Regulation of Platelet-derived Growth Factor-A Chain by Krüppel-like Factor 5
The transcription factor Krüppel-like factor 5 (KLF5) and its genetically downstream target gene platelet-derived growth factor-A (PDGF-A) chain are key factors in regulation of cardiovascular remodeling in response to stress. We show that KLF5 mediates a novel distinct delayed persistent induction of PDGF-A chain in response to the model agonist, phorbol ester, through a cis-element previously shown to mediate phorbol ester induction on to PDGF-A chain through the early growth response factor (Egr-1). Interestingly, the nuclear factor-κB (NF-κB) p50 subunit further cooperatively activates PDGF-A chain through protein-protein interaction with KLF5 but not Egr-1. RNA interference analysis confirmed that KLF5 and p50 are important for induction of PDGF-A chain. Collectively, we identify a novel regulatory pathway in which PDGF-A chain gene expression, under the control of KLF5, is cooperatively activated by the NF-κB p50 subunit and a pathophysiological stimulus. The transcription factor Krüppel-like factor 5 (KLF5) and its genetically downstream target gene platelet-derived growth factor-A (PDGF-A) chain are key factors in regulation of cardiovascular remodeling in response to stress. We show that KLF5 mediates a novel distinct delayed persistent induction of PDGF-A chain in response to the model agonist, phorbol ester, through a cis-element previously shown to mediate phorbol ester induction on to PDGF-A chain through the early growth response factor (Egr-1). Interestingly, the nuclear factor-κB (NF-κB) p50 subunit further cooperatively activates PDGF-A chain through protein-protein interaction with KLF5 but not Egr-1. RNA interference analysis confirmed that KLF5 and p50 are important for induction of PDGF-A chain. Collectively, we identify a novel regulatory pathway in which PDGF-A chain gene expression, under the control of KLF5, is cooperatively activated by the NF-κB p50 subunit and a pathophysiological stimulus. Adaptation to external stimuli is the hallmark feature of cardiovascular pathogenic processes ranging from atherosclerosis to heart failure (1.Carmeliet P. Collen D. J. Pathol. 2000; 190: 387-405Crossref PubMed Scopus (115) Google Scholar, 2.Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (6940) Google Scholar). Insult in the form of ischemia, infection, or physical stress brings about a remodeling process of cardiovascular cells and tissues to repair and compensate through activation of vascular endothelial and smooth muscle cells as well as cardiac interstitial cells. Recent investigations by our group using genetically engineered mice have shown that the transcription factor Krüppel-like factor 5 (KLF5) 1The abbreviations used are: KLF5, Krüppel-like factor 5; PDGF-A, platelet-derived growth factor-A; Egr-1, early growth response factor-1; RT, reverse transcriptase; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; SEAP, secreted alkaline phosphatase. 1The abbreviations used are: KLF5, Krüppel-like factor 5; PDGF-A, platelet-derived growth factor-A; Egr-1, early growth response factor-1; RT, reverse transcriptase; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; SEAP, secreted alkaline phosphatase. is an important mediator of cardiovascular remodeling as manifested by a hypertrophic cardiac response to angiotensin II stimulus and a vascular inflammatory remodeling response to mechanical stress (3.Shindo T. Manabe I. Fukushima Y. Tobe K. Aizawa K. Miyamoto S. Kawai-Kowase K. Moriyama N. Imai Y. Kawakami H. Nishimatsu H. Ishikawa T. Suzuki T. Morita H. Maemura K. Sata M. Hirata Y. Komukai M. Kagechika H. Kadowaki T. Kurabayashi M. Nagai R. Nat. Med. 2002; 8: 856-863Crossref PubMed Scopus (327) Google Scholar). Two important findings of these past studies that are relevant to the present study were that (1.Carmeliet P. Collen D. J. Pathol. 