Keiko Kawai-Kowase

Krüppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling.

We recently isolated a Krüppel-like zinc-finger transcription factor 5 (KLF5; also known as BTEB2 and IKLF), which is markedly induced in activated vascular smooth-muscle cells and fibroblasts. Here we describe our analysis of the in vivo function of KLF5 using heterozygous KLF5-knockout mice (Klf5+/−). In response to external stress, Klf5+/− mice showed diminished levels of arterial-wall thickening, angiogenesis, cardiac hypertrophy and interstitial fibrosis. Also, angiotensin II induced expression of KLF5, which in turn activated platelet-derived growth factor-A (PDGF-A) and transforming growth factor-β (TGF-β) expression. In addition, we determined that KLF5 interacted with the retinoic-acid receptor (RAR), that synthetic RAR ligands modulated KLF5 transcriptional activity, and that in vivo administration of RAR ligands affected stress responses in the cardiovascular system in a KLF5-dependent manner. KLF5 thus seems to be a key element linking external stress and cardiovascular remodeling.

BTEB2, a Krüppel-Like Transcription Factor, Regulates Expression of the SMemb/Nonmuscle Myosin Heavy Chain B (SMemb/NMHC-B) Gene

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a lambdagt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione S-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.

Regulation of Platelet-derived Growth Factor-A Chain by Krüppel-like Factor 5 NEW PATHWAY OF COOPERATIVE ACTIVATION WITH NUCLEAR FACTOR-κB

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.

HERP1 Inhibits Myocardin-Induced Vascular Smooth Muscle Cell Differentiation by Interfering With SRF Binding to CArG Box

Objective—Myocardin is a coactivator of serum response factor (SRF) required for vascular smooth muscle cell (VSMC) differentiation. HERP1 is a transcriptional repressor, which is abundantly expressed in vascular system and is known to function as a target gene of Notch. However, the role of HERP1 in the pathogenesis of vascular lesions remains unknown. The present study characterizes the expression of HERP1 in normal and diseased vessels, and tests the hypothesis that HERP1 inhibits SRF/myocardin-dependent SMC gene expression. Methods and Results—Immunohistochemistry revealed that HERP1 and myocardin expression was localized to SMC in the neointima of balloon-injured rat aorta and in human coronary atherosclerotic lesions. Expression of both HERP1 and myocardin was elevated in cultured VSMCs compared with medial SMC. Overexpressed HERP1 inhibited the myocardin-induced SMC marker gene expression in 10T1/2 cells. HERP1 protein interfered with the SRF/CArG–box interaction in vivo and in vitro. Immunoprecipitation assays showed that HERP1 physically interacts with SRF. Conclusions—HERP1 expression was associated with the SMC proliferation and dedifferentiation in vitro and in vivo. HERP1 may play a role in promoting the phenotypic modulation of VSMCs during vascular injury and atherosclerotic process by interfering with SRF binding to CArG-box through physical association between HERP1 and SRF.

Basic Fibroblast Growth Factor Antagonizes Transforming Growth Factor-β1–Induced Smooth Muscle Gene Expression Through Extracellular Signal–Regulated Kinase 1/2 Signaling Pathway Activation

