Significance of the transcription factor KLF5 in ... - Wiley Online Library

Journal of Thrombosis and Haemostasis, 3: 1569–1576

REVIEW ARTICLE

Significance of the transcription factor KLF5 in cardiovascular remodeling R . N A G A I , T . S U Z U K I , K . A I Z A W A , T . S H I N D O and I . M A N A B E Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

To cite this article: Nagai R, Suzuki T, Aizawa K, Shindo T, Manabe I. Significance of the transcription factor KLF5 in cardiovascular remodeling. J Thromb Haemost 2005; 3: 1569–76.

Introduction Summary. Structural remodeling of the heart and blood vessels is an important pathologic process in the development of many cardiovascular diseases. However, transcriptional regulation of altered gene expression during cardiovascular remodeling is not well understood. We previously isolated KLF5/basic transcription element-binding (BTEB)2, a Kru¨ppel-like factor, as a transcription factor that binds the promoter of the embryonic smooth muscle myosin heavy chain gene (SMemb). KLF5 activates many genes inducible during cardiovascular remodeling, such as platelet-derived growth factor (PDGF)-A/B, Egr-1, plasminogen activator inhibitor-1 (PAI-1), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor (VEGF) receptors. KLF5 is abundantly expressed in embryonic smooth muscles and is down-regulated with vascular development, but reinduced in proliferative neointimal smooth muscles in response to vascular injury. In KLF5 gene-targeted mice, homozygotes die at an early embryonic stage whereas heterozygotes are apparently normal. However, in response to external stress, arteries of heterozygotes exhibit diminished levels of smooth muscle and adventitial cell activation. Furthermore, angiotensin II-induced cardiac hypertrophy and fibrosis are attenuated in heterozygotes. KLF5 activities are regulated by many transcriptional regulators and nuclear receptors, such as retinoic acid receptor-a (RARa), NF-jB, PPARc, p300, and SET. Interestingly, RARa agonist suppresses KLF5 and cardiovascular remodeling, whereas RARa antagonist activates KLF5 and induces angiogenesis. These results indicate that KLF5 is an essential transcription factor in cardiovascular remodeling and a potential therapeutic target for cardiovascular disease. Keywords: cardiac hypertrophy, cardiovascular remodeling, KLF5, platelet-derived growth factor-A, transcription factor.

Correspondence: Ryozo Nagai, Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: 81-3-5800-6526; fax: 81-3-3815-2087; e-mail: nagai-tky@ umin.ac.jp. 2005 International Society on Thrombosis and Haemostasis

Structural remodeling of the heart and blood vessels underlies heart failure, atherosclerosis, and postangioplasty restenosis. Histopathologically, cardiovascular remodeling involves hyperplasia, interstitial fibrosis, accumulation of extracellular matrices, and/or angiogenesis. Physical stress, ischemia or infection induces a remodeling process of the cardiovascular system through activation of cardiac myocytes, fibroblasts, endothelial cells, and smooth muscle cells [1]. Locally expressed growth factors including platelet-derived growth factor (PDGF), bFGF and vascular endothelial growth factor (VEGF) are known to play key roles in these processes. However, little is known about the underlying transcriptional regulatory mechanisms. We previously identified KLF5 as a transcription factor that regulates the SMemb/NMHC-B gene, which is a molecular marker of phenotypically modulated smooth muscle cells and myofibroblasts [2–5]. KLF5 belongs to the Kru¨ppel-like transcription factor family. Members of this family contain three Kru¨ppel-like C2H2-type zinc (Zn)-finger domains, recognize GC boxes, and have diverse functions in cell proliferation, differentiation, and embryonic development (Fig. 1) [6–8]. KLF5 was originally identified as basic transcription elementbinding (BTEB)2, based on similarity to BTEB protein that binds GC-rich sequences [9]. KLF5 was also isolated from the intestine and named intestinal-enriched Kru¨ppel-like factor (IKLF) [10]. In the cardiovascular system, KLF5 is abundantly expressed in embryonic vascular smooth muscles, but is downregulated in adult vessels. Its expression, however, is strongly reinduced in activated smooth muscle cells and myofibroblasts in athero- and arterio-sclerosis [5]. We present here our recent studies on the role of KLF5 in cardiovascular remodeling and regulation of transcriptional activities of KLF5 [11]. Reduced vascular responses to external stresses in KLF5+/) mice Exons 2–3 of the murine KLF5 gene were deleted in 129/Svderived embryonic stem cells as described [12]. Male chimeras were crossbred with C57BL/6 females. Analysis of the embryos

