Effects Of Histone Modifications And SWI/SNF Chromatin Remodeling Complex On BMSCs Differentiating To SMCs

Background and aimsAdult stem cells(ASCs)could be induced in vitro to differentiate into smooth muscle cells(SMCs) by several factors including cytokines stimulation, genetic modification and mechanical stretch. Indirect contact co-culture is an easy and economic method to induce the committed differentiation of stem cells. However, whether ASCs could be induced to differentiate into SMCs via the indirect contact co-culture has being kept inconsistent to date. Bone marrow mesenchymal stem cells(BMSCs) are considered an ideal transplantation seed due to the easy obtaining, no ethical concerns and little immunogenicity. Thus, we aimed to identify whether the co-culture system could induce BMSCs to differentiate into SMCs in vitro to provide a novel strategy to induce stem cell differentiation.Low differentiation efficiency has impeded the application of stem cell transplantation. Sodium butyrate(NaB) is an inhibitor of histone deacetylase(HDACi) and can induce mouse embryonic stem cells(ESCs) differentiating to pancreatic and hepatic progenitor cells. However, whether NaB could induce BMSCs differentiating to SMCs kept unknown. Here, we aimed to establish an effective way to promote BMSCs to SMCs via the effects of NaB combining co-culture strategy, which will provide experimental data for the clinical application of stem cell transplantation.SMCs specific genes expression in BMSCs is considered to be the marker to indicate that BMSCs have differentiated to SMCs. However, whether and how these genes expression is controlled in the process kept unknown. Serum response factor(SRF), a transcription factor(TF) of SMC specific genes, could combine the CArG Box in DNA sequence to activate these genes transcription, which is regulated by epigenetic regulation. Histone methylation and acetylation could loose the condensed chromatin construction to make TF combining DNA sequence easily. Some researches have proved that histone specific site methylation and acetylation could adjust SMCs marker gene expression. Thus, we aimed to explore what the effect of histone specific site methylation and acetylation on the differentiation of SMCs from BMSCs pretreated with or without NaB treatment. Dysfunction of the differentiated stem cells was another roadblock of stem cell transplantation. Here, we evaluated the contraction function of differentiated BMSCs and aimed to provide experimental data for the application of stem cell transplantation.ATP-dependent chromatin remodeling complexes could loose the chromatin construction depending on the energy of ATP hydrolysis and make TF to recognize the specific DNA sequence. BRG1/BRM was the core subunit of SWI/SNF complexes and involved in SMCs marker genes expression during SMC differentiation. However, the mechanism that BRG1/BRM regulated SMCs specific genes expression kept unknown. Thus, we aimed to explore the mechanism that BRG1/BRM regulated SMCs marker genes transcription and supply the reference for other stem cells differentiation.Methods1 BMSCs could differentiate to SMCs in vitro in a non-cell-cell contact manner.Four-week-old female Sprague Dawley(SD) rats were sacrificed by neck-breaking, BMSCs were aseptically isolated from the femurs and tibias and SMCs were harvested from the bladder. BMSCs were cultured in DMEM/F12 medium with 10% FBS, SMCs were in DMEM-HG medium with 10% FBS and the third or fourth passage cells were used in the experiment. Flow cytometry(FCM) assay determined the surface marker expression of BMSCs and confocal laser scanning microscope(CLSM) assay identified the level of α-SMC, Calponin and SM-MHC proteins in SMCs. SMCs were cultured in the chamber outsert and BMSCs were seeded in the insert. BMSCs were then harvested at the different time points for further analysis. Finally, quantity reverse transcription-polymerase chain reaction(q-PCR) was used to measure the expression level of SMCs specific genes in the co-cultured BMSCs at