Molecular Cell
Article
Innate Antiviral Host Defense Attenuates
TGF-b Function through IRF3-Mediated
Suppression of Smad Signaling
Pinglong Xu,1,2,*Samantha Bailey-Bucktrout,3Ying Xi,4Daqi Xu,3Dan Du,2Qian Zhang,1Weiwen Xiang,1Jianming Liu,2 Andrew Melton,5Dean Sheppard,5Harold A.Chapman,4Jeffrey A.Bluestone,3and Rik Derynck2,*
1Life Sciences Institute and Innovation Center for Cell Biology,Zhejiang University,Hangzhou,Zhejiang310058,China
2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research,Department of Cell and Tissue Biology
3Diabetes Center and the Department of Medicine
4Department of Medicine and Cardiovascular Research Institute
5Lung Biology Center and the Department of Medicine
University of California at San Francisco,CA94143,USA
*Correspondence:xupl@https://www.doczj.com/doc/739917428.html,(P.X.),rik.derynck@https://www.doczj.com/doc/739917428.html,(R.D.)
https://www.doczj.com/doc/739917428.html,/10.1016/j.molcel.2014.11.027
SUMMARY
TGF-b signaling is essential in many processes,
including immune surveillance,and its dysregulation
controls various diseases,including cancer,?brosis,
and in?ammation.Studying the innate host defense,
which functions in most cell types,we found that RLR
signaling represses TGF-b responses.This regula-
tion is mediated by activated IRF3,using a dual
mechanism of IRF3-directed suppression.Activated
IRF3interacts with Smad3,thus inhibiting TGF-b-induced Smad3activation and,in the nucleus, disrupts functional Smad3transcription complexes
by competing with coregulators.Consequently,
IRF3activation by innate antiviral signaling represses
TGF-b-induced growth inhibition,gene regulation
and epithelial-mesenchymal transition,and the
generation of Treg effector lymphocytes from naive
CD4+lymphocytes.Conversely,silencing IRF3
expression enhances epithelial-mesenchymal transi-
tion,TGF-b-induced Treg cell differentiation upon
virus infection,and Treg cell generation in vivo.
We present a mechanism of regulation of TGF-b
signaling by the antiviral defense,with evidence for
its role in immune tolerance and cancer cell behavior. INTRODUCTION
The immune system is a central context in which TGF-b controls cell differentiation and function(Li and Flavell,2008;Yang et al., 2010).For example,TGF-b regulates the activation of naive T cells following antigen recognition and their differentiation into effector T cells to combat pathogens.Speci?cally,TGF-b controls differentiation of regulatory T(Treg)lymphocytes and T helper-17(Th17)effector T cell subsets,while restricting the generation of Th1and Th2cells(Li and Flavell,2008).In the pres-ence of interleukin(IL)-2,TGF-b induces expression of the tran-scription factor Foxp3,which drives Treg cell differentiation from naive T cells(Chen et al.,2003),and exposure to TGF-b with IL-6 induces Th17cells differentiation(Bettelli et al.,2006).
TGF-b signaling also controls cancer progression by inducing an epithelial plasticity response that often leads to epithelial-mesenchymal transition(EMT)(Ikushima and Miyazono,2010; Heldin et al.,2012).EMT dissolves epithelial junctions,downre-gulates epithelial and activates mesenchymal gene expression, and increases motility and invasion.Increased TGF-b signaling and EMT,or at a minimum an epithelial plasticity response,are increasingly also seen as prerequisites in the development of ?brosis(Chapman,2011).
The different roles of TGF-b derive from the versatility of TGF-b signaling and its regulation by other signaling pathways(Feng and Derynck,2005;Massague′,2012;Xu et al.,2012).TGF-b ini-tiates signaling through cell surface complexes of two pairs of transmembrane kinases.Upon ligand binding,the T b RII kinases phosphorylate and induce conformation changes in T b RI, enabling recruitment of Smads and phosphorylation of two C-terminal serines by T b RI.The receptor-activated(R-)Smads then dissociate from the receptors and form trimers with one Smad4that translocate into the nucleus,where they activate or repress transcription of target genes through association with high-af?nity DNA binding transcription factors at regulatory gene sequences and recruitment of coactivators or corepres-sors(Feng and Derynck,2005;Massague′,2012).Activation of transcription requires direct R-Smad interactions with the his-tone acetyltransferases CBP or p300that are stabilized by Smad4,which consequently also serves as coactivator(Feng et al.,1998;Janknecht et al.,1998;Feng and Derynck,2005). GRIP1,discovered as a coactivator of the glucocorticoid recep-tor,also acts as Smad3coactivator in activating gene responses (Li et al.,2006).The cooperation of Smads with other transcrip-tion factors sets the stage for extensive versatility in transcrip-tion,and crosstalk with other signaling pathways(Feng and Derynck,2005),and explains the context-dependent responses of hundreds of target genes(Koinuma et al.,2009).TGF-b also induces non-Smad signaling pathways,such as MAP kinase
pathways or the PI3K-Akt-TOR pathway,that target Smad sig-naling for further regulation and activate nontranscription re-sponses(Derynck and Zhang,2003;Zhang,2009). Metazoans developed innate defense mechanisms to recog-nize pathogens and defend against infection.Viral double-stranded RNA can be sensed by Toll-like receptors(TLRs)in endosomes or cytoplasmic RIG-I-like receptors(RLRs)(Akira et al.,2006).Binding of viral dsRNA to these receptors leads to activation of the kinases TBK1and/or IKKεthat C-terminally phosphorylate,and thus activate,the signaling mediator IRF3 (Fitzgerald et al.,2003;Sharma et al.,2003).Following dimeriza-tion and nuclear translocation,activated IRF3acts as DNA-bind-ing transcription factor(Belgnaoui et al.,2011;Kawasaki et al., 2011).TLR and RLR activation by dsRNA also induces the NF-k B pathway.IRF3and NF-k B then cooperate to activate inter-feron-b expression,which initiates an antiviral response through IRF7expression,IRF7activation by TBK1or IKKε,and coordi-nate regulation of IRF7-and IRF3-responsive genes(Belgnaoui et al.,2011).
The transactivation domain of IRF3has substantial structural similarity with the transactivation domain,i.e.,the MH2domain, of Smads,in organization of a helices and b sheets,and three-dimensional structure,albeit much less in sequence;however, IRF3forms dimers,while Smads form trimers.IRF3and the related IRF7also show similarities in activation mechanism with R-Smads(Qin et al.,2003;Takahasi et al.,2003).IRF3and IRF7 are activated by phosphorylation of multiple C-terminal serines, resulting in reorganization of auto-inhibitory elements and func-tional unmasking of the transactivation domain(Qin et al.,2003), while R-Smads are activated by phosphorylation of two C-termi-nal serines(Chacko et al.,2004).These similarities raise the ques-tion whether Smads can associate with IRF3or IRF7,thus enabling functional crosstalk between innate immune signaling through IRF3and TGF-b signaling through Smad activation. Here we show that IRF3activation in response to RLR signaling regulates the activation of Smad signaling in response to TGF-b.We propose a dual mechanism for IRF3-mediated in-hibition of Smads,i.e.,by preventing association of Smad3with the T b RI receptor,thus decreasing TGF-b-induced Smad3acti-vation,and by interfering with functional Smad transcription complexes in the nucleus.The repression of TGF-b signaling by antiviral immune signaling controls TGF-b physiology, apparent by the decreased target gene activation by TGF-b, impaired EMT,and decreased Treg leukocyte differentiation.
RESULTS
RLR Signaling Suppresses TGF-b-Induced Smad Responses
Transfection of double-stranded poly(I:C)RNA,designated TpIC,and infection with Sendai virus(SeV)are commonly used to activate RLR signaling(Belgnaoui et al.,2011;Kato et al., 2011;Kawasaki et al.,2011).To study the effect of RLR signaling on TGF-b signaling,we used human HepG2hepatoma cells and mouse NMuMG mammary epithelial cells,which are generally used in studies of TGF-b signaling mechanisms.TpIC and SeV induced luciferase expression from an IRF3/7-responsive re-porter(Qin et al.,2003),with TpIC acting more strongly than SeV infection(Figure1A).Accordingly,TpIC activated IRF3 much more ef?ciently than SeV,as shown by immunoblotting for C-terminally phosphorylated IRF3(Figure1B).These results indicate that RLR signaling activates IRF3in epithelial cells. Conversely,SeV infection,but not TpIC,induced IRF3activation ef?ciently in human CD4+cells(Figure S1A).The lack of IRF3 activation by TpIC in these cells was due to a failure of liposomal poly(I:C)delivery(data not shown).TGF-b induced transcription from a Smad3-responsive promoter(Zhang et al.,1998)in HepG2and NMuMG cells,which was inhibited by the T b RI ki-nase inhibitor SB431542(Laping et al.,2002)(Figure1C). Combining RLR activation with TGF-b signaling,TpIC inhibited TGF-b/Smad3-induced transcription from a Smad3-dependent reporter(Figure1C).Smad signaling directly controls Smad7 and c-Myc expression,with Smad7mRNA expression increased and c-Myc mRNA expression decreased in response to TGF-b (Nakao et al.,1997;Seoane et al.,2001).TpIC inhibited the induction of endogenous Smad7mRNA expression,and repres-sion of c-Myc mRNA expression,in response to TGF-b(Fig-ure1D).The decreased basal Smad7mRNA and increased basal c-Myc mRNA expression in response to TpIC(Figure1D)are consistent with the autocrine TGF-b signaling control of gene expression in cultured cells.
IRF3Activation Represses TGF-b-Induced Transcription RLR-induced IRF3activation results from the activities of TBK1 or IKKεthat phosphorylate and thus activate IRF3(Fitzgerald et al.,2003;Sharma et al.,2003).BX795,an inhibitor of TBK1 and IKKε(Clark et al.,2009),inhibited the activation of IRF3(Fig-ure2A,4th lane),and restored the inhibition of TGF-b-induced transcription by TpIC(Figure2B,lanes3,4).In contrast,BAY 11-7082and Celastrol,which inhibit the NF-k B pathway,failed to reverse the TpIC-induced repression of TGF-b/Smad3 signaling(Figure2B,lanes7and8).These results suggested that RLR signaling inhibits TGF-b/Smad signaling through IRF3 activation.Accordingly,silencing IRF3expression using siRNA (Figure2A,5th lane)largely rescued the repression of the TGF-b/Smad pathway by RLR activation(Figures2B,lanes3,5,6, and S2A).
