer signalling pathways converge on nuclear transcription factors,which then orchestrate the expression of a tumour-promoting transcriptome,targeting these transcription factors in combination with upstream-activating factors may be an attractive approach.Indeed,this has come to the fore in terms of emerging anti-tumour strategies 6–9In cancer cells,one of the transcription factors that sit at the hub of tumour cell signalling pathways is the cyclic-AMP response element binding (CREB)protein,a serine/threonine kinase-regulated transcription factor in which phosphorylation of CREB in the N-terminal kinase-inducible domain recruits transcriptional co-activators such as CREB-binding protein and transducers of regulated CREB activity to activate CREB target gene transcription.10,11CREB has been implicated in the growth and progression of multiple cancers,including
1
Department of Pathology,The University of Melbourne,Parkville,Victoria,Australia;2Department of Anatomical Pathology,The Royal Melbourne Hospital,Parkville,Victoria,Australia;3NICTA Victorian Research Laboratories,Centre for Neural Engineering,The University of Melbourne,Carlton,Australia;4Laboratory of Physiology,Faculty of Medicine,University of Patras,Patras,Greece;5Surgical Oncology Research Laboratory,Peter MacCullum Cancer Centre,Melbourne,Victoria,Australia;6Sir Peter MacCallum Department of Oncology,The University of Melbourne,Parkville,Victoria,Australia and 7Differentiation and Transcription Laboratory,Peter MacCallum Cancer Centre,Melbourne,Victoria,Australia.Correspondence:Dr T Mantamadiotis,Department of Pathology,Faculty of Medicine,Dentistry and Health Sciences,The University of Melbourne,Parkville,Victoria 3010,Australia.
E-mail:theom@https://www.doczj.com/doc/5c15601893.html,.au
Received 3February 2014;revised 29April 2014;accepted 15May 2014
Citation:Oncogenesis (2014)3,e108;doi:10.1038/oncsis.2014.21
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leukemia,12,13colorectal cancer 14and breast cancer.15CREB lies at the hub of multiple oncogenic signalling pathways initiated by ligand-growth factor receptor interactions such as the platelet-derived growth factor receptor and EGFR receptor tyrosine kinases and is activated by numerous downstream kinase pathways,including PI3K and MAPK,and deactivated by tumour suppressors,including PTEN.In melanoma cells harbouring BRAF(V600E)mutations,CREB is a key factor involved in MAPK pathway-mediated drug resistance 16In GBM,CREB function appears to regulate growth of tumour cells via transcriptional control of miRNA-23a 17and Nf-1.18Previous work has shown that loss of CREB function during brain development leads to neuronal death 19,20and that CREB is required for ef?cient neuronal stem and progenitor cell proliferation 21,22Based on these ?ndings,we hypothesized that CREB activation is a critical step in gliomagenesis and that CREB function sits downstream of key cancer signalling pathways.
We show that CREB expression and activation correlates with glioma grade and that activation occurs via both the PI3K and MAPK pathways and that CREB promotes glioma cell proliferation.Furthermore,we show that the mechanism by which CREB promotes glioma cell proliferation includes the regulation of key cell cycle factors cyclin B1,D1and proliferating cell nuclear antigen (PCNA).
RESULTS
CREB is constitutively activated in human GBM cells lines and human brain tumours exhibit grade-dependent pCREB expression Although CREB is expressed throughout the normal brain during all stages of life,constitutively phosphorylated CREB (pCREB)is progressively restricted to neurogenic zones.It is within these zones that neural stem/progenitor cell (NSPC)populations reside and CREB gene/expression disruption studies in mice and zebra?sh show that NSPC proliferation and survival depends on CREB function.21,22Based on data showing that CREB has oncogenic roles in the hemopoietic system 12and other tissues 23and as the CREB functions required by NSPCs may be important in GBM biology,we explored CREB expression,activation and function in brain tumour tissue and cells.Assessment of CREB expression and activation in human GBM tumour cell lines using CREB and pCREB antibodies revealed that GBM tumour lines showed robust CREB and pCREB expression.Non-tumour brain tissue exhibited weaker total CREB expression and undetectable levels of pCREB (Figure 1a).
Using whole GBM surgical tumour specimens prepared from formalin-?xed,paraf?n-embedded tissue,we examined the mor-phological landscape of a further 15patient specimens diagnosed with grade IV GBM.GBM tumour morphologies associated with pCREB expression are shown in Figures 1b-i–vi.pCREB expression was evident in most tumour cells examined with a clustering of strong pCREB expression in highly vascularized tumour regions (Figure 1b-i)and in regions with strong nestin expression (Figure 1b-vi).pCREB-positive cell density and expression was high in hypercellular pseudopalisading regions adjacent to necrotic regions,where migrating proliferating tumour cells are seen (Figure 1b-iii,arrows).Of note,tumour morphologies corresponding to GBM regions associated with less malignant characteristics exhibited weaker pCREB expression.Giant cells in GBM showed relatively weaker pCREB expression (Figure 1b-ii,arrows),and cells in tumour regions with oligodendroglial features,characterized by the so-called ‘chicken wire’or ‘fried egg’morphology showed the weakest pCREB expression (Figures 1b-iv and -v,arrows).
Interrogation of glioma tissue microarray specimens harbouring 40patient glioma tissue cores (3mm diameter)in duplicate showed that while ‘normal’non-tumour and ‘normal tissue adjacent to tumour’showed little-to-no CREB activation.By contrast,tumour tissue exhibited robust grade-dependent levels of pCREB expression (Figure 1c).Grade II tumours showed the lowest levels of pCREB,and WHO grade IV tissues exhibited the strongest pCREB positivity.Both pCREB intensity and the number of cells expressing pCREB correlated with tumour grade,where grade IV GBM showed the most intense and highest density of pCREB-positive https://www.doczj.com/doc/5c15601893.html,ing a ‘t’scale where ‘t’is just detectable staining/1%to o 5%of cell/?eld positive and ‘ttttt’is strong dark staining/almost 100%cells/?eld positive,grade II tumours showed between zero and ‘tt’;grade III tumours showed between ‘t’and ‘ttt’and grade IV/GBM showed between ‘ttt’and ‘ttttt’.Three oligodendroglioma specimens showed ‘t’,‘t’and ‘tt’,while all ‘adjacent to tumour’and ‘normal’brain tissue showed zero staining.
Using the GBM cell line T98G to examine CREB activation,we show that pCREB coincides with cells in cycle.Speci?cally,pCREB is present in cells undergoing S-phase of the cell cycle as shown by co-labelling with PCNA and newly divided cells labelled with
the
Figure 1.CREB is hyperactivated in glioma cell lines and tissue.(a )Cells from a range of human glioma cell lines were lysed and analysed from CREB expression and activation.All GBM cell lines tested showed an abundance of CREB protein,which was phosphorylated/activated.Non -tumour mouse brain tissue shows expression of CREB but no detectable pCREB.(b )Immunohisto-chemical analysis of human GBM tissue specimens show differential pCREB expression.(i)pCREB-positive tumour cells surround a tumour blood vessel;(ii)Giant cells in GBM show moderate-to-strong pCREB labelling;(iii)pseudopalidaing areas (arrows)in GBM show dense pCREB-positive tumour cells;(iv)and (v)two different patient tumours featuring oligodendroglial features show weak pCREB expression (arrows indicate oligodendroglial tumour cells);and (vi)double-labelling immunohistochemistry showed dense pCREB-positive cells (brown)surrounding foci with strong nestin expression (blue).(c )Brain tumour tissue microarray (US Biomax)with pCREB immunostaining.Top image shows the complete array with tumour grade layout indicated and cores magni?ed in the lower panel.
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nucleotide analogue bromodeoxyuridine (BrdU;Figure 2).In contrast,cells labelled with the M-phase marker phospho-histone 3(pH3)demonstrated poor colocalization to pCREB-positive cells.PI3K and MAPK but not protein kinase A (PKA)signalling pathways modulate CREB activity in GBM cells
Many signalling pathways relevant to GBM converge onto CREB,suggesting that constitutive CREB activation as observed in GBM is the result of cooperation between multiple upstream factors.To determine which pathways activate CREB,we evaluated cells grown in stimulatory and non-stimulatory conditions for differ-ences in kinase activation.Several serine/threonine kinases that are known to phosphorylate CREB at Serine133were investigated.Analysis of cell lines T98G,LN18and U118demonstrated activation of CREB,AKT and MAPK upon serum stimulation.By contrast,we did not observe activation of PKA,an activator of CREB in other cell types (Figure 3a),showing that there is a selective use of upstream components regulating CREB activity.To link the speci?city of AKT and MAPK activation with CREB activation,the PI3K pathway inhibitor LY294002and the MAPK pathway inhibitor U0126were used on T98G and U118(Figures 3b and c).These cell lines were chosen for further analysis for two reasons.First,they showed the greatest CREB-dependent change in proliferation (Figures 4b and c),and second,these cell lines differ in the tumour-suppressor PTEN status,where T98G has intact PTEN function and U118is PTEN de?cient.One of the PTEN’s major functions is to act as the major phosphatase that inhibits the activation of the PI3K pathway 24Signi?cant variability was seen between the cell lines tested with respect to the contribution of either the PI3K or MAPK pathways on CREB activation.In U118cells,inhibition of the PI3K pathway resulted in no change in CREB activation,while inhibition of the MAPK pathway resulted in knockdown of CREB activation to basal ‘serum-deprived’levels,suggesting that MAPK signalling is the predominant CREB activation pathway in U118cells (Figures 3c and e).By contrast,inhibition of either or both the PI3K or MAPK pathways in T98G cells did not result in signi?cant changes in CREB activation (Figures 3b and d),highlighting the diversity and complex interdependence between signalling pathways involved in CREB activation in GBM cells.
