Kruppel-like Factor 4 Abrogates Myocardin-induced Activation of Smooth Muscle Gene Expression

Platelet-derived growth factor BB (PDGF-BB) has been shown to be an extremely potent negative regulator of smooth muscle cell (SMC) differentiation. Moreover, previous studies have demonstrated that the Kruppel-like transcription factor (KLF) 4 potently represses the expression of multiple SMC genes. However, the mechanisms whereby KLF4 suppresses SMC gene expression are not known, nor is it clear whether KLF4 contributes to PDGF-BB-induced down-regulation of SMC genes. The goals of the present studies were to determine the molecular mechanisms by which KLF4 represses expression of SMC genes and whether it contributes to PDGF-BB-induced suppression of these genes. Results demonstrated that KLF4 markedly repressed both myocardin-induced activation of SMC genes and expression of myocardin. KLF4 was rapidly up-regulated in PDGF-BB-treated, cultured SMC, and a small interfering RNA to KLF4 partially blocked PDGF-BB-induced SMC gene repression. Both PDGF-BB and KLF4 markedly reduced serum response factor binding to CArG containing regions within intact chromatin. Finally, KLF4, which is normally not expressed in differentiated SMC in vivo, was rapidly up-regulated in vivo in response to vascular injury. Taken together, results indicate that KLF4 represses SMC genes by both down-regulating myocardin expression and preventing serum response factor/myocardin from associating with SMC gene promoters, and suggest that KLF4 may be a key effector of PDGF-BB and injury-induced phenotypic switching of SMC. Platelet-derived growth factor BB (PDGF-BB) has been shown to be an extremely potent negative regulator of smooth muscle cell (SMC) differentiation. Moreover, previous studies have demonstrated that the Kruppel-like transcription factor (KLF) 4 potently represses the expression of multiple SMC genes. However, the mechanisms whereby KLF4 suppresses SMC gene expression are not known, nor is it clear whether KLF4 contributes to PDGF-BB-induced down-regulation of SMC genes. The goals of the present studies were to determine the molecular mechanisms by which KLF4 represses expression of SMC genes and whether it contributes to PDGF-BB-induced suppression of these genes. Results demonstrated that KLF4 markedly repressed both myocardin-induced activation of SMC genes and expression of myocardin. KLF4 was rapidly up-regulated in PDGF-BB-treated, cultured SMC, and a small interfering RNA to KLF4 partially blocked PDGF-BB-induced SMC gene repression. Both PDGF-BB and KLF4 markedly reduced serum response factor binding to CArG containing regions within intact chromatin. Finally, KLF4, which is normally not expressed in differentiated SMC in vivo, was rapidly up-regulated in vivo in response to vascular injury. Taken together, results indicate that KLF4 represses SMC genes by both down-regulating myocardin expression and preventing serum response factor/myocardin from associating with SMC gene promoters, and suggest that KLF4 may be a key effector of PDGF-BB and injury-induced phenotypic switching of SMC. Alterations in the differentiated state of the smooth muscle cell (SMC), 1The abbreviations used are: SMC, smooth muscle cell; SRF, serum response factor; PDGF-BB, platelet-derived growth factor BB; KLF4, Kruppel-like transcription factor 4; TCE, TGFβ control element; RT, reverse transcription; dn, dominant negative; si, small interfering; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; MADS, (MCM-1, agamous, deficiens, SRF). or phenotypic switching, has been shown to play a key role in the repair of tissue damage and in the development of a variety of major human diseases, including atherosclerosis, restenosis, and asthma (1Owens G.K. Kumar M.S. Wamhoff B.R. Physiol. Rev. 2004; 84: 767-801Crossref PubMed Scopus (2646) Google Scholar, 2Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19470) Google Scholar). A hallmark feature of this phenotypic switching is coordinate repression of the expression of the normal repertoire of genes that distinguish