Evaluating several cell lines,human epithelial HaCaT cells showed constitutive IRF3activation(Figure S1B),allowing us to study its control of TGF-b signaling.Transduction of cells with siRNA for IRF3,thus decreasing IRF3protein(Figure2C) and mRNA expression(Figure2D),enhanced the TGF-b respon-siveness,apparent by increased p15Ink4B and Smad7mRNA expression from direct TGF-b/Smad3responsive genes(Nakao et al.,1997;Seoane et al.,2001)(Figure2D)and increased transcription from a Smad3-dependent reporter(Figure2E). Silencing IRF3expression also enhanced their basal levels without adding TGF-b(Figures2D and2E),consistent with auto-crine TGF-b responsiveness.Blocking IRF3activation using BX795similarly increased the basal and TGF-b-induced tran-scription from the Smad3-responsive luciferase promoter (Figure2E).
Our data using BX795implicated IRF3activation in the sup-pression of the TGF-b pathway by RLR signaling.We therefore evaluated,in the absence of RLR activation,the effects of an activated form of IRF3,known as IRF35SD,in which the
Molecular Cell IRF3Activation Attenuates TGF-b Signaling
activating phosphorylation of ?ve serines is mimicked by replac-ing these with aspartic acids (Lin et al.,1998).Wild-type IRF3did not repress TGF-b -induced,Smad3-dependent activity,but IRF35SD expression resulted in >90%inhibition of the TGF-b /Smad response (Figure 2F;Figure S1C).Expression of IKK ε,which activates IRF3,also inhibited TGF-b -induced transcrip-tion,and this inhibition was enhanced when wild-type IRF3was coexpressed (Figure 2F).The differential effects of IRF35SD versus wild-type IRF3were striking in a dose-dependent comparison using TGF-b /Smad3-activated transcription as read-out (Figure 2G).IRF35SD repressed transcription at low levels (>80%using 10ng plasmid DNA),progressing to >97%repression at high levels,and wild-type IRF3did not repress TGF-b /Smad3responsiveness even at 100ng DNA (Figure 2G).Finally,we compared wild-type IRF3and IRF35SD for their ef-fects on TGF-b target genes,and measured basal,i.e.,autocrine TGF-b -dependent,and TGF-b -induced mRNA expression of Smad7,p15Ink4B and p21Cip1,three direct Smad3targets that are induced by TGF-b (Feng et al.,2000;Moustakas and Kardas-sis,1998;Nakao et al.,1997),and c-Myc,which is directly repressed by Smad3in response to autocrine or added TGF-b (Seoane et al.,2001).In HaCaT and 293T cells,IRF35SD abol-ished or decreased the TGF-b -induced increase of Smad7,p15Ink4B and p21Cip1,and decrease of c-Myc mRNA expression (Figures 2H and 2I).In contrast,wild-type IRF3expression had only a minimal effect (Figure 2I).These results indicate that IRF3phosphorylation is critical for repression of Smad-depen-dent TGF-b activation by innate antiviral
signaling.
A B
C
D
Figure 1.RLR Signaling Suppresses TGF-b Responsiveness
(A)Transfection of poly (I:C)(TpIC)or Sendai virus (SeV)infection increased transcription from an IRF3/7-responsive promoter in HepG2and NMuMG cells.N =3experiments.*and **p <0.001,compared with control,by Student’s t test.
(B)Immunoblotting of C-terminally phosphorylated IRF3and total IRF3revealed IRF3activation in response to TpIC or SeV infection in HepG2or NMuMG cells.Note the slightly decreased electorophoretic mobility of human IRF3,but not mouse IRF3,after C-terminal phosphorylation.
(C)TGF-b -induced transcription from a Smad3-responsive promoter in HepG2or NMuMG cells,and inhibition of this induction by TpIC.SB431542blocked TGF-b -induced transcription.N =4experiments.*and **p <0.001,compared with control,by Student’s t test.
(D)TpIC blocked TGF-b -induced Smad7mRNA expression and TGF-b -induced repression of c-Myc mRNA expression in HepG2cells.TpIC also repressed the basal Smad7mRNA and enhanced the basal c-Myc mRNA expression under autocrine TGF-b signaling control.mRNA levels were quanti?ed by qRT-PCR.N =3experiments.*p <0.001,and **p <0.01,compared with control with TGF-b treatment,by Student’s t tests.
Molecular Cell
IRF3Activation Attenuates TGF-b Signaling
A D G H I
B C
Figure 2.IRF3Activation Controls TGF-b Signaling
(A)Immunoblotting for C-terminally phosphorylated IRF3revealed IRF3activation,and inhibition of IRF3activation by BX795,or siRNA-mediated depletion of IRF3,in HepG2cells.
(B)BX795and silencing IRF3expression (si-IRF3),but not NF-k B inhibition by BAY11-7082or Celastrol,inhibited TGF-b -induced transcription from a Smad3-responsive promoter.N =3experiments.*p <0.001,compared with control with TGF-b treatment;**and ***p <0.001,compared with samples with TGF-b and TpIC treatments,by Student’s t test.
(C–E),HaCaT cells,transfected with control or IRF3siRNA,were treated or not with TGF-b ,and subjected to immunoblotting for IRF3(C),qRT-PCR quanti?cation of IRF3,p15Ink4B or Smad7mRNA (D),or reporter assay of Smad3-responsive transcription (E).In (D),siRNA to IRF3mRNA enhanced basal and TGF-b -induced gene expression.N =3experiments.*p <0.01,compared with control siRNA with TGF-b treatment,by Student’s t test.In (E),TGF-b -induced,Smad3-mediated transcription was enhanced by silencing IRF3expression,or preventing IRF3activation by BX795.N =3experiments.*p <0.001,compared with control siRNA without TGF-b treatment;**p <0.01,compared with control siRNA with TGF-b treatment,by Student’s t test.
(F)TGF-b -induced transcription is inhibited in HepG2cells by activated IRF35SD,but not wild-type IRF3,or IRF3activation by IKK ε.N =3experiments.*and **p <0.01,compared with control with TGF-b treatment with vector or wild-type IRF3,by Student’s t test.
(G)Dose-dependent inhibition of Smad3-mediated transcription by IRF35SD,but not wild-type IRF3.N =3experiments.*p <0.001,compared with wild-type IRF3with TGF-b treatment by Student’s t test.
(H and I)IRF35SD,but not wild-type IRF3,suppressed TGF-b -induced activation of Smad7,p15Ink4B and p21Cip1mRNA expression or repression of c-Myc mRNA expression.In (H),HaCaT cells,stably expressing GFP-tagged IRF35SD,were used.In (I),TGF-b signaling was activated in 293T cells by coexpressing activated T b RI (caT b RI).N =3experiments.*p <0.01,compared with control with TGF-b treatment by Student’s t test.
Molecular Cell
IRF3Activation Attenuates TGF-b Signaling
IRF3Activation Represses TGF-b-Induced Smad3 Activation
The repression of Smad3-mediated transcription in response to TGF-b by RLR-activated IRF3might result from gene regu-lation by IRF3,e.g.,from the expression of IRF7(Marie′et al., 1998).However,TpIC did not induce IRF7expression in HepG2cells(Figure S2B),and siRNA to IRF7did not affect the repression of Smad3-mediated transcription by TpIC(Fig-ure S2C).Furthermore,TpIC did not affect much the inter-feron-b mRNA expression,which can be directed by IRF3 (Wathelet et al.,1998)(Figure S2B).Increased expression of Smad7,an inhibitory Smad that binds T b RI,that can be induced by Jak/STAT signaling(Ulloa et al.,1999)might also inhibit TGF-b-induced Smad3activation;however,TpIC decreased Smad7mRNA expression(Figure1D).Moreover,a derivative of IRF35SD defective in DNA binding,IRF3 5SD_nDB,retained its ability to inhibit TGF-b/Smad3-induced transcription(Figure3A,middle,right panels),even though it was transcriptionally inactive(Figure3A,left).This result sug-gests that activated IRF3itself,and not one or several IRF3 target genes,confers the inhibition.
The inhibition of TGF-b-induced transcription by activated IRF3might be due to either direct repression of Smad3activa-tion,or inhibition of Smad3-mediated transcription.We?rst explored whether IRF3activation decreased the C-terminal phosphorylation of Smad3by T b RI.Silencing IRF3expression in HaCaT cells with their constitutive IRF3activation enhanced TGF-b-induced Smad3activation,determined by immuno-blotting of nuclear phospho-Smad3(Figure3B).Conversely, treatment of HepG2cells with TpIC decreased TGF-b-induced Smad3activation,concomitantly with activation of IRF3, apparent from its slower migration on gel(Figure3C)or immuno-blotting for phospho-IRF3(Figures1B and2A).Since IRF3acti-vation correlated with decreased Smad3activation and Smad3-mediated transcription(Figures1C,2B,2F,and3C),we compared the effects of wild-type IRF3and IRF35SD on Smad3activation.Transfected wild-type IRF3did not affect Smad3activation in293T cells,but similar levels of activated IRF35SD strongly inhibited Smad3activation(Figures3D and S3A),not requiring IRF30s DNA binding domain(Figure S3A). Additionally,activated IRF35SD repressed TGF-b-induced Smad3activation in HaCaT cells(Figure3E),and TpIC induced wild-type IRF3,but not a mutant IRF3SA that cannot be acti-vated,to repress Smad3activation(Figure S3B).These results correlate IRF3activation with repression of Smad3activation. The transactivation domain of IRF3,which in its non-activated form is masked by an auto-inhibitory conformation(Qin et al., 2003),and mediates IRF3dimerization(Takahasi et al.,2003), structurally resembles the MH2transactivation domain,which mediates Smad trimerization(Chacko et al.,2004).Additionally, IRF7can associate with Smad3through its MH2domain(Qing et al.,2004).Given the striking similarities between IRF3and Smad3in structure and activation through C-terminal phosphor-ylation(Qin et al.,2003;Takahasi et al.,2003),we hypothesized that IRF3activation may inhibit Smad3activation by T b RI through association with Smad3.Indeed,when coexpressed with Smad3,activated IRF35SD,but not wild-type IRF3,associ-ated with Smad3(Figure3F,lanes3versus4).This occurred pre-dominantly in the cytoplasm,but was also seen in the nucleus (Figure S3C).Activated IRF35SD did not interact with Smad4, while wild-type IRF3did(Figure S3D).Furthermore,TpIC pro-moted cytoplasmic IRF3association with Smad3(Figure S3E) and dissociation from Smad4(Figure S3D)in transfected cells, and association of endogenous IRF3and Smad3in HepG2cells (Figure3G).However,T b RI activation,which confers Smad3 activation,reduced the IRF35SD association with Smad3(Fig-ure3F,lanes4versus6,Figure S3F).These results strongly indi-cate that,upon activation,IRF3interacts with non-activated Smad3.
