Opposite Smad and Chicken Ovalbumin Upstream Promoter Transcription Factor Inputs in the Regulation of the Collagen VII Gene Promoter by Transforming Growth Factor-β
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m402178200
ISSN1083-351X
AutoresMarı́a Julia Calonge, Joan Seoane, Joan Massagué,
Tópico(s)Cancer-related gene regulation
ResumoA critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-β (TGF-β). The gene, COL7A1, is activated by TGF-β via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-β may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-β-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-β-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-β-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-β addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-β signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs. A critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-β (TGF-β). The gene, COL7A1, is activated by TGF-β via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-β may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-β-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-β-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-β-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-β addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-β signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs. Type VII collagen belongs to an extensive family of closely related proteins involved in cell anchoring to extracellular matrix and cartilage formation. Although some collagens have a widespread distribution, type VII collagen is found exclusively in the basement membrane of stratified squamous epithelia (1Sakai L.Y. Keene D.R. Morris N.P. Burgeson R.E. J. Cell Biol. 1986; 103: 1577-1586Crossref PubMed Scopus (435) Google Scholar, 2Uitto J. Chung-Honet L.C. Christiano A.M. Exp. Dermatol. 1992; 1: 2-11Crossref PubMed Scopus (62) Google Scholar). Its subunit, the type VII collagen α-chain (COL7A1), is expressed in both dermal fibroblasts and epidermal keratinocytes (3Woodley D.T. Briggaman R.A. Gammon W.R. O'Keefe E.J. Biochem. Biophys. Res. Commun. 1985; 130: 1267-1272Crossref PubMed Scopus (29) Google Scholar, 4Koenig B.B. Cook J.S. Wolsing D.H. Ting J. Tiesman J.P. Correa P.E. Olson C.A. Pecquet A.L. Ventura F. Grant R.A. Chen G.-X. Wrana J.L. Massagué J. Rosenbaum J.S. Mol. Cell. Biol. 1994; 14: 5961-5974Crossref PubMed Scopus (312) Google Scholar, 5Ryynanen J. Sollberg S. Parente M.G. Chung L.C. Christiano A.M. Uitto J. J. Clin. Investig. 1992; 89: 163-168Crossref PubMed Scopus (70) Google Scholar). COL7A1 forms homotrimers that are assembled into fibrils (6Christiano A.M. Greenspan D.S. Lee S. Uitto J. J. Biol. Chem. 1994; 269: 20256-20262Abstract Full Text PDF PubMed Google Scholar). These fibrils are thought to anchor the epidermal basement membrane to the underlying dermal extracellular matrix (7Uitto J. Pulkkinen L. Mol. Biol. Rep. 1996; 23: 35-46Crossref PubMed Scopus (79) Google Scholar). Mutations that cause structural alterations or defective expression of COL7A1 lead to dystrophic epidermolysis bullosa, a group of inherited skin disorders in which blisters form between the basement membrane and the papillary dermis (8Pulkkinen L. Uitto J. Matrix Biol. 1999; 18: 29-42Crossref PubMed Scopus (234) Google Scholar). Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; COUP-TF, chicken ovalbumin upstream promoter transcription factor; FBS, fetal bovine serum; SBE, Smad binding element.1The abbreviations used are: TGF-β, transforming growth factor-β; COUP-TF, chicken ovalbumin upstream promoter transcription factor; FBS, fetal bovine serum; SBE, Smad binding element. in particular is a potent inducer of COL7A1 expression in fibroblast and keratinocytes (9Vindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar, 10Verrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (159) Google Scholar, 11Naso M. Uitto J. Klement J.F. J. Investig. Dermatol. 