Sp1-dependent Activation of HDAC7 Is Required for Platelet-derived Growth Factor-BB-induced Smooth Muscle Cell Differentiation from Stem Cells

Abstract

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. Embryonic stem (ES)4 cells are the most promising pluripotent stem cell sources and are derived from the inner cell mass of blastocysts. They have unlimited cell growth and the capacity to self-renew and differentiate into all types of mature tissue cells. For instance, ES cells may serve as a source of vascular smooth muscle cells (SMCs) that is essential for vascular tissue engineering. In addition, stem/progenitor cells were reported to differentiate into vascular cells to repair damaged vascular tissues and form neointima in vivo (1-4). Our previous studies demonstrated that ES cells can differentiate into SMCs (5-8) and endothelial cells in response to growth factor stimulation (3) and mechanical force (9). We also demonstrated that extracellular matrix protein collagen IV plays an important role in SMC differentiation from ES cells and that the collagen type IV-integrin signaling pathways are involved in SMC differentiation (6). However, the detailed molecular mechanisms of SMC differentiation from stem cells have not been fully clarified.Platelet-derived growth factor-BB (PDGF-BB) has been identified as one of the key modulat...

Functional Impact of Heterogeneous Nuclear Ribonucleoprotein A2/B1 in Smooth Muscle Differentiation from Stem Cells and Embryonic Arteriogenesis

“…In parallel with SMC-specific gene inductions reported previously (2)(3)(4)(5)(6)(7)(8), the expression levels of hnRNPA2/B1, one of the several up-regulated nuclear proteins found during early SMC differentiation as demonstrated by our nuclear proteomics analysis (data not shown), were transiently elevated when ES cells were cultivated on collagen IV-coated plates in the absence of leukemia inhibitory factor to allow for SMC differ-entiation ( Fig. 1, A and B).…”

“…Cell Culture and SMC Differentiation-Detailed protocols for mouse ES cell (ES-D3 cell line, CRL-1934; ATCC, Manassas, VA) culture and SMC differentiation were described in our previous studies (2)(3)(4)(5)(6)(7)(8). For SMC differentiation, undifferentiated ES cells were seeded on mouse collagen IV (5 g/ml)-coated flasks or plates in differentiation medium (DM), ␣-minimal essential medium (Invitrogen) supplemented with 10% FBS, 0.05 mM ␤-mercaptoethanol, 100 units/ml penicillin, and 100 g/ml streptomycin for 3-7 days prior to further treatments.…”

“…Other Procedures-Experiments of nucleofection, siRNA experiments, indirect immunofluorescent staining for cells, and real time quantitative PCRs (RT-qPCR) were performed according to the standard procedures as described previously (2)(3)(4)(5)(6)(7)(8). Whole mount immunohistochemistry and in situ hybridization were carried out as described previously (7).…”

“…Chromatin Immunoprecipitation (ChIP) Assays-As described previously (3,5,7,8), differentiating ES cells transfected with pCMV5 or pCMV5-Hnrnpa2/b1 were treated with 1% (v/v) formaldehyde at room temperature for 10 min and then quenched with glycine at room temperature. After removing the medium, cells were harvested for sonication.…”

Upregulated sirtuin 1 by miRNA-34a is required for smooth muscle cell differentiation from pluripotent stem cells

“…Detailed protocols for mouse ESCs (mESCs) (ES-D3 cell line, CRL-1934; ATCC, Manassas, VA, USA) culture and SMC differentiation were described in our previous studies. [11][12][13][14]17,22,34,43,44 The Shef-1, 2, 3, 6, and 7 human ESC lines were obtained from the United Kingdom Stem Cell Bank (UKSCB, Hertfordshire, UK) and maintained in our Laboratory as described in our previous study. 18 Briefly, undifferentiated ESCs were dissociated into single cells and seeded onto collagen I/IV (5 μg/ml)-coated flasks or plates in differentiation medium (DM, MEM alpha medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin) for 0-9 days prior to further treatment.…”