As a further and independent measure of the contribution of the PI3K pathway to CREB activation,we used a mutant NSPC that has a constitutively activated PI3K pathway,due to the combined conditional mutation of the Pik3ca gene,which encodes for the major catalytic p110alpha subunit of PI3K,and has a conditional loss of tumour-suppressor PTEN.These NSPCs were derived from a conditional mutant mouse shown to develop tumours in various tissues tested.25–27CREB activation (pCREB)was signi?cantly elevated along with AKT activation (pAKT),compared with parental NSPCs from the same mouse in which the PI3K pathway mutations were not activated (Figure 3f).
CREB function contributes to GBM cell proliferation
As CREB is strongly associated with proliferative zones within the normal adult brain,we examined the effect of CREB knockdown on GBM cell proliferation.We tested siRNA-mediated CREB knockdown in ?ve human GBM cell lines (T98G,U118,U373,ANGM-CSS,U87),which represent cells with an array of GBM-associated genetic alterations.The combined use of three CREB siRNAs targeting different mRNA regions allowed robust CREB expression knockdown (490%)over at least 120h (Figure 4a)in T98G cells.All ?ve cell lines tested showed a reduction in cell proliferation (n ?1,not shown).Further experiments were performed with selected cell lines as indicated.The greatest reduction in proliferation was seen in cell lines T98G and U118,with a 58%and 52%reduction compared with scrambled siRNA-transfected controls after 120h (Figures 4b and c).As we
have
Figure 2.CREB is activated in dividing cells.T98G cells were analysed for the colocalization of pCREB(Ser 133)with markers of cell proliferation:(a )PCNA,(b )phospho -histone (pH3),nd (c )BrdU.
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Figure 3.The PI3K and MAPK -dependent signalling pathways but not the PKA pathway activate nuclear CREB in glioma cells.(a )GBM cell lines T98G,LN18and U118were serum starved for 24h then exposed to serum and protein lysate immunoblotted for the indicated antibodies.(b ,c )GBM cell lines T98G and U118were pretreated with a PI3K inhibitor LY294002,a MAPK inhibitor U0126or both inhibitors,and then exposed to serum,protein lysate collected after 4h and analysed for the presence of the indicated antibodies (d ,e )Quanti?cation of the effects of inhibitor combinations on pCREB levels in T98G and U118GBM cells.*P o 0.05,**P o 0.005.(f )Western blotting showing the level of pCREB and pAKT in mouse NSPCs grown as neurospheres,with an activated Pik3ca H1047R mutation and PTEN deletion (M)or parental NSPCs with ‘wild-type’Pik3ca and PTEN (C).All western blottings were performed at least three times,and where applicable,total CREB,AKT and MAPK were imaged,then membranes were stripped and reprobed for pCREB,pAKT and pMAPK
detection.
Figure 4.CREB is required for ef?cient human glioma cell line proliferation.(a )T98G cells were transfected with either CREB1-speci?c siRNA (siCREB)or scramble siRNA and then lysate analysed by western blotting for ef?ciency of CREB knockdown.*P ?0.05,**P ?0.005,***P ?0.0005.(b ,c )Scrambled or siCREB was transfected into GBM cell lines T98G and U118,and proliferation was analysed every 24h using an MTT assay.(d ,e )Scrambled or siCREB was transfected into GBM cell lines T98G and U118,and apoptosis was determined by FACs determination of AnnexinV uptake.
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previously shown that loss of CREB function in zebra?sh and mouse brain as well as mouse neural NSPCs leads to compromized cell survival,we tested whether this was the case in GBM cells.Notably,cell survival was not affected by CREB knockdown using two different assay systems,measuring lactate dehydrogenase released from dead cells and measuring early apoptosis by measuring appearance and accumulation of Annexin V (Figures 4d and e)following CREB knockdown.Thus reduced cell numbers can be attributed to reduced proliferation.
CREB knockdown leads to aberrant GBM cell cycle kinetics due to inhibition of cell cycle factor expression
To determine the mechanism by which CREB regulates GBM cell proliferation,cell cycle parameters were measured using DNA content by ?ow cytometry in T98G and U118cells,which were the cell lines showing the greatest CREB-dependent decrease in proliferation.CREB knockdown increased the proportion of cells in G0/G1phase in both cell lines tested (Figures 5a and b).siCREB T98G cells also showed signi?cantly reduced proportions in cell cycle phase distribution in S-and G2/M phases compared with control cells,whereas siCREB U118cells exhibited increased G0/G1and fewer G2/M phase cells only.To explore the molecular basis underlying disruption of the cell cycle,we measured the expression of cell cycle/proliferation factors previously reported to be transcriptionally regulated by CREB,including cyclins B1,D128and PCNA.14,29Protein expression of cyclins B1,D1and PCNA was assessed in T98G and U118cells in CREB knockdown or scrambled control siRNA-treated GBM cell lines (Figures 5c and d).Cells were serum deprived for 24h before the addition of serum to synchronize cell cycle activation,and cyclin and PCNA protein levels were determined every 12h,over 48h.The dynamics of expression seen
was consistent with the reported cyclin and PCNA expression pro?les for mammalian cells,where PCNA and cyclin D1are consistently expressed throughout the cell cycle while cyclin B1expression peaks at G2/M.30Upon treatment with serum (triggering cell cycle entry),we observed inhibition of protein expression of cyclin B1,cyclin D1and PCNA in both T98G and U118cell lines as early as 12h (not shown)with maximal inhibition reached at 24h,compared with no-serum cells (Figures 5c and d).T98G cells showed a consistent and almost complete block in cyclin D1,cyclin B1and PCNA expression over 48h in CREB knockdown cells,in contrast to U118cells,which exhibited a more modest inhibition of cyclin D1,over 48h.In U118cells,cylin B1protein expression was maximally inhibited at 24h,but little effect was seen in PCNA expression (Figure 5d).Moreover,in U118cells cyclin B1inhibition was not sustained,with expression approaching control levels beyond 24h.Given that cyclin B1,D1and PCNA harbour cAMP-resonsive elements (CREB-binding sequences)in their promoters (see Supplementary Data),we tested whether CREB exerted its in?uence on these target genes directly;we performed reverse transcriptase–PCR (RT–PCR)at 24h following siCREB treatment to measure mRNA levels of cyclin B1,D1and PCNA.CREB knockdown robustly inhibited cyclin D1mRNA expression in both T98G and U118cells,while cyclin B1mRNA expression was signi?cantly reduced in T98G cells only.Surprisingly,PCNA mRNA expression was unaf-fected by CREB knockdown in both cell lines (Figures 5e and f),implying that CREB exerts its affect on PCNA protein expression indirectly.The transcriptional in?uence CREB exerts in the expres-sion of these genes re?ects the context of the cAMP-resonsive elements in their respective promoters (see Supplementary Data),with cyclin D1showing the best context with a full (8-base pair consensus)cAMP-resonsive element positioned closest to the transcription start
site.
Figure 5.Knockdown of CREB alters human GBM cell cycle kinetics through regulation of cyclin B1,cyclin D1and PCNA.(a ,b )Synchronized GBM cell lines T98G and U118were treated with either siCREB or scramble control siRNA and then stimulated with serum for 24h before being analysed for cell cycle proportions using DNA-content analysis.(c ,d )Synchronized GBM cell lines T98G and U118were treated with either siCREB or scramble control siRNA,stimulated with serum and harvested every 12h for analysis for cyclin B1,cyclin D1and PCNA expression.(e ,f )Synchronized GBM cell lines T98G and U118were treated with either siCREB or scrambled control siRNA and then stimulated with serum for 24h before harvesting and analysis using qRT–PCR for CCNB1,CCND1and PCNA mRNA expression.***P o 0.0005,**P o 0.005,*P o 0.05.All western blottings were performed at least three times,and blottings for total CREB assay were imaged,then membranes were stripped and reprobed for pCREB detection.
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Selective CREB regulation of cell cycle factors by canonical and non-canonical interations with the PI3K pathway
To investigate the mechanism of the differential regulation of cyclin D1,cyclin B1and PCNA by CREB in T98G compared with U118cells,we looked at whether CREB knockdown affected upstream signalling components,given that previous studies have shown a link between CREB and upstream signalling components of the PI3K and MAPK pathways,such as insulin growth factor receptor (IGFR)and insulin receptor substrate-1/2(IRS-1/2).31,32Crucially,T98G and U118differ in PTEN status (T98G PTEN tand
U118PTEN à),which may account for the differential CREB-mediated regulation of cell cycle factors via interaction with the MAPK and PI3K pathways.33,34Treatment of T98G cells with siCREB resulted in an almost complete block of AKT activation,which was not rescued by the addition of serum (Figure 6a).In comparison,CREB knockdown in U118cells showed reduced AKT activation only in serum-free conditions (Figure 6b).Upon the addition of serum,AKT activation in U118cells increased to levels identical to control cells (Figure 6b).
To determine whether the cell-speci?c regulation of cyclin B1and PCNA by CREB could be due to effects on AKT activation and/or PTEN status,we used the PI3K inhibitor LY294002in conjuction with siCREB in U118cells (Figure 6c).This experiment con?rmed the dependence of cyclin D1expression on CREB,with siCREB treatment alone showing almost complete attenuation of cyclin D1expression.Furthermore,the expression of cyclin B1and PCNA were found to be dependent on both CREB and PI3K signalling.Neither siCREB nor the PI3K inhibitor LY294002alone could block cyclin B1or PCNA expression in U118cells.