2003; 121: 1469-1478Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). A multifunctional cytokine, TGF-β critically regulates cell adhesion and extracellular matrix production among other cellular functions (12Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg, Germany1990: 419-472Google Scholar, 13Massagué J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3002) Google Scholar). In addition to activating the production of collagen VII and other types of interstitial collagens, TGF-β controls the expression of fibronectin, extracellular matrix proteoglycans, integrin cell adhesion receptors, pericellular proteases, and protease inhibitors. The effects of TGF-β on genes encoding the cell adhesion apparatus, along with its effects on cell proliferation and differentiation, exert a profound influence on tissue development and homeostasis. TGF-β activates COL7A1 expression at the transcriptional level (9Vindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar, 10Verrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (159) Google Scholar, 11Naso M. Uitto J. Klement J.F. J. Investig. Dermatol. 2003; 121: 1469-1478Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Although a general signal transduction pathway for transcriptional regulation by TGF-β has been established, little is known about the specific mechanisms involved in the COL7A1 gene response. Activated TGF-β receptors directly phosphorylate Smad2 and Smad3, inducing their accumulation in the nucleus to regulate the expression of a large set of genes (14Shi Y. Massagué J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4795) Google Scholar). Receptor-phosphorylated Smads associate with Smad4, which in most instances is indispensable for transcriptional regulation. Smad proteins recognize the sequence CAGAC, commonly referred to as the Smad binding element (SBE). To regulate specific target genes, however, activated Smad complexes must additionally interact or functionally cooperate with other transcription factors. In the case of COL7A1 activation by TGF-β, previous studies have shown the involvement of Smad and AP1 transcription factors (9Vindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar, 10Verrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (159) Google Scholar, 11Naso M. Uitto J. Klement J.F. J. Investig. Dermatol. 2003; 121: 1469-1478Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The TGF-β-responsive region in the COL7A1 contains a canonical SBE. Mutation of this element inhibits the activation of the COL7A1 promoter by TGF-β in fibroblasts (9Vindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar). Paradoxically, this SBE is not required for this response in keratinocytes (11Naso M. Uitto J. Klement J.F. J. Investig. Dermatol. 2003; 121: 1469-1478Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) suggesting that the cellular context and other complexities play an important role in the regulation of COL7A1 expression by TGF-β. To address these questions, we investigated the role of various elements present in the TGF-β-responsive region of the COL7A1 promoter in keratinocytes and fibroblasts. We report the existence of a second Smad binding site in the COL7A1 promoter that provides cell type-dependent redundancy, clarifying previous controversies. Our results also suggest that COL7A1 activation in response to TGF-β involves not only activation by a Smad complex but also the relief of inhibition by COUP-TF transcriptional repressors. Cell Lines—HaCaT, NIH 3T3, and COS1 cells were maintained in Dulbecco's modified Eagle's medium plus 10% FBS (Invitrogen). Plasmids—The human COL7A1 promoter fragment spanning from -496 to +92 was generated by PCR, using human DNA as template and oligonucleotides primers with flanking SalI/HindIII sites. This fragment was cloned into the XhoI/HindIII sites of the low basal activity luciferase reporter plasmid pGL2-basic (Promega). Mutant forms of the wild type promoter were obtained by site-directed mutagenesis using oligonucleotides carrying the indicated mutations. FLAG-tagged versions of the COUP-TFI and -TFII cDNAs were generated and cloned into the pCMV5 vector. FLAG-tagged COUP-Dim was generated by PCR, amplifying the C-terminal region of COUP-TFII (residues 300-414) comprising the dimerization domain, and was cloned into the pCMV5 vector. COS1 cells were transiently transfected with FLAG-tagged COUP-TFI or -TFII or COUP-Dim using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Transcriptional Assays—HaCaT cells were transfected by using the DEAE-dextran method as described previously (15Iavarone A. Massagué J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (329) Google Scholar). NIH 3T3 cells were transfected using LipofectAMINE or the calcium-phosphate precipitation method. Briefly, 3 μg of plasmid DNA were diluted in 250 μl of 2 m CaCl2. This solution was mixed with 250 μl of 2× HEPES-buffered saline (50 mm HEPES, 280 mm NaCl, 1.5 mm Na2HPO4, to final pH 7.1). 