Nuclear factor of activated T cells 5 regulates vascular smooth muscle cell phenotypic modulation.
ABSTRACT: The tonicity-responsive transcription factor, nuclear factor of activated T cells 5 (NFAT5/tonicity enhancer binding protein [TonEBP]), has been well characterized in numerous cell types; however, NFAT5 function in vascular smooth muscle cells (SMCs) is unknown. Our main objective was to determine the role of NFAT5 regulation in SMCs.We showed that NFAT5 is regulated by hypertonicity in SMCs and is upregulated in atherosclerosis and neointimal hyperplasia. RNAi knockdown of NFAT5 inhibited basal expression of several SMC differentiation marker genes, including smooth muscle ? actin (SM?A). Bioinformatic analysis of SM?A revealed 7 putative NFAT5 binding sites in the first intron, and chromatin immunoprecipitation analysis showed NFAT5 enrichment of intronic DNA. Overexpression of NFAT5 increased SM?A promoter-intron activity, which requires an NFAT5 cis element at +1012, whereas dominant-negative NFAT5 decreased SM?A promoter-intron activity. Because it is unlikely that SMCs experience extreme changes in tonicity, we investigated other stimuli and uncovered 2 novel NFAT5-inducing factors: angiotensin II, a contractile agonist, and platelet-derived growth factor-BB (PDGF-BB), a potent mitogen in vascular injury. Angiotensin II stimulated NFAT5 translocation and activity, and NFAT5 knockdown inhibited an angiotensin II-mediated upregulation of SM?A mRNA. PDGF-BB increased NFAT5 protein, and loss of NFAT5 inhibited PDGF-BB-induced SMC migration.We have identified NFAT5 as a novel regulator of SMC phenotypic modulation and have uncovered the role of NFAT5 in angiotensin II-induced SM?A expression and PDGF-BB-stimulated SMC migration.
Project description:Platelet-derived growth factor (PDGF)-BB is a well-known smooth muscle (SM) cell (SMC) phenotypic modulator that signals by binding to PDGF alphaalpha-, alphabeta-, and betabeta-membrane receptors. PDGF-DD is a recently identified PDGF family member, and its role in SMC phenotypic modulation is unknown. Here we demonstrate that PDGF-DD inhibited expression of multiple SMC genes, including SM alpha-actin and SM myosin heavy chain, and upregulated expression of the potent SMC differentiation repressor gene Kruppel-like factor-4 at the mRNA and protein levels. On the basis of the results of promoter-reporter assays, changes in SMC gene expression were mediated, at least in part, at the level of transcription. Attenuation of the SMC phenotypic modulatory activity of PDGF-DD by pharmacological inhibitors of ERK phosphorylation and by a small interfering RNA to Kruppel-like factor-4 highlight the role of these two pathways in this process. PDGF-DD failed to repress SM alpha-actin and SM myosin heavy chain in mouse SMCs lacking a functional PDGF beta-receptor. Importantly, PDGF-DD expression was increased in neointimal lesions in the aortic arch region of apolipoprotein C-deficient (ApoE(-/-)) mice. Furthermore, human endothelial cells exposed to an atherosclerosis-prone flow pattern, as in vascular regions susceptible to the development of atherosclerosis, exhibited a significant increase in PDGF-DD expression. These findings demonstrate a novel activity for PDGF-DD in SMC biology and highlight the potential contribution of this molecule to SMC phenotypic modulation in the setting of disturbed blood flow.