DISCUSSION
CREB-dependent brain functions have been extensively explored with most previous studies focussed on neurological functions ranging from learning and memory,35opiate withdrawal 36to feeding behavior.37Other work has shown that CREB is critical for neuronal survival and that CREB disruption in vivo leads to a neurodegenerative phenotype 20and reduction in neural stem cell survival and proliferation 21,22In parallel,a body of evidence has accumulated showing that CREB imparts oncogenic properties on cells outside the central nervous system.23,38We focussed our investigations on de?ning CREB-dependent mechanisms orchestrating malignant GBM cell proliferation.Analysis of a panel of human GBM cell lines shows that CREB is not only highly expressed but also constitutively hyperactivated (Figure 1a).This is in contrast to non-tumour brain tissue,which show highly regulated and transient activation,dependent on external stimuli.11Cells in adult mouse brain showing robust constitutive phosphorylated CREB expression are enriched in the neurogenic zones,22,39so the constitutive expression of pCREB in GBM tumour cells is consistent with the generally held view that tumour cells exhibit immature stem/progenitor cell-like characteristics.In GBM tissue examined,pCREB was evident in many GBM hallmark morphological features,including near tumour angiogenic regions (Figure 1b-i),peripheral to necrotic foci characterized by pseudopalisading regions (Figure 1b-iii).This is interesting as GBM regions with pseudopalisading features harbour migrating tumour cells adjacent to hypoxic necrotic regions with high levels of pro-angiogenic factors,including hypoxia-inducible factor-1,vascular endothelial growth factor and interleukin-8,which are all CREB target genes.40–42CREB may therefore be intimately involved in regulating the angiogenic tumour niche in GBM.In one GBM tumour examined,strong pCREB-positive cells were clustered in foci exhibiting strong nestin co-labelling (Figure 1b-vi).Nestin,an intermediate ?lament protein,is highly expressed in immature NSPCs and,similar to pCREB,is associated with brain tumour grade 43–45This suggests that CREB may contribute to immature tumour cell biology by regulating CREB target genes involved in tumour cell growth and suppression of differentiation/promotion of immaturity;this is supported by studies showing that PI3K regulation of CREB is crucial for NSPC function.46Our data also shows that less aggressive GBM tumour subtypes,known to be associated with better survival,express lower levels of activated CREB.Giant cell GBM and oligodendroglial regions in GBM tissue show low to almost no pCREB labelling in most tumour cells (Figures 1b-iv and -v).It may be that pCREB is a biomarker associated with GBM malignancy and may also actively promote malignant properties in the more aggressive types of brain
cancer.
Figure 6.Selective dependence of cyclin expression on CREB is determined by the PI3K pathway and tumour-suppressor PTEN status in glioma cells.(a ,b )T98G and U118cells were treated with either scramble or siCREB for 24h before exposure to serum-free or serum conditions for 12h.Cell lysate was then analysed for markers of PI3K activation.(c )U118cells were treated with either LY294002,siCREB or both,and cell lysate was then immunoblotted for cyclin B1,cyclin D1and PCNA expression changes.All western blottings were performed at least three times,and where applicable,total CREB and AKT were imaged,and then membranes were stripped and reprobed for pCREB and pAKT detection.
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These putative roles of CREB relate to its position in a complex signalling network where CREB sits at a convergence point of multiple oncogenic signalling pathways and is activated by multiple factors,including the PI3K and MAPK pathways which are essential contributors to the cancer phenotype.As such,CREB activation may be re?ective of oncogenic pathway https://www.doczj.com/doc/5c15601893.html,e of the PI3K inhibitor LY294002and MAPK inhibitor U0126to dissect the contribution of these core pathways established a causal relationship between CREB activation and the PI3K and MAPK pathways;however,our data also demonstrate that CREB is activated by multiple pathways in GBM and that these pathways vary between GBM cell lines(Figures3b and c).The regulation of CREB by the PI3K and MAPK pathways appears to be complex and there is some redundancy in this regulation.Furthermore,CREB is able to regulate cell proliferation(Figure4),an important characteristic of GBM cells to which multiple pathways are able to contribute.47–49Taken together,our data suggest that disruption of oncogenic functions regulated by the PI3K and MAPK pathways in GBM may be achieved by direct targeting of CREB rather than the redundant signalling pathways.
One mechanism by which CREB regulates proliferation is via modulation of the cell cycle,a previously reported role conserved across multiple cancer and non-cancer cell types.28,50,51 Knockdown of CREB in GBM cell lines T98G and U118resulted in attenuation of the cell cycle however the effect on cell cycle progression was much greater in T98G than U118.The sensitivity to CREB knockdown differed in the two cells tested,with CREB loss having a greater effect on cyclin expression in T98G cells compared with U118(Figures5a and b).Cyclin D1exhibited robust CREB dependence in both cell lines,whereas CREB knockdown reduced expression of cyclin B1and PCNA in T98G but not in U118cells(Figures5c and d).Cyclin D1,cyclin B1and PCNA are known markers of poor survival in a multitude of cancers,such as hepatocellular carcinoma,52anorectal malignant melanoma53epithelial ovarian carcinoma54and breast cancer.55 Differences observed in the regulation of cell cycle genes by CREB are likely due to differences in the genetic background of GBM where PTEN status was most notable in the GBM cell lines tested(T98G PTENt,U118PTENà).The presence or absence of PTEN can in?uence many cellular responses,including proliferation, with PTEN not only acting as a negative regulator of the canonical PI3K pathway but in?uencing many other PI3K-independent effects,including roles in cell cycle and DNA integrity.56 Mechanistically,a difference in PTEN status likely explains the ability for CREB to differentially regulate AKT activity in T98G and U118(Figures6a and b).A feedback loop involving CREB and AKT may occur through direct CREB-dependent regulation of insulin receptor substrate2(IRS1/2)expression,adaptor proteins down-stream of IGFR/IR which directly associates with the p85subunit of PI3K to activate the PI3K/AKT pathway.57In the presence of PTEN, the activation of the PI3K pathway is antagonized,resulting in the upregulation of IR/IGFR/IRS1/IRS58in an attempt to maintain AKT activation.Importantly,CREB has been shown to regulate the expression of IRS components,59suggesting a mechanism by which CREB is able to regulate the activity of AKT.In T98G cells, the presence of PTEN would inhibit the phosphorylation of IRS1, while the knockdown of CREB would cause loss of IRS2,leading to the sustained downregulation of PI3K pathway(Figure6a).By contrast,U118cells that are PTEN null would have no PTEN-dependent inhibitory in?uence on IRS1,thus allowing a serum-dependent increase in PI3K activity(Figure6b).In line with this, the consequence of CREB loss on cell cycle progression and regulation of cell cycle genes cyclin B1and PCNA was dependent on the regulation of PI3K by CREB in a PTEN-dependent manner. Importantly,co-inhibition of CREB and PI3K activity was necessary for the downregulation of cyclin B1and PCNA(Figure6c), highlighting the key role for CREB in promoting cell proliferation, even when the PI3K pathway is inhibited.This has considerable therapeutic signi?cance,as redundancy in oncogenic signalling networks is a major mechanism by which the clinical impact of targeted therapies is negated.Indeed,recent reports have demonstrated that CREB has a major role mediating resistance to targeted therapies in melanoma,16which when considered with the
data presented here,highlights the need for further investigation of CREB and PI3K pathway target genes in tumour cells.
Overall,this study shows that pCREB is a marker of glioma grade
and that CREB activation in GBM cells appears to modulate GBM tumour cell proliferation through the integration of signals from
PI3K and MAPK pathways(Figure7).We also show that CREB acts
as a master transcriptional regulator of cyclin D1in GBM.This observation is consistent with the spectrum of tumour cell types where CREB has been shown to regulate cyclin D1expression, including malignant T-cells60gastric cancer cells,61breast cancer cells,62embryonic carcinoma cells63and prostate cancer cells. Furthermore,we establish that CREB may be part of a complex signalling feedback loop modulating the expression of cell cycle factors.Given the roles CREB has in regulating GBM cell proliferation and the convergence of multiple redundant cancer signalling pathways upon CREB,the use of anti-tumour therapeutic approaches incorporating CREB inhibitors would be expected to provide more effective anti-tumour responses
and
Figure7.CREB-dependent cell cycle factor expression signalling network in GBM tumour cells.CREB lies downstream of both the
PI3K and MAPK pathways and modulates cell cycle factor expres-sion.Cyclin D1is completely dependent on CREB activity while cyclin B1and PCNA appear to require activation of both CREB and
non-CREB mechanisms.The width of the arrow indicates the relative strength of the signal.The broken arrows pointing from CREB to AKT indicates the putative CREB-dependent regulation of the PI3K pathway.
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may bypass the inherent redundancy and associated drug resistance that promote tumour cell proliferation. MATERIALS AND METHODS
Cell culture and treatments
Human GBM cell lines(T98G,LN18and U118)were purchased from the American Type Culture Collection(ATCC,Manassas,VA,USA).Cells were maintained at371C and5%CO2in complete media(Dulbecco’s Modi?ed Eagle Medium(DMEM)F12tGlutaMAX(Invitrogen,Carlsbad,CA,USA) supplemented with10%fetal bovine serum(Fisher Biotech,Wembley,WA, Australia)and penicillin/streptomycin(Invitrogen)diluted to50units/ml of penicillin and50m g/ml of streptomycin.Cells were synchronized before treatment with serum and/or inhibitors by serum starvation for24h. Inhibitor experiments were performed on serum-starved cells using25m M of LY294002(Genesearch)and/or U0126(Genesearch,Danvers,MA,USA) or the equivalent concentration of dimethyl sulphoxide.Cells were exposed to treatments for30min before the addition of serum and then harvested as necessary.
siRNA transfection
Twenty picomolar CREB1-speci?c siRNA or scramble siRNA(Santa Cruz, Dallas,TX,USA)was incubated with2m l/ml Lipofectamine RNAiMax (Invitrogen)in unsupplemented DMEM F12for20min at371C to allow complex formation.Cells were incubated with siRNAs overnight at371C before washing in phosphate-buffered saline(PBS)and culturing in complete media(as above).
MTT and FACS analysis
Cell lines were transfected with CREB siRNA or scramble siRNA and then plated onto96-well dishes in complete media the next day.Cell growth was determined every24h by incubation with MTT reagent(Invitrogen) at 1.2m M?nal concentration,for4h before lysis in MTT lysis buffer (isopropanol,1%Triton-X,1drop glacial HCl/50ml).Absorbance at570nm was then measured using a Wallac1420VICTOR2plate reader(GMI, Ramsey,MN,USA).