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were thin-walled and dilated (Fig. 2D), which was in striking contrast to the wild-type animals, which showed thickened intima composed of proliferative smooth muscle cells (Fig. 2C). We also found that KLF5+/) mice showed impaired angiogenic activity in a hind limb ischemia model, in which the femoral arteries were ablated, and that angiogenic responses to implanted tumors were also markedly attenuated.

KLF6/ ZF9 KLF7/ UKLF ZNF741 KLF12/AP-2rep KLF3/BKLF

KLF5/BTEB2/IKLF KLF4/ GKLF KLF2/ LKLF KLF1/EKLF KLF11/ TIEG2 KLF10/ TIEG1 KLF9/ BTEB1 KLF5 Sp2 NH2 Sp4 Sp3 Sp1

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Fig. 1. Kru¨ppel-like zinc-finger transcription factors. Members of this family recognize the GT/CACC box, contain Zn-finger motifs, and are involved in tissue-specific gene cell differentiation including erythropoiesis, and angiogenesis.

from timed KLF5+/) intercrosses showed that KLF5)/) homozygotes died before embryonic day 8.5. KLF5+/) mice, on the other hand, were apparently normal and survived until adulthood, although expression of KLF5 was reduced to about half that in wild-type mice. In order to examine the structural changes in the arterial walls in response to external stress, a polyethylene tube cuff was placed around femoral arteries and left for 4 weeks. In KLF5+/+ mice a thick layer of granulation tissue developed around the cuff (Fig. 2A). The KLF5+/) mice, by contrast, developed markedly less granulation tissue and showed less angiogenesis (Fig. 2B). Moreover, within the cuffs, the arteries of the heterozygotes A

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Reduced cardiac hypertrophy and fibrosis in KLF5+/) mice We next examined the role of KLF5 in cardiac response to angiotensin II, a potent growth factor known to play a central role in both cardiac hypertrophy and vascular remodeling [13–15]. The hearts of wild-type mice were significantly heavier than those of heterozygous mice following continuous 14-day infusion of angiotensin II. When induced by abdominal aortic banding, KLF5+/) mice also developed cardiac hypertrophy to a significantly less extent than wild-type mice. These results clearly indicate that KLF5 plays a critical role in cardiovascular remodeling, involving mesenchymal cell activation, development of interstitial fibrosis, and angiogenesis. Angiotensin II-induced expression of the KLF5 and PDGF-A genes The PDGF, a homo- or hetero-dimer of A- and B-chains, is a mediator of cardiovascular remodeling and wound healing processes as an inducible factor controlling cell proliferation B

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Fig. 2. Vascular injury model entailing placement of a cuff around the femoral artery in KLF5+/+ (wild type) and KLF5+/) mice. In wild-type mice, a thick layer of highly vascular granulation tissue surrounds the polyethylene cuff, making it barely visible, whereas KLF5+/) mice exhibits markedly less granulation tissue, less angiogenesis, and less severe fibrosis. Cuff placement also causes intimal hyperplasia in wild-type mice, but not in KLF+/) [11]. 2005 International Society on Thrombosis and Haemostasis

Cardiovascular remodeling by KLF5 1571

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Egr-1 KLF5 PDGF-A 18S Fig. 3. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses of KLF5, Egr-1, and platelet-derived growth factor (PDGF)-A expression in angiotensin II-stimulated cardiac fibroblasts. Up-regulation of Egr-1 and KLF5 is detected from 0.5 h and 2 h, respectively, after angiotensin II application. Expression of Egr-1 persists for only 2 h, while KLF5 expression is sustained for more than 4 h and followed by up-regulation of PDGF-A expression [11].