2000; 190: 387-405Crossref PubMed Scopus (115) Google Scholar) the mitogenic growth factor platelet-derived growth factor-A (PDGF-A) chain gene was shown to be an endogenous target gene of KLF5 and (2.Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (6940) Google Scholar) there was an attenuated inflammatory wound-healing response to vascular stimulus (cuff-injury). PDGF, a homo- or hetero-dimer of A and B chains, is a well known mediator of cardiovascular remodeling and wound healing processes as an inducible factor controlling cell proliferation and migration (4.Forsberg K. Valyi-Nagy I. Heldin C.H. Herlyn M. Westermark B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 393-397Crossref PubMed Scopus (204) Google Scholar, 5.Lindahl P. Johansson B.R. Leveen P. Betsholtz C. Science. 1997; 277: 242-245Crossref PubMed Scopus (1740) Google Scholar, 6.Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9971) Google Scholar, 7.Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19200) Google Scholar). PDGF-A chain is a target gene of KLF5 based on the biochemical analysis of the knockout mouse and on the partial resemblance of the phenotypes (e.g. intestine) of the PDGF-A chain and KLF5 knockout mice (8.Karlsson L. Lindahl P. Heath J.K. Betsholtz C. Development. 2000; 127: 3457-3466PubMed Google Scholar). Despite these observations, we know little about how the PDGF-A chain is regulated by KLF5, which is important to understand a variety of pathophysiological mechanisms. We undertook the present study to dissect the mechanisms whereby KLF5 acts on PDGF-A chain, and particularly to understand their regulation in response to pathophysiological stimulus. Plasmid Construct Preparation—Luciferase reporter deletion constructs were made by cleaving the PDGF-A chain luciferase promoter construct (PDGF-900) (3.Shindo T. Manabe I. Fukushima Y. Tobe K. Aizawa K. Miyamoto S. Kawai-Kowase K. Moriyama N. Imai Y. Kawakami H. Nishimatsu H. Ishikawa T. Suzuki T. Morita H. Maemura K. Sata M. Hirata Y. Komukai M. Kagechika H. Kadowaki T. Kurabayashi M. Nagai R. Nat. Med. 2002; 8: 856-863Crossref PubMed Scopus (327) Google Scholar) into the following restriction fragments; SacI-HindIII (–630 to +10), XhoI-HindIII (–260 to +10), SacI-HindIII (–71 to +10), and SacI-HindIII (–55 to +10) fragments. The resultant plasmids were named PDGF-630, PDGF-260, PDGF-71, and PDGF-55, respectively. The KLF5 expression vector pCAG-KLF5 has been described (3.Shindo T. Manabe I. Fukushima Y. Tobe K. Aizawa K. Miyamoto S. Kawai-Kowase K. Moriyama N. Imai Y. Kawakami H. Nishimatsu H. Ishikawa T. Suzuki T. Morita H. Maemura K. Sata M. Hirata Y. Komukai M. Kagechika H. Kadowaki T. Kurabayashi M. Nagai R. Nat. Med. 2002; 8: 856-863Crossref PubMed Scopus (327) Google Scholar), and p50 and p65 expression vectors were a kind gift of Dr. Kunsch (9.Kunsch C. Rosen C.A. Mol. Cell Biol. 1993; 13: 6137-6146Crossref PubMed Google Scholar). Cell Culture and Transfection Assays—HeLa cells (1 × 105 cells) were transfected with 100 ng reporter and 1 μg expression plasmids by Tfx-20 reagent (Promega). Cells were incubated for 48 h and then subjected to luciferase assay (Promega) by luminometry (Lumat LB9507, Berthold). Luciferase activity was normalized to protein concentration of cell lysates. Shown are the results of a representative assay as done in duplicate and reproduced in at least two other occasions. Error bars denote standard deviation. Protein Expression and Purification—The recombinant His6-tagged KLF5 DNA-binding domain protein (KLF5DBD) was generated by PCR on pBK-IKLF (4.Forsberg K. Valyi-Nagy I. Heldin C.H. Herlyn M. Westermark B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 393-397Crossref PubMed Scopus (204) Google Scholar) (a kind gift of Dr. Teng) using primers (5′-CCGGATCCGGAGAAACGACGCATCCACTAC-3′ and 5′-CGGGATCCTCAGTTCTGGTGCCTCTTC-3′) containing BamHI restriction endonuclease sites, inserted into the vector 6HIS-pET11d, and then expressed and purified essentially according to described methods (10.Suzuki T. Kimura A. Nagai R. Horikoshi M. Genes Cells. 