Cell cycle and AnnexinV analysis was performed on siRNA-transfected cells using the MUSE cell analyser(Millipore,Billerica,MA,USA).For AnnexinV experiments,cells were harvested48h after transfection, resuspended in100m l complete media and100m l AnnexinV buffer solution(Millipore),incubated for15min at room temperature and then analysed on the MUSE.For cell cycle analysis,siRNA-transfected cells were serum starved for24h to synchronize cells before the addition of complete media.Twenty-four hours after the addition of complete media,cells were harvested and then?xed in ice-cold80%ethanol overnight.Cells were then washed and incubated with cell cycle assay buffer(Millipore)for 30min before being analysed on a MUSE cell analyser(Millipore). Protein extraction and western blotting
Total cellular protein was extracted using RIPA buffer(150m M sodium chloride,1%Triton-X,0.5%sodium deoxycholate,50m M Tris,pH8.0) supplemented with Pierce complete protease and phosphatase inhibitor tablet(Pierce,Rockford,IL,USA)and then protein quanti?ed using a BCA protein assay kit(Pierce).In all,15–20m g of protein was run on a10% polyacrylamide gel by electrophoresis and then transferred onto a polyvinylidene di?uoride membrane(Millipore)before being blocked in 1%Tween-20Tris-buffered saline pH6.8(TBS-T)supplemented with2% skim milk.Membranes were probed for speci?c proteins using antibodies as listed below.
Immunohistochemistry
Fixed(4%buffered paraformaldehyde)paraf?n-embedded tissue was used to section at7m m and then dewaxed with ethanol and histolene. Immunohistochemical labelling was performed using antibodies as listed below,with overnight incubation of primary antibodies at41C,followed by 1h incubation of secondary antibodies and solutions for colour develop-ment provided in a VectoLabs Vectastain ABC kit(Vector Laboratories, Burlingame,CA,USA)used according to the manufacturer’s instructions. Double labelling immunohistochemistry was performed sequentially,each primary antibody being individually incubated and developed.Quantitative RT–PCR(qRT–PCR)
Synchronized cells were treated with either scrambled(control)or CREB-speci?c siRNA and then treated with serum for24h.Cellular RNA was harvested by the addition of300m l of Trizol(Ambion,Foster City,CA,USA) directly to the plate before addition of100m l of chloroform(Sigma,St Louis,MO,USA)and subsequent centrifugation for15min at12000r.p.m. for phase separation.The upper RNA phase was collected before being passed through a NucleoSpin RNA Clean-up XS column(Macherey-Nagel, D-52313,Du¨ren,Germany).Eight hundred nanograms of RNA was reverse transcribed using a Superscript III?rst-strand reverse transfection kit (Invitrogen)and then ampli?ed with primers speci?c to CCND1(Forward-50-GATCAAGTGTGACCCGGACTG-30;Reverse-50-CCTTGGGGTCCATGTTCT GC-30),CCNB1(Forward-50-TGGTGAATGGACACCAACTC-30;Reverse-50-TTC TTAGCCAGGTGCTGCAT-30)or PCNA(Forward-50-GCCCTGGTTCTGGAGGT AAC-30;Reverse-50-TAGCTGGTTTCGGCTTCAGG-30).Ampli?cation was per-formed on a LightCycler480(Roche,Basel,Switzerland)using LightCycler 480SYBR Green I Mastermix(Roche)under the following conditions: preincubation501C for2min,951C for2min,ampli?cation at951C for15s and then at55–651C for30s.
Immuno?uorescence
Cells were cultured on poly-d-lysine and?bronectin-coated glass cover-slips and then incubated with or without BrdU(3m g/ml)for4h before being?xed in4%paraformaldehyde(Sigma)for15min on ice.Cells were then washed in PBS,and BrdU slides were incubated in HCl(1N)for 10min.Cells were permeabilized and blocked in staining buffer1(DPBS supplemented with0.1%triton-X,10%fetal calf serum)for1h before incubation with primary antibodies overnight at41C.Slides were then washed3?in DPBS and then co-labelled with Alexa-Fluor secondary antibodies(Invitrogen)for1h.Slides were washed in PBS again before a10-min incubation with100ng/ml DAPI solution(Sigma). Slides were then washed a?nal time in distilled water and then mounted with Fluorogel aqueous anti-fade media(ProSciTech,Townsville City,QLD, Australia).
Antibodies used
The antibodies used were CREB(Genesearch no.9197L),pCREB Serine133 (Genesearch no.9197),BrdU(Genesearch no.5292),pH3(Genesearch no. 9701S),AKT(Genesearch no.9272),pAKT Threonine308(Genesearch no. 9275S),MAPK(Genesearch no.9102),pMAPK Threonine202/Tyrosine 204(Genesearch no.9101),PKA-C(Genesearch no.4782),pPKA-C Threonine197(Genesearch no.4781),Cyclin D1(Abcam no.ab95281), Cyclin B1(Abcam no.ab2949),PCNA(Genesearch no.2586),and GAPDH (Genesearch no.2118S).All primary antibodies were used at1:2000 dilutions for western blottings and1:100for immuno?uorescence or immunohistochemistry.Blots were then developed with enhanced chemiluminescence reagent(Bio-Rad,Hercules,CA,USA)and imaging performed on a Microchemi(DNR BioImaging Systems,South San Francisco,CA,USA).
Statistical analysis