different time points.2 Mechanisms of histone modifications in NaB promoting BMSCs differentiating to SMCs.To explore the effects of NaB on BMSCs proliferation, Cell Counting Kit-8(CCK8) was used to measure the proliferation of BMSCs treated with different concentration of NaB at 24 h, 48 h and 72 h. To reveal what effect of NaB on the SMCs differentiation from BMSCs, q-PCR was employed to test the expression level of SMCs specific genes in BMSCs treated with different concentration of Na B for 48 h. Furthermore, CLSM assay determined the level of SMCs specific genes protein in BMSCs treated with NaB. To establish an effective method to improve BMSCs differentiating to SMCs, we treated BMSCs with different concentration of NaB for 48 h, and then co-cultured with SMCs and finally harvested BMSCs at different time points. Q-PCR was used to investigate the expression level of SMC specific genes. Furthermore, Western blot(WB) and CLSM assay determined the level of these proteins in BMSCs.To explored the effects of histone methylation and acetylation on the differentiation of BMSCs to SMCs, chromatin immunoprecipitation quantitative PCR(ChIP-qPCR) was used to investigate the enrichment of H3K43 met, H3K27 met, H3K9 ace, H3 ace and H4 ace at the promoter of SMCs marker genes. Briefly, 1×107 cells were fixed with 1% formaldehyde for 10 min at room temperature. The cross-linked DNA was sheared to 200-500 bp in length by sonication. ChIP grade antibodies were incubated with each immunoprecipitation reaction at 4°C overnight. Normal rat IgG was used as a negative control. After reversing the crosslinks, the DNA was purified and analyzed using qPCR. Next, we employed CLSM assay to compare the difference of HDAC1 and HDAC2 in BMSCs with or without NaB treatment and ChIP-qPCR to the enrichment of HDAC1 and HDAC2 at the promoter of SMCs specific genes. Finally, to explore whether histone acetylation regulated SMCs marker genes expression through serum response factor(SRF), we used ChIP-qPCR to investigate SRF recruitment at these genes in the co-cultured BMSCs pretreated with or without NaB.To evaluate the contraction function of the differentiated BMSCs, we loaded the differentiated BMSCs with Fluo-8/AM and CLSM assay measured the intracellular Ca2+ changes in these samples. Furthermore, we suspended these BMSCs in the rat tail collagen and observed the collagen contraction area.3 Effects of SWI/SNF chromatin remodeling complex on BMSCs differentiating to SMCs.We designed siRNA(small interfering RNA) to observe the effects of BRG1/BRM on the SMCs specific genes expression during the differentiation of SMCs from BMSCs. Firstly, three duplexed siRNA targeting BRG1 and BRM corresponding to the respective coding regions were designed and synthesized. Transfections were carried out using LipofectamineTM 2000 according to the manufacturer instruction. To choose the best duplexed siRNA, q-PCR was used to measure BRG1 and BRM expression level in BMSCs transfected with siBRG1 or siBRM. Secondly, to explore what the effect of BRG1 and BRM on the BMSCs differentiation to SMCs, q-PCR and CLSM were used to compare α-SMA and SM-MHC genes expression in the co-cultured BMSCs transfected with or without siBRG1 or si BRM. Thirdly, to identify whether BRM could combine H3K9 ace, H3 ace and H4 ace to regulated the SMCs specific genes expression, CLSM were used to compared H3K9 ace, H3 ace and H4 ace protein level and ChIP-qPCR to the recruitmentof H3K9 ace, H3 ace and H4 ace at the promoter of SMCs-specific genes in the co-cutured BMSCs with or without siBRM. Finally, to identify whether BRM combining histone acetylation modifications regulated SMCs-specific genes expression throuth SRF during BMSCs to SMCs, ChIP-qPCR was used to test SRF enrichment at the promoter of SMCs marker genes in the co-cultured BMSCs with or without siBRM.Results1 BMSCs could differentiate to SMCs in vitro in a non-cell-cell contact manner.Primary SMCs were polygon and had many vacuoles in the cytoplasm. BMSC were positive for CD29 expression and negative for CD31, CD34 and CD45. The level of SMCs-specific genes expression such as α-SMA, Calponin and SM-MHC in the co-cultured BMSCs increased significantly, which implied that BMSCs could be induced to differentiate to SMCs through co-culturing with SMCs in the transwell chamber.2 Mechanisms of histone modifications in NaB promoting BMSCs differentiating to SMCs.