80-μl aliquots of this precipitate were overlaid on wells of a 12-well dish containing 1 ml of freshly added Dulbecco's modified Eagle's medium plus 10% FBS. After transfection cells were incubated in medium containing 10% FBS for 6-8 h. The medium was changed to 0.2% FBS, and cells were incubated with 100 pm TGF-β for 20-24 h. Cell lysates were then subjected to luciferase assays (Promega) in a Berthold luminometer (Nashua, NH). A cytomegalovirus promoter Renilla luciferase plasmid (Promega) was used as a control to normalize the transfection efficiency and was assayed as described previously (16Calonge M.J. Massagué J. J. Biol. Chem. 1999; 274: 33637-33643Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Oligonucleotide Precipitation Assays—Cells were treated with TGF-β for 1 h under normal culture conditions and then lysed by sonication in buffer (10 mm Hepes, pH 7.9, 100 mm KCl, 10% glycerol, 1 mm dithiothreitol, 0.5% Nonidet P-40) with phosphatase and protease inhibitors. Cell debris was removed by 5-min centrifugation at 10,000 × g at 4 °C. Cell extracts were incubated for 16 h with 1 μg of biotinylated double-strand oligonucleotide corresponding to the wild type or mutant forms of the COL7A1 -495/-431 promoter region. DNA-bound proteins were collected with streptavidin-agarose beads for 1 h, washed with lysis buffer, separated on a SDS-polyacrylamide gel, and identified by Western blotting. RNA Assays—Exponentially growing cells were incubated with 100 pm TGF-β for the indicated time. Cells were harvested, and total RNA was extracted by using Qiagen (Chatsworth, CA) RNeasy minikit. 100 mg of total RNA was then used to obtain poly(A) RNA using a Clontech kit. The poly(A) RNA obtained was run on parallel denaturing gel and subjected to Northern analysis. Blots were probed with probes corresponding to human or mouse COL7A1, actin, COUP-TFI, or COUP-TFII. Yeast One-hybrid Screening—A NIH 3T3 cDNA library in the pGAD10 fusion vector (Clontech), which provides an N-terminal GAL4 fusion transcriptional activation domain, was transformed into a yeast strain bearing four consecutive copies of the -495/-431 region of the COL7A1 promoter upstream of both HIS3 and a LacZ reporter gene. cDNA clones that allowed growth in -His plates and gave a strong β-galactosidase activity in a colony lift assay were identified as positive. Role of Two Smad Binding Elements in the COL7A1 Promoter—To investigate the transcriptional activation of COL7A1 by TGF-β, we generated luciferase reporter constructs driven by the -496/+92 region of the human COL7A1 (9Vindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar) (Fig. 1A). Versions of this promoter were generated containing mutations that target the previously described SBE site (9Vindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar), which we refer to as 5′-SBE, and an adjacent AP1 site (10Verrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (159) Google Scholar) as well as other sites in this region that are conserved in the human and mouse genes (Fig. 1A). TGF-β addition strongly stimulated the expression of the wild type COL7A1 promoter in mouse NIH 3T3 fibroblasts, and this response was diminished by mutations targeting the 5′-SBE (Fig. 1B). These results are in full agreement with those reported in human dermal fibroblasts (9Vindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar). TGF-β also stimulated the expression of the wild type COL7A1 promoter in HaCaT human skin keratinocytes. Surprisingly, however, this response was not diminished by mutations targeting the 5′-SBE (Fig. 1B). Several lines of evidence suggested that this tolerance for a mutant 5′-SBE in HaCaT keratinocytes reflected a genuine difference between fibroblasts and keratinocytes and not an anomaly of the HaCaT cell line. HaCaT cells are well characterized in terms of their responsiveness to TGF-β (17Kang Y. Chen C.R. Massagué J. Mol. Cell. 2003; 11: 915-926Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Further, we verified that TGF-β stimulates the expression of the endogenous COL7A1 gene at the mRNA level (Fig. 1C). Moreover, the lack of an effect of mutations in this SBE has been reported recently in mouse keratinocytes as well (11Naso M. Uitto J. Klement J.F. J. Investig. Dermatol. 