Data are presented as mean±s.e.m.of at least three independent experiments.P-values were determined using a pairwise two-tailed Student’s t-test,with P o0.05considered statistically signi?cant.
CONFLICT OF INTEREST
The authors declare no con?ict of interest.
ACKNOWLEDGEMENTS
We thank the members of the Department of Pathology,TheUniversityofMelbourne, Parkville,Victoria,Australia for the use of equipment and reagents,especially Dr Genevieve Evin,Dr Vicki Lawson,Laura Ellett and Janine James.We also thank Professor Paul Waring for?nancial support for PD,GF and DVB and the Cell Signaling Laboratory.Part of this work was also supported by an EU-FP7-PEOPLE Marie Curie IRG(Neurogencreb231032)grant to TM.WAP,RGR and JM were supported,in part,by project grants from the National Health and Medical Research Council of Australia.
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CREB-mediated cell cycle factor expression in GBM cells
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Hippo信号通路 一、Hippo信号通路概述 Hippo 信号通路，也称为Salvador / Warts / Hippo（SWH）通路，命名主要源于果蝇中的蛋白激酶Hippo（Hpo），是通路中的关键调控因子。该通路由一系列保守激酶组成，主要是通过调控细胞增殖和凋亡来控制器官大小。 Hippo信号通路是一条抑制细胞生长的通路。哺乳动物中，Hippo信号通路上游膜蛋白受体作为胞外生长抑制信号的感受器，一旦感受到胞外生长抑制信号，就会激活一系列激酶级联磷酸化反应，最终磷酸化下游效应因子YAP和TAZ。而细胞骨架蛋白会与磷酸化后的YAP和TAZ结合，使它滞留在细胞质内，降低其细胞核活性，从而实现对器官大小和体积的调控。 二、Hippo信号通路家族成员 虽然Hippo信号通路在各个物种中保守性很高，但是相同功能的调控因子或效应因子在不同物种间还是存在着差异，下表中我们对比了果蝇与哺乳动物中Hippo信号通路相同功能的关键因子[1]。
Expanded(Ex) FRMD6/Willin 含有FERM结构域的蛋白，能与Kibra及Mer结合，调控Hippo信号通路的上游信号 Dachs(Dachs) 肌浆球蛋白myosin的一种，能结合Wts 并促进其降解 Kibra(Kibra) WWC1 含有WW结构域的蛋白，能与Ex及Mer 结合，调控Hippo信号通路的上游信号 Merlin(Mer) NF2 含有FERM结构域的蛋白，能与Kibra及Ex结合，调控Hippo信号通路的上游信号 Hippo(Hpo) MST1,MST2 Sterile-20-样激酶，磷酸化并激活Wts Salvador(Sav) WW45(SAV1) 含有WW结构域的蛋白，能起到一个脚手架蛋白的作用，易化Hippo对Warts的磷酸化 Warts（Wts）LATS1,LATS2 核内DBF-2相关激酶，能磷酸化Yki并使之失活 Mob as tumor suppressor(Mats) MOBKL1A,MOBKL1B 能与Wts结合的激酶，与Wts结合后能 促进Wts的催化活性 Yorkie(YKi) YAP,TAZ 转录共激活因子，能在非磷酸化的激活状态下与转录因子Sd结合，并激活下游靶基因的转录。这些受调控的下游靶基因主要参与了细胞的增殖、生长并抑制凋亡的发生 Scalloped(Sd) TEAD1,TEAD2,TEAD3, TEAD4 能与Yki结合的转录因子，与Yki共同 作用，调控靶基因的转录 三、Hippo信号通路的功能 近十年相关研究结果表明，无论是果蝇还是哺乳动物，Hippo信号通路都可以通过调节细胞增殖、凋亡和干细胞自我更新能力实现对器官大小的调控。Hippo信号通路异常会导致大量组织过度生长。此外，大量研究证实，Hippo信号通路在癌症发生、组织再生以及干细胞功能调控上发挥着重要功能[2][3][4]。 a.Hippo信号通路在器官大小控制中的作用 起初，关于Hippo信号通路的研究主要集中在器官大小的调控。大量研究表明，Hippo 途径主要通过抑制细胞增殖并促进细胞凋亡，继而实现对器官大小的调控。激酶级联反应是该信号传导的关键。Mst1/2激酶与SA V1形成复合物，然后磷酸化LATS1/2；活化后的LATS1/2激酶随即磷酸化Hippo信号通路下游关键效应分子——Y AP和TAZ，同时抑制了
1 PPAR信号通路：过氧化物酶体增殖物激活受体( PPARs) 是与维甲酸、类固醇 和甲状腺激素受体相关的配体激活转录因子超家族核激素受体成员。它们作为脂 肪传感器调节脂肪代谢酶的转录。PPARs由PPARα、PPARβ和PPARγ 3种亚型组成。PPARα主要在脂肪酸代谢水平高的组织，如：肝、棕色脂肪、心、肾和骨骼肌表达。他通过调控靶基因的表达而调节机体许多生理功能包括能量代谢、生 长发育等。另外，他还通过调节脂质代谢的生物感受器而调节细胞生长、分化与 凋亡。PPARa同时也是一种磷酸化蛋白，他受多种磷酸化酶的调节包括丝裂原激活蛋白激酶( ERK-和p38．M APK) ，蛋白激酶A和C( PKA，PKC) ，AM PK和糖原合成酶一3( G SK3) 等调控。调控PPARa生长信号的酶报道有M APK、PKA和G SK3。PPARβ广泛表达于各种组织，而PPAR γ主要局限表达在血和棕色脂肪，其他组织如骨骼肌和心肌有少量表达。PPAR-γ在诸如炎症、动脉粥样硬化、胰岛素抵抗和糖代谢调节，以及肿瘤和肥胖等方面均有着举足轻重的作用， 而其众多生物学效应则是通过启动或参与的复杂信号通路予以实现。鉴于目前人 们对PPAR—γ信号通路尚不甚清，PPARs通常是通过与9-cis维甲酸受体( RXR)结合实现其转录活性的。 2 MAPK信号通路:mapk简介:丝裂原激活蛋白激酶（mitogen—activated protein kinase，MAPK）是广泛存在于动植物细胞中的一类丝氨酸/苏氨酸蛋白激酶。作用主要是将细胞外刺激信号转导至细胞及其核内，并引起细胞的生物化学反应（增殖、分化、凋亡、应激等）。 MAPKs家族的亚族 :ERKs（extracellular signal regulated kinase)：包括ERK1、ERK2。生长因子、细胞因子或激素激活此通路，介导细胞增殖、分化。 JNKs(c-Jun N-terminal kinase)包括JNK1、JNK2、JNK3。此亚族成员能使 Jun转录因子N末端的两个氨基酸磷酸化而失活，因此称为Jun N末端激酶(JNKs)。物理、化学的因素引起的细胞外环境变化以及致炎细胞因子调节此通路。P38 MAPKs：丝氨酸/络氨酸激酶，包括p38 α、p38β、p38γ、p38δ。p38 MAP K参与多种细胞内信息传递过程 ,能对多种细胞外刺激发生反应,可磷酸化其它细胞质蛋白,并能从胞浆移位至细胞核而调节转录因子的活性来改变基因的表达水平 ,从而介导细胞生长、发育、分化及死亡的全过程。 ERK5:是一种非典型的MAPK通路，也叫大MAPK通路，只有一个成员。它可被各种刺激因素激活。不仅可以通过磷酸化作用使底物活化，并且通过C端的物理性结合作用激活底物。 3 ERBB信号途径:ErbB 蛋白属于跨膜酪氨酸激酶的 EGF 受体家族成员。ErbB 的命名来源于在禽红白血病 B( v-Erb-B) 发现的 EGF 受体的突变体,因而 EGF 受体 亦称为“ ErbB1”。人源 ErbB2 称为HER2, 特指人的 EGF 受体。ErbB 家族的
干货细胞信号通路图解之MAPK通路【值得珍藏】 科研小助手原创，转载请注明来源。公众号内回复“Cell Signaling Pathway”获取全套信号通路图本文由百度贴吧nosce吧吧主黄杰投稿一、MAPK信号通路： （1）有丝分裂原激活的蛋白激酶(MAPK)是一族在真核生物中非常保守的丝/苏氨酸蛋白激酶，在许多细胞活动中起作用，如生长增殖，细胞分化，细胞运动或死亡。MAPK级联信号传导由3 个不同层次的分子所组成。MAPK被MAPK的激 酶( MAPKK)磷酸化后激活，MAPKK被MAPKK的激酶(MAPKKK )磷酸化而激活。而MAPKKK通过与小GTPase 和/或其他蛋白酶相互作用而被激活，从而将MAPK和细胞 表面的受体以及胞外的信号联系在一起。 （2）许多参与生长和分化的受体都能够激活MAPK/ERK信号通路，比如说受体酪氨酸激酶(RTK)，整合素，和离子通道。响应特定信号所涉及到的具体分子会相差很大，但通路的结构是一致的，那就是接头分子(adaptor，如Shc, GRB2, Crk等)将鸟苷酸交换因子(SOS, C3G 等)和受体连接在一起，然后把信号向小GTP 结合蛋白(Ras, Rap1)传递，后者又激活核心的级联反应，这是由一个MAPKKK( Raf) ，一个MAPKK( MEK1/2)和MAPK( Erk)所构成的。活化的ERK 二聚体能调节胞浆中的目标分子，也可以转移到细胞核中，然
后对一系列转录因子进行磷酸化以调节基因表达。SciRes（3）很多外部的刺激都能够激活G蛋白偶联受体(GPCR)。在受体活化以后，G 蛋白将GDP 转换成GTP ，然后结合了GTP的α和β/γ亚基从受体脱离开，启动信号向胞内的传导。与不同亚型的异质三聚体G 蛋白结合的受体可以采取不同 的手段激活小G 蛋白/MAPK级联反应，至少有三个不同家族的酪氨酸激酶参与其中。Src家族激酶响应活化的PI3Kγ，而后者被β/γ亚基激活。它们还能够响应受体的内化，受体酪氨酸激酶的交叉活化，以及有Pyk2 和/或FAK参与的整 合素途径信号。GPCRs同样可以通过PLCβ去激活PKC 和CaMKII ，对下游的MAPK通路可以有激活或抑制的影响。SciRes（4）压力激活的蛋白激酶(Stress-activated protein kinase, SAPK)或称Jun氨基端激酶(Jun amino-terminal kinase, JNK) 是MAPK的家族成员，能被一系列的环境压力，炎症细胞因子，生长因子和GPCR激动剂所激活。压力信号通过Rho家族的小GTP 酶(small GTPase)向这条级联通路传导，这些小GTP酶包括(Rac, Rho, cdc42) 。和其他的MAPK情况一样，靠近膜的激酶是一个MAPKKK，一般 是MEKK1-4 ，或者是一个混合激酶去磷酸化并激活 MKK4(SEK)或MKK7，它们是SAPK/JNK的激酶。另外，MKK4/7也可以被生发中心激酶(germinal center kinase, GCK)以一种GTPase 依赖的方式激活。活化后的