(1) CCK8 analysis results showed that 1.0 mmol/L and 1.5 mmol/L NaB could remarkably inhibit BMSCs proliferation. q-PCR assay exhibited that 1.0mmol/L NaB promoted significantly the expression level of α-SMA, Calponin and SM-MHC in BMSCs.(2) q-PCR and WB analysis results showed that 1.0mmol/L NaB could improve the expression levels of SMC specific genes in the co-cultured BMSCs and the peak level for each target gene was reached after 2 days of co-culturing when the cells were pretreated with 1.0 mmol/L NaB. CLSM assay indicated that 1.0 mmol/L NaB remarkably increased the expression of SMC specific genes in co-cultural BMSCs.(3) H3K43 met enrichment at α-SMC increased in co-cultural BMSCs pretreated with NaB, despite increasing in the normal co-culture system, which implied that NaB might improve α-SMA level via H3K43 met. The enrichment of H3K43 met and H3K27 met at Calponin and SM-MHC decreased in BMSCs co-cultured with SMCs and reduced even more in the co-cultured BMSCs pretreated with NaB, which implied that H3K43 met and H3K27 met might suppress Calponin and SM-MHC expression during SMC from BMSCs treated with or without NaB. Results showed that H3K9 ace and H4 ace enrichment at α-SMA、Calponin and SM-MHC increased in the co-cultured BMSCs and improved even more after treating with NaB, which implied that NaB promoted SMCs-specific genes expression during BMSCs to SMCs via H3K9 aceand H4ace. The enrichment of H3 ace at SMCs-specific genes increased significantly in BMSCs co-cultured with SMCs and decreased after treating with NaB.(4) To explore whether H3K9 ace and H4 ace were adjusted by HDAC1 and HDAC2, we employed CLSM and WB assays to compare the level of HDAC1 and HDAC2 protein in BMSCs pretreated with or without NaB. Results showed that HDAC1 increased and HDAC2 decreased in BMSCs treated with NaB, which implied that NaB might increase HDAC1 expression level and decreased HDAC2 during BMSCs to SMCs. To further reveal the relationship between the change of HDAC expression and the SMCs-specific genes transcription, we used ChIP-qPCR to test the enrichment of HDAC1 and HDAC2 at the promoter of SMCs-specific genes in BMSCs with or without NaB treatment. Results showed that HDAC1 enrichment increased and HDAC2 decreased significantly at the promoter of these genes in BMSCs treated with NaB, which implied that NaB might increase H3K9 ace and H4 ace during BMSCs to SMCs through decreasing HDAC2 recruitment to the promoters of these genes.(5) To test whether SMCs specific genes expression that regulated by histone acetylation were triggered by SRF, we used CLSM assay to measure SRF in the co-cultured BMSCs pretreated with or without NaB. Results showed SRF recruitment increased remarkably in the differentiated BMSCs with NaB treatment. To further reveal the relationship between SRF and SMCs-specific expression, we used ChIP-qPCR to test SRF enrichment at the promoter of SMCs-specific genes in the co-cultured BMSCs pretreated with or without NaB and results showed that SRF enrichment increased significantly remarkably in co-cultured BMSCs pretreated with NaB, which implied that histone acetylation might promote the SMCs-specific genes expression via SRF during NaB promoting BMSCs to SMCs.(6) We loaded the differentiated BMSCs with Fluo-8/AM and employed CLSM to measure the intracellular Ca2+ changes in these samples. Results showed that the intracellular Ca2+ level of differentiated MSCs increased significantly upon carbachol stimulation. Furthermore, we suspended BMSCs in the rat tail collagen and observed the collagen contraction area. Results showed that the collagen area containing the differentiated BMSCs decreased significantly. These results indicated that NaB-induced differentiated MSCs displayed the appropriate contractile function.3 Effects of SWI/SNF chromatin remodeling complex on BMSCs differentiating to SMCs.