2000; 5: 29-41Crossref PubMed Scopus (142) Google Scholar). Gel-shift DNA-binding Assay—Gel-shift DNA-binding assays were done essentially as described previously (10.Suzuki T. Kimura A. Nagai R. Horikoshi M. Genes Cells. 2000; 5: 29-41Crossref PubMed Scopus (142) Google Scholar). A DNA oligomer containing the –71 bp to –55 bp sequence (5′-GGGGGGGGCGGGGGCGGGGGCGGGGGAGGG-3′ and 5′-CCCTCCCCCGCCCCCGCCCCCGCCCCCCCC-3′) was used for analysis of KLF5 DNA binding. For mutant analysis, the underlined nucleotides GG were substituted by TT. The NF-κB probe was purchased from Promega. Super-shift experiments were done using anti-His probe (Santa Cruz) and anti-p50 (Santa Cruz) antibodies. See legends to Figs. 1D and 3D for details.Fig. 3KLF5 and NF-κB p50 subunit cooperatively activate the PDGF-A chain promoter.A, co-transfection reporter analysis showing cooperative activation of PDGF-A chain promoter by KLF5 and NF-κB. Note that NF-κB p65/c-rel subunit does not show cooperative activation (lane 6) as seen for p50 with KLF5 (lane 7). B, the NF-κB inhibitor, parthenolide, inhibits cooperative activation by KLF5 and p50 (lane 3 versus 6). C, mapping the KLF5-p50-activated region of the PDGF-A chain promoter. Note that there is a significant decrease in cooperative activation between PDGF-71 and PDGF-55 (lane 12 versus 15). D, gel-shift assay showing KLF5 and p50 form a DNA-protein complex on PDGF-A chain promoter between –71 bp and –55 bp. Note that under conditions that KLF5 binds this region (lane 2) and p50 does not (lane 3), reaction with both proteins results in a slower mobility DNA-protein complex (lane 4), which is supershifted by anti-p50 antibody (lane 5). E, co-immunoprecipitation assay showing binding of KLF5 and p50. Under conditions in which cell lysate was immunoprecipitated with control IgG (lane 2) and p50 (lane 3) antibodies, immunoblot with KLF5 showed interaction with p50. Similar analysis was done with Egr-1 showing lack of association. F, specificity of cooperative activation between KLF5 and p50. Co-transfection analysis to test the specificity of KLF5, Sp1, and Egr-1 in combination with p50 on the PDGF-A chain promoter as well as various other promoters as shown (see supplemental figure for raw data). Combinations showing cooperative activation are shown in bold.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Reverse Transcriptase-PCR (RT-PCR) Analysis—Rat cardiac muscle fibroblasts were isolated and cultured as described previously (3.Shindo T. Manabe I. Fukushima Y. Tobe K. Aizawa K. Miyamoto S. Kawai-Kowase K. Moriyama N. Imai Y. Kawakami H. Nishimatsu H. Ishikawa T. Suzuki T. Morita H. Maemura K. Sata M. Hirata Y. Komukai M. Kagechika H. Kadowaki T. Kurabayashi M. Nagai R. Nat. Med. 2002; 8: 856-863Crossref PubMed Scopus (327) Google Scholar) using 100 ng/ml of phorbol 12-myristate 13-acetate (PMA). Quantitative multiplex PCR was then done as described previously (11.Manabe I. Owens G.K. Circ. Res. 2001; 88: 1127-1134Crossref PubMed Scopus (142) Google Scholar). Previously unpublished primers are KLF2(LKLF) forward, 5′-TGCCGTCCTTTGCCACTTTCGCCAG-3′, reverse, 5′-CGGGGCGCAGCAGCTCTGTTCCCAG-3′; KLF4(GKLF) forward, 5′-CTGGCGAGTCTGACATGGCTGTCAG-3′, reverse, 5′-CGCCACTCTCCAGGTCTGTGGCCAC-3′; KLF6-(GBF) forward, 5′-CACGACCAAATTTACCTCTG-3′, reverse, 5′-CATGAGCATCTGTAAGGC-3′; Egr-1 forward, 5′-CCAAGTTCTTCACCTCTATC-3′, reverse, 5′-ATGCAAATTATCTTCACATC-3′. Construction of Recombinant Adenovirus KLF5 Expression Vector— Full-length KLF5 cDNA was inserted into the cosmid vector pAxCAwt (Takara) at the SwaI site. 293 cells were co-transfected with the cosmid vector and restriction enzyme-treated DNA-terminal protein complex by the calcium phosphate method. Homologous recombination resulted in recombinant adenovirus (pAxCA-KLF5). Viral titer was determined using the plaque method. Co-immunoprecipitation