differentiated mature SMC from other cell types. However, the mechanisms that regulate this process are very poorly understood. Indeed, major challenges for the field have been to define regulatory cis-elements within the promoter enhancer regions of SMC marker genes, such as SM α-actin, SM22α, and SM myosin heavy chain, to identify factors that bind and regulate the activity of these regulatory regions and to elucidate environmental factors/cues that influence these regulatory processes in response to tissue injury and/or pathological circumstances. The model that has emerged is that regulation of SMC differentiation is extremely complex and involves constant interplay between environmental cues and the genetic program that controls the coordinate expression of genes characteristic of the SMC lineage (reviewed by Owens et al. (1Owens G.K. Kumar M.S. Wamhoff B.R. Physiol. Rev. 2004; 84: 767-801Crossref PubMed Scopus (2646) Google Scholar)). It is of particular interest that expression of virtually all SMC marker genes characterized to date is dependent on CArG box elements and SRF (reviewed by Miano (3Miano J.M. J. Mol. Cell. Cardiol. 2003; 35: 577-593Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar)). Moreover, an especially exciting recent discovery was the demonstration (by our laboratory and others (4Yoshida T. Sinha S. Dandre F. Wamhoff B.R. Hoofnagle M.H. Kremer B.E. Wang D.Z. Olson E.N. Owens G.K. Circ. Res. 2003; 92: 856-864Crossref PubMed Scopus (312) Google Scholar, 5Du K.L. Ip H.S. Li J. Chen M. Dandre F. Yu W. Lu M.M. Owens G.K. Parmacek M.S. Mol. Cell. Biol. 2003; 23: 2425-2437Crossref PubMed Scopus (313) Google Scholar, 6Wang Z. Wang D.Z. Pipes G.C. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7129-7134Crossref PubMed Scopus (436) Google Scholar, 7Li S. Wang D.Z. Wang Z. Richardson J.A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9366-9370Crossref PubMed Scopus (307) Google Scholar, 8Chen J. Kitchen C.M. Streb J.W. Miano J.M. J. Mol. Cell. Cardiol. 2002; 34: 1345-1356Abstract Full Text PDF PubMed Google Scholar)) that the SMC/cardiac myocyte-restricted SRF co-activator myocardin is required for the expression of multiple SMC marker genes by inducing CArG/SRF-dependent transcription of SMC genes in SMC and many embryonic stem cells/fibroblasts and, most importantly, that it is necessary for SMC differentiation in vivo. However, so far, it is not clear whether down-regulation of SMC marker genes associated with phenotypic switching is due to the decreased expression of a transcriptional activator, such as myocardin, and/or stimulation of the expression of cell-selective gene repressors. The most potent and efficacious inhibitor of SMC differentiation identified to date is PDGF-BB (1Owens G.K. Kumar M.S. Wamhoff B.R. Physiol. Rev. 2004; 84: 767-801Crossref PubMed Scopus (2646) Google Scholar). Indeed, a series of studies by our laboratory and others demonstrated that treatment of cultured SMC with PDGF-BB was associated with profound selective down-regulation of SMC gene expression through a combination of transcriptional (9Van P.V. Li X. Maselli J. Nemenoff R.A. Circ. Res. 1994; 75: 1126-1130Crossref PubMed Scopus (30) Google Scholar) and post-transcriptional mechanisms (10Corjay M.H. Blank R.S. Owens G.K. J. Cell. Physiol. 1990; 145: 391-397Crossref PubMed Scopus (53) Google Scholar, 11Blank R.S. Owens G.K. J. Cell. Physiol. 1990; 142: 635-642Crossref PubMed Scopus (92) Google Scholar, 12Holycross B.J. Blank R.S. Thompson M.M. Peach M.J. Owens G.K. Circ. Res. 1992; 71: 1525-1532Crossref PubMed Scopus (140) Google Scholar). It is of particular significance that we also demonstrated that these effects were not a direct function of the mitogenic properties of PDGF-BB, in that chronic treatment of cultured SMC with PDGF-BB in post-confluent cultures was associated with sustained, but reversible, repression of SMC gene expression in the absence of sustained mitogenesis (11Blank R.S. Owens G.K. J. Cell. Physiol. 