2003; 121: 1469-1478Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Searching for a possible basis for this difference between keratinocytes and fibroblasts, we noticed a perfect inverted SBE (3′-SBE) ∼50 base pairs downstream of the 5′-SBE (Fig. 1A). This site was not investigated in previous studies (9Vindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar, 10Verrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (159) Google Scholar, 11Naso M. Uitto J. Klement J.F. J. Investig. Dermatol. 2003; 121: 1469-1478Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). We tested the effect of mutating this site alone or in combination with the 5′-SBE. Mutation of the 3′-SBE affected the response of the COL7A1 promoter to TGF-β in a manner similar to mutation of the 5′-SBE site, as the 3′-SBE mutations diminished the TGF-β response in fibroblasts but not in keratinocytes (Fig. 1B). Remarkably, the simultaneous mutation of the 5′-SBE and 3′-SBE sites completely eliminated the TGF-β response not only in fibroblasts but also in keratinocytes (Fig. 1B). These results suggested that the 5′-SBE and 3′-SBE in the COL7A1 promoter are important for the TGF-β response in both cell types, acting redundantly in keratinocytes but not in fibroblasts. Cell Type-dependent Cooperation with AP1—AP1 sites and the Fos-Jun complexes that recognize these sites have been shown to play a role in certain TGF-β gene responses, including the COL7A1 response (10Verrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (159) Google Scholar). An AP1 site that is adjacent to the 5′-SBE (see Fig. 1A) has been implicated in the TGF-β response of the COL7A1 promoter in fibroblasts (10Verrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (159) Google Scholar). We found that the requirement of this site for the TGF-β response is cell type-dependent. Mutation of the AP1 site had no effect on the TGF-β response of the COL7A1 promoter in the keratinocytes, but it inhibited this response in fibroblasts (Fig. 1B). To investigate the interaction of endogenous Smad and AP1 proteins with these sites in the COL7A1 promoter, we conducted DNA-mediated precipitation assays using biotinylated double-stranded oligonucleotides corresponding to the human COL7A1 -495/-431 promoter region. Extracts from control and TGF-β-treated cells were incubated with wild type or mutant probes, and bound complexes were collected using streptavidin-agarose beads. Proteins of interest were investigated by Western immunoblotting of these precipitates using specific antibodies. Both HaCaT and NIH 3T3 cells showed a TGF-β-dependent formation of a Smad complex capable of binding to the wild type COL7A1 probe, as determined using anti-Smad4 antibody (Fig. 2). Binding of this complex to the probe was not affected by mutations that disrupt the AP1 site. Mutation of the 5′-SBE or the 3′-SBE diminished the TGF-β-dependent binding of Smad4, and a combination of these mutations essentially eliminated Smad4 binding to the probe (Fig. 2). To investigate the binding of AP1 complexes to these probes, we used antibodies that cross-react with various members of the Jun family. The wild type COL7A1 probe bound endogenous Jun proteins, and the level of binding was similar in control cells and TGF-β-treated cells (Fig. 2). Mutation of either or both SBE sites did not affect the binding of Jun proteins to the probe, whereas mutation of the AP1 site completely prevented Jun binding (Fig. 2). Collectively these results suggest that Smad and AP1 complexes may bind to the SBE and AP1 sites in the COL7A1 promoter independently of each other, and this binding is dependent on TGF-β stimulation in the case of the Smad site but independent in the case of the AP1 site. Furthermore, this protein binding behavior was the same regardless of whether the SBE sites were functionally redundant in keratinocytes or were non-redundant and cooperative with AP1 in fibroblasts. COUP-TF Binding to the TGF-β Regulatory Region of the COL7A1 Promoter—To determine whether additional factors may control the COL7A1 response to TGF-β, we tested the role of other segments in this region that are conserved in the human and mouse genes. We generated a reporter construct containing mutations in a conserved segment around