Objective—Transforming growth factor-β1 (TGFβ1) and fibroblast growth factor (FGF) families play a pivotal role during vascular development and in the pathogenesis of vascular disease. However, the interaction of intracellular signaling evoked by each of these growth factors is not well understood. The present study was undertaken to examine the molecular mechanisms that mediate the effects of TGFβ1 and basic FGF (bFGF) on smooth muscle cell (SMC) gene expression. Methods and Results—TGFβ1 induction of SMC gene expression, including smooth muscle protein 22-&agr; (SM22&agr;) and smooth muscle &agr;-actin, was examined in the pluripotent 10T1/2 cells. Marked increase in these mRNA levels by TGFβ1 was inhibited by c-Src-tyrosine kinase inhibitors and protein synthesis inhibitor cycloheximide. Functional studies with deletion and site-directed mutation analysis of the SM22&agr; promoter demonstrated that TGFβ1 activated the SM22&agr; promoter through a CC(A/T-rich)6GG (CArG) box, which serves as a serum response factor (SRF)–binding site. TGFβ1 increased SRF expression through an increase in transcription of the SRF gene. In the presence of bFGF, TGFβ1 induction of SMC marker gene expression was significantly attenuated. Transient transfection assays showed that bFGF significantly suppressed induction of the SM22&agr; promoter–driven luciferase activity by TGFβ1, whereas bFGF had no effects on the TGFβ1-mediated increase in SRF expression and SRF:DNA binding activity. Mitogen-activated protein kinase kinase-1 (MEK1) inhibitor PD98059 abrogated the bFGF-mediated suppression of TGFβ1-induced SMC gene expression. Conclusion—Our data suggest that bFGF-induced MEK/extracellular signal-regulated kinase signaling plays an antagonistic role in TGFβ1-induced SMC gene expression through suppression of the SRF function. These data indicate that opposing effects of bFGF and TGFβ1 on SMC gene expression control the phenotypic plasticity of SMCs.

Runx2 represses myocardin-mediated differentiation and facilitates osteogenic conversion of vascular smooth muscle cells.

ABSTRACT Phenotypic plasticity and the switching of vascular smooth muscle cells (SMCs) play a critical role in atherosclerosis. Although Runx2, a key osteogenic transcription factor, is expressed in atherosclerotic plaques, the molecular mechanisms by which Runx2 regulates SMC differentiation remain unclear. Here we demonstrated that Runx2 repressed SMC differentiation induced by myocardin, which acts as a coactivator for serum response factor (SRF). Myocardin-mediated induction of SMC gene expression was enhanced in mouse embryonic fibroblasts derived from Runx2 null mice compared to wild-type mice. Forced expression of Runx2 decreased the expression of SMC genes and promoted osteogenic gene expression, whereas the reduction of Runx2 expression by small interfering RNA enhanced SMC differentiation in human aortic SMCs. Runx2 interacted with SRF and interfered with the formation of the SRF/myocardin ternary complex. Thus, this study provides the first evidence that Runx2 inhibits SRF-dependent transcription, as a corepressor independent of its DNA binding. We propose that Runx2 plays a pivotal role in osteogenic conversion tightly coupled with repression of the SMC phenotype in atherosclerotic lesions.

Notch Signaling Induces Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells: Role of Msx2 Gene Induction via Notch-RBP-Jk Signaling

Objective—Vascular calcification is closely correlated with cardiovascular morbidity and mortality. Here, we demonstrate the role of Notch signaling in osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). Methods and Results—The Msx2 gene, a key regulator of osteogenesis, was highly induced by coculture with Notch ligand-expressing cells or overexpression of Notch intracellular domains (NICDs) in human aortic SMCs (HASMCs). Furthermore, the Notch1 intracellular domain (N1-ICD) overexpression markedly upregulated alkaline phosphatase (ALP) activity and matrix mineralization of HASMCs. A knockdown experiment with a small interfering RNA confirmed that Msx2 mediated N1-ICD–induced osteogenic conversion of HASMCs. Interestingly, Msx2 induction by N1-ICD was independent of bone morphogenetic protein–2 (BMP-2), an osteogenic morphogen upstream of Msx2. The transcriptional activity of the Msx2 promoter was significantly enhanced by N1-ICD overexpression. The RBP-Jk binding element within the Msx2 promoter was critical to Notch-induced Msx2 gene expression. Correspondingly, N1-ICD overexpression did not induce the Msx2 expression in RBP-Jk–deficient fibroblasts. Immunohistochemistry of human carotid artery specimens revealed localization of Notch1, Jagged1 and Msx2 to fibrocalcific atherosclerotic plaques. Conclusion—These results imply a new mechanism for osteogenic differentiation of vascular SMCs in which Notch/RBP-Jk signaling directly induces Msx2 gene expression and suggest its crucial role in mediating vascular calcification.