1990; 142: 635-642Crossref PubMed Scopus (92) Google Scholar). In addition, we found that PDGF-BB could induce suppression of SMC marker genes at concentrations that were 1–2 orders of magnitude lower than that required to induce maximal mitogenesis (11Blank R.S. Owens G.K. J. Cell. Physiol. 1990; 142: 635-642Crossref PubMed Scopus (92) Google Scholar). Given that PDGF-BB levels are increased dramatically following vascular injury (13Golden M.A. Au Y.P. Kirkman T.R. Wilcox J.N. Raines E.W. Ross R. Clowes A.W. J. Clin. Investig. 1991; 87: 406-414Crossref PubMed Scopus (118) Google Scholar, 14Wilcox J.N. Smith K.M. Williams L.T. Schwartz S.M. Gordon D. J. Clin. Investig. 1988; 82: 1134-1143Crossref PubMed Scopus (367) Google Scholar), it has been hypothesized, although not yet proven, that PDGF-BB is a key mediator of SMC phenotypic switching in response to vascular injury in vivo. Surprisingly, however, despite the fact that it has been over a decade since we first demonstrated the differentiation repressing activity of PDGF-BB in cultured SMC, virtually nothing is known as to how it mediates this effect. Of interest, based on a yeast one-hybrid screen for factors that bound to a novel TGFβ control element (TCE) found just 3′ to a required CArG element within the SM α-actin promoter, we identified a potent repressor of SMC gene expression termed KLF4. This Kruppel-like factor potently inhibited the expression of multiple SMC promoter-reporter genes in co-transfection studies (15Adam P.J. Regan C.P. Hautmann M.B. Owens G.K. J. Biol. Chem. 2000; 275: 37798-37806Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). However, KLF4 was not expressed in SMC tissues in vivo based on either in situ (16Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) or RT-PCR analyses (15Adam P.J. Regan C.P. Hautmann M.B. Owens G.K. J. Biol. Chem. 2000; 275: 37798-37806Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) and was expressed only at low levels in vitro (15Adam P.J. Regan C.P. Hautmann M.B. Owens G.K. J. Biol. Chem. 2000; 275: 37798-37806Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 17Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar); and it is unknown whether KLF4 is up-regulated in response to PDGF-BB in vitro or injury in vivo. The goals of the present studies were: 1) to determine molecular mechanisms whereby KLF4 inhibits expression of SMC marker genes, including testing the hypothesis that it interferes with CArG/SRF/myocardin-dependent gene expression and recruitment of SRF to CArG containing regions of SMC genes within intact chromatin and 2) to determine whether KLF4 expression is induced by treatment of cultured SMC with PDGF-BB and/or within phenotypically modulated SMC in vivo following vessel injury. Cell Cultures and Stimulation of SMC by PDGF-BB—Rat aortic SMC and NIH3T3 were cultured as described previously (17Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 200 μg/ml l-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). SMCs were plated at 1.5 × 104/cm2 and starved the next day with serum-free medium, ISFM (Dulbecco's modified Eagle's medium/F12 supplemented with 200 μg/ml l-glutamines, 6.25 ng/ml sodium selenite, 5 μg/ml transferrin, and 200 μm ascorbic acid). Roughly 16–17 h after starvation, SMCs were treated with 30 ng/ml PDGF-BB (Upstate Biotechnology) or vehicle (10 mm acetic acid and 2 mg/ml fatty acid-free bovine serum albumin) for different time points (18Dandre F. Owens G.K. Am. J. Physiol. 2004; 286: H2042-H2051Crossref PubMed Scopus (107) Google Scholar). Treating SMCs with cycloheximide (Sigma) was described previously (19Hautmann M.B. Madsen C.S. Owens G.K. J. Biol. Chem. 1997; 272: 10948-10956Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Generation of Constructs and Adenovirus—SM α-actin promoter/luciferase reporter constructs with (PPITCE-Luc) or without (PPI-Luc) mutation of the TCE and expression plasmids pcDNA-KLF4 were constructed as described previously (17Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). pcDNA-FLAG-KLF4 was generated by putting a FLAG tag sequence after KLF4 ATG via PCR-based mutagenesis, and the construct was verified by sequencing. pCGN-SRF