Assay—Full-length KLF5 was PCR-mutagenized and subcloned into p3xFLAG-myc-CMV (Sigma). Transfected HeLa cells were incubated for 48 h then lysed with lysis buffer (50 mm Tris HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100). One μg of anti p50 (Santa Cruz) or control rabbit IgG antibody (Santa Cruz) was bound to 10 μl of protein G-Sepharose (Amersham Biosciences) by rotating for 6 h in lysis buffer at 4 °C then washed. Immunoprecipitate was subjected to SDS-PAGE and immunoblotted using anti-KLF5 (KM1785) or anti-Egr-1 antibody (Santa Cruz). Small Interfering RNA (siRNA)—siRNA were constructed according to the manufacturer's protocol for the Silencer™ siRNA Construction kit (Ambion). 1.5 μg of double-stranded 21-mer siRNA was transfected into 2 × 105 10T1/2 cells using Polyfect (Qiagen) according to the manufacturer's protocol. After 72 h of incubation, cells were harvested and RNA was extracted using RNeasy (Qiagen) and then subjected to RT-PCR analysis as described (KLF2, KLF4, KLF6, and Egr-1) (3.Shindo T. Manabe I. Fukushima Y. Tobe K. Aizawa K. Miyamoto S. Kawai-Kowase K. Moriyama N. Imai Y. Kawakami H. Nishimatsu H. Ishikawa T. Suzuki T. Morita H. Maemura K. Sata M. Hirata Y. Komukai M. Kagechika H. Kadowaki T. Kurabayashi M. Nagai R. Nat. Med. 2002; 8: 856-863Crossref PubMed Scopus (327) Google Scholar, 11.Manabe I. Owens G.K. Circ. Res. 2001; 88: 1127-1134Crossref PubMed Scopus (142) Google Scholar). The siRNA oligonucleotide probes (numbers are arbitrary) were as follows: KLF5#1 sense 5′-AAGCUCACCUGAGGACUCAUU-3′; KLF-5#1 antisense 5′-UGAGUCCUCAGGUGAGCUUUU-3′; KLF5#2 sense 5′-CCCACACCCCUCUGCUGUUUU-3′; KLF5#2 antisense 5′-AACAGCAGAGGGGUGUGGGUU-3′; KLF5#3 sense 5′-UUCGCCAACUCUCCCACCUUU-3′; KLF5#3 antisense 5′-AGGUGGGAGAGUUGGCGAAUU-3′; p50#1 sense 5′-GCACGGAUGACAGAGGCGUUU-3′; p50#1 antisense 5′-ACGCCUCUGUCAUCCGUGCUU-3′; p50#2 sense 5′-GGAGAUGGACCUGAGCGUGUU-3′; p50#2 antisense 5′-CACGCUCAGGUCCAUCUCCUU-3′; p50#3 sense 5′-GAAAAUGGCGGAGUUUGGGUU-3′; p50#3 antisense 5′-CCCAAACUCCGCCAUUUUCUU-3′; SEAP (secreted alkaline phosphatase) sense 5′-AGGGCAACUUCCAGACCAUUU-3′; SEAP antisense 5′-AUGGUCUGGAAGUUGCCCUUU-3′. Transcriptional Activation of the PDGF-A Chain Promoter by KLF5—We first investigated whether KLF5 transcriptionally regulates PDGF-A chain. Using reporter constructs harboring the PDGF-A chain promoter upstream of a luciferase reporter, cell co-transfection studies showed that KLF5 activates the PDGF-A chain promoter (containing 900 bp upstream of the transcription initiation site) (Fig. 1, A and B). To next determine the cis-element responsible for mediating the KLF5 response, serial deletion PDGF-A chain promoter-reporter constructs were made and subjected to co-transfection analysis. Results showed that a region between –71 bp and –55 bp of the transcription initiation site mediated the KLF5 response as shown by a dramatic decrease in promoter activation for constructs not containing this region. This region between –71 bp and –55 bp is well characterized as an element that mediates various stimuli including phorbol ester on to the PDGF-A chain promoter and is known to bind the Egr-1 and Sp1 transcription factors (Fig. 1C) (12.Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13.Khachigian L.M. Collins T. Circ. Res. 1997; 81: 457-461Crossref PubMed Scopus (146) Google Scholar). As KLF5 belongs to the Sp/KLF-family of zinc finger transcription factors that have a common property to bind similar GC-rich binding sites (14.Shi H. Zhang Z. Wang X. Liu S. Teng C.T. Nucleic Acids Res. 1999; 27: 4807-4815Crossref PubMed Scopus (62) Google Scholar, 15.Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (142) Google Scholar, 16.Philipsen S. Suske G. Nucleic Acids Res. 1999; 27: 2991-3000Crossref PubMed Scopus (533) Google Scholar, 17.Bieker J.J. J. Biol. Chem. 2001; 276: 34355-34358Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar), we thought that KLF5 would also bind the Sp1-binding sites as well as the similar Egr-1-binding sites. Using gel-shift analysis, we show that KLF5 binds this region between –71 bp and –55 bp of the PDGF-A chain promoter in a sequence-specific manner (Fig. 1D) thus showing that KLF5 has the ability to directly bind this cis-element and mediate promoter activation. KLF5 Mediates Activation of PDGF-A Chain by Phorbol Ester—Phorbol ester, a model agonist to investigate inducible pathophysiological stimulation, is known to stimulate PDGF-A chain promoter activation by the Egr-1 transcription factor, which is transiently and rapidly expressed at one hour after stimulation through this cis-element (12.Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13.Khachigian L.M. Collins T. Circ. Res. 1997; 81: 457-461Crossref PubMed Scopus (146) Google Scholar). KLF5 is also transcriptionally activated by phorbol ester stimulation. We therefore investigated KLF5 mediate phorbol ester stimulation on to the PDGF-A chain promoter K. Kurabayashi M. Y. Y. Nagai R. Circ. Res. 1999; PubMed Scopus Google Scholar, R. X. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Under conditions in which the region between –71 bp and –55 bp of the transcription initiation site mediated phorbol ester activation of PDGF-A chain with studies (Fig. co-transfection analysis showed that KLF5 activates PDGF-A chain promoter in with phorbol ester (Fig. Expression analysis of KLF5 after phorbol ester stimulation showed that KLF5 expression showed a that was at after stimulation, in to Egr-1 that was transiently in the first hour after stimulation (Fig. PDGF-A chain expression the expression of To test the between KLF5 and PDGF-A chain expression, we whether expression of KLF5 would endogenous PDGF-A gene of of KLF5, expression of KLF5 was by phorbol ester with expression of KLF5 PDGF-A chain gene expression as shown by RT-PCR analysis (Fig. Collectively, KLF5 mediates phorbol ester stimulation of PDGF-A chain in a manner that is as with the induction mediated by Egr-1. This a pathway for the regulation of PDGF-A chain by KLF5 in response to a pathophysiological stimulus. Activation of PDGF-A Chain by KLF5 and mediates a persistent induction of PDGF-A chain in response to phorbol ester stimulation an important in mediating stress. the other that are activated by phorbol ester, the NF-κB pathway is well known for mediating external particularly in inflammatory T. J. 2001; PubMed Scopus Google Scholar). a pathway NF-κB and PDGF-A chain has not been As the KLF5 knockout mice showed a in inflammatory vascular response, we that there be cooperative interaction between these downstream factors to transcription in the We first whether NF-κB with KLF5 in PDGF-A chain Using co-transfection reporter analysis, we show that NF-κB p50 and p65 or not activate the PDGF-A chain promoter-reporter cooperative activation of the PDGF-A chain promoter is seen the p50 but not the p65/c-rel subunit of is with KLF5 (Fig. This is not to the of p50 on KLF5 expression as shown by of on immunoblot analysis. of an NF-κB inhibitor, J. 1999; Google Scholar), this cooperative activation with p50 (Fig. To determine the cis-element which mediates this response, PDGF-A chain promoter deletion constructs were which showed that the cooperative activation was mediated by the –71 bp to –55 bp region (Fig. This region mediates KLF5 activation through DNA but as not a we that p50 through protein-protein to cooperatively activate the PDGF-A chain promoter. Using gel-shift analysis, under conditions in which recombinant KLF5 but not p50 binds this region of the PDGF-A chain a DNA-protein complex of slower mobility was seen both KLF5 and p50 were that p50 and KLF5 form a complex on Super-shift experiments using antibody p50 confirmed that was this complex (Fig. A recombinant form of the DNA-binding domain of KLF5 was for this Co-immunoprecipitation analysis further showed that KLF5 bound p50 (Fig. Egr-1 not