Notch Signaling Pathway Enhances Bone Morphogenetic Protein 2 (BMP2) Responsiveness of Msx2 Gene to Induce Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells

Vascular calcification is regulated in a process similar to bone formation. BMP2 (bone morphogenetic protein 2) is essential for osteoblastic differentiation of mesenchymal progenitor cells and thus has been implicated in the development of vascular calcification. Here we examined whether Notch signaling interacts with BMP2 signaling to regulate osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). BMP2 alone scarcely induced the expression of alkaline phosphatase (ALP), an ectoenzyme crucially required for active biomineralization, in human aortic SMCs (HASMCs), despite its strong induction in osteoblast precursor MC3T3-E1 cells. Notably, overexpression of the Notch1 intracellular domain (N1-ICD) markedly enhanced BMP2-mediated induction of ALP activity and mineralization of HASMCs. In HASMCs, expression of Msx2 gene, a well documented BMP2 target gene in osteoblasts, was barely induced by BMP2 alone, and N1-ICD clearly enhanced the BMP2-driven Msx2 gene expression. Deletion and site-directed mutation analysis of Msx2 gene promoter revealed that the RBPJk-binding site was necessary for BMP2 responsiveness. Using the RBPJk-deficient cells and siRNA for RBPJk, we showed that RBPJk was required for BMP2 induction of Msx2 gene expression and ALP activity. Moreover, we showed that Smad1, a transcription factor downstream of BMP2 signaling, interacted with N1-ICD to form a complex within the Msx2 promoter. Immunohistochemistry of human calcifying atherosclerotic plaques revealed colocalized expression of Notch1, BMP2, and Msx2. These results indicate that the Notch intracellular domain·RBPJk complex enhances the BMP2-induced Msx2 gene expression by cooperating with Smad1 and suggest that Notch signaling makes vascular SMC responsive to BMP2 and promotes vascular calcification.

PIAS1 Mediates TGFβ-Induced SM α-Actin Gene Expression Through Inhibition of KLF4 Function-Expression by Protein Sumoylation

Objective—TGF&bgr; and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) &agr;-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGF&bgr; activates SM &agr;-actin through PIAS1. Methods and Results—An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGF&bgr;-induced expression of SM &agr;-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM &agr;-actin promoter activity in a TGF&bgr; control element (TCE)-dependent manner. Because the TCE within the SM &agr;-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmunoprecipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM &agr;-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation. Conclusions—These results provide evidence that PIAS1 promotes TGF&bgr;-induced activation of SM &agr;-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.

Fibroblast growth factor-2 induces osteogenic differentiation through a Runx2 activation in vascular smooth muscle cells

Expression of bone-associated proteins and osteoblastic transcription factor Runx2 in arterial cells has been implicated in the development of vascular calcification. However, the signaling upstream of the Runx2-mediated activation of osteoblastic program in vascular smooth muscle cells (VSMC) is poorly understood. We examined the effects of fibroblast growth factor-2 (FGF-2), an important regulator of bone formation, on osteoblastic differentiation of VSMC. Stimulation of cultured rat aortic SMC (RASMC) with FGF-2 induced the expression of the osteoblastic markers osteopontin (OPN) and osteocalcin. Luciferase assays showed that FGF-2 induced osteocyte-specific element (OSE)-dependent transcription. Downregulation of Runx2 by siRNA repressed the basal and FGF-2-stimulated expression of the OPN gene in RASMC. FGF-2 produced hydrogen peroxide in RASMC, as evaluated by fluorescent probe. Induction of OPN expression by FGF-2 was inhibited not only by PD98059 (MEK1 inhibitor) and PP1 (c-Src inhibitor), but also by an antioxidant, N-acetyl cysteine. Nuclear extracts from FGF-2-treated RASMC exhibited increased DNA-binding of Runx2 to its target sequence. Immunohistochemistry of human coronary atherectomy specimens and calcified aortic tissues showed that expression of FGF receptor-1 and Runx2 was colocalized. In conclusion, these results suggest that FGF-2 plays a role in inducing osteoblastic differentiation of VSMC by activating Runx2 through mitogen-activated protein kinase (MAPK)-dependent- and oxidative stress-sensitive-signaling pathways.