and pCGN-dnSRF were kindly provided by Dr. R. Misra (Medical College of Wisconsin) (20Spencer J.A. Misra R.P. J. Biol. Chem. 1996; 271: 16535-16543Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21Zhang X. Chai J. Azhar G. Sheridan P. Borras A.M. Furr M.C. Khrapko K. Lawitts J. Misra R.P. Wei J.Y. J. Biol. Chem. 2001; 276: 40033-40040Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), pcDNA-myocardin was a generous gift from Dr. E. Olson (University of Texas Southwestern Medical Center at Dallas) (22Wang D.Z. Chang P.S. Wang Z.G. Sutherland L. Richardson J.A. Small E. Krieg P.A. Olson E.N. Cell. 2001; 105: 851-862Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar), and pcDNA-KLF5 was provided courtesy of Dr. Masahiko Kurabayahi at the Gunma University (23Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar). Adenovirus constructs Ad-FLAG-myocardin and Ad-FLAG-KLF4 were constructed as described previously (4Yoshida T. Sinha S. Dandre F. Wamhoff B.R. Hoofnagle M.H. Kremer B.E. Wang D.Z. Olson E.N. Owens G.K. Circ. Res. 2003; 92: 856-864Crossref PubMed Scopus (312) Google Scholar, 17Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and adenovirus overexpressing myocardin and KLF4 were amplified and purified by the Gene Transfer Vector Core at The University of Iowa (Iowa city, Iowa). KLF4 siRNA construct (pM-KLF4) was generated by inserting a siRNA-generating sequence against KLF4 after a mouse H1 promoter in the pMighty vector. 2M. H. Hoofnagle and G. K. Owens, unpublished data. Transient Transfection and Infection—Cultured NIH3T3 cells were transfected with reporter PPI-Luc or PPITCE-Luc and effectors pcDNA-KLF4, pcDNA-SRF, pcDNA-KLF5, and/or pcDNA-FLAG-myocardin in triplicate using Superfect reagent (Qiagen) according to the manufacturer's protocol. The transfected cells were incubated 48 h before harvesting. The cultured SMCs were fed with fresh serum-free medium (ISFM), and transfected with PPI-Luc and pM-KLF4 in triplicate using FuGENE 6 according to the manufacturer's instructions 1 day after plating. Between 20 and 21 h after transfection, the SMCs were treated with PDGF-BB (Upstate Biotechnology) or vehicle for 24 h. The transfected cells were harvested with passive lysis buffer (Promega). Luciferase activity was measured with luciferase assay substrate (Promega) and was normalized to total protein (Coomassie Plus protein assay reagent, Pierce). Transfections were repeated at least three times, and the relative luciferase activities were presented as mean ± S.E. COS-7 cells were transfected with pcDNA-FLAG-KLF4 and pCGN-SRF or pCGN-dnSRF using FuGENE 6 according to the manufacturer's instructions. The transfected cells were harvested 48 h after transfection with cold radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mm NaCl, and 1 mm EDTA supplemented with stock solution made with protease inhibitor mixture (Roche Applied Science)). SMCs were infected with KLF4 adenovirus (Ad-CMV-KLF4) or control virus (Ad-CMV) 3 days after plating at a multiplicity of infection of 5 plaque-forming units/cell. The infection efficiency was demonstrated to be almost 100% based on infection with an EGFP expression adenovirus construct, i.e. Ad-CMV-EGFP (data not shown). Twenty-four hours after infection, SMCs were fixed with 1% formaldehyde at 37 °C for 10 min and harvested with cold phosphate-buffered saline supplemented with protease inhibitor mixture solution and subjected to coimmunoprecipitation; or RNA samples were prepared with TRIzol reagent (Invitrogen) and then subjected to real-time RT-PCR analyses as described previously (17Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The relative changes were presented as mean ± S.E. The experiments were repeated three times. RNA Preparation and Real-time RT-PCR—Total RNA was prepared from PDGF-BB-stimulated or Ad-CMV-KLF4- or AD-CMV-myocardin-infected SMCs, and subjected to reverse transcription and real-time RT-PCR assays for KLF4, myocardin, SM α-actin, and SM MHC. The results were normalized against GAPDH or 18 S as described previously (17Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The sequences for SM α-actin and SMC primers and probes were published previously (17Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and the primers and probes for KLF4 and Myocardin are: KLF4-For, 5′-ctttcctgccagaccagatg-3′; KLF4-Rev, 5′-ggtttctcgcctgtgtgagt-3′; KLF4 probe, 5′-attatcaagagctcatgccaccggg-3′; myocardin-008, 5′-cggattcgaagctgttgtctt-3′; myocardin-009, 5′-aaaccaggccccctccc-3′; and myocardin probe, 5′-actctgacaccttgagatcatccaggtttgg-3′ (IDT). Co-immunoprecipitation Assays—Cell extract derived from transfected COS-7 cells or infected SMCs was precipitated with 5 μl of SRF antibody (Santa Cruz Biotechnology) or 3 μl of FLAG antibody (Sigma) for 2 h at 4 °C, and antibody-protein complex was pulled down with protein A-agarose for 5 h at 4 °C. Precipitants were washed three times with radioimmune precipitation assay buffer (10 min, 4 °C, each time) before being eluted from agarose bead and then subjected to Western blotting analyses. Quantitative Chromatin Immunoprecipitation Assay—The experiment procedure was described in detail by Wamhoff et al. (24Wamhoff B.R. Bowles D.K. McDonald O.G. Sinha S. Somlyo A.P. Somlyo A.V. Owens G.K. Circ. Res. 2004; 95: 406-414Crossref PubMed Scopus (156) Google Scholar). Briefly, cultured cells were grown to the desired density on 100-mm plates and treated with 1% formaldehyde for 10 min at 37 °C to cross-link protein/DNA and protein/protein interactions within intact chromatin. Chromatin was purified as described previously (25Manabe I. Owens G.K. J. Clin. Investig. 2001; 107: 823-834Crossref PubMed Scopus (126) Google Scholar). The cross-linked chromatin was sonicated to shear chromatin fragments to 200–600 bp. The sonicated chromatin was immunoprecipitated with 5 μl of immunoserum to SRF (Santa Cruz Biotechnology), whereas negative control/input DNA was immunoprecipitated with no antibody, and immune complexes were recovered with agarose beads (Upstate Biotechnology). Cross-links were reversed, chromatin subjected to proteinase K digestion to remove protein from the DNA, and the DNA was purified via phenol-chloroform extraction. Recovered DNA was quantitated by fluorescence with picogreen reagent (Molecular Probes) according to manufacturer's recommendations. Real-time PCR was performed on genomic DNA from chromatin immunoprecipitation experiments as described in Litt et al. (26Litt M.D. Simpson M. Recillas-Targa F. Prioleau M.N. Felsenfeld G. EMBO J. 2001; 20: 2224-2235Crossref PubMed Scopus (323) Google Scholar), with minor modifications. Rat Carotid Injury—Balloon-injured Sprague-Dawley rat carotids and their uninjured contralateral control vessels were acquired from Zivic Miller Laboratories, Inc. (Pittsburgh, PA). Six samples were obtained at each time point (0, 1, 2, 6, and 24 h). The Fast Prep FP120 (Q-Biogene) was used to homogenize tissues, and the RNeasy kit (Qiagen) was used to purify total RNA from independent samples. Gene expression in each injured vessel was normalized to its contralateral uninjured control carotid. KLF4 Dramatically Repressed SRF/Myocardin-induced Activation of SM α-Actin Promoter, and the Repression Could Be Rescued by Overexpression of SRF—Given that KLF4 was originally identified based on its binding to TCEs, which are found adjacent to CArG elements within SMC promoters (19Hautmann M.B. Madsen C.S. Owens G.K. J. Biol. Chem. 1997; 272: 10948-10956Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), we hypothesized that KLF4 may act, in part, through direct inhibition of SRF/CArG-dependent transcription. As an initial test of this hypothesis, cultured SMC was co-transfected with myocardin and with KLF4 in conjunction with the SM α-actin promoter-luciferase constructs. Consistent with previous reports, myocardin dramatically activated expression of the wild-type SM α-actin promoter inducing a nearly 600-fold increase (Fig. 1a) as compared with control constructs. Interestingly, although absolute transcription rates were decreased by mutation of the TCE, myocardin-induced activation was unaffected, eliciting