and migration [16–19]. We found that PDGF-A chain is a target gene of KLF5 based on the immunohistologic analysis of the knockout mouse and on the partial resemblance of the phenotypes (e.g. intestine) of the PDGF-A chain and KLFknockout mice [20]. KLF5 and PDGF-A mRNA were induced when cultured cardiac fibroblasts were treated with angiotensin II. Up-regulation of KLF5 was detected within 2 h after the start of angiotensin II stimulation, sustained for >4 h, and followed by up-regulation of PDGF-A (Fig. 3). The PDGF-A promoter activity was activated by KLF5 in transiently transfected cells. Our analyses showed that a region between )71 and )55 bp of the transcription initiation site of PDGF-A gene mediated the KLF5 response. This region has been well characterized as an element, which mediates various stimuli including phorbol ester onto the PDGF-A chain promoter, and is known to bind the Egr-1 and Sp1 transcription factors [21,22]. To further confirm that KLF5 controls the PDGF-A gene promoter in response to angiotensin II, we carried out a series of chromatin immunoprecipitation (ChIP) assays using antiKLF5 antibody. When cultured cardiac fibroblasts treated with 1 lM angiotensin II for 3.5 h and untreated cells were formalin-fixed and subjected to ChIP analysis, angiotensin II was found to clearly increase the binding of KLF5 to the promoter region of the PDGF-A gene. Cooperative activation of PDGF-A chain by KLF5 and NF-jB p50 subunit KLF5 mediates a persistent induction of PDGF-A chain in response to phorbol ester stimulation. Among the other signaling pathways that are activated by phorbol ester, the NF-jB pathway is well-known for mediating external stimuli, 2005 International Society on Thrombosis and Haemostasis

particularly in inflammatory responses [23]. However, a pathway linking NF-jB and PDGF-A chain has not been described. As the KLF5-knockout mice showed a defect in inflammatory vascular response, we hypothesized that there may be cooperative interaction between these downstream factors. Co-transfection reporter analysis revealed that while NF-jB p50 and p65 either alone or together did not activate the PDGF-A chain promoter–reporter construct, cooperative activation of the PDGF-A chain promoter was seen specifically when the p50 subunit, but not the p65/c-rel subunit of NF-jB, was co-expressed with KLF5 (Fig. 4A). Administration of an NF-jB inhibitor, parthenolide [24], blocked this cooperative activation with p50. This cooperative activation of KLF5 with the p50 subunit of NF-jB was mediated by the )71 to )55 bp region where no consensus NF-jB-binding site exists. Thus, we reasoned that p50 might act through protein–protein interactions with KLF5 to cooperatively activate the PDGF-A chain promoter. Gelshift analysis and super-shift experiment using anti-p50 antibody demonstrated that KLF5 and p50 form a complex on DNA. The DNA-binding domain (DBD) of KLF5 was sufficient for this reaction. Co-immunoprecipitation analysis further showed that KLF5 bound p50 (Fig. 4B). Under conditions in which KLF5 and p50 cooperatively activated the PDGF-A chain promoter, neither Sp1 nor Egr-1 showed cooperative action with p50. Other KLF5-responsive promoters such as VCAM-1, inducible nitric oxide synthase (iNOS), and SMemb did not show cooperative activation, but tissue factor and plasminogen activator inhibitor-1 (PAI-1) also showed this phenomenon. Collectively, KLF5 and the p50 subunit of NF-jB show cooperative activation of PDGF-A chain by forming a complex on the KLF5-binding element of the PDGF-A chain promoter. Retinoic acid receptor-a binds to KLF5 and RAR ligands can modulate KLF5 transcriptional activities KLF5 co-immunoprecipitated with retinoic acid receptora (RARa), indicating direct physical interaction between KLF5 and RARa (Fig. 5B). We screened compounds for their ability to modulate KLF5 activity using a PDGF-A promoter activation as an indicator. We found that LE135, a synthetic RAR antagonist [25,26], enhanced PDGF-A promoter activity in cells overexpressing KLF5. Conversely, Am80, a synthetic RAR agonist [25,26], inhibited PDGF-A promoter activity in cells overexpressing KLF5 and RARa (Fig. 5A), but did not significantly affect PDGF-A promoter activity if either KLF5 or RARa was not overexpressed. We further administered LE135 or Am80 to wild type and KLF5+/) mice. When administered to KLF5+/) mice with cuffed femoral arteries, LE135 enhanced development of granulation tissue and neointimal hyperplasia. PDGF-A expression was also increased in LE135-treated mice. On the contrary, Am80 reduced development of granulation tissue and neointima in wild-type mice as well as suppressed angiotensin II-induced cardiac hypertrophy (Fig. 5C). RARa

