TGF?1-induced Baf60c regulates both smooth muscle cell commitment and quiescence.
ABSTRACT: Smooth muscle cells (SMCs) play critical roles in a number of diseases; however, the molecular mechanism underlying their development is unclear. Although the role of TGF?1 signaling in SMC development is well established, the downstream molecular signals are not fully understood. We used several rat multipotent adult progenitor cell ((r)MAPC) lines that express levels of Oct4 mRNA similar to hypoblast stem cells (HypoSC), and can differentiate robustly to mesodermal and endodermal cell types. TGF?1 alone, or with PDGF-BB, induces differentiation of rMAPCs to SMCs, which expressed structural SMC proteins, including ?-smooth muscle actin (?SMA), and contribute to the SMC coat of blood vessels in vivo. A genome-wide time-course transcriptome analysis revealed that transcripts of Baf60c, part of the SWI/SNF actin binding chromatin remodeling complex D-3 (SMARCD3/BAF60c), were significantly induced during MAPC-SMC differentiation. We demonstrated that BAF60c is a necessary co-regulator of TGF?1 mediated induction of SMC genes. Knock-down of Baf60c decreased SMC gene expression in rMAPCs whereas ectopic expression of Baf60c was sufficient to commit rMAPCs to SMCs in the absence of exogenous cytokines. TGF?1 activates Baf60c via the direct binding of SMAD2/3 complexes to the Baf60c promoter region. Chromatin- and co-immunoprecipitation studies demonstrated that regulation of SMC genes by BAF60c is mediated via interaction with SRF binding CArG box-containing promoter elements in SMC genes. We noted that compared with TGF?1, Baf60c overexpression in rMAPC yielded SMC with a more immature phenotype. Similarly, Baf60c induced an immature phenotype in rat aortic SMCs marked by increased cell proliferation and decreased contractile marker expression. Thus, Baf60c is important for TGF?-mediated commitment of primitive stem cells (rMAPCs) to SMCs and is associated with induction of a proliferative state of quiescent SMCs. The MAPC-SMC differentiation system may be useful for identification of additional critical (co-)regulators of SMC development.
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.
Project description:Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells with a high growth rate suitable for therapeutical applications as three-dimensional (3D) aggregates. Combined applications of osteogenically differentiated MAPC (OD-MAPC) aggregates and adeno-associated viral vectors (AAV) in bone bioengineering are still deferred until information with regard to expansion technologies, osteogenic potential, and AAV cytotoxicity and transduction efficiency is better understood. In this study, we tested whether self-complementary AAV (scAAV) can potentially be used as a gene delivery system in an OD-MAPC-based 'in vivo' bone formation model in the craniofacial region. Both expansion of rat MAPC (rMAPC) and osteogenic differentiation with dexamethasone were also tested in 3D aggregate culture systems 'in vitro' and 'vivo'. rMAPCs grew as undifferentiated aggregates for 4 days, with a population doubling time of 37?h. After expansion, constant levels of Oct4 transcripts, and Oct4 and CD31 surface markers were observed, which constitute a hallmark of undifferentiated stage of rMAPCs. Dexamethasone effectively mediated rMAPC osteogenic differentiation by inducing the formation of a mineralized collagen type I network, and facilitated the activation of the wnt/?-catenin, a crucial pathway in skeletal development. To investigate the genetic modification of rMAPCs grown as 3D aggregates before implantation, scAAV serotypes 2, 3 and 6 were evaluated. scAAV6 packaged with the enhanced green fluorescent protein expression cassette efficiently mediated long-term transduction (10 days) 'in vitro' and 'vivo'. The reporter transduction event allowed the tracing of OD-rMAPC (induced by dexamethasone) aggregates following OD-rMAPC transfer into a macro-porous hydroxyapatite scaffold implanted in a rat calvaria model. Furthermore, the scAAV6-transduced OD-rMAPCs generated a bone-like matrix with a collagenous matrix rich in bone-specific proteins (osteocalcin and osteopontin) in the scaffold macro-pores 10 days post-implantation. Newly formed bone was also observed in the interface between native bone and scaffold. The collective work supports future bone tissue engineering applications of 3D MAPC cultures for expansion, bone formation and the ability to alter genetically these cells using scAAV vectors.
Project description:Recently, our group has contrasted an endothelial cell-smooth muscle cell (EC-SMC) co-culture model with 3D-cultured SMCs and found that SMCs could respond to high shear stress (SS), which has not been explored before. SMCs were not directly exposed to the flow but were under an EC monolayer; therefore, it is necessary to explore the influence of EC on SMC behaviors under high SS for understanding the mechanism of SMC response to various magnitudes of SS. In the present study, TGF-?1 expression in ECs in an EC-SMC co-culture model was suppressed by an siRNA transfection method. Next, phenotypic changes were observed and MMP-2 and -9 productions were measured in SMCs in the co-culture model after 72-h flow exposure to different SS levels. We confirmed that TGF-?1 expression in ECs could influence SMC phenotypic change under SS conditions and that TGF-?1 expression in ECs could also change MMP-2 production but not MMP-9 production in SMCs under SS conditions in the co-culture model. These results could be useful for understanding the mechanisms of SMC response to SS, particularly for understanding signal transduction emanating from ECs.
Project description:Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-?1 (TGF?1), human umbilical cord-derived MSCs abundantly express the SMC markers ?-smooth muscle actin (?SMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGF?1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and ?SMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts.