a similar enhancement of expression over control groups as seen with the wild-type promoter. The activating effect of myocardin peaked with 0.4 μg of plasmid for both wild-type and TCE mutation promoter. It is of major interest that the results showed that overexpression of KLF4 virtually abolished myocardin-induced activation of the SM α-actin promoter (Fig. 1a). This effect was concentration-dependent in that increasing the myocardin plasmid concentration relative to the KLF4 plasmid concentration was associated with reduced KLF4-induced repression (Fig. 1b). Also of interest is that the repression of the activation by myocardin could be rescued by wild-type SRF (20Spencer J.A. Misra R.P. J. Biol. Chem. 1996; 271: 16535-16543Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) but not by SRF containing a mutated MADS domain (21Zhang X. Chai J. Azhar G. Sheridan P. Borras A.M. Furr M.C. Khrapko K. Lawitts J. Misra R.P. Wei J.Y. J. Biol. Chem. 2001; 276: 40033-40040Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), which has been shown by Wang et al. (22Wang D.Z. Chang P.S. Wang Z.G. Sutherland L. Richardson J.A. Small E. Krieg P.A. Olson E.N. Cell. 2001; 105: 851-862Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar) to be required for its binding to myocardin (Fig. 1c). KLF4 Specifically Bound to SRF in Immunoprecipitation Assays—The preceding results suggest that KLF4 and SRF may physically interact with each other. To assess this possibility, a series of immunoprecipitation assays were performed. Because no antibody is available that is completely specific for KLF4 and works in immunoprecipitation assays, we constructed a FLAG epitope-tagged KLF4 plasmid (pcDNA-FLAG-KLF4) and an adenovirus with which to overexpress a FLAG-tagged KLF4 (Ad-FLAG-KLF4). In this manner, we could perform both SRF immunoprecipitation assays, test for binding of FLAG-tagged KLF4, or perform KLF4 immunoprecipitation assays and test for the presence of SRF. Results showed that wild-type SRF (but not SRF containing a mutated MADS domain) specifically interacted with KLF4 within COS-7 cells (Fig. 2a). Subsequent experiments were then done to test for interaction between KLF4 and SRF within cell lysates derived from SMC infected by Ad-FLAG-KLF4. Results showed that the endogenous SRF bound to the FLAG epitope-tagged KLF4 in SMCs (Fig. 2b). The specificity of the association was confirmed by reciprocal precipitations with either SRF antibody or FLAG antibody. Taken together, these results provide compelling evidence that SRF bound to KLF4 and that the MADS domain of SRF was required for this binding. PDGF-BB Up-regulated KLF4 Expression in Cultured SMC, and Both PDGF-BB and KLF4 Repressed Myocardin Expression—Results of the current (Fig. 1) and previous studies (15Adam P.J. Regan C.P. Hautmann M.B. Owens G.K. J. Biol. Chem. 2000; 275: 37798-37806Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 17Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) provide compelling evidence that KLF4 potently represses expression of multiple SMC differentiation marker genes. However, although KLF4 has the capacity to repress SMC genes, there was no direct evidence as to whether it actually contributes to SMC gene expression under either physiological or pathological circumstances. Interestingly, KLF4 is expressed in cultured SMCs that are known to be phenotypically switched (27Owens G.K. Physiol. Rev. 1995; 75: 487-517Crossref PubMed Scopus (1411) Google Scholar), but it is not normally expressed in fully differentiated SMC in vivo (15Adam P.J. Regan C.P. Hautmann M.B. Owens G.K. J. Biol. Chem. 2000; 275: 37798-37806Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 16Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Because both PDGF-BB and KLF4 potently repress SMC marker genes in vitro (9Van P.V. Li X. Maselli J. Nemenoff R.A. Circ. Res. 1994; 75: 1126-1130Crossref PubMed Scopus (30) Google Scholar, 10Corjay M.H. Blank R.S. Owens G.K. J. Cell. Physiol. 1990; 145: 391-397Crossref PubMed Scopus (53) Google Scholar, 11Blank R.S. Owens G.K. J. Cell. Physiol. 