Association of activated IRF3with non-activated Smad3(Fig-ure3F)explains the decreased Smad3phosphorylation upon IRF3activation(Figures3C–3E).Since Smad3activation results from transient interaction with T b RI,which cannot be visualized at endogenous levels,we compared the effects of wild-type or activated IRF3on the interaction of tagged Smad3with activated T b RI.Activated IRF35SD,but not wild-type IRF3or IRF3SA, which cannot be activated,strongly decreased the association of Smad3with the receptor(Figures3H,S3G,and S3H). Enhanced Smad3-T b RI association in the presence of IRF3SA (Figure S3H)may re?ect interference with autocrine inhibition by endogenous IRF3.Collectively,these data show that IRF3 activation controls Smad3phosphorylation,through association of activated IRF3with non-activated Smad3,thus interfering with the Smad3interaction with T b RI that is required for TGF-b-induced Smad activation.
Activation of IRF3Disrupts the Smad3Transcription Complex
Since activated IRF3was also seen to associate with Smad3in the nucleus(Figure S3C),we evaluated whether IRF3disrupts the Smad3transcription complex.For this purpose,we used an activated Smad3with the C-terminal serine phosphorylation mimicked through substitution for glutamic acid(Chipuk et al., 2002).In reporter assays,IRF35SD,but not wild-type IRF3, repressed transcription directed by activated Smad3(Figure4A), or Smad3fused to a Gal4DNA binding domain at a Gal4 sequence-controlled promoter(Figure4B).Enhanced expres-sion of the Smad coactivators Smad4,p300or GRIP1partially rescued the repression of Smad3-mediated transcription by activated IRF3(Figure4C),supporting the notion that in these as-says the repression by activated IRF3results from inhibition of the transcription complex.
Visualizing the subcellular localization of endogenous Smad3 and IRF3,combined treatment with TGF-b and TpIC did not induce colocalization of activated Smad3with activated IRF3 in a punctate pattern(Figure4D).This is consistent with our ob-servations that Smad3activation by T b RI strongly decreased the association of activated IRF3with non-activated Smad3(Fig-ure3F,lanes4and6),and suggests that no signi?cant com-plexes of activated IRF3with activated Smad3are formed. Since Smad4,p300and GRIP1partially rescued the inhibition of Smad3-mediated transcription(Figure4C),we explored whether IRF3activation disrupts the Smad3transcription com-plex by displacing these coactivators.Shown by coimmunopre-cipitation,IRF35SD interfered with the Smad3-p300association (Figures4E and4F,top panels),whereas wild-type IRF3or IIRF3
Molecular Cell
IRF3Activation Attenuates TGF-b Signaling
A
C
F
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D
E
B
Figure3.IRF3Activation Enables Smad3Association and Inhibits TGF-b-Induced Smad3Activation
(A)Activated IRF3(IRF35SD),but not its derivative that lacks DNA binding(IRF35SD_nDB),activates an IRF3-responsive transcription reporter(left),yet both IRF3mutants block Smad3-mediated transcription from the4SBE(middle)or3TP(right)reporter.N=3experiments.*p<0.001,compared with IRF35SD; **p<0.001,compared with IRF3WT coexpression,by Student’s t test.
(B)Depletion of IRF3expression using IRF3siRNA enhanced TGF-b-induced Smad3activation in HaCaT cells.Smad3activation was shown by immunoblotting of phospho-Smad3(top panel)versus total Smad3in the nuclear fraction(second panel).IRF3in the nuclear fraction was detected by anti-IRF3immunoblotting (third panel),and GRIP1levels(lowest panel)served as loading control of nuclear fraction proteins.
(C)TpIC induced a decrease in Smad3activation in HepG2cells,assessed by immunoblotting for phospho-Smad3(top panel)versus total Smad3(second panel) in the nuclear fraction,and increased IRF3accumulation in the nuclear fraction.GRIP1levels(lowest panel)served as loading control of nuclear proteins. (D and E)Activated IRF35SD,but not wild-type IRF3,reduced Smad3activation,assessed by immunoblotting for phospho-Smad3,in293T cells expressing activated T b RI(caT b RI)(D),or HaCaT cells treated with TGF-b(E).
(F)Flag-tagged Smad3associated with Myc-tagged activated IRF3(IRF35SD),but not Myc-tagged wild-type IRF3in transfected293T cells.This association was reduced when Smad3was activated by caT b RI.
(G)Time-dependent association of endogenous Smad3with endogenous IRF3in HepG2cells that were treated with TpIC.
(H)Activated,but not wild-type IRF3interfered with Smad3association with caT b RI,shown by coimmunoprecipitation of HA-tagged Smad3with Flag-tagged caT b RI.
Molecular Cell
IRF3Activation Attenuates TGF-b Signaling
SA had much less effect(Figures4E and S4A).While decreasing the Smad3-p300interaction,activated IRF3interacted with p300 (Figures4E and4F,second panels;S4B;and S4C),consistent with the roles of CBP and p300as IRF3coactivators(Wathelet et al.,1998;Weaver et al.,1998;Lin et al.,2001),and the struc-ture of this interface(Qin et al.,2005).Similarly to the Smad3-p300interaction,activated IRF35SD,but not wild-type IRF3, interfered with the association of Smad3with GRIP1(Figure4G) or Smad4(Figure4H),shown in two-hybrid assays.Finally,IRF3 5SD decreased the TGF-b-induced interaction of Smad3at endogenous Smad7and Snail promoter regulatory sequences (Figure4I),further showing that IRF3activation impacts the func-tional integrity of the Smad3transcription complex.
The interference of activated IRF35SD with Smad3-coactiva-tor interactions likely involves a basic amino acid patch in IRF30s transactivation domain that provides a structural interface with several coregulators(Qin et al.,2003;Takahasi et al.,2003). Indeed,replacement of four basic amino acids in this patch with alanines,thus generating IRF35SDm,abolished most inhi-bition of activated Smad3-mediated transcription by IRF35SD (Figure4J,last two lanes),without affecting its ability to inhibit Smad3recruitment to T b RI(Figure S4D)or Smad3activation (Figure S4E),and decreased the interaction of IRF35SD with p300(Figure4E,compare last two lanes).
Finally,we evaluated the effect of TpIC on the integrity of the Smad3transcription complex.As reported(Feng et al.,1998; Janknecht et al.,1998),TGF-b induced the interaction of Smad3with Smad4and with p300.TpIC treatment decreased the TGF-b-induced Smad3association with p300(Figure4K, top panel),concomitantly with association of IRF3with p300 (Figure4K,second panel),and of Smad3with Smad4(Figure4L). Together,these results illustrate that activation of IRF3directly impacts the integrity of the functional Smad3nucleoprotein complex,required for TGF-b-induced transcription activation, through interference with Smad3-coactivator interactions.
IRF3Activation Represses TGF-b-Induced EMT
Since IRF3controls TGF-b-induced gene expression,we evalu-ated whether IRF3activation regulates TGF-b-induced EMT.Ha-CaT cells transition into a mesenchymal phenotype in response to TGF-b,with increased expression of Slug,a Snail-related tran-scription factor that drives EMT,dispersion of E-cadherin from junctions,decreased epithelial and increased mesenchymal gene expression,and changes in actin organization and cell shape(Lamouille and Derynck,2007;Thuault et al.,2006). TGF-b-activated Smad3directly controls Slug and several other EMT-regulated genes(Brandl et al.,2010;Xu et al.,2009).In contrast to many cell lines,HaCaT cells do not repress E-cad-herin expression during EMT(data not shown).
Compared to control HaCaT cells,downregulation of IRF3 expression enhanced the TGF-b-induced mesenchymal ex-pression of Slug,?bronectin,N-cadherin and vimentin(Fig-ure5A),and the responsiveness to TGF-b-induced morphology changes.Without adding TGF-b,control HaCaT cells had an epithelial cobblestone-like morphology,and increasing TGF-b levels resulted in loss of the epithelial phenotype and change to-ward an elongated cell shape(Figure5B).Cells with silenced IRF3expression acquired a?broblast phenotype at lower TGF-b concentrations than control HaCaT cells(Figure5B), and showed less cortical actin and more stress?ber formation (Figure5C),indicating a more robust EMT response to TGF-b when IRF3signaling is silenced.
Consistent with the effect of silencing IRF3expression in Ha-CaT cells,RLR signaling in response to TpIC or SeV infection (Figure5D),or activated IRF35SD expression(Figure5E)atten-uated the EMT responses in NMuMG cells.Thus,TpIC and SeV attenuated the decrease in E-cadherin,and increases in Snail, N-cadherin and vimentin expression that accompany EMT in NMuMG cells(Figure5D),and the change in cell morphology to-ward an elongated spindle phenotype(Figure5F).For example, at10ng/ml TGF-b NMuMG cells had a spindle cell phenotype, whereas IRF3activation enabled the cells to largely maintain their cuboidal epithelial phenotype.These data demonstrate that IRF3activation controls the TGF-b-induced EMT response in HaCaT and NMuMG cells.