资深PI最新文章解析信号通路 ------------------------------------------------------------------------------------------------------------------------------------ 摘要：来自新加坡分子与细胞生物学研究院，癌症与发育细胞生物学部的研究人员获得了YAP－TEAD4复合物在YAP因子N端结构域相互作用，以及在TEAD4 C端结构域与YAP相互作用的晶体结构，从中研究人员认为YAP中的PXXΦP片段是与TEAD4相互作用的关键结构，这为研究Hippo信号通路提供了重要的分子机理线索。这一研究成果公布在《Genes Development》杂志上。 生物通报道：来自新加坡分子与细胞生物学研究院，癌症与发育细胞生物学部的研究人员获得了YAP－TEAD4复合物在YAP因子N端结构域相互作用，以及在TEAD4 C端结构域与YAP相互作用的晶体结构，从中研究人员认为YAP中的PXXΦP片段是与TEAD4相互作用的关键结构，这为研究Hippo信号通路提供了重要的分子机理线索。这一研究成果公布在《Genes Development》杂志上。 领导这一研究的是新加坡分子与细胞生物学研究院宋海卫博士，其早年毕业于河南大学化学系，之后进入中科院生物物理研究院进行分子生物学方面的学习，1998年获得利兹大学（The University of Leeds）分子生物学专业博士学位。目前任新加坡分子与细胞生物学研究所资深研究员。 Hippo信号转导通路是几年前发现的一个信号转导通路。研究发现Hippo信号通路是参与调控器官大小发育的关键信号通路，这一观点首先在果蝇中被发现，后来的研究发现在哺乳动物的发育过程中Hippo有相同的功能。06年Cell发表的一篇文章证实Hippo 是一种细胞分裂和死亡的控制开关。Hippo信号转导通路通过促进细胞调亡和限制细胞
目录 actin肌丝 (5) Wnt/LRP6 信号 (7) WNT信号转导 (7) West Nile 西尼罗河病毒 (8) Vitamin C 维生素C在大脑中的作用 (10) 视觉信号转导 (11) VEGF，低氧 (13) TSP-1诱导细胞凋亡 (15) Trka信号转导 (16) dbpb调节mRNA (17) CARM1甲基化 (19) CREB转录因子 (20) TPO信号通路 (21) Toll-Like 受体 (22) TNFR2 信号通路 (24) TNFR1信号通路 (25) IGF-1受体 (26) TNF/Stress相关信号 (27) 共刺激信号 (29) Th1/Th2 细胞分化 (30) TGF beta 信号转导 (32) 端粒、端粒酶与衰老 (33) TACI和BCMA调节B细胞免疫 (35) T辅助细胞的表面受体 (36) T细胞受体信号通路 (37) T细胞受体和CD3复合物 (38) Cardiolipin的合成 (40) Synaptic突触连接中的蛋白 (42) HSP在应激中的调节的作用 (43) Stat3 信号通路 (45) SREBP控制脂质合成 (46) 酪氨酸激酶的调节 (48) Sonic Hedgehog (SHH)受体ptc1调节细胞周期 (51) Sonic Hedgehog (Shh) 信号 (53) SODD/TNFR1信号 (56) AKT/mTOR在骨骼肌肥大中的作用 (58) G蛋白信号转导 (59) IL1受体信号转导 (60) acetyl从线粒体到胞浆过程 (62) 趋化因子chemokine在T细胞极化中的选择性表达 (63) SARS冠状病毒蛋白酶 (65) SARS冠状病毒蛋白酶 (67) Parkin在泛素-蛋白酶体中的作用 (69)
收稿日期:2008 07 01 作者简介:刘顺会(1971 ),男,湖北荆州市人,博士,主要从事生物信息学研究。 基金项目:国家自然科学基金(30572124)、广东省科技厅(2004B31201001)、教育部科学技术研究重点项目(205116)、广东省自然科学基金(5002855)联合资助。 *广东药学院临床医学院 文章编号:1004 4337(2008)06 0641 06 中图分类号:R392 4 文献标识码:A 医学数学模型探讨 T 细胞受体信号转导通路的动力学分析 刘顺会 肖兰凤 * 黄树林 (广东药学院生命科学与生物制药学院 广州510006) 摘 要: 目的:建立T 细胞受体信号转导途径的动力学模型,通过模型仿真揭示T 细胞受体信号途径各分子间的动态调控过程,简要分析模型的动力学特性。方法:根据数据库KEGG 及相关中英文文献,提取T 细胞受体信号转导各条通路相关分子作用的方式及数量关系,利用M atlab 7.0的S imulin k 工具箱构建信号途径的动力学模型并仿真。结果:模型仿真结果与文献符合得较好,能够从数量上反映T 细胞受体信号转导途径中各分子间复杂的调控关系,并能通过模型仿真发现和验证该信号途径中的关键节点分子。结论:模型基本反映了T 细胞受体信号转导途径的动力学特征,可以作为后续的精确定量关系研究的基础。 关键词: T 细胞受体; 信号转导; 动力学模型 T 细胞特异性抗原或T CR/CD3的特异性抗体可引起T 细胞跨膜受体以及膜附近的其它信号分子的活化,并引起T 细胞形态改变、细胞因子分泌、细胞粘附性改变等免疫应答,从而调节T 细胞的增殖、分化或凋亡,该过程涉及一系列下游信号转导和基因表达调控。T 细胞活化时信号传递由T 细胞表面抗原识别受体(T cell receptor ,T CR)介导,外来信号经受体及相关蛋白传递给胞内的蛋白酪氨酸激酶,此后启动三条下游信号途径:一为磷脂酶C 1(Phospholipase C 1,PL C 1)介导的信号途径,二为Ras M A PK 途径,三为共刺激分子介导的磷酸肌醇激酶 3(PI3K)辅助信号途径。同时,为保持免疫应答的平衡,避免过度活化,T 细胞的活化过程还受到抑制性信号通路的调节,这些通路彼此交织,构成一个十分复杂的T 细胞活化调控网络[1]。 随着各种复杂的信号转导网络中各个分子通道的确定,如何从系统水平上定量地分析各信号转导网络的动态特征就成为当前系统生物学的研究重点。除各种并行、高通量的实验技术外,数学建模和仿真是另外一种研究复杂生化网络的强有力手段,比如在细胞代谢研究领域已经很广泛地利用数学模型分析具有多个调控环的代谢网络[2]。实践表明,通过建立生化网络的数学模型并进行计算机仿真能够拟合现有的实验数据,解释实验中观测到的现象,预测一些不能通过当前实验手段获得的结果,减少实验的强度和数量,并验证实验提出的可能机制。 本研究建立了T 细胞受体信号转导途径的动力学模型,通过模型仿真揭示了T 细胞受体信号途径各分子间的动态调控过程,并对模型的动力学特性进行了简要分析。1 材料与方法 1 1 T 细胞受体信号转导途径 T 细胞受体信号转导途径(图1)摘自K EG G 数据库(ht tp://ww w.g eno me.jp/keg g/)。本研究根据相关中英文文献,对图中涉及的信号转导相关分子之间的作用方式及数量关系进行了详细研究和确证,并据此定义整个信号转导途径的变量(包括自变量和因变量)和变量之间的关系。1 2 动力学建模 本研究采用S 系统方程(S system equations )[2]来描述信号转导的生化级联反应过程。对于含有n 个因变量X 1,X 2,!,X n (其数量随时间而变化)和m 个自变量X n +1 ,X n +2,!, X n +m (一般设定为某些不变常量)的生化级联反应系统,其动 力学时变方程可以表达为: d X i /d t =V +i -V -i i =1,2,!,n (1) 式中X i 的生产函数V i +=V i +(X 1,!,X n ,X n +1,!,X n +m )和消耗函数V i -=V i -(X 1,!,X n ,X n +1,!,X n +m )是所有变量的函数,式(1)用S 系统方程可表达为: d X i /d t = i n +m j =1 X g ij j - i n +m j =1 X h ij j i =1,2,!,n (2) 式(2)中 i 和 i ( i 、 i >0)分别表示生产函数和消耗函数的速率常数,g ij 和h ij 分别表示变量X j 在因变量X i 的生产和消耗过程中的动力学阶,其中g ij 或h ij >0表示X j 在X i 的生产或消耗过程中起"正"调控作用,反之,如果g ij 或h ij <0则表示X j 在X i 的生产或消耗过程中起?负#调控作用,而当g ij 或h ij =0时则表示X j 在X i 的生产或消耗过程中不起任何调控作用。 641
mTOR信号通路图 mTOR可对细胞外包括生长因子、胰岛素、营养素、氨基酸、葡萄糖等多种刺激产生应答。它主要通过PI3K/Akt/mTOR途径来实现对细胞生长、细胞周期等多种生理功能的调控作用。正常情况下，结节性脑硬化复合物-1(TSC-1)和TSC-2形成二聚体复合物，是小GTP 酶Rheb(Ras-homolog enriched in brain)的抑制剂，而Rheb是mTOR活化所必需的刺激蛋白，因此TSC-1/TSC-2在正常情况下抑制mTOR的功能。当Akt活化后，它可磷酸化TSC-2的Ser939和Thr1462，抑制了TSC-1/TSC-2复合物的形成，从而解除了对Rheb 的抑制作用，使得mTOR被激活。活化的mTOR通过磷酸化蛋白翻译过程中的某些因子来参与多项细胞功能，其中最主要的是4EBP1和P70S6K。
在整个PI3K/Akt/mTOR信号通路中，有一条十分重要的负反馈调节剂就是10号染色体上缺失与张力蛋白同源的磷酸酶基因(phosphatase and tensin homology deleted on chromosome 10, PTEN)。PTEN是一个肿瘤抑制基因，位于人染色体10q23。它有一个蛋白酪氨酸磷酸酶结构域，在这条通路中可以将PI-3，4-P2与PI-3，4，5-P3去磷酸化，从而负调节PI3K下游AKt/mTOR信号通路的活性。 本信号转导涉及的信号分子主要包括 IRS-1，PI3K，PIP2，PIP3，PDK1，PTEN，Akt，TSC1，TSC2，Rheb，mTOR，Raptor，DEPTOR，GβL，p70S6K，ATG13，4E-BP1，HIF-1，PGC-1α，PPARγ，Sin1，PRR5，Rictor，PKCα，SGK1，PRAS40，FKBP12，Wnt，LRP，Frizzled，Gαq/o，Dvl，Erk，RSK，GSK-3，REDD1，REDD2，AMPK，LKB1，RagA/B，RagC/D等。