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Relative luciferase activty

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Fig. 4. Cooperative activation of the platelet-derived growth factor (PDGF)-A chain gene by KLF5 and NF-jB p50 subunit. Co-transfection reporter analysis shows that NF-jB p50 and p65 alone or together does not activate the PDGF-A chain promoter–reporter construct. Cooperative activation of the PDGF-A chain promoter is observed specifically when the p50 subunit, but not the p65/c-rel subunit of NF-jB, is co-expressed with KLF5 (lane 7; A). Co-immunoprecipitation analysis shows that KLF5 binds p50 (lane 3; B) [38].

ligands thus appear to modulate KLF5 function both in vitro and in vivo. Regulation of KLF5 activities by transcriptional cofactors, p300 and SET As a mechanism underlying transcriptional regulation of KLF family members, we show that interaction and modification, namely through the DBD to be important [27]. To identify cofactors of KLF5, we affinity purified interacting factors that

bind the DBD region of KLF5 from nuclear extract of vascular smooth muscle cells. Matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry/peptide mass fingerprinting with a computer search of the National Center for Biotechnology Information (NCBI) mammalian proteome database revealed one band of apparent molecular weight of 39 kDa to be the SET oncoprotein, the product of the SET oncogene whose translocation has been implicated in leukemia [28,29]. SET is a histone chaperone that can displace and/or assemble nucleosomal histones in an ATP-independent manner [30–33]. We found that SET-bound KLF5 DBD and inhibited the DNA-binding activity of KLF5. SET showed dose-dependent inhibition of KLF5-mediated transactivation of the KLF5responsive embryonic vascular smooth muscle myosin heavy chain (SMemb) promoter and PDGF-A promoter (Fig. 6). Furthermore, under conditions in which adenovirus-mediated forced expression of KLF5-stimulated cell growth of smooth muscle cells, expression of SET inhibited cell growth to almost basal levels. We also found that the coactivator/acetylase p300 immunoprecipitated with KLF5 (Fig. 7A), coactivated KLF5 transcriptional activity (Fig. 7B) and monoacetylated a lysine residue at amino acid number 369 in the first Zn finger of the KLF5 DBD region. As one of the functions of SET has been recently shown to be inhibition of histone acetylation [34], we examined whether acetylation of KLF5 by p300 was inhibited in the presence of SET, which indeed it was. An arginine substitution of this lysine (K369R substitution) resulted in loss of acetylation showing that it is the solely acetylated residue. SET can bind both K369R and wild-type KLF5 suggesting that interaction is not impaired by this mutation. K369R mutation of KLF5 showed no effects on PDGF-A chain promoter activation when compared with wild-type KLF5 when transfected alone. However, a 20% decrease in PDGF-A chain promoter activity was seen when p300 was co-transfected with the K369R mutant KLF5 construct when compared with the KLF5 wild type construct showing that acetylation is important for transactivation of PDGF-A chain reporter activity by p300 on KLF5 (Fig. 8A). Furthermore, in cells transfected by wild type or the K369R mutant by adenovirus-mediated transfer, the K369R mutant transfected cells showed a reduction in cell number (Fig. 8B). BrdU assay also showed reduced uptake in the K369R mutant transfected cells further showing that cell replication is decreased in these cells. Discussion Cardiovascular remodeling is a complex process involving activation of mesenchymal cells, production of extracellular matrix, and angiogenesis, all of which contribute to the pathogenesis of atherosclerosis and heart failure. Following initial activation of mesenchymal cells (e.g. smooth muscle cells and myofibroblasts) by an external stress such as physical injury, pressure overload, and ischemia, several immediate 2005 International Society on Thrombosis and Haemostasis