position -450 (-450 conserved box; Fig. 1A). This construct had much higher basal activity than the wild type construct in transcriptional assays in keratinocytes (Fig. 3A). The activity of this construct was further increased by TGF-β, suggesting that a repressor factor may bind to this region under basal conditions and may determine the overall responsiveness of the COL7A1 promoter to TGF-β. To identify factors that may regulate COL7A1 from the -495/-431 promoter region, we screened a cDNA expression library for gene products that bind to this region. To this end, we generated a yeast strain expressing HIS3 and LacZ under the control of four copies of this promoter region and then used these cells to screen a mouse fibroblast cDNA library fused to the GAL4 transcriptional activation domain. In this approach, cDNAs encoding GAL4 fusion proteins that bind to the -495/-431 promoter region would confer HIS3 phenotype and activate LacZ expression. Only four cDNA clones were isolated that fulfilled these criteria. One of these cDNAs encoded the full-length COUP-TFI (also known as EAR3), and the other three encoded the full-length COUP-TFII (also known as ARP-1) (Fig. 3B). COUP-TFI and COUP-TFII are closely related orphan members of the nuclear/steroid receptor family (18Qiu Y. Krishnan V. Pereira F.A. Tsai S.Y. Tsai M.J. J. Steroid Biochem. Mol. Biol. 1996; 56: 81-85Crossref PubMed Scopus (81) Google Scholar, 19Cooney A.J. Lee C.T. Lin S.C. Tsai S.Y. Tsai M.J. Trends Endocrinol. Metab. 2001; 12: 247-251Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). They consist of an N-terminal DNA binding domain containing two zinc finger motifs and a C-terminal transcription regulatory domain containing a dimerization region (Fig. 3B). COUP-TFI and COUP-TFII bind to DNA as dimers that recognize two direct TGACC(C/T) repeats separated by a single base pair spacer (20Cooney A.J. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Cell. Biol. 1992; 12: 4153-4163Crossref PubMed Scopus (336) Google Scholar). COUP-TFI and -TFII are thought to act primarily as transcriptional repressors (20Cooney A.J. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Cell. Biol. 1992; 12: 4153-4163Crossref PubMed Scopus (336) Google Scholar, 21Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar, 22Achatz G. Holzl B. Speckmayer R. Hauser C. Sandhofer F. Paulweber B. Mol. Cell. Biol. 1997; 17: 4914-4932Crossref PubMed Scopus (53) Google Scholar, 23Robinson C.E. Wu X. Nawaz Z. Onate S.A. Gimble J.M. Endocrinology. 1999; 140: 1586-1593Crossref PubMed Scopus (21) Google Scholar, 24Kobayashi S. Shibata H. Kurihara I. Saito I. Saruta T. Endocr. Res. 2002; 28: 579Crossref PubMed Scopus (3) Google Scholar, 25Smirnov D.A. Hou S. Ricciardi R.P. Virology. 2000; 268: 319-328Crossref PubMed Scopus (40) Google Scholar). Examination of the COL7A1 -495/-431 region revealed that the sequence of the -450 conserved box is similar to the COUP-TF consensus binding sequence (Fig. 3C). As mutations of the -450 conserved box augmented the activity of the COL7A1 promoter, we hypothesized that this effect might be due to a loss of endogenous COUP-TF binding to this region. To determine whether this region can bind COUP-TF proteins, we carried out DNA precipitation assays using wild type and mutant biotinylated COL7A1 -495/-431 oligonucleotide probes (Fig. 3C). A FLAG-tagged COUP-TFII construct expressed in COS cells bound to the wild type probe but not to two different probes that contain mutations in the -450 conserved box (Fig. 3D). COUP-TFII binding to the wild type promoter was not affected by cell treatment with TGF-β and was not disrupted by SBE or AP1 site mutations that block the binding of Smad or Jun, respectively (Fig. 3E). We looked for, but could not find, evidence of Smad3 or Smad4 binding to COUP-TFI or COUPTFII. In extracts from cells overexpressing Smads and COUPTFs, these proteins neither enhanced nor interfered with the binding of each other to the COL7A1 -495/-431 probe (data not shown). These results suggest that the -450 conserved box is a COUP-TF site that binds COUP-TF proteins independently of Smad and AP1. Down-regulation of COUP-TF Expression by TGF-β—Interestingly, GeneChip transcriptomic profiling data in HaCaT cells revealed that TGF-β treatment decreases the level of COUP-TFII transcripts (17Kang Y. Chen C.R. Massagué J. Mol. Cell. 