IRF3Signaling Controls the Growth Inhibitory
Effect of TGF-b
Since TGF-b also inhibits epithelial cell proliferation,we ad-dressed the effect of IRF3activation on the growth inhibitory response of TGF-b.Silencing IRF3expression enhanced the in-hibition of HaCaT cell proliferation by TGF-b(Figure5G),and,the expression of the cdk inhibitor p15Ink4B(Figure5H).These effects of IRF3siRNA were already seen without adding TGF-b,sug-gesting either regulation of autocrine TGF-b signaling by IRF3, or a direct effect of IRF3on growth control,as proposed(Kim et al.,2007).Since IRF3siRNA did not affect basal cell prolife-ration(Figure5G)or basal p15Ink4B mRNA expression(Figure5H) in the presence of the T b RI kinase inhibitor SB431542,we con-clude that IRF3controls growth inhibition through its control of TGF-b signaling.
RLR-IRF3Activation Regulates Treg Lymphocyte Differentiation
TGF-b controls the differentiation of multiple immune cell line-ages.Notably,TGF-b induces Treg cell differentiation from naive CD4+T cells,marked by the expression of Foxp3,which drives Treg cell differentiation(Li and Flavell,2008;Yang et al.,2010). Since antiviral signaling mobilizes the immune response through interferon induction and in?ammation,we evaluated whether RLR signaling through IRF3affects induced(i)Treg lymphocyte development.
As in primary human CD4+cells(Figure S5A),SeV infection eli-cited robust IRF3activation in primary mouse CD4+T cells,as-sessed by immunoblotting for C-terminally phosphorylated IRF3(Figure6A).We activated TCR signaling in naive CD4+ T cells using anti-CD3and anti-CD28antibodies,and treated the cells with TGF-b and IL-2,which resulted in activation of Foxp3,CTLA4and PD-1expression,as a measure of CD4+ T cell differentiation into iTreg cells.iTreg cell induction was blocked by SB431542(Figures6B and6C).SeV infection prior to this treatment prevented induction of Foxp3,CTLA4and PD-1mRNA expression,and induced IRF7mRNA expression, known to result from IRF3activation(Figures6B and6C),indi-cating that SeV-induced RLR signaling represses the generation of iTreg cells in culture.
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IRF3Activation Attenuates TGF-b Signaling
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IRF3Activation Attenuates TGF-b Signaling
To validate the role of RLR-IRF3signaling in Treg differentia-tion,we also isolated primary CD4+naive T cells from age-and sex-matched Irf3à/àmice.As T cells from this strain lacked IRF3expression and SeV was therefore unable to induce IRF3activa-tion (Figure 6A),SeV induced only a minimal level of IRF7mRNA expression,compared to wild-type CD4+cells (Figure 6C,right).TGF-b and IL-2induced Foxp3mRNA expression in Irf3à/àcells to a higher level than in wild-type CD4+T cells (Figure 6C,left),and,unlike wild-type cells in which SeV infection repressed iTreg differentiation,the TGF-b -induced Foxp3,CTLA4,and PD-1mRNA levels were only minimally reduced by SeV infection (Figure 6C).These results argue that RLR signaling represses TGF-b -induced differentiation of Treg cells through IRF3.
We also assessed the Treg generation in the colonic lamina propria,which is inherently prone to RLR activation and TGF-b signaling (Curotto de Lafaille and Lafaille,2009;Sheridan and Le-franc ?ois,2011).The number of CD4+Foxp3expressing Treg cells was increased in Irf3à/àmice,compared with wild-type mice (Figure 6D),with an increase in peripheral Treg cells and propor-tional decrease in thymic-derived Treg cells (Figure 6E).These results are consistent with increased TGF-b /Smad signaling in Irf3à/àmice,and illustrate attenuation of TGF-b -induced Treg cell generation by activated IRF3in vivo,with effects on periph-eral and thymic T cells.
Viral Infection Represses TGF-b -Induced Gene Expression in Mice
To evaluate whether viral infection represses the TGF-b response in vivo,we took advantage of the high basal TGF-b ac-tivity that is normally seen in the lung and is required for normal lung physiology and remodeling (Sheppard,2006).RLR-IRF3antiviral signaling was induced in lung cells by intranasal delivery of in?uenza A virus (strain A/Puerto Rico/8/1934;H1N1)(Kumar et al.,2006).As shown in Figure 6F,intranasal delivery of the virus induced a dramatic increase in IRF7and ISG54mRNA
expression in pulmonary immune cells obtained by bronchoal-veolar lavage,indicating strong activation of IRF3.Much weaker activation of IRF7and ISG54expression was observed in Irf3à/àmice (Figure 6F),consistent with our data in Irf3à/àCD4+T cells (Figure 6C,right).
Among the genes known to be targeted by TGF-b -activated Smads,we found that the genes encoding Smad7(Nakao et al.,1997),MMP11(Barrasa et al.,2012)and TIMP2(unpub-lished data)are expressed in lung epithelium.Their mRNA expression was signi?cantly attenuated following in?uenza virus infection in the lungs of wild-type mice,but not in lungs of Irf3à/àmice (Figure 6G),consistent with the decreased Smad3activa-tion in response to in?uenza infection in wild-type,but not Irf3à/àlungs (Figure S5B).These data support the inhibitory crosstalk of virus-induced IRF3signaling on TGF-b -induced gene responses in vivo.DISCUSSION
TGF-b signaling plays pervasive roles in the regulation of cell pro-liferation,differentiation and functions,with the outcome depen-dent on other signaling pathways,and cell and tissue type.The functional availability of TGF-b receptors and the Smad activities are regulated through posttranslational modi?cations,and coop-eration of Smads with DNA-binding transcription factors and co-regulators sets the stage for signaling crosstalk of Smads at nucleoprotein complexes.We now present a novel mode of regulation of TGF-b /Smad responsiveness,i.e.,through activa-tion of IRF3in response to RLR signaling.This crosstalk ema-nates from the innate antiviral host response,and represses TGF-b -induced Smad signaling.Considering the high expression of IRF3and RLRs in many cell types,we surmise that IRF3-medi-ated repression affects many TGF-b responses in many cell types,dependent on the level of IRF3and its activation in response to extracellular cues.
Figure 4.IRF3Activation Decreases Smad3Transcription Complex Formation and Promoter Binding
(A)IRF35SD,but not wild-type IRF3,inhibits transcription induced by constitutively activated Smad3(caSmad3)in 293T cells (left)or HepG2cells (right).N =3experiments.*p <0.001,compared with control with caSmad3expression,by Student’s t test.
(B)IRF35SD,but not wild-type IRF3,inhibits Gal4-responsive transcription from a nuclear Gal4DNA binding domain-fused Smad3(Gal4-Smad3)in HepG2cells.N =3experiments.*p <0.001,compared with control Gal4-Smad3expression,and **p <0.001,compared with Gal4-Smad3expression and TGF-b treatment,by Student’s t test.
(C)Inhibition of Smad3-mediated transcription by activated IRF35SD in HepG2cells is partially rescued by expression of the coregulators p300,GRIP1or Smad4.N =3experiments.*p <0.01,compared with TGF-b treatment and IRF35SD coexpression.
(D)Subnuclear distribution,assessed by confocal immuno?uorescence,of endogenous IRF3and Smad3in HepG2cells,treated with TGF-b and TpIC to activate both IRF3and Smad3.The punctate IRF3and Smad3patterns showed minimal overlap,as illustrated with Imageplus analysis.
(E)Activated,Flag-tagged Smad3(caSmad3)associated with HA-tagged p300in 293T cells,and this interaction was disrupted by activated IRF35SD,but not by wild-type IRF3or the basic patch mutant of IRF35SD (IRF35SDm)(top panel).Under these conditions,activated IRF35SD,but not wild-type IRF3or IRF35SDm,interacted strongly with p300(2nd panel).
(F)Increasing levels of IRF35SD disrupt the interaction of Flag-Smad3with HA-p300in 293T cells,with parallel formation of an IRF35SD complex with p300.(G)IRF35SD disrupts the interaction of VP16-fused Smad3(VP16-Smad3)with Gal4DNA binding domain-fused GRIP1(Gal4-GRIP1)in HepG2cells.
(H)Activated T b RI promotes the interaction of VP16-Smad3with Gal4DNA binding domain-fused Smad4(Gal4-Smad4)in HepG2cells,and this increase is prevented by activated IRF35SD,but not wild-type IRF3.For (H)and (I),N =3experiments;*p <0.01,compared with both controls,**p <0.01,compared with coexpression of fusion proteins,by Student’s t test.
(I)CHIP assay revealed that IRF35SD largely abolishes the TGF-b -induced Smad3association with Smad7(left)or Snail (right)promoter sequences in NMuMG cells.N =3experiments.*p <0.01,compared with control without TGF-b treatment,and **p <0.01,compared with control with TGF-b treatment but without IRF35SD coexpression,by Student’s t test.
(J)Activated IRF35SD,but not its basic patch mutant IRF35SDm,inhibited Smad3-mediated transcription by activated Smad3(caSmad3).
(K and L)TGF-b -induced association of endogenous Smad3with p300(K)or Smad4(L)in HepG2cells is disrupted (K)or decreased (L)upon IRF3activation in response to TpIC,concomitant with endogenous p300-IRF3complex formation (K).
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IRF3Activation Attenuates TGF-b Signaling
A B
C
D
E G Figure 5.IRF3Controls TGF-b -Induced Epithelial-Mesenchymal Transition and Growth Inhibition
(A–C)HaCaT cells,transfected with control or IRF3siRNA,were treated or not with TGF-b to induce EMT.(A)qRT-PCR quanti?cation of Slug,N-cadherin,vimentin and ?bronectin mRNA at 2hr after adding TGF-b .N =3experiments.*p <0.01,compared with control siRNA,by Student’s t test.
(B)Changes in cell morphology show transition from a cuboidal epithelial to an elongated mesenchymal appearance in response to increasing TGF-b levels for 48hr.Suppressed IRF3expression using siRNA allows for EMT at lower TGF-b levels.
(C)F-actin staining in HaCaT cells treated with 2.5ng/ml TGF-b for 48hr.At this concentration,control HaCaT cells showed cortical actin organization,char-acteristic of epithelial cells,whereas suppression of IRF3expression allowed actin reorganization into stress ?bers,as seen in mesenchymal cells.