Project description:The molecular mechanisms behind phenotypic modulation of smooth muscle cells (SMCs) remain unclear. In our recent paper, we reported the establishment of novel culture system of gizzard SMCs (Hayashi, K., H. Saga, Y. Chimori, K. Kimura, Y. Yamanaka, and K. Sobue. 1998. J. Biol. Chem. 273: 28860-28867), in which insulin-like growth factor-I (IGF-I) was the most potent for maintaining the differentiated SMC phenotype, and IGF-I triggered the phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB(Akt)) pathway. Here, we investigated the signaling pathways involved in de-differentiation of gizzard SMCs induced by PDGF-BB, bFGF, and EGF. In contrast to the IGF-I-triggered pathway, PDGF-BB, bFGF, and EGF coordinately activated ERK and p38MAPK pathways. Further, the forced expression of active forms of MEK1 and MKK6, which are the upstream kinases of ERK and p38MAPK, respectively, induced de-differentiation even when SMCs were stimulated with IGF-I. Among three growth factors, PDGF-BB only triggered the PI3-K/PKB(Akt) pathway in addition to the ERK and p38MAPK pathways. When the ERK and p38MAPK pathways were simultaneously blocked by their specific inhibitors or an active form of either PI3-K or PKB(Akt) was transfected, PDGF-BB in turn initiated to maintain the differentiated SMC phenotype. We applied these findings to vascular SMCs, and demonstrated the possibility that the same signaling pathways might be involved in regulating the vascular SMC phenotype. These results suggest that changes in the balance between the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine phenotypes of visceral and vascular SMCs. We further reported that SMCs cotransfected with active forms of MEK1 and MKK6 secreted a nondialyzable, heat-labile protein factor(s) which induced de-differentiation of surrounding normal SMCs.
Project description:Smooth muscle cells (SMCs) are key regulators of vascular disease and circulating smooth muscle progenitor cells may play important roles in vascular repair or remodelling. We developed enhanced protocols to derive smooth muscle progenitors from murine bone marrow and tested whether factors that are increased in atherosclerotic plaques, namely platelet-derived growth factor-BB (PDGF-BB) and monomeric collagen, can influence the smooth muscle specific differentiation, proliferation, and survival of mouse bone marrow-derived progenitor cells. During a 21 day period of culture, bone marrow cells underwent a marked increase in expression of the SMC markers ?-SMA (1.93 ± 0.15 vs. 0.0008 ± 0.0003 (ng/ng GAPDH) at 0 d), SM22-? (1.50 ± 0.27 vs. 0.005 ± 0.001 (ng/ng GAPDH) at 0 d) and SM-MHC (0.017 ± 0.004 vs. 0.001 ± 0.001 (ng/ng GAPDH) at 0 d). Bromodeoxyuridine (BrdU) incorporation experiments showed that in early culture, the smooth muscle progenitor subpopulation could be identified by high proliferative rates prior to the expression of smooth muscle specific markers. Culture of fresh bone marrow or smooth muscle progenitor cells with PDGF-BB suppressed the expression of ?-SMA and SM22-?, in a rapidly reversible manner requiring PDGF receptor kinase activity. Progenitors cultured on polymerized collagen gels demonstrated expression of SMC markers, rates of proliferation and apoptosis similar to that of cells on tissue culture plastic; in contrast, cells grown on monomeric collagen gels displayed lower SMC marker expression, lower growth rates (319 ± 36 vs. 635 ± 97 cells/mm2), and increased apoptosis (5.3 ± 1.6% vs. 1.0 ± 0.5% (Annexin 5 staining)). Our data shows that the differentiation and survival of smooth muscle progenitors are critically affected by PDGF-BB and as well as the substrate collagen structure.
Project description:Calcineurin (Cn) and the nuclear factor of activated T cells (NFAT) family of transcription factors are critical in vascular smooth muscle cell (SMC) development and pathology. Here, we used a genomics approach to identify and validate NFAT gene targets activated during platelet-derived growth factor-BB (PDGF-BB)-induced SMC phenotypic modulation.Genome-wide expression arrays were used to identify genes both (1) differentially activated in response to PDGF-BB and (2) whose differential expression was reduced by both the Cn inhibitor cyclosporin A and the NFAT inhibitor A-285222. The 20 most pharmacologically sensitive genes were validated by quantitative reverse transcription-polymerase chain reaction analysis of PDGF-BB-stimulated SMCs in the presence of Cn/NFAT inhibitors, including the VIVIT peptide. In all experiments, protein C receptor (PROCR) gene activation was reduced. We showed that PROCR expression was virtually absent in untreated, quiescent SMCs. PDGF-BB stimulation, however, induced significant PROCR promoter activation and downstream protein expression in a Cn/NFAT-dependent manner. Mutation of a species-conserved, NFAT binding motif significantly attenuated PDGF-BB-induced PROCR promoter activity, thereby distinguishing NFAT as the first PROCR transcriptional activator to date. Moreover, SMC PROCR expression was upregulated in the neointima as early as 7 days following acute vascular injury in rat carotid arteries.We hereby report PROCR as a novel, NFAT-dependent gene that may be implicated in vascular restenosis and consequent inward remodeling.