Project description:Transforming growth factor-? (TGF-?) signaling is critical for the differentiation of smooth muscle cells (SMCs) into quiescent cells expressing a full repertoire of contractile proteins. Heterozygous mutations in TGF-? receptor type II (TGFBR2) disrupt TGF-? signaling and lead to genetic conditions that predispose to thoracic aortic aneurysms and dissections (TAADs). The aim of this study is to determine the molecular mechanism by which TGFBR2 mutations cause TAADs.Using aortic SMCs explanted from patients with TGFBR2 mutations, we show decreased expression of SMC contractile proteins compared with controls. Exposure to TGF-?1 fails to increase expression of contractile genes in mutant SMCs, whereas control cells further increase expression of these genes. Analysis of fixed and frozen aortas from patients with TGFBR2 mutations confirms decreased in vivo expression of contractile proteins relative to unaffected aortas. Fibroblasts explanted from patients with TGFBR2 mutations fail to transform into mature myofibroblasts with TGF-?1 stimulation as assessed by expression of contractile proteins.These data support the conclusion that heterozygous TGFBR2 mutations lead to decreased expression of SMC contractile protein in both SMCs and myofibroblasts. The failure of TGFBR2-mutant SMCs to fully express SMC contractile proteins predicts defective contractile function in these cells and aligns with a hypothesis that defective SMC contractile function contributes to the pathogenesis of TAAD.
Project description:Tissue-specific transcription factors initiate differentiation toward a specialized cell type by inducing transcription-permissive chromatin modifications at target gene promoters, through the recruitment of the SWI/SNF chromatin-remodeling complex (1, 2). The molecular mechanism that regulates the chromatin re-distribution of SWI/SNF in response to differentiation signals is currently unknown. Here we show that the muscle determination factor MyoD and the SWI/SNF structural sub-unit, BAF60c (SMARCD3), form a complex on the regulatory elements of MyoD-target genes in undifferentiated myoblasts, prior to the activation of gene expression. MyoD-BAF60c complex is devoid of the ATP-dependent enzymatic sub-units Brg1 and Brm, is required for stable MyoD binding to Ebox sequences, and marks the chromatin for signal-dependent recruitment of the SWI/SNF core complex to muscle loci. BAF60c phosphorylation on a conserved threonine by differentiation-activated p38 signalling promotes the incorporation of MyoD-BAF60c into a Brg1-based SWI/SNF complex, which is competent to remodel the chromatin and activates transcription of MyoD-target genes. Our data support an unprecedented two-step model, by which pre-assembled BAF60c-MyoD complex directs the SWI/SNF complex chromatin re-distribution to muscle loci in response to differentiation cues. Differentiation of C2C12 cells individually interfered for BRG1, BAF60B, BAF60C
Project description:Tissue-specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodelling complex. However, the molecular mechanism that regulates SWI/SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD-target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal-dependent recruitment of the SWI/SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation-activated p38? kinase is the signal that promotes incorporation of MyoD-BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Our data support an unprecedented two-step model by which pre-assembled BAF60c-MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues.
Project description:OBJECTIVE:Elastin gene deletion or mutation leads to arterial stenoses due to vascular smooth muscle cell (SMC) proliferation. Human induced pluripotent stem cells-derived SMCs can model the elastin insufficiency phenotype in vitro but show only partial rescue with rapamycin. Our objective was to identify drug candidates with superior efficacy in rescuing the SMC phenotype in elastin insufficiency patients. Approach and Results: SMCs generated from induced pluripotent stem cells from 5 elastin insufficiency patients with severe recurrent vascular stenoses (3 Williams syndrome and 2 elastin mutations) were phenotypically immature, hyperproliferative, poorly responsive to endothelin, and exerted reduced tension in 3-dimensional smooth muscle biowires. Elastin mRNA and protein were reduced in SMCs from patients compared to healthy control SMCs. Fourteen drug candidates were tested on patient SMCs. Of the mammalian target of rapamycin inhibitors studied, everolimus restored differentiation, rescued proliferation, and improved endothelin-induced calcium flux in all patient SMCs except one Williams syndrome. Of the calcium channel blockers, verapamil increased SMC differentiation and reduced proliferation in Williams syndrome patient cells but not in elastin mutation patients and had no effect on endothelin response. Combination treatment with everolimus and verapamil was not superior to everolimus alone. Other drug candidates had limited efficacy. CONCLUSIONS:Everolimus caused the most consistent improvement in SMC differentiation, proliferation and in SMC function in patients with both syndromic and nonsyndromic elastin insufficiency, and offers the best candidate for drug repurposing for treatment of elastin insufficiency associated vasculopathy.
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:Although soluble factors, such as transforming growth factor ?1 (TGF-?1), induce mesenchymal stem cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage, the role of adherens junctions in this process is not well understood. In this study, we found that cadherin-11 but not cadherin-2 was necessary for MSC differentiation into SMCs. Cadherin-11 regulated the expression of TGF-?1 and affected SMC differentiation through a pathway that was dependent on TGF-? receptor II (TGF?RII) but independent of SMAD2 or SMAD3. In addition, cadherin-11 activated the expression of serum response factor (SRF) and SMC proteins through the Rho-associated protein kinase (ROCK) pathway. Engagement of cadherin-11 increased its own expression through SRF, indicative of the presence of an autoregulatory feedback loop that committed MSCs to the SMC fate. Notably, SMC-containing tissues (such as aorta and bladder) from cadherin-11-null (Cdh11(-/-)) mice showed significantly reduced levels of SMC proteins and exhibited diminished contractility compared with controls. This is the first report implicating cadherin-11 in SMC differentiation and contractile function in vitro as well as in vivo.