磷脂酰肌醇3-激酶(PI3Ks)信号通路 磷脂酰肌醇3-激酶(PI3Ks)信号通路相关 磷脂酰肌醇3-激酶(PI3Ks)蛋白家族参与细胞增殖、分化、凋亡和葡萄糖转运等多种细胞功能的调节。PI3K活性的增加常与多种癌症相关。PI3K磷酸化磷脂酰肌醇PI（一种膜磷脂）肌醇环的第3位碳 原子。PI在细胞膜组分中所占比例较小，比磷脂酰胆碱、磷脂酰乙醇胺和磷脂酰丝氨酸含量少。但在脑细胞膜中，含量较为丰富，达磷脂总量的10%。 PI的肌醇环上有5个可被磷酸化的位点，多种激酶可磷酸化PI肌醇环上的4th和5th位点，因而通常在这两位点之一或两位点发生磷酸化修饰，尤其发生在质膜内侧。通常，PI-4,5-二磷酸（PIP2）在磷脂酶C的作用下，产生二酰甘油（DAG）和肌醇-1,4,5-三磷酸。PI3K转移一个磷酸基团至位点3， 形成的产物对细胞的功能具有重要的影响。譬如，单磷酸化的PI-3-磷酸，能刺激细胞迁移（cell trafficking)，而未磷酸化的则不能。PI-3,4-二磷酸则可促进细胞的增殖（生长）和增强对凋亡的抗性，而其前体分子 PI-4-磷酸则不然。PIP2转换为PI-3,4,5-三磷酸，可调节细胞的黏附、生长和存活。 PI3K的活化 PI3K可分为3类，其结构与功能各异。其中研究最广泛的为I类PI3K, 此类PI3K为异源二聚体，由一个调节亚基和一个催化亚基组成。调节亚基含有SH2和SH3结构域，与含有相应结合位点的靶蛋白相作用。该亚基通常称为 p85, 参考于第一个被发现的亚型（isotype）,然而目前已知的6种调节亚基，大小从50至110kDa不等。催化亚基有4种，即p110α, β,δ,γ，而δ仅限于白细胞，其余则广泛分布于各种细胞中。 PI3K的活化很大程度上参与到靠近其质膜内侧的底物。多种生长因子和信号传导复合物，包括成纤维细胞生长因子（FGF)、血管内皮生长因子(VEGF)、人生长因子（HGF）、血管位蛋白I(Ang1)和胰岛素都能启始PI3K的激活过程。这些因子激活受体酪氨酸激酶（RTK），从而引起自磷酸化。受体上磷酸化的
山东农业大学学报(自然科学版),2015,46(4):514-518VOL.46N0.42015 Journal of Shandong Agricultural University(Natural Science Edition)doi:10.3969/j.issn.1000-2324.2015.04.007 细胞凋亡的信号通路 谢昆,李兴权 红河学院生命科学与技术学院,云南蒙自661199 摘要:细胞凋亡是细胞程序性死亡的一种方式，与自噬和坏死有明显的区别。细胞凋亡的信号途径比较复杂，在凋亡诱导因子的刺激下经历不同的信号途径。本文就细胞凋亡的三条信号通路——线粒体途径、内质网途径和死亡受体途径做一综述，以便为人们进一步了解细胞凋亡发生的机制，从而对癌症及其他一些相关疾病的治疗奠定基础。关键词:细胞凋亡;信号通路;线粒体途径;内质网途径;死亡受体途径 中图法分类号:R329.2+8文献标识码:A文章编号:1000-2324(2015)04-0514-05 The Signal Pathway of Apoptosis XIE Kun,LI Xing-quan Department of Life Science and Technology/Honghe University,Mengzi661199,China Abstract:Apoptosis is a process of programmed cell death which distinguishes from autophagy and necrosis.The signal pathways of apoptosis are complex and different under apoptosis induced factor stimulating.Three kinds of signal pathways of apoptosis including Mitochondrial pathway,Endoplasmic Reticulum pathway and Death Receptor pathway were summarized in this review in order to make people further comprehend the mechanism of apoptosis，so that it should make a basis for us all to treat cancer and other related diseases. Keywords:Apoptosis;signal pathway;Mitochondrial pathway;Endoplasmic Reticulum pathway;Death Receptor pathway 细胞凋亡是细胞程序性死亡（Program cell death，PCD）中特有的一种细胞死亡方式，是细胞在一系列内源性基因调控下发生的自然或生理性死亡过程。Kerr等1972年最早提出了凋亡（apoptosis）和坏死（necrosis）的概念[1]，随后Paweletz等对其进行了详细的描述[2,3]。在形态学上，凋亡表现为核浓缩、细胞质密度增高、染色质凝聚、核膜破裂、核内DNA断裂、细胞集聚成团、形成凋亡小体(Apoptosome)等特征，这些凋亡小体最终被巨噬细胞清除，但不会引起周围细胞的炎症反应，另外，凋亡发生在单个细胞之间[4,5]。坏死，通常是由相邻的多个细胞之间发生细胞肿胀，细胞核溶解，细胞膜破裂，细胞质流入到细胞间质中，并伴发一系列的炎症反应，从而与凋亡表现为本质性区别[6,7]。 目前认为，凋亡发生的途径分为三种。第一种是线粒体途径，也称为内源性途径，该途径包括两类，第一类需要通过激活Caspase通路促进凋亡，在一序列凋亡诱导因素刺激下，线粒体中的Cyt C（细胞色素C）释放至细胞质中，从而与Apaf-1(Apoptosis protease activating factor1，凋亡蛋白酶活化因子1)结合形成多聚体，形成的多聚体再进一步与凋亡起始分子Caspase-9结合形成凋亡小体，凋亡小体激活Caspase-9，从而激活下游的凋亡执行分子Caspase-3，Caspase-6和Caspase-7等诱导细胞凋亡的级联反应；第二类是不依赖于Caspase途径的，通过线粒体释放AIF（Apoptosis induce factor，凋亡诱导因子）直接诱导凋亡的发生。但是在细胞内，直接检测AIF比较困难，而且AIF的变化不一定能代表凋亡发生的程度，因为引起凋亡发生的途径不一。第二种是死亡受体途径（也称为外源性途径），经由死亡受体（如TNF，Fas等）与FADD的结合而激活Caspase-8和caspase-10，进一步激活凋亡执行者caspase-3,6,7，从而促进凋亡的发生；第三条途径是内质网途径，内质网应激（蛋白质错误折叠或未折叠、内质网胁迫）会导致细胞内钙超载或钙离子稳态失衡一方面激活caspase-12，caspase-12进一步激活caspase-9而促进凋亡的发生，另一方面诱导Bcl-2(B细胞淋巴瘤蛋白)家族中促凋亡蛋白Bax和Bak的激活诱导凋亡[8]。 1凋亡的线粒体途径 在哺乳动物中，由于凋亡的激活需要线粒体中细胞色素C（CytC）的释放，因此CytC由线粒体膜间隙释放到细胞质中的多少可以作为判断凋亡发生强弱的指标之一。有研究认为，CytC的释放是通过Bcl-2家族调控线粒体膜透化（Mitochondrial outer membrane permeabilization，MOMP），科学 收稿日期:2013-03-07修回日期:2014-09-11 基金项目:云南省科技厅应用基础研究面上项目(2010ZC151) 作者简介:谢昆(1975-),男,云南富民人,博士研究生,研究方向为动物生物化学与分子生物学.E-mail:xk_biology2@https://www.doczj.com/doc/5c15601893.html, 数字优先出版:2015-06-03https://www.doczj.com/doc/5c15601893.html,
TGF-β信号通路概述 转化生长因子β信号通路是通过转化生长因子所介导的一系列信号传递的过程。TGF-β信号通路在细胞和组织的生长、发育、分化中起关键作用，对细胞的增殖、细胞间质产生、分化、调亡，胚胎发育，器官的形成，免疫功能，炎性反应，创伤修复等有重要的调节作用。 1. TGF-β信号通路的过程： 首先，TGF-βRⅡ需要自身磷酸化其氨基酸残基中Ser213、Ser409才能被激活，其后与TGF-βRⅠ相互作用并激活TGF-βRⅠ[1]。在与TGF-β反应之后，TGF-βRⅠ也能发生酪氨酸残基的磷酸化[2]，在不存在Ⅱ型受体的情况下，Ⅰ型受体无法独立与TGF-β结合。被TGF-β活化的Ⅱ型受体磷酸化Ⅰ型受体的GS功能区（一个高度保守的甘氨酸及丝氨酸残基结构域），该区域在TGF-βRⅠ激酶活化中起着重要作用。活化的Ⅰ型受体可以磷酸化其下游信号分子-受体活化的Smad2和Smad3。Smad2和Smad3被SARA(smad-anchor for receptor activation)募集到Ⅰ型受体上。被磷酸化的Smad2和Smad3接着与Smad4形成三聚体复合物，这一复合物可进入细胞核，在DNA结合辅助因子的帮助下与DNA上被称为Smad结合元件(Smad-binding element)的区域结合后诱导转录，从而调节细胞的增殖、分化、移行、凋亡。完成转录之后，Smad复合物能够解离，磷酸化的R-Smads被细胞核内的磷酸酶(例如PPM1A /PP2C)脱去磷酸基，使这些R-Smads分子重新回到细胞质中，形成一个“Smad循环”[3]
2.TGF-β1/Smads信号通路的影响因子： 在生物体中，TGF-β信号通路受多种因素控制，如微环境条件[4] [5]、激素[6]、细胞因子和生长因子[7]、microRNAs(MiRNAs) [8]、长的非编码RNA[9]、磷酸化和去磷酸化激酶[3]，泛素连接酶和去泛素酶[10]以及其他因子。 TGF-β受体：目前发现的TGF-β 超家族受体主要有转化生长因子TGF-βRⅠ、TGF-βR Ⅱ和TGF-βRⅢ型受体3种亚型，均包含胞外区、跨膜区和胞内区。其中两个TGF-βRⅠ和两个TGF-βRⅡ分子组成的异源四聚体包含功能性受体。TGF-βⅢ型受体属于辅助型受体，不直接参与信号传导，其主要功能是增加细胞表面上TGF-β的结合，并将其提供给Ⅰ型和Ⅱ型受体[11]。TGF-βRⅢ还能抑制肿瘤细胞的转移、浸润、生长和血管的发生，在肿瘤治疗中的潜在的应用价值[12]。Smads蛋白：Smads蛋白是细胞内重要的TGF-β信号传导和调节分子，可以将TGF-β信号由细胞膜直接传导进入细胞核内。调节型Smad（receptor-regulated Smad, R-Smads），是Ⅰ型受体激酶的直接作用底物，与信号通路的特异性有关；通用型Smad（common-mediator Smad, Co-Smad），是所有TGF-β家族信号转位入细胞核所必须的，参与所有TGF-β超家族成员的信号转导抑制型Smads