Project description:We have previously demonstrated that histone deacetylase 7 (HDAC7) expression and splicing play an important role in smooth muscle cell (SMC) differentiation from embryonic stem (ES) cells, but the molecular mechanisms of increased HDAC7 expression during SMC differentiation are currently unknown. In this study, we found that platelet-derived growth factor-BB (PDGF-BB) induced a 3-fold increase in the transcripts of HDAC7 in differentiating ES cells. Importantly, our data also revealed that PDGF-BB regulated HDAC7 expression not through phosphorylation of HDAC7 but through transcriptional activation. By dissecting its promoters with progressive deletion analysis, we identified the sequence between -343 and -292 bp in the 5'-flanking region of the Hdac7 gene promoter as the minimal PDGF-BB-responsive element, which contains one binding site for the transcription factor, specificity protein 1 (Sp1). Mutation of the Sp1 site within this PDGF-BB-responsive element abolished PDGF-BB-induced HDAC7 activity. PDGF-BB treatment enhanced Sp1 binding to the Hdac7 promoter in differentiated SMCs in vivo as demonstrated by the chromatin immunoprecipitation assay. Moreover, we also demonstrated that knockdown of Sp1 abrogated PDGF-BB-induced HDAC7 up-regulation and SMC differentiation gene expression in differentiating ES cells, although enforced expression of Sp1 alone was sufficient to increase the activity of the Hdac7 promoter and expression levels of SMC differentiation genes. Importantly, we further demonstrated that HDAC7 was required for Sp1-induced SMC differentiation of gene expression. Our data suggest that Sp1 plays an important role in the regulation of Hdac7 gene expression in SMC differentiation from ES cells. These findings provide novel molecular insights into the regulation of HDAC7 and enhance our knowledge in SMC differentiation and vessel formation during embryonic development.
Project description:The phenotype of smooth muscle cells (SMCs) plays an important role in vascular function in health and disease. We investigated the mechanism of modulation of SMC phenotype (from contractile to synthetic) induced by the synergistic action of a growth factor (platelet-derived growth factor, PDGF-BB) and a cytokine (interleukin, IL-1beta). Human aortic SMCs grown on polymerized collagen showed high expression levels of contractile markers (smooth muscle alpha-actin, myosin heavy chain, and calponin). These levels were not significantly affected by PDGF-BB and IL-1beta individually, but decreased markedly after the combined usage of PDGF-BB and IL-1beta. PDGF/IL-1beta costimulation also induced a sustained phosphorylation of Akt and p70 ribosomal S6 kinase (p70S6K). The effects of PDGF/IL-1beta costimulation on contractile marker expression and Akt and p70S6K phosphorylation were blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 and by adenovirus expressing a dominant-negative Akt, and they were mimicked by constitutively active Akt. PDGF-BB/IL-1beta induced a sustained phosphorylation of PDGF receptor (PDGFR)-beta and its association with IL-1 receptor (IL-1R1). Such activation and association of receptors were blocked by a PDGFR-beta neutralizing antibody (AF385), an IL-1R1 antagonist (IL-1ra), as well as a specific inhibitor of PDGFR-beta phosphorylation (AG1295); these agents also eliminated the PDGF-BB/IL-1beta-induced signaling and phenotypic modulation. PDGF-BB/IL-1beta inhibited the polymerized collagen-induced serum response factor DNA binding activity in the nucleus, and this effect was mediated by the PDGFR-beta/IL-1R1 association and phosphatidylinositol 3-kinase/Akt/p70S6K pathway. Our findings provide insights into the mechanism of SMC phenotypic modulation from contractile to synthetic, e.g., in atherosclerosis.