1990; 142: 635-642Crossref PubMed Scopus (92) Google Scholar, 12Holycross B.J. Blank R.S. Thompson M.M. Peach M.J. Owens G.K. Circ. Res. 1992; 71: 1525-1532Crossref PubMed Scopus (140) Google Scholar), we hypothesized that the effects of PDGF-BB might be mediated at least in part by the induction of KLF4. Consistent with this, results of real-time RT-PCR analyses showed that treatment of cultured SMC with PDGF-BB was associated with rapid induction of KLF4 mRNA expression. Indeed, expression was increased by 2-fold within 15 min of treatment, peaked at an increase of 5-fold at 45 min, and subsequently decreased to control levels by 8 h (Fig. 3a). Furthermore, treatment of cultured SMC with the protein synthesis inhibitor cycloheximide failed to block PDGF-BB-induced increases in KLF4 expression (Fig. 3b). Of major interest is that PDGF-BB induced marked repression of myocardin (Fig. 3c), with levels decreasing by 75% of levels in untreated control SMC at 4 h. These results suggest that PDGF-BB-induced expression of KLF4 may represent an early and direct effector pathway for PDGF-BB-induced repression of myocardin. In support of this, adenoviral-mediated overexpression of KLF4 dramatically repressed endogenous myocardin mRNA expression in SMCs, as measured by real-time RT-PCR (Fig. 3d). Suppression of KLF4 Expression by Interference RNA Partially Blocked PDGF-BB-induced Down-regulation of SM α-Actin Promoter Activity—To directly test the role of KLF4 in PDGF-BB-induced repression of SMC marker genes in cultured SMCs, we constructed a unique KLF4 siRNA plasmid with which to inhibit PDGF-BB-induced expression of KLF4. The plasmid contains a mouse H1 promoter driving the expression of siRNA targeted to KLF4. Because an antibody to KLF4 is not available, the efficacy of the KLF4 siRNA plasmid was documented by Western blot analysis of the effects of the KLF4 siRNA plasmid on expression of FLAG epitope-tagged KLF4 in cultured SMC co-transfected with our FLAG epitope-tagged KLF4 expression plasmid. Results showed that expression of FLAG-KLF4 was significantly (although not completely) inhibited by pM-KLF4 as compared with multiple control siRNAs, including an empty vector (pMighty) or pM-Scramble (Fig. 4a). Significantly, PDGF-BB-induced repression of SM α-actin was partially abrogated by transfection with the KLF4 siRNA plasmid (Fig. 4b), suggesting that KLF4 was at least one effector of PDGF-BB-induced transcriptional repression in cultured SMCs. Similar results were obtained with several independent siRNA-targeting constructs, whereas no effect was seen with the scrambled siRNA or with an siRNA targeting EGFP. PDGF-BB Treatment or Overexpression of KLF4 Virtually Abrogated SRF Binding to the CArG Region of SM α-Actin— Because the MADS domain of SRF was reported to be necessary for DNA binding (28Norman C. Runswick M. Pollock R. Treisman R. Cell. 1988; 55: 989-1003Abstract Full Text PDF PubMed Scopus (709) Google Scholar), we tested the possibility that KLF4-induced repression might be the result of the inhibition of SRF binding to CArG elements. Significantly, although KLF4 had no effect on SRF binding in in vitro gel shift assays (data not shown), results of quantitative chromatin immunoprecipitation assays showed that KLF4 overexpression, as well as PDGF-BB treatment, of cultured SMC were associated with marked reductions in SRF binding to CArG-containing regions of the SM α-actin promoter within intact chromatin (Fig. 5, a and b). Importantly, these effects were selective in that SRF binding to the c-fos CArG was unchanged by KLF4 and increased by PDGF-BB. Consistent with these results, our previous transfection studies (18Dandre F. Owens G.K. Am. J. Physiol. 2004; 286: H2042-H2051Crossref PubMed Scopus (107) Google Scholar) showed that PDGF-BB treatment was associated with repression of the SM α-actin promoter but activation of the c-fos promoter, whereas KLF4 significantly repressed expression of the SM α-actin promoter (Fig. 5c). KLF4 Expression Was Markedly Increased in Vivo following Vascular Injury—The KLF4 knock-out