(1) To reveal what the effect of BRG1/BRM on the SMCs-specific gene expression in SMCs differentiated from BMSCs, we used q-PCR to test the expression level of α-SMC and SM-MHC in the co-cultured BMSCs transfected with siBRG1 or siBRM. Results showed that these genes expression level decreased significantly in the co-cultured BMSCs with transfected siBRM. However, α-SMC and SM-MHC expression level did not changed in the BMSCs with siBRG1. Furthermore, CLSM assay results showed that α-SMA and SM-MHC protein reduced remarkably in the co-cultured BMSCs with siBRM. These results indicated that it was BRM but not BRG1 that could promote the SMCs-specific gene expression during BMSCs to SMCs.(2) To explore whether BRM promote SMCs-specific genes expression via SRF and Myocardin during BMSCs to SMCs, CLSM assay was used to test SRF and myocardin protein in co-cultured BMSCs with or without siBRM. Results showed that SRF and Myocardin decreased in BMSCs transfected with siBRM. To further reveal the relationship between SRF and SMCs-specific genes expression, we employed ChIP-qPCR to measure the enrichment of SRF at the promoter of α-SMA and SM-MHC in co-cultured BMSCs interfered with or without siBRM. Results showed that SRF enrichment at α-SMA and SM-MHC decreased significantly in co-cultured BMSCs with siBRM, which implied that SRF might promote SMCs-specific genes expression during BMSCs to SMCs.(3) To reveal whether BRM combining histone acetylation regulated the expression of SMCs-specific genes, we used ChIP-qPCR to compare the enrichment of H3K9 ace, H3 ace and H4 ace at the promoter of α-SMA and SM-MHC in co-cultured BMSCs interfered with or without siBRM. Results showed that the enrichment of H3K9 ace, H3 ace and H4 ace at SMCs-specific genes reduced in co-cultured BMSCs interfered with siBRM, which implied that BRM might combine H3K9 ace, H3 ace and H4 ace to promote SMCs-specific genes expression during BMSCs to SMCs.Conclusions1. BMSCs could be induced to differentiate into SMCs in vitro, in a non-cell-cell contact manner.2. BMSCs could differentiate to SMCs in the co-cultured system at 4d. Furthermore, SMCs-specific genes expression level could be promoted remarkably in the co-cultured BMSCs treated with 1.0 mmol/L NaB at 2 day and the contraction function of the differentiated BMSCs could be enhanced greatly.3. H3K43 met and H3K27 met might suppress SMC-specific genes expression in co-cultured BMSCs. However, H3K9 ace, H3 ace and H4 ace might promote these genes expression in BMSCs, co-cultured with SMCs. NaB-mediated decrease of HDAC2 expression and its recruitment to SMC specific genes in MSCs, which further induces high levels of H3K9 ace and H4 ace and increased significantly SRF-dependent SMCs marker genes expression.4. It was BRM but not BRG1 that could increase significantly SMCs-marker genes expression in BMSCs co-culturing with SMCs. BRM might combine H3K9 ace, H3 aceand H4ace to improve SRF-dependent SMCs marker genes expression.In summary, our results established a non-cell-cell contact co-culture model to induce BMSCs differentiating to SMCs. Furthermore, we improved the efficiency and enhanced the contraction function of the co-cultured BMSCs pretreated with NaB. Furthermore, we revealed that NaB increased H3K9 ace and H4 ace during BMSCs to SMCs through decreasing HDAC2 recruitment to the promoters of the SRF-dependent SMCs-specific genes. Simultaneously, it was BRM but not BRG1 that might combine H3K9 ace, H3 ace and H4 ace, promoted SRF, Myocardin-dependent SMCs-specific genes expression during BMSCs to SMCs. Our research established the method that promoted stem cell differentiation and revealed the effect of epigenetic regulation on SMCs-specific genes expression and supplied experiment data for stem cell transplantion treating SUI and other defect of tissue and organ diseases.