Project description:In this study we demonstrate that CD34(+) cells derived from human embryonic stem cells (hESCs) have higher smooth muscle cell (SMC) potential than CD34(-) cells. We report that from all inductive signals tested, retinoic acid (RA) and platelet derived growth factor (PDGF(BB)) are the most effective agents in guiding the differentiation of CD34(+) cells into smooth muscle progenitor cells (SMPCs) characterized by the expression of SMC genes and proteins, secretion of SMC-related cytokines, contraction in response to depolarization agents and vasoactive peptides and expression of SMC-related genes in a 3D environment. These cells are also characterized by a low organization of the contractile proteins and the contractility response is mediated by Ca(2+), which involves the activation of Rho A/Rho kinase- and Ca(2+)/calmodulin (CaM)/myosin light chain kinase (MLCK)-dependent pathways. We further show that SMPCs obtained from the differentiation of CD34(+) cells with RA, but not with PDGF(BB,) can be maturated in medium supplemented with endothelin-1 showing at the end individualized contractile filaments. Overall the hESC-derived SMCs presented in this work might be an unlimited source of SMCs for tissue engineering and regenerative medicine.
Project description:Smooth muscle cell- (SMC-) based tissue engineering provides a promising therapeutic strategy for SMC-related disorders. It has been demonstrated that human dental pulp stem cells (DPSCs) possess the potential to differentiate into mature bladder SMCs by induction with condition medium (CM) from bladder SMC culture, in combination with the transforming growth factor-?1 (TGF-?1). However, the molecular mechanism of SMC differentiation from DPSCs has not been fully uncovered. The canonical Wnt signaling (also known as Wnt/?-catenin) pathway plays an essential role in stem cell fate decision. The aim of this study is to explore the regulation via GSK3? and associated downstream effectors for SMC differentiation from DPSCs. We characterized one of our DPSC clones with the best proliferation and differentiation abilities. This stem cell clone has shown the capacity to generate a smooth muscle layer-like phenotype after an extended differentiation duration using the SMC induction protocol we established before. We further found that Wnt-GSK3?/?-catenin signaling is involved in the process of SMC differentiation from DPSCs, as well as a serial of growth factors, including TGF-?1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor-homodimer polypeptide of B chain (BB) (PDGF-BB), and vascular endothelial growth factor (VEGF). Pharmacological inhibition on the canonical Wnt-GSK3?/?-catenin pathway significantly downregulated GSK3? phosphorylation and ?-catenin activation, which in consequence reduced the augmented expression of the growth factors (including TGF-?1, HGF, PDGF-BB, and VEGF) as well as SMC markers (especially myosin) at a late stage of SMC differentiation. These results suggest that the canonical Wnt-GSK3?/?-catenin pathway contributes to DPSC differentiation into mature SMCs through the coordination of different growth factors.
Project description:A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [alpha-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.
Project description:Smooth muscle formation and function are critical in development and postnatal life. Hence, studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function. TGF-beta1 alone or combined with PDGF-BB in serum-free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Furthermore, SMCs derived from MAPCs (MAPC-SMCs) demonstrated functional L-type calcium channels. MAPC-SMCs entrapped in fibrin vascular molds became circumferentially aligned and generated force in response to KCl, the L-type channel opener FPL64176, or the SMC agonists 5-HT and ET-1, and exhibited complete relaxation in response to the Rho-kinase inhibitor Y-27632. Cyclic distention (5% circumferential strain) for 3 weeks increased responses by 2- to 3-fold, consistent with what occurred in neonatal SMCs. These results provide evidence that MAPC-SMCs are phenotypically and functionally similar to neonatal SMCs and that the in vitro MAPC-SMC differentiation system may be an ideal model for the study of SMC development. Moreover, MAPC-SMCs may lend themselves to tissue engineering applications.