(D)NMuMG cells,exposed or not to TpIC or SeV,were treated or not with TGF-b to induce EMT.qRT-PCR quanti?ed Snail,E-cadherin,N-cadherin,and vimentin mRNA at 2hr after adding TGF-b .N =3experiments.*p <0.01,compared with samples treated with TGF-b only,by Student’s t test.
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IRF3Activation Attenuates TGF-b Signaling
Mechanism for IRF3-Mediated Inhibition of Smad Signaling
Inhibition of TGF-b /Smad signaling by IRF3involves a dual mechanism,i.e.,inhibition of Smad3activation in response to TGF-b ,and functional interference with Smad transcription com-plexes (Figure 7).Interference with Smad3activation by T b RI re-sults from association of activated IRF3,but not inactive IRF3,with non-activated Smad3,thus preventing Smad3recruitment to T b RI,and decreasing Smad3activation.The structural basis for this association is suggested by the remarkably similar three-dimensional structures of the IRF3and Smad3transacti-vation domains (Qin et al.,2003;Takahasi et al.,2003).Both have a central b sandwich and a loop-helix region that binds phosphoserines,and are followed by C-terminal serines.Phos-phorylation of two C-terminal serines by T b RI results in Smad activation and trimer formation (Chacko et al.,2004),and phos-phorylation of multiple serines by TBK1or IKK εconfers IRF3activation,through reorganization of auto-inhibitory elements,and dimerization (Qin et al.,2003;Takahasi et al.,2003).Smad trimer and IRF3dimer formation involve in either case a con-served basic cleft with negatively charged amino acids (Chacko et al.,2004;Qin et al.,2003;Takahasi et al.,2003).Association of activated IRF3with Smad3is consistent with the interaction of the Smad3MH2domain with the transactivation domain of the IRF3-related IRF7(Qing et al.,2004).
The interference of activated IRF3with Smad3transcription complexes may result from shared use of CBP or p300(Feng et al.,1998;Janknecht et al.,1998;Qing et al.,2004;Wathelet et al.,1998;Weaver et al.,1998),and GRIP1(Li et al.,2006;Reily et al.,2006)as transcription coactivators.Activated IRF3repressed Smad3-mediated transcription by interfering with the interactions of Smad3with p300,GRIP1,and Smad4,which stabilizes R-Smad-CBP/p300interactions.Conversely,TGF-b -induced Smad3activation did not inhibit IRF3activation,and activated Smad3did not interfere with the IRF3-p300associa-tion,nor repress IRF3-mediated transcription.Our ?ndings com-plement previous results showing TGF-b /Smad3-mediated increase of transcription by IRF7at IRF3/7-binding gene se-quences,as found in the interferon-b gene (Qing et al.,2004).Collectively,the structural similarities between the transactiva-tion domains of Smads and IRF3/7enable multiple levels of crosstalk that may extend to other IRFs.
The inhibition of TGF-b /Smad signaling by IRF3activation re-sembles the dual repression by inhibitory Smads,i.e.,Smad6and Smad7.Inhibitory Smads interact with R-Smads and acti-vated type I receptors,thus antagonizing R-Smad recruitment to the receptor,and inhibiting R-Smad activation (Hayashi et al.,1997;Imamura et al.,1997).Similarly,activated IRF3inter-acted with Smad3,interfering with Smad3recruitment to T b RI,although we did not detect IRF3association with T b RI.Inhibitory Smads can also directly repress transcription at gene regulatory
sequences (Miyake et al.,2010;Zhang et al.,2007),providing a second mode of repression,although the underlying mechanism at endogenous genes requires further study.IRF3-mediated repression of TGF-b /Smad signaling is as effective as repression by inhibitory Smads,with the latter not known to be inducible and the former mostly dependent on activation of RLR or other antiviral signaling.
Roles of IRF3Activation in the Control of EMT and T Cell Differentiation
The repression of TGF-b responsiveness by IRF3may substan-tially affect many processes that are controlled by TGF-b .RLRs,TBK1and/or IKK ε,and IRF3are widely expressed,and IRF3can be activated by DNA damage,membrane fusion,and ER stress,in addition to virus infection.Additionally,pathogens and extra-cellular stimuli other than viruses also activate IRF3via STING,TLR and RLR signaling (Goubau et al.,2013;Collins and Moss-man,2014).
Among the TGF-b -regulated processes,the repression of EMT-associated gene reprogramming may be particularly rele-vant in epithelial healing,?brosis and cancer progression.In wound healing,epithelial cells undergo a transient epithelial plas-ticity response that enables wound closure (Heldin et al.,2012;Xu et al.,2009).Epithelial plasticity also contributes to ?brosis,with EMT-associated reprogramming activated by injury and in?am-mation (Chapman,2011).In carcinomas,EMT,or at a minimum an epithelial plasticity response,is seen as prerequisite for tumor cell invasion (Heldin et al.,2012),whereas EMT is also integral to the generation of cancer stem cells (Katsuno et al.,2013;Scheel and Weinberg,2012).In all three contexts,increased TGF-b signaling is thought to drive EMT.Reprogramming of gene expression during EMT involves Smad3-mediated activation of Snail or other transcription factors that drive EMT,and Smad3-mediated repression of epithelial genes and activation of mesen-chymal genes (Heldin et al.,2012;Xu et al.,2009).Accordingly,RLR signaling leading to IRF3activation represses gene reprog-ramming during EMT.We hypothesize that RLR or TLR signaling affects wound healing,?brosis and carcinoma progression,through repression of Smad signaling by activated IRF3.
TGF-b also controls suppression of immune surveillance,and regulates the differentiation and functions of T cell lineages.TGF-b -induced Smad signaling drives Foxp3induction,and suppression of IL-2,IL-4,and interferon-g expression.CD4+CD25+Foxp3+Treg lymphocytes differentiate from naive T cells in response to TGF-b and IL-2,and prevent pathological self-reactivity,i.e.,autoimmune disease (Li and Flavell,2008;Yang et al.,2010).They also enforce tumor immune surveillance and are critical for preventing cancer metastasis (Curiel,2007).We show that IRF3activation represses Smad3-mediated control of T cell differentiation,and postulate that the antiviral defense attenuates immunosuppressive functions of TGF-b , e.g.,in
(E and F)NMuMG cells expressing activated IRF35SD were analyzed for EMT marker mRNA expression at 2hr after adding TGF-b (E),or for cell morphology at 24hr after adding TGF-b (F).IRF35SD expression renders the cells less sensitive to EMT.
(G and H)Silencing IRF3expression enhances TGF-b -induced growth inhibition (G)and p15Ink4B mRNA expression (H)in HaCaT cells.In (G),cell proliferation was assessed by BrdU incorporation after 48hr of TGF-b treatment,normalized to untreated,control cells.N =3experiments.*p <0.05,compared with control cells treated with the same TGF-b concentration,by Student’s t test.In (H),p15Ink4B mRNA was quanti?ed by qRT-PCR after 72hr of TGF-b treatment,and mormalized to untreated control cells.p <0.05,compared with control cells,by Student’s t test.
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IRF3Activation Attenuates TGF-b Signaling
A D E F
G
B
Figure 6.Virus-Induced IRF3Activation Represses Treg Lymphocyte Differentiation and TGF-b /Smad3Target Genes in Mice
(A)SeV induced IRF3activation,assessed by immunoblotting for C-terminally phosphorylated IRF3,in primary Irf3+/+or Irf3à/àmouse CD4+cells at 12hr after infection.
(B)In vitro Treg cell differentiation of naive CD4+T cells from wild-type mice with or without SeV infection for 1hr.TGF-b -induced Foxp3mRNA expression,detected by qRT-PCR after 48hr,was largely abolished by the short exposure to SeV that resulted in IRF7mRNA expression.*and **p <0.001,compared with TGF-b -treated control cells,by Student’s t test.
(C)Effect of SeV-induced RLR signaling on Treg differentiation of naive CD4+T cells isolated from wild-type or Irf3à/àmice.Expression and activation of endogenous IRF3in these cells were shown in (A).Foxp3,CTLA4,PD-1and IRF7mRNA levels were determined by qRT-PCR after 48hr of TGF-b treatment.*p <0.01,compared with wild-type cells without SeV infection,**and ***p <0.01,compared with wild-type cells after SeV infection,by Student’s t test.
(D)Increased CD4+Foxp3+Treg cells in the colonic lamina propria of Irf3à/àmice,compared to wild-type control B6mice.Treg lymphocytes were quanti?ed by ?ow cytometer following labeling with tagged,?uorescence-conjugated antibodies.N =3mice/group.*p <0.05,by Student’s t test.
(E)Flow cytometry using ?uorescence-conjugated antibodies distinguished thymic-derived (tTreg,Nrp-1-positive)and periphery-derived (pTreg,Nrp-1-nega-tive)Treg lymphocytes from the lamina propria.Irf3à/àmice showed decreased tTreg cell frequency (left)and increased pTreg cell number (right).N =3mice/group.*p <0.05,**p <0.001,by Student’s t test.
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IRF3Activation Attenuates TGF-b Signaling
immune tolerance or tumor surveillance,by inhibiting Smad-dependent Treg cell generation and function.
In conclusion,we unveiled a role of innate antiviral host de-fense in Treg cell differentiation and EMT,and a novel mode of functional control of TGF-b -induced Smad signaling,i.e.,through RLR signaling resulting in IRF3activation,enabling innate antiviral signaling to suppress the TGF-b pathway.This repression may potently affect many processes that are regu-lated by TGF-b ,perhaps with most relevance to epithelial plasticity responses,as seen in wound healing and cancer pro-gression,and to the regulation of immune responses.
EXPERIMENTAL PROCEDURES
Luciferase Reporter and Mammalian Two-Hybrid Assays
HepG2,NMuMG or HaCaT cells were transfected and used for luciferase as-says.In brief,cells were cultured for 12hr post transfection,stimulated by transfection with poly(I:C),then after 8hr treated overnight with TGF-b at the indicated concentration,and/or pharmacological inhibitors.Luciferase assays were performed using a dual luciferase system,quanti?ed with SpectraMax M5luminometer,and normalized to the internal Renilla luciferase control.Mamma-lian two-hybrid assays were performed as described (Feng et al.,1998),by co-
expressing Gal4-DBD-fused GRIP1or Smad4with VP16-fused Smad3,and the luciferase reporter,using the Mammalian Matchmaker two-hybrid kit.Quantitative RT-PCR Assay
Total RNA was extracted using an RNAeasy extraction kit.cDNA was gener-ated using the one-step iScript cDNA synthesis kit,and quantitative real-time PCR was performed using the iQ SYBR green supermix and CFX96real-time PCR system.Relative quanti?cation was expressed as 2-^Ct,where ^Ct is the difference between the main Ct value of triplicates of the sample and that of an endogenous L19or GAPDH mRNA control.