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KLF5 Am80 Fig. 5. Effects of retinoid on KLF5 activity. The retinoic acid receptor-a (RAR)a agonist Am80 inhibits KLF5 activity (A). RARa co-immunoprecipitates with KLF5 (B). Effect of Am80 administration (given orally from 1 week before to 5 weeks after cuff placement) on formation of granulation tissue and neointima in cuffed femoral arteries of wild-type mice (C) [11].

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early response genes are activated followed by sustained remodeling processes. Cardiac hypertrophy also develops from external workload through hypertrophy of myocytes, proliferation of non-myocytes, and deposition of extracellular matrices [35,36]. In this series of studies, we identified KLF5 as an important transcription factor involved in cardiovascular remodeling. We originally isolated KLF5 from a smooth muscle cell cDNA library as a transcription factor for SMemb/NMHC-B, which is markedly induced in proliferating smooth muscle cell and cardiac myofibroblasts in pressure-overloaded heart. In vitro analyses showed KLF5 to activate many types of gene promoters such as PDGF-A/-B, iNOS, PAI-1, and VEGF receptors, which are known to be induced during cardiovascular remodeling. The significance of KLF5 in cardiovascular remodeling was clearly shown in animal models using KLF5+/) mice, in which inflammatory vascular response and angiotensin II-induced cardiac hypertrophy were attenuated. The amount of KLF5 expression may be important for development of cardiovascular remodeling because attenuated cellular responses in KLF5+/) mice was associated with reduced expression of KLF5, suggesting KLF5 as a haploinsufficiency gene. Which cell types with KLF5 expression are most critical in cardiovascular remodeling remains to be elucidated. A tissue-specific knockout of KLF5 gene, which is currently in progress in our laboratory, will be highly useful in answering these questions. Genes regulated by KLF5 have not been well characterized. In our hands, PDGF-A gene was found to be one of the

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Fig. 6. Effects of SET on KLF5-transactivation. Co-transfection analysis of effects of SET on KLF5-transcriptional activation. SET dose dependently inhibits KLF5-mediated transactivation of the KLF5-responsive platelet-derived growth factor (PDGF)-A promoter (lanes 5 and 6; modified from Ref. [39] with permission).

KLF5-regulated genes because ChIP assay revealed KLF5 binding to the PDGF-A gene promoter. PDGF-B gene is also reported to be regulated by KLF5 [37]. Transforming growth factor (TGF)-b may be also regulated by KLF5 because TGF-b expression in KLF5+/) mice is reduced in injured

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Fig. 7. Interaction and activation of KLF5 by the coactivator/acetylase p300. Interaction of KLF5 and p300 in vivo. p300 was immunoprecipitated from cells followed by immunoblotting against KLF5 (lane 2; A). Effects of p300 on KLF5 transactivation as assessed by reporter co-transfection assay (lanes 4 and 5). Effects of an acetyltransferase region-deleted mutant of p300 (DHAT) on KLF5-mediated transcriptional activation were also examined (lanes 6, 7; B; modified from Ref. [39] with permission).

artery. Systematic identification of KLF5-regulated genes will be important for elucidating the functions of KLF5 as well as for understanding the molecular mechanisms underlying its effects on cardiovascular remodeling. Of interest is that KLF5 interacts with nuclear receptors and other transcription factors or cofactors. NF-jB p50 subunit mediates cooperative activation of the PDGF-A chain gene by KLF5. Given the well-known role of NF-jB to mediate inflammatory responses secondary to pathologic stimuli, activation of KLF5 by NF-jB p50 subunit may contribute to inflammation-induced tissue remodeling. Our finding that p50 cooperatively activates the PDGF-A chain promoter through protein–protein interaction with KLF5 has not been previously reported. This implies not only a direct pathway involving activation of PDGF-A chain through a delayed