bind to p50 under similar conditions to the specificity of the reaction (Fig. To further the specificity of this reporter assays using various promoters and factors were Under conditions in which KLF5 and p50 cooperatively activated the PDGF-A chain Sp1 Egr-1 showed cooperative with p50. promoters as vascular cell inducible and not show cooperative but factor and also showed this which be of the of this (Fig. and supplemental Collectively, we show that KLF5 and the p50 subunit of NF-κB show cooperative activation of PDGF-A chain by a complex on the element of the PDGF-A chain promoter. To our this is the first of regulation of PDGF-A chain. one stimulation of KLF5 activation by phorbol ester and mediated by the p50 subunit of NF-κB on KLF5, therefore on KLF5 to activate the PDGF-A chain promoter. of KLF5 and p50 for PDGF-A Chain Expression in that KLF5 and p50 mediate PDGF-A chain expression in we used an RNA interference to the of these two factors in mediating this We first conditions in which KLF5 be by siRNA (Fig. of the constructs were by of on RNA and the The specificity of the siRNA constructs KLF5 were confirmed by lack of on factors KLF4, KLF6, and Egr-1 (Fig. We next conditions in which p50 be by siRNA (Fig. and then whether KLF5 and p50 siRNA constructs PDGF-A chain expression by RT-PCR analysis. KLF5 and p50 siRNA constructs PDGF-A chain expression and constructs were and further used to show that PDGF-A promoter activation by reporter and that these factors at the of transcription (Fig. findings that KLF5 and p50 siRNA constructs PDGF-A chain expression and the of these two factors in expression of PDGF-A chain. is to further experiments using but at of this in with to of of PDGF-A Chain by present study that KLF5 transcriptionally regulates the PDGF-A chain with cooperative activation by phorbol Interestingly, phorbol ester expression of KLF5 in a delayed persistent manner that a response as mediated by Egr-1 (Fig. expression of KLF5 in combination with phorbol ester was to PDGF-A chain expression in the which that this combination of factors is relevant in the pathophysiological We that this regulation of PDGF-A chain expression as manifested by stimulation by Egr-1 by that of KLF5 for and of of regulation on PDGF-A chain. A bp region between –71 bp and –55 bp of the PDGF-A chain promoter these this region GC-rich binding sites that have been shown to bind Egr-1 and KLF5, both of these zinc finger transcription factors bind GC-rich binding sites. we the that both Egr-1 and KLF5 in on the PDGF-A chain promoter (e.g. their distinct expression in response to phorbol ester stimulation that We therefore that Egr-1 acts as the factor to mediate the early KLF5 mediates at for activation of the PDGF-A chain promoter. are mediated by Egr-1 and is important to understand the regulation and that on Egr-1 and KLF5 to understand their novel in the present study that the NF-κB p50 subunit mediates cooperative activation on PDGF-A chain by KLF5 but not by Egr-1 be one of the to this important the well known of NF-κB to mediate inflammatory to is an nuclear factor to the inducible PDGF-A chain. that p50 cooperatively activates the PDGF-A chain promoter through protein-protein interaction with KLF5 was therefore a This not a pathway activation of PDGF-A chain through a delayed in expression of KLF5, but also a cooperative as manifested by of the NF-κB on to KLF5 to further this cooperative response is not seen with Egr-1, which that this is a response that is for activation of PDGF-A chain in response to stimuli in a complex pathophysiological we have a novel pathway regulation of PDGF-A chain by KLF5 (Fig. of on to KLF5 (e.g. phorbol ester and the as well as of the regulation of PDGF-A chain. This pathway through KLF5 is a target for at PDGF-A chain and its in the cardiovascular cooperative activation between NF-κB and KLF5 also be a target for of inducible pathophysiological Download with