Coimmunoprecipitations,Nuclear Extract Preparation,and Immunoblotting
HepG2or 293T cells,transfected with plasmids encoding Myc-,Flag-,or HA-tagged Smad3,caSmad3,IRF3,caT b RI or p300,were treated with TGF-b and/or TpIC,lysed,and subjected to immunoprecipitation using anti-Flag or anti-HA antibodies for transfected proteins,or anti-Smad2/3,anti-IRF3,or anti-p300antibodies for endogenous proteins.After extensive washing,adsorbed proteins were analyzed by gradient SDS-PAGE and immunoblotting.Nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear and Cyto-plasmic Extraction kit.
RNAi
HaCaT or HepG2cells were transfected with double stranded siRNA targeting the human IRF3mRNA using Lipofectamine RNAiMAX for 48hr before assay,or 24hr before adding TGF-b in the EMT assay.Reverse transfection was used to reach optimal ef?ciency.
Immuno?uorescence and Microscopy
C2C12cells were treated as indicated,?xed,permeabilized,blocked,and incubated sequentially with primary antibodies (anti-Smad2/3or anti-IRF3)and Alexa-labeled secondary antibodies with extensive washing.Slides were then mounted with Vectorshield and stained with DAPI.Images were ob-tained and analyzed using a Leica SP5AOBS Upright 2laser scanning confocal microscope.For F-actin staining,HaCaT cells were ?xed,washed,and then incubated with Alexa-546phalloidin at a 1:500dilution.Images were obtained and analyzed by a Leica DMI 4000B inverted microscope.Chromatin Immunoprecipitation Assays
NMuMG cells were treated with 2ng/ml TGF-b for 1hr and ?xed,then resus-pended in SDS lysis buffer.After sonication,samples were incubated over-night at 4 C with anti-Smad3antibody or control IgG.After adding Protein A Dynabeads,immunoprecipitates were sequentially washed once with low-salt buffer,then high-salt buffer,and LiCl buffer,and twice with TE buffer.DNA-protein complexes were eluted and crosslinked by heating,digested with proteinase K and RNase A.DNA was recovered and subjected to RT-PCR analysis using primers for the Smad7or Snail promoter regions.In Vitro Treg Cell Differentiation
Naive CD4+CD25-cells were isolated from wild-type or Irf3–/–C57BL/6mice as described (Fantini et al.,2007),and seeded in 24-well plates coated with 10m g/ml anti-CD3antibody in the presence of 2m g/ml anti-CD28antibody.Af-ter 48hr,cells were infected with SeV for 1hr,with or without inhibitors and/or TGF-b 1(10ng/ml),as indicated.Induction of Foxp3,CTLA4,PD-1and IRF7mRNA expression after 48hr was quanti?ed by RT-PCR.
Leukocyte Isolation from Colonic Lamina Propria and FACS
Colons were harvested from 8–10week old wild-type or Irf3–/–C57BL/6mice,sliced into pieces,and digested as described (Yadav et al.,2012).Cells were
(F)In?uenza virus infection of wild-type and Irf3à/àmice induced IRF7and ISG56mRNA expression in immune cells of broncho-alveolar lavage,detected by qRT-PCR 48hr after infection.
(G)In?uenza viral infection of wild-type or Irf3à/àmice changed lung mRNA expression of TGF-b /Smad3target genes encoding MMP11,Smad7,and TIMP2,detected by qRT-PCR at 48hr after infection.N =4mice/group,*p <0.001,compared with wild-type mice without viral infection,and **p <0.05,compared with wild-type mice after viral infection,by Student’s t
test.
Figure 7.Model for the Roles of RLR-Induced IRF3Activation in Suppression of TGF-b Signaling
Without activation of innate antiviral signaling,IRF3resides primarily in the cytosol and is not associated with Smad3.Upon RLR activation by virus RNA,IRF3is C-terminally phosphorylated by TBK1or IKK ε.Activated IRF3then mostly forms a dimer,translocates into the nucleus to activate transcription from IRE-containing promoters.Some activated IRF3attenuates TGF-b signaling by dual mechanisms,based on the remarkable structural similarity of its transactivation domain with the Smad3MH2domain,thus preventing its association with T b RI and attenuating TGF-b -induced Smad3activation.In the nucleus,activated IRF3competes with Smad3/4coregulators,thus disrupting the formation of functional Smad complexes and their binding to Smad-responsive promoters.
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IRF3Activation Attenuates TGF-b Signaling
then resuspended in40%Percoll and carefully underlaid with80%Percoll. After centrifuging,the interface containing the leukocytes was collected for surface staining for CD4and Nrp-1,and intracellular staining for Foxp3. Stained cells were FACS analyzed on LSR II and by FlowJo software.
Cell Proliferation Assays
HaCaT cells were transfected with siRNA to IRF3mRNA or control siRNA,and were seeded24hr later in a48-well plate without or with TGF-b or SB431542. After72hr,cells were incubated with BrdU for8hr,and incorporated BrdU was measured using a BrdU Cell Proliferation Assay kit.
Statistics
Quanti?ed data from at least three independent experiments are presented as mean±SEM.Data shown as fold change or percentage were log-transformed before statistical analysis.When appropriate,statistical differences between groups were analyzed using an unpaired Student’s t test by Sigmaplot10.0. Differences were considered signi?cant at*p<0.05.
SUPPLEMENTAL INFORMATION
Supplemental Information includes?ve?gures and Supplemental Experi-mental Procedures and can be found with this article online at http://dx.doi. org/10.1016/j.molcel.2014.11.027.
ACKNOWLEDGMENTS