1PPAR信号通路：过氧化物酶体增殖物激活受体(PPARs)是与维甲酸、类固醇和甲状腺激素受 体相关的配体激活转录因子超家族核激素受体成员。它们作 为脂肪传感器调节脂肪代谢酶的转录。PPARs由PPARα、PPARβ和PPARγ3种亚型组成。PPARα主要在脂肪酸代谢水平高的组织，如：肝、棕色脂肪、心、肾和骨骼肌表达。他通过 调控靶基因的表达而调节机体许多生理功能包括能量 代谢、生长发育等。另外，他还通过调节脂质代谢的生物感受器而调节细胞生长、分化与凋亡。PPARa同时也是一种磷酸化蛋白，他受多种磷酸化酶的调节包括丝裂原激活蛋白激酶(ERK-和 p38．MAPK)，蛋白激酶A和C(PKA，PKC)，AMPK和糖原合成酶一3(GSK3)等调控。调控PPARa 生长信号的酶报道有MAPK、PKA和GSK3。PPARβ广泛表达于各种组织，而PPARγ主 要局限表达在血和棕色脂肪，其他组织如骨骼肌和心肌有少量表达。PPAR-γ在诸如炎症、动 脉粥样硬化、胰岛素抵抗和糖代谢调节，以及肿瘤和肥胖等方面 均有着举足轻重的作用，而其众多生物学效应则是通过启动或参与的复杂信号 通路予以实现。鉴于目前人们对PPAR—γ信号通路尚不甚清，PPARs通常是通过与9-cis维 甲酸受体(RXR)结合实现其转录活性的。 2MAPK信号通路:mapk简介:丝裂原激活蛋白激酶（mitogen—activatedproteinkinase，MAPK）是广泛存在于动植物细胞中的一类丝氨酸/苏氨酸蛋 白激酶。作用主要是将细胞外刺激信号转导至细胞及其核内，并引起细胞的生物化学反 应（增殖、分化、凋亡、应激等）。 MAPKs家族的亚族:ERKs（extracellularsignalregulatedkinase) ：包括 ERK1、ERK2。生长因子、细胞因子或激素激活此通路，介导细胞增殖、分化。 JNKs(c-JunN-terminalkinase)包括JNK1、JNK2、JNK3。此亚族成员能使Jun转录因子N末 端的两个氨基酸磷酸化而失活，因此称为JunN末端激酶(JNKs)。物理、化学的因素引起的 细胞外环境变化以及致炎细胞因子调节此通 路。 P38MAPKs：丝氨酸/络氨酸激酶，包括p38α、p38β、p38γ、p38δ。p38MAPK参与多种细胞内信息传递过程,能对多种细胞外刺激发生反应,可磷酸化 其它细胞质蛋白,并能从胞浆移位至细胞核而调节转录因子的活性来改变基因的表达水平,从而 介导细胞生长、发育、分化及死亡的全过程。 ERK5:是一种非典型的MAPK通路，也叫大MAPK通路，只有一个成员。它可 被各种刺激因素激活。不仅可以通过磷酸化作用使底物活化，并且通过C端的物理性结合作用 激活底物。 3ERBB信号途径:ErbB蛋白属于跨膜酪氨酸激酶的EGF受体家族成员。ErbB的命名来源于在禽 红白血病B(v-Erb-B)发现的EGF受体的突变体,因而EGF
最新细胞各种信号通路《Cell》 SnapShots are handy reference guides, carefully designed to highlight the key information on a particular topic on one page. SnapShots present up-to-date tables of nomenclature and glossaries, full signaling pathways, and schematic diagrams of cellular processes.Snapshots in red are FREE[/B]. Actin Regulators I[/url] Actin Regulators II[/url]
Antibiotic Inhibition of Protein Synthesis I[/url] Antibiotic Inhibition of Protein Synthesis II[/url] ENHANCED[/url]
Auxin Signaling and Transport Bacterial Appendages I Bacterial Appendages II B7/CD28 Costimulation
BCL-2 Proteins Ca2+-Calcineurin-NF A T Signaling
Ca2+-Dependent Transcription in Neurons Cell-Cycle Regulators I
细胞信号传导通路 1. 信息传导通路的基本组成 人体细胞之间的信息转导可通过相邻细胞的直接接触来实现，但更重要的也是更为普遍的则是通过细胞分泌各种化学物质来调节自身和其他细胞的代谢和功能，因此在人体中，信息传导通路通常是由分泌释放信息物质的特定细胞、信息物质（包含细胞间与细胞内的信息物质和运载体、运输路径等）以及靶细胞 （包含特异受体等）等构成。 信号转导通常包括以下步骤： 释放信息物质→信息物质经扩散或血循 环到达靶细胞→与靶细胞的受体特异性 结合→受体对信号进行转换并启动细胞 内信使系统→靶细胞产生生物学效应 【1】。通过这一系列的过程，生物体对外界刺激作出反应。 3. 信息物质及其分类 信息物质可分为细胞间信息物质与细胞内信息分子。 凡由细胞分泌的调节靶细胞生命活动的化学物质统称为细胞间信息物质，即第一信使，按照细胞分泌信息物质的方式又可将细胞间信息物质分为神经递质、内分泌激素、局部化学介质和气体信号分子。在细胞内传递细胞调控信号的化学物质称为细胞内信息物质，其组成多样化。通常将Ca2+、cAMP、cGMP、DAG、IP3、Cer、花生四烯酸及其代谢物等这类在细胞内传递信息的小分子化合物称为第二信使。责细胞核内外信息传递的物质称为第三信使，能与靶基因特异序列结合，发挥着转录因子或转录调节因子的作用。 研究发现一些信息物质能与位于分泌细胞自身的受体结合而起调节作用，称为自分泌信号。如肝癌细胞能分泌多种血管生成因子，其中VEGF是目前发现的刺激肿瘤血管形成最重要的促进因子，研究表示，肿瘤细胞分泌的VEGF除选择性作用于肿瘤血管内皮细胞上的特异性VEGF受体（Flt-1和KDR），通过酪氨酸激酶介导的信号转导，调控内皮细胞分化和血管形成外，肿瘤细胞自身也有VEGF受体的表达，而且针对VEGF及其受体的干预措施可以改变这些肿瘤细胞的体外增殖活性和其他生物学特征，这些研究表示肿瘤中存在VEGF的自分泌机制【2】。自分泌所产生的信息物质也具有其独特而重要的生理功能。4. 受体分类及与受体相关的信息转导途径 受体是细胞膜上或细胞内能识别生物活性分子并与之结合的成分，他能把识别和接受的信号正确无误地放大并传递到细胞内部，进而引起生物学效应。存在于细胞质膜上的受体称为膜受体，化学本质绝大部分是糖镶嵌蛋白；位于胞液和细胞核中的受体称为胞内受体，它们
KEGG上的信号通路图怎么看？ 提示：请点击标题下方蓝色“实验万事屋”，添加关注后，发“嗯”可以查看我们之前的文章。未经允许，其他公众号不得转载哦！ 想要把自己研究的分子扯上明星分子或者明星通路？那是不难，难的是具体到底要怎么去扯，芯片结果啊，生信结果啊，都会给你提示，但真的要具体扯上去，还得看懂那些七七八八的信号通路图。 KEGG Pathway上有着大量的信号通路图，画得一个复杂啊！巨坑爹有没有？曾经有师弟说我之前曾经把Wnt通路描述错了，他师兄告诉他，应该是GSK-3β磷酸化抑制β-Catenin降解，并促进它入核的。在这里，我们只能默默地祝福这位师兄了…… 那我们就用Wnt通路来做例子吧。先上KEGG下载一个Wnt的信号通路图，如下：
绝壁是很高大上的不是么？这要咋看呢？其实这张图上把三个Wnt通路都画上去了，也就是Wnt/β-Catenin（经典Wnt通路），Wnt/PCP（平面的细胞极性途径）和Wnt/Ca2+（Wnt/钙离子）三条信号通路组成，我们就删减一下，就光看经典的Wnt通路，就变成了下面这个模样： 感觉还是很高大上有木有？那就再删减一下，把它变成经典Wnt信号通路的骨架会是什么样呢？就是这样： 简洁明快了吧，但要怎么来看懂这样的图呢？我们来看一下KEGG Pathway的具体图例：
把这些图例用来解释经典Wnt信号通路骨架图，就变成了： 看懂了么？那给你从左到右解释一下： 1）Wnt激活膜上受体，将信号传递到第二信使Dvl，活化的Dvl抑制由Axin、APC 和GSK-3β组成的复合物的活性,使β-catenin不能被GSK-3β磷酸化。 2）磷酸化的β-catenin才可通过泛素化(ubiquitination)而被胞浆内的蛋白酶体所降解，由于非磷酸化的β-catenin不能被蛋白酶体降解，从而导致β-catenin在胞浆内积聚，并移向核内。
1 PPAR 信号通路：过氧化物酶体增殖物激活受体( PPARs) 是与维甲酸、类固醇和甲状腺激素受体相关的配体激活转录因子超家族核激素受体成员。它们作为脂肪传感器调节脂肪代谢酶的转录。PPARs由PPAR a、PPAR B和PPAR 丫3种亚型组成。PPAR a主要在脂肪酸代谢水平高的组织，如：肝、棕色脂肪、心、肾和骨骼肌表达。他通过调控靶基因的表达而调节机体许多生理功能包括能量代谢、生长发育等。另外，他还通过调节脂质代谢的生物感受器而调节细胞生长、分化与凋亡。PPARa 同时也是一种磷酸化蛋白，他受多种磷酸化酶的调节包括丝裂原激活蛋白激酶(ERK-和p38 . M APK)，蛋白激酶A和C( PKA , PKC) , AM PK和糖原合成酶一3( G SK3) 等调控。调控PPARa 生长信号的酶报道有M APK 、PKA和G SK3。PPAR B广泛表达于各种组织，而PPAR 丫主要局限表达在血和棕色脂肪，其他组织如骨骼肌和心肌有少量表达。PPAR- 丫在诸如炎症、动脉粥样硬化、胰岛素抵抗和糖代谢调节，以及肿瘤和肥胖等方面均有着举足轻重的作用，而其众多生物学效应则是通过启动或参与的复杂信号通路予以实现。鉴于目前人们对PPAR―丫信号通路尚不甚清，PPARs通常是通过与9-cis维甲酸受体(RXR) 结合实现其转录活性的。 2 MAPK信号通路:mapk简介丝裂原激活蛋白激酶(mitogen —activated protein kinase ，MAPK )是广泛存在于动植物细胞中的一类丝氨酸/苏氨酸蛋白激酶。作用主要是将细胞外刺激信号转导至细胞及其核内，并引起细胞的生物化学反应(增殖、分化、凋亡、应激等)。 MAPKs 家族的亚族:ERKs (extracellular signal regulated kinase) ：包括ERK1、 ERK2。生长因子、细胞因子或激素激活此通路，介导细胞增殖、分化。 JNKs(c-Jun N-terminal kinase) 包括JNK1、JNK2、JNK3。此亚族成员能使 Jun 转录因子N 末端的两个氨基酸磷酸化而失活，因此称为Jun N 末端激酶(JNKs)。物理、化学的因素引起的细胞外环境变化以及致炎细胞因子调节此通路。 P38 MAPKs :丝氨酸/ 络氨酸激酶，包括p38 a、p38 B、p38 丫、p38 3。p38 MAP K 参与多种细胞内信息传递过程,能对多种细胞外刺激发生反应,可磷酸化其它细胞质蛋白,并能从胞浆移位至细胞核而调节转录因子的活性来改变基因的表达水平,从而介导细胞生长、发育、分化及死亡的全过程。 ERK5:是一种非典型的MAPK通路，也叫大MAPK通路，只有一个成员。它可被各种刺激因素激活。不仅可以通过磷酸化作用使底物活化，并且通过 C 端的物理性结合作用激活底物。 3 ERBB信号途径：ErbB蛋白属于跨膜酪氨酸激酶的EGF受体家族成员。ErbB的命名来源于在禽红白血病B( v-Erb-B) 发现的EGF 受体的突变体,因而EGF 受体亦称为“ ErbB1 ” 。人源ErbB2 称为HER2, 特指人的EGF 受体。ErbB 家族的另外两个