Cell number ratio (%)

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–10 Empty –5.7% KLF5 K369R –15% –20 Fig. 8. Effects of KLF5 K369R mutation. Effect of KLF5 K369R on platelet-derived growth factor (PDGF)-A chain promoter transcriptional activation compared with KLF5 wild type in the presence of p300 (A). Effects of KLF5 K369R on cell growth. KLF5 wild type and K369R by adenovirus were counted on day 6 in comparison with non-treated cells (B; modified from Ref. [39] with permission).



increase in expression amounts of KLF5, but also a cooperative effect as manifested by convergence of the NF-jB signal onto KLF5 to further provide regulation. Interaction of KLF5 with RARa, and modulation of interaction by retinoid ligand was intriguing as this allows for indirect but potent therapeutic modulation of KLF5 activities. Drugs affecting transcriptional processes have remained elusive because of the necessity to deliver and compartmentalize the agent to the nucleus. Therapeutic targeting of protein–protein interaction represents a viable and potent method to control specific transcription factors and their activities. We are presently testing other retinoids to determine which most potently and specifically can target KLF5 activities. We also showed that the coactivator/acetylase p300 positively regulates KLF5 whereas the oncogenic regulator/histone chaperone SET negatively regulates its activities. Interaction was coupled with acetylation. As these interactions and modifications are fundamental processes associated with eukaryotic chromatin processes, these mechanisms of action help us to understand how KLF5 and other transcription factors act at the level of chromatin and higher ordered DNA processes. As selective modulation of chromatin structure is vital for therapeutic intervention, our described mechanisms will likely play an important role in further developing ÔpinpointÕ drugs. We are presently analyzing the crystal structure of KLF5 and its interactors to design specific modulating compounds. In conclusion, the results of the present study provide strong evidence that KLF5 is a crucial determinant of the cardiovascular response to external stresses, playing a key role in mediating tissue remodeling. This study provides a clear basis for the development of drugs to control cardiovascular remodeling and angiogenesis by modulating KLF5 function. References 1 Libby P. Changing concepts of atherogenesis. J Intern Med 2000; 247: 349–58. 2 Kuro-o M, Nagai R, Tsuchimochi H, Katoh H, Yazaki Y, Ohkubo A, Takaku F. Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms. J Biol Chem 1989; 264: 18272–5. 3 Manabe I, Kurabayashi M, Shimomura Y, Kuro-o M, Watanabe N, Watanabe M, Aikawa M, Suzuki T, Yazaki Y, Nagai R. Isolation of the embryonic form of smooth muscle myosin heavy chain (SMemb/ NMHC-B) gene and characterization of its 5¢-flanking region. Biochem Biophys Res Commun 1997; 239: 598–605. 4 Watanabe N, Kurabayashi M, Shimomura Y, Kawai-Kowase K, Hoshino Y, Manabe I, Watanabe M, Aikawa M, Kuro-o M, Suzuki T, Yazaki Y, Nagai R. BTEB2, a Kruppel-like transcription factor, regulates expression of the SMemb/Nonmuscle myosin heavy chain B (SMemb/NMHC-B) gene. Circ Res 1999; 85: 182–91. 5 Shiojima I, Aikawa M, Suzuki J, Yazaki Y, Nagai R. Embryonic smooth muscle myosin heavy chain SMemb is expressed in pressureoverloaded cardiac fibroblasts. Jpn Heart J 1999; 40: 803–18. 6 Bieker JJ. Kruppel-like factors: three fingers in many pies. J Biol Chem 2001; 276: 34355–8. 7 Dang DT, Pevsner J, Yang VW. The biology of the mammalian Kruppel-like family of transcription factors. Int J Biochem Cell Biol 2000; 32: 1103–21.


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