We are grateful to Dr.John Hiscott for IRF3plasmids,Dr.Tadatsugu Taniguchi for Irf3à/àmice and Dr.Jeffrey Gotts for technical support.This research was sponsored by NIH grants RO1-CA63101&-CA136690to R.D,MOST973Plan 2015CB553802,NSFC Project81472665,and the Fundamental Research Funds for the Central Universities2014QN81002to P.X.,NIH grants RO1-AI50834and P30-DK63720to J.A.B,RO1-HL44712to H.A.C.,and RO1-HL64353,-HL53949,-HL083950and-AI024674to D.S.S.B.-B.was supported by an American Diabetes Association Mentor Based Award,J.L.was sup-ported by a Muscular Dystrophy Association Scientist Development Award, D.D.was supported by a Human Frontiers Science Program postdoctoral fellowship.
Received:February26,2014
Revised:October2,2014
Accepted:November21,2014
Published:December18,2014
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Molecular Cell
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1 目录 No. 序号Description 说明 Page 页 2 门机调整 2 2.1 K100门机系统的介绍 2 2.2 CTU2板的介绍 3 2.2.1 CTU2板实物图及示意图 3 2.2.2 CTU2板端口定义 5 2.2.3 CTU2板性能指标 6 2.2. 4 CTU2板的指示灯说明7 2.3 门机变频器Altivar 28的介绍8 2.3.1 门机变频器Altivar 28控制面板介绍8 2.3.2 门机变频器Altivar 28菜单访问10 2.4 门机变频器Altivar 31的介绍11 2.4.1 门机变频器Altivar 31控制面板介绍11 2.4.2 门机变频器Altivar 31菜单访问13 2. 5 K100型门机系统的调试13 2.5.1 检查和确认13 2.5.2 门机的接线13 2.5.3 CTU2板菜单操作及状态显示说明1 6 2.5.4 CTU2板的参数设置18 2.5.5 门机的运行位置----速度参数图20 2.5.6 门机自学习21 2.5. 7 门机通电后的第一次运行21 2.5. 8 门机的正常运行21 2.5. 9 门机的手动运行21 2.5.10 如何确认K100门机调试结束21 2.6 CTU2板故障代码说明23 2.7 相关故障说明24 2.8 CTU2板软件版本号25
2.2 CTU2板的介绍 2.2.1 CTU2板实物图及示意图
图2 K100门机板实物图
2.2.2 CTU2板端口定义 ●JP4各端口定义 JP4.1 光耦隔离输入端口1，模拟开关门使能信号输入 JP4.2 光耦隔离输入端口2，变频器过力矩信号输入 JP4.3 光耦隔离输入端口3，用作关门输入信号端口 JP4.4 光耦隔离输入端口4，用作开门输入信号端口 JP4.5 光耦隔离输入的+24V输入端 JP4.6 光耦隔离输入的公共端 JP4.7 编码器+12V输出端 JP4.8 编码器0 V公共端 JP4.9 编码器A相输入端 JP4.10 编码器B相输入端 ●JP5各端口定义 JP5.1 继电器1输出端，用于开门到位信号 JP5.2 继电器2输出端，用于关门到位信号 JP5.3 继电器3输出端，用于过力矩限制，即重开门输出信号JP5.4 继电器公共端 JP5.5～JP5.8 备用 ●JP2各端口定义 JP2.1 TXV+ JP2.2 TXV- JP2.3 TXA+ JP2.4 TXA- ●JP1端口定义 RS485通讯接口 ●J1短接帽： J1.1-J1.2：选用Altivar 28变频器时短接 J1.2-J1.3：选用Altivar 31变频器时短接 ●DB1串行通讯口（备用）
第四章 调试 1 工作程序的编写 分散型控制系统的CPU 需使用V90以后版本的程序； 新装电梯的MC3电路板中一般均已装有工作程序； 电梯改装时，可经EPROM 装载或下载有关工作程序； EPROM 装载指通过4只主印EPROM 组将工作程序载入闪速EPROM 中，MC3中的SO501及SO502插口即用于EPROM 装载或闪速编程。 EPROM 装载的存储芯片版本号如下： 1.1闪速EPROM 的EPROM 装载 1) 将轿厢驶入顶层，接通控制柜内的电动召回S01； 2) 断开控制柜内的Q00开关； 3) 插入短接块J501，J502及J503； 4) 把第1只主印ROM 组插入S0501及S0502插口中； 5) 连接诊断器1； 6) 接通电源开关Q00，诊断器会显示如下信息： 表1： 在第3步中，有可能不显示FFOE ，而显示如下错误信息： 表2： EPROM 装载的正式程序版本存储芯片
之后按同样方法对主EPROM组2，3，4进行编程，只有在4只EPROM组均已载入程序后闪速程序才能运行； 将短接块J501，J502及J503插入原来位置； 再次插入电梯特性程序。 1.2闪速EPROM的下载 下载是指用诊断器5或6经串行接口将工作程序写入闪速EPROM中，闪速EPROM中需先装有下载程序； 将第6代远程监控下载到MH3时亦采用此方法。 下载步骤： —轿厢驶入顶层，接通应急运行开关S01； —将诊断器连接至MC3电路板的X1接头上； —打开诊断器电源开关并双击主菜单上的Window load； —在MC3窗口上点击―下载目的地‖； —点击―选择文件夹‖； —在窗口上选择并点击所需软件； —选择并点击MC3软件； —选择并点击工作程序版本号； —点击下载； 系统会自动执行下载，时间为10分钟左右，中间不能中断。 —下载完毕后系统会显示―下载结束‖； —按下OK键。 1.3电梯特性程序 MC的特性程序需插入S0501插口中并向左对齐。 1.选层器 需使用标准选层器码板； 已设计第2条选层器轨道（用于将选层器码板装在护脚板上）。
FOR ELECTRICAL ADJUSTMENT 8. 电梯故障代码
FOR ELECTRICAL ADJUSTMENT Catalogue 目录 No. 序号Description 说明 Page 页 8.1 SM-01主板故障代码8-3 8.2 安川变频器（Yaskawa Varispeed 616G5）故障代码8-5 8.2.1 故障检查8-5 8.2.2 警告（报警）检查8-8 8.2.3 操作出错8-10 8.3 富士变频器（Fuji Frenic 5000G11UD）故障代码8-11 8.4 变频门机RCF-1板故障代码8-12
FOR ELECTRICAL ADJUSTMENT 8. 电梯故障代码 8.1 SM-01主板故障代码 故障代码显示内容原因对策 02 运行中厅门锁脱开（急停）1．运行中门刀擦门球 2．门锁线头松动 1．调门刀与门球的间 隙 2．压紧线头 03 错位（超过45CM），撞到上强停时修正（急停）1．上限位开关误动作 2．限位开关移动后未进行井道教入 3．编码器损坏 1．查限位开关 2．重新进行井道教入 3．更换编码器 04 错位（超过45CM），撞到下强停时修正（急停）1．下限位开关误动作 2．限位开关移动后未进行井道教入 3．编码器损坏 1．限位开关 2．重新进行井道教入 3．换编码器 05 电梯到站无法开门1．门锁短接 2．门电机打滑 3．门机不工作 1．停止短接 2．检查皮带 3．检查门机控制器 06 关门受阻时间超过120秒1．关门时门锁无法合上2．安全触板动作 3．外呼按钮卡死 4．门电机打滑 5．门机不工作 08 SM-02-B和SM-03A轿厢控制器通讯中断（不接收 指令） 1．通讯受到干扰 2．通讯中断 3．终端电阻未短接 1．检查通讯线是否远离 强电 2．连接通讯线 3．短接终端电阻 09 调速器出错变频器故障对应变频器故障代码表处理 10 错位（超过45CM），撞到上多层强慢时修正1．上行多层减速开关误动作 2．多层减速开关移动后未进行井道教入 3．编码器损坏 1．检查多层减速开关 2．重新井道教入 3．更换编码器 11 错位（超过45CM），撞到下多层强慢时修正1．下行多层减速开关误动作 2．多层减速开关移动后未进行井道教入 编码器损坏 1．检查多层减速开关 2．重新井道教入 3．更换编码器 12 错位（超过45CM），撞到上单层强慢时修正1．上行单层减速开关误动作 2．单层减速开关移动后未进行井道教入 3．编码器损坏 1．检查单层减速开关 2．重新井道教入 3．更换编码器 13 错位（超过45CM），撞到下单层强慢时修正1．下行单层减速开关误动作 2．单层减速开关移动后未进行井道教入 编码器损坏 1．检查单层减速开关 2．重新井道教入 3．更换编码器
DIAGNOSTIC UNIT I Function 01 00 Display of error stack as per: 05.00 显示故障堆栈 Example: Error Explanation Weighting BW 14 03 AAjob-specific error messag任务特性故障信息N emergency stop 紧急停梯 XX landing 楼层S stopping 停止 YY undefined 未定义M spontaneous message 自发信息 NN explanations from page 21 解释见21页 B lift blocked 电梯被锁定 ZZ N umber of marking flag 标志编号 ..B o peration phase 运行阶段Significance: Frequency levels of error: 含义：故障频繁度 Error code number 故障代码Level 10 frequent 10度频繁发生 Error Error description Causes, remedy or instructions BW 01 XX Interlock contact RK or RKD is jamming in landing XX. 门锁触点RK或RKD在XX楼层处卡 住/在XX层检查门锁触点或门的机械部件。在任务Check interlock contact or mechanical parts of door in landing XX. A correct door type must be programmed in the job- 6 Start/Stop button LK sensor Run: IS/RS2) UP direction with IS DOWN direction with IS Program selector wheel LN sensor W/W1 contactor WO/WU Schütz Seven-segment-function display flashing1) Pulses Check-back SR module Safety circuit EK HK TK KT
蒂森电梯变频器调试的基本步骤 一、蒂森电梯配件的变频器带电机空载运行 1.设置电机的功率、极数,要综合考虑蒂森电梯配件的变频器的工作电流。 2.设定蒂森电梯配件的变频器的最大输出频率、基频、设置转矩特性。通用蒂森电梯配件的变频器均备有多条VPf曲线供用户选择,用户在使用时应根据负载的性质选择合适的VPf曲线。如果是风机和泵类负载,要将蒂森电梯配件的变频器的转矩运行代码设置成变转矩和降转矩运行特性。为了改善蒂森电梯配件的变频器启动时的低速性能,使电机输出的转矩能满足生产负载启动的要求,要调整启动转矩。在蒂森电梯配件的异步电机变频调速系统中,转矩的控制较复杂。在低频段,由于电阻、漏电抗的影响不容忽略,若仍保持VPf为常数,则磁通将减小,进而减小了电机的输出转矩。为此,在低频段要对电压进行适当补偿以提升转矩。一般蒂森电梯配件的变频器均由用户进行人工设定补偿。 3.将蒂森电梯配件的变频器设置为自带的键盘操作模式,按运行键、停止键,观察电机是否能正常地启动、停止。 4.熟悉变频器运行发生故障时的保护代码,观察热保护继电器的出厂值,观察过载保护的设定值,需要时可以修改。蒂森电梯配件的变频器的使用人员可以按变频器的使用说明书对变频器的电子热继电器功