2003; 11: 915-926Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). We verified by Northern analysis that TGF-β addition causes a rapid and profound decrease in the level of COUP-TFII mRNA in HaCaT and NIH 3T3 cells (Fig. 4). COUP-TFI expression, which was present in NIH 3T3 cells but barely detectable in HaCaT cells, was also inhibited by TGF-β (Fig. 4). The down-regulation of COUP-TFII by TGF-β in HaCaT cells was rapid (t½ ≤ 2 h; Fig. 4) and preceded the up-regulation of COL7A1 expression (t½ = 4 h; refer to Fig. 1C). Thus, TGF-β action down-regulates the expression of a putative COL7A1 repressor. Repression of COL7A1 Promoter by COUP-TF—We investigated whether COUP-TFs can act as repressors of the COL7A1 promoter in transfected HaCaT cells. Indeed, both COUP-TFI and COUP-TFII markedly inhibited the basal activity of the wild type COL7A1 promoter as well as its activation by TGF-β (Fig. 5A). The promoter construct containing mutations in the -450 conserved box (the COUP-TF binding element) was not only hyperactive under basal conditions but also completely resistant to inhibition by exogenous COUP-TFI or COUP-TFII (Fig. 5A). As no anti-COUP-TF antibodies could be obtained for these studies, we resorted to alternative approaches to determine whether the COL7A1 promoter is sensitive to endogenous COUP-TFs. First, we mutated the COUP-TF binding region to bring this sequence closer to the optimal COUP-TF binding sequence (COUP+ mutant in Fig. 3C) (20Cooney A.J. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Cell. Biol. 1992; 12: 4153-4163Crossref PubMed Scopus (336) Google Scholar). When tested in oligonucleotide precipitation assays, a COL7A1 probe containing this mutant sequence bound COUP-TFII with higher affinity than did the wild type probe (Fig. 5B). A COL7A1 promoter construct bearing this mutation showed a significantly decreased activity in HaCaT cells, which was suggestive of a response to endogenous COUP-TFs (Fig. 5C). Thus, the COUP+ mutation hypersensitizes the COL7A1 promoter to COUP-TF repression. To test further this hypothesis, we generated a FLAG-tagged mutant COUP-TF construct designed to inhibit the dimerization and thus the function of endogenous COUP-TFs. This construct, COUP-Dim, encodes the dimerization domain of COUP-TFII (Fig. 6A). The COUP-Dim product was able to completely inhibit the binding of COUP-TFII to the COL7A1 promoter (Fig. 6A), demonstrating that it can act as a dominant-negative construct. When tested in COL7A1 promoter activity assays, COUP-Dim increased the activity of this promoter while still allowing a further activation by TGF-β (Fig. 6C). Taken together, these results suggest that the COL7A1 promoter is repressed by COUP-TF or a closely related factor in HaCaT cells. The ability of TGF-β to sharply decrease the expression of endogenous COUP-TFs in these cells while simultaneously inducing the formation of a COL7A1-activating Smad complex suggests that COL7A1 induction by TGF-β involves a combination of promoter activation and deinhibition inputs. The results of this work suggest that the activation of COL7A1 expression by TGF-β is a complex process involving two inputs (schematically summarized in Fig. 7). One input is provided by TGF-β-dependent Smad-transactivating factors and the other by the relief of COUP-TF-mediated transcriptional repression of the COL7A1 promoter. This complex regulatory process has several features that distinguish it from previously characterized TGF-β-regulated promoters. We provide evidence that regulation of the COL7A1 promoter by TGF-β-activated Smad factors occurs via two SBEs in the TGF-β-responsive region of this promoter. The SBE, or CAGAC sequence, is the best characterized of the various sequences implicated in DNA binding by signal-activated Smad factors (14Shi Y. Massagué J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4795) Google Scholar, 26Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar, 27Shi Y. Wang Y.-F. Jayaraman L. Yang H. Massagué J. Pavletich N. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar). In many TGF-β target promoters characterized to date, the responsive region contains only one SBE. The COL7A1 promoter was previously thought to be in this class, with only one identified functional SBE (referred to here as the 5′-SBE) (9Vindevogh
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