能进行设定。当变频器的输出电流超过其容许电流时,变频器的过电流保护将切断变频器的输出。因此,变频器电子热继电器的门限最大值不超过变频器的最大容许输出电流。 二、带载试运行 1.手动操作变频器面板的运行停止键,观察电机运行停止过程及蒂森电梯配件的变频器 的显示窗,看是否有异常现象。 2.如果启动P停止电机过程中蒂森电梯配件的变频器出现过流保护动作,应重新设定加速P减速时间。电机在加、减速时的加速度取决于加速转矩,而蒂森电梯配件的变频器在启、制动过程中的频率变化率是用户设定的。若电机转动惯量或电机负载变化,按预先设定的频率变化率升速或减速时,有可能出现加速转矩不够,从而造成电机失速,即电机转速与蒂森电梯配件的变频器输出频率不协调,从而造成过电流或过电压。因此,需要根据电机转动惯量和负载合理设定加、减速时间,使蒂森电梯配件的变频器的频率变化率能与电机转速变化率相协调。检查此项设定是否合理的方法是先按经验选定加、减速时间进行设定,若在启动过程中出现过流,则可适当延长加速时间;若在制动过程中出现过流,则适当延长减速时间。另一方面,加、减速时间不宜设定太长,时间太长将影响生产效率,特别是频繁启、制动时。 3.如果蒂森电梯配件的变频器在限定的时间内仍然保护,应改变启动P停止的运行曲线,从直线改为S形、U形线或S形、反U形线。电机负载惯性较大时,应该采用更长的启动停止时间,并且根据其负载特
蒂森克虏伯电梯（上海）有限公司 现场服务说明 TE030-TS 版本：A 更改码：00 生效日期：2004.10.8 1 目的 将已出厂的K100门机SM-05板及其附属芯片全部换回，用CTU2板和新的芯片替换。 2 替换步骤 1) 将SM-05板断电； 2) 将SM-05板拆下，保留其原有的插件（不要拆任何线）； 3) 将CTU2板装上（CTU2板与SM-05板大小及安装尺寸均一样）； 4) CTU2板与SM-05板的端口定义的区别 按上述说明将05板原有插件插至CTU2板上，唯一不同的是05板上的JP3； 5) 将门机编码器屏蔽线从JP4.3上拆下接至03板安装板上的固定螺丝上，现在屏蔽线必须可靠接地， 以前是接在05板JP4.3上（接0V ）； 6) 485通讯线 ● 使用Altivar 28 门机变频器的系统，通讯线可以继续使用 ● 使用Altivar 31 门机变频器的系统，通讯线必须更换（通讯线详情见TE026-TS 中的13、14 页） 7) 确认CTU2板芯片软件版本： TEDC_SER-V1.1 8) 确认门机变频器的型号后，将CTU2板上的J1正确跳线； 9) 将CTU2板上电，设置门机变频器参数： ● Altivar 28 门机变频器： Set- ACC 2.0s SM-05板 CTU2板 JP1 JP1 JP2 JP2 JP3 JP5 JP4 JP4
蒂森克虏伯电梯（上海）有限公司现场服务说明 TE030-TS 版本：A 更改码：00 生效日期：2004.10.8 dEC 2.0s Altivar 31 门机变频器： Set- 参数设定值 ACC 1.0 dEC 1.2 ItH 1.5 Ctd 0.2 DrC- 参数设定值 bFr 50 UnS 100 FrS 25 nCr 1.0 nSP 662 COS 0.7 UFt L Fun- 菜单参数设定值 rPC rPt S StC Stt FST FSt no dCF 2 dC1 no IdC 0.7 AdC AdC Ct tdC1 1
I 型 诊 断 仪 七 段 数 字 显 示 屏起 / 停 按 钮 (闪 烁) 脉 冲 感 应 器 行：IS/RS 2) 上 行 SR 模 块 检 查 回 应 下 行 序 选 择 轮 安 全 回 路 感 应 器 井道 轿厢 接 厅门 接 触 器 用 I 型 诊 断 道地地仪 总 共 可 以 查 询 或 处 理 16 种 功 能。 各 项 具 体 的 功 能 可 以 用 程 序 选 择 轮 来 选 定。被 选 定 的 功 能 就 会 出 现 在 七 段 数 字 显 示 屏 上 ( 闪 烁 显 示)。 所 有 各 项 功 能 将 在 以 下 各 页 说 明： TCI/TCM 1) 功 能 功 能 号 0100 至 1400 2) 教 入 简 介 功 能 号 1500 3) 教 入 功 能 号 1500 4) 存 储 地 址 功 能 号 0000 F2/F3 5) 门 机 功 能 号 0100, 1400, 1500 LSM1 6) 称 重 装 置 功 能 号 1300, 1400, 1500 ________________________________________________________________ 1) 当 七 段 显 示 屏 闪 烁 时， 上 图 侧 面 所 示 功 能 将 由 A 列 及 B 列 发 光 二 极 管 指 示 出 来。 2) IS ... 检 修 运 行 RS ... 应 急 电 动 运 行 用 於 TCI 及 TCM 的 诊 断 仪 当 TCI 采 用 从 04.86/3 版 起 的 工 作 程 序 时， 或 者 当 TCM 采 用 从 V1 版 起 的 工 作 程 序 时 可 选 择 的 功 能 (有 效 程 序 版 本 表 自 26 页 起)。 EK HK TK KT
版本：A 更改码：00 蒂森克虏伯电梯（上海）有限公司 MF3-S 技术说明书 TE036-SM 生效日期：6/13/2006 1 概述 参考MA12/6510/70 TCM Single-Board Lift Control MC2等相关文档；MF3为TCM-MC2系统中的轿厢控制板。 根据我工厂实际情况及市场实际需求，MF3-S 为在MF3的基础上，本地化设计的产品。 2 特性描述 2.1 电源输入：典型使用 24VDC ；2.2 工作电压范围DC 9～40V; 2.3 使用功耗：不包括输入/输出，24VDC 电压输入时，小于等于50mA; 3 功能描述3.1 CAN BUS 通信： MF3-S 与TCM 主板（如MC2）进行实时的CAN BUS 串行通信，以交互轿厢控制与电梯控制主板（如TCM-MC2）之间的数据； 3.2 内召按钮控制： MF3-S 已设计1-8层站的召唤按钮控制，如电梯高于8层站的情况，需MF4-S 板进行扩展； 3.3 轿内显示控制： MF3-S 板解码来自主板的CAN BUS 数据包，再通过特定的数据接口，以向轿内显示板（如GMA9-S ）提供正确的显示信息； 3.4 轿内输入/输出信号： MF3-S 板上提供一些基本的输入信号，MF3-S 处理后发送给电梯控制主板，以如轿内基本操作信号：轿内优先、检修上/下、开关门到位等；同时MF3-S 也设计了一些基本的输出信号，MF3-S 通过CAN-BUS 接收控制主板信息，并正确输出，如超载运行、开/关门信号、上/下到站钟等等。
版本：A 更改码：00 蒂森克虏伯电梯（上海）有限公司 MF3-S 技术说明书 TE036-SM 生效日期：6/13/2006 4 线路板安装及布局图
警告 DIC-S1 20P4 型门机变频器仅供资质认证的专业人员使用。为了您本人及他人的安全，请不要尝试任何未经资质认证的操作。 所有操作必须符合最新版的国家电气规范，电梯安全守则及所有与之相关的当地法规。 本说明书中所有的图纸和资料都为蒂森克虏伯电梯（上海）有限公司的专有财产，蒂森克虏伯电梯（上海）有限公司有权随时收回。任何单位和个人都不得擅自公开发行，复制，直接或间接引用本说明书的内容，或以其他任何方式损害蒂森克虏伯电梯（上海）有限公司的利益。 我们尽力保证本说明书内容准确并及时更新，但蒂森克虏伯电梯（上海）有限公司并不为因错误或遗漏而产生的后果承担任何责任。如发现说明书中出现的问题，请及时反馈给蒂森克虏伯电梯（中国制造）工程中心。
目 录 序言....................................................................................................................................................................III 重要标识：.....................................................................................................................................V DIC – S1 20P4型门机变频器安装手册.. (1) 1 注意事项 (1) 1.1变频器适用范围及特点 (1) 1.2变频器的使用环境 (2) 1.3注意事项 (2) 2 产品介绍 (3) 2.1型号说明 (3) 2.2技术规范 (3) 3 安装及配线说明 (5) 3.1开箱检查 (5) 3.2外形尺寸 (5) 3.3安装说明 (6) 3.4端子定义 (7) 3.4.1 主回路端子定义 (7) 3.4.2 控制端子定义 (8) 3.5变频器基本接线图 (10) 3.5.1 DIC-S1 20P4变频器基本接线图 (10) 3.6跳线接线图 (12) 3.7接线规格表 (13) 4 参数表 (14) 4.1功能参数表 (14) 4.2监视参数表 (18) 5 功能参数说明 (19) 6 操作器 (25) 6.1面板操作器外形图 (25) 6.2 面板操作器键盘 (25) 6.3 面板操作器U参数表 (35) 6.4 面板操作举例： (37)
蒂森电梯易驱变频器在扶梯上的应用 蒂森电梯易驱变频器工艺要求： 自动扶梯多采用单速交流电机作为主机，链轮链条式传动，梯级额定速度大多为0.5m/s。这种扶梯即使在无乘客使用时仍以额定速度运行，具有耗能大、机械磨损严重、使用寿命低等缺点。 有人踏上扶梯前电梯要高速平稳运行，当无人时扶梯以低速运行。 系统要求蒂森电梯变频器启动运行平稳，加速性能好，启动转矩大，过载能力强，同时应具备当蒂森电梯变频器调速系统出现故障时，控制系统自动切换到工频运行，保障扶梯输送功能的正常实施。 蒂森电梯易驱变频器系统方案： 在电梯首尾处各安装一支红外传感器开关，乘客通过电梯时，红外传感器开关被触发并发出开关信号给蒂森电梯变频器 有客流时，红外传感开关被触发，蒂森电梯变频器立即加速到多段速频率1，并使电梯高速平稳运行;无乘客通过时蒂森电梯变频器将自动切换到多段速频率2，进行低速运行对电梯上行和下行，外围控制采用开关互锁，保证扶梯系统的正常工作 为消耗下行或者制动过程产生的多余能量，需在蒂森电梯变频器上加装制动电阻。 系统优势： 在开环矢量控制模式下具有低频启动扭矩大、响应速度快等特点，在0.5Hz启动时其输出转矩可达150%额定转矩，完全能够满足电梯的工艺要求 蒂森电梯变频器的软启动功能可消除电机启动时的电流冲击 蒂森电梯变频器为容性负载，可有效改善电机的功率因数，减少无功损耗 蒂森电梯变频器具有过流、过压、过载、过热等多种电子保护装置，并具有丰富的故障报警输出功能，可有效保证整个系统的正常运作 蒂森电梯易驱变频器无客流量时电梯运行速度很低，机械磨损大为降低 蒂森电梯易驱变频器变频拖动系统的启动、停止及速度转换过程平稳顺畅，舒适感较好。 有“手动”“自动”两种工作模式，在蒂森电梯变频器出现故障的情况下，仍可按手动工作方式继续运行。 基本接线图： 蒂森电梯易驱变频器参数设置： 功能号出厂值设定值
蒂森门机控制器说明 1 电源的接线将相线接到端子1，中线接到端子2，地线接到外壳接地标志处好端子3 2 控制线的连接各控制输入端子4,5,6,7 与公共端子11之间应为无源触点(常开触点) 3 输出线的连接可采用无源器件耦合输出到电梯控制柜，根据参数II1的设置，无源器件可为常开触点或常闭触点。 4 电机的连接输出端子U,V,W必须与电机的U,V,W耦合，电机线尽可能短，电机输出端子不得短路接地。 5 控制器端子描述 端子号端子符号功能描述控制LED 1 PH 电源相线 2 N 电源中线 3 E 接地 4 11 开门指令L1 5 12 关门指令L2 6 13 光幕指令L3 7 14 强制关门指令L4 8 15 无功能L5 9 16 无功能L6’ 10 17 无功能L7
11 C 输入信号11到17的公共端 12,13 继电器1 开门输出触点L9 14,15 继电器2 开门（重开门）输出触点L10 16,17 继电器3 门位置输出触点L11 18,19 继电器4 关门到位输出触点L12 20 D 编码器A相脉冲输入L15 21 TH 编码器B脉冲输入L16 22 + 编码器电源电压+20V 23 C 编码器电源接地GND 3a Earth 编码器电缆屏蔽端 24,25,26 U.V,W 电机接线端 6 编码器接线指导 黄色A相脉冲输出，绿色B相脉冲输出，棕色电源电压（DC20V）白色电源电压（0V） 7控制器的调试与操作 @检查总电源，检查现场电压是否符合门机控制器的电源要求（220V,50HZ） @将轿门定位到半开状态，用手将轿门拉到半开状态，以便接通电源盒输入指令后确定门的运行方向 @接通电源，检查门运行方向，接通电源，屏幕显示“。。。 —”，按参数值+几次，注意轿门是否向关门方向移动。 如果轿门向关门方向移动，则说明电机接线正确。如果