Previous studies have demonstrated that myocardin-related transcription factor A (MRTF-A) generates a link between the dynamics of the actin cytoskeleton and gene expression with its coregulator, serum response factor (SRF). MRTF-A has also been suggested as a regulator of stem cell differentiation. However, the role of MRTF-A in human mesenchymal stem cell differentiation remains understudied. We aimed to elucidate whether MRTF-A is a potential regulator of human adipose stem cell (hASC) differentiation towards adipogenic and osteogenic lineages. To study the role of MRTF-A activity in the differentiation process, hASCs were cultured in adipogenic and osteogenic media supplemented with inhibitor molecules CCG-1423 or CCG-100602 that have been shown to block the expression of MRTF-A/SRF-activated genes. Our results of image-based quantification of Oil Red O stained lipid droplets and perilipin 1 staining denote that MRTF-A inhibition enhanced the adipogenic differentiation. On the contrary, MRTF-A inhibition led to diminished activity of an early osteogenic marker alkaline phosphatase, and export of extracellular matrix (ECM) proteins collagen type I and osteopontin. Also, quantitative Alizarin Red staining representing ECM mineralization was significantly decreased under MRTF-A inhibition. Image-based analysis of Phalloidin staining revealed that MRTF-A inhibition reduced the F-actin formation and parallel orientation of the actin filaments. Additionally, MRTF-A inhibition affected the protein amounts of α-smooth muscle actin (α-SMA), myosin light chain (MLC), and phosphorylated MLC suggesting that MRTF-A would regulate differentiation through SRF activity. Our results strongly indicate that MRTF-A is an important regulator of the balance between osteogenesis and adipogenesis of hASCs through its role in mediating the cytoskeletal dynamics. These results provide MRTF-A as a new interesting target for guiding the stem cell differentiation in tissue engineering applications for regenerative medicine.

中文翻译：

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心肌相关转录因子A（MRTF-A）调节人脂肪干细胞成脂与成骨之间的平衡

以前的研究表明，心肌相关转录因子A（MRTF-A）与肌动蛋白细胞骨架的动力学和基因表达之间存在联系，其核心调节因子为血清反应因子（SRF）。MRTF-A也被建议作为干细胞分化的调节剂。但是，MRTF-A在人类间充质干细胞分化中的作用仍未得到研究。我们旨在阐明MRTF-A是否是人类脂肪干细胞（hASC）向成脂和成骨谱系分化的潜在调节剂。为了研究MRTF-A活性在分化过程中的作用，将hASCs在添加有抑制剂分子CCG-1423或CCG-100602的成脂和成骨培养基中进行培养，这些抑制剂分子已被证明可阻断MRTF-A / SRF激活基因的表达。我们基于图像的油红O染色脂质滴和perilipin 1染色定量结果表明，MRTF-A抑制作用增强了脂肪形成分化。相反，MRTF-A抑制导致早期成骨标记碱性磷酸酶的活性降低，并输出I型胶原和骨桥蛋白的细胞外基质（ECM）蛋白。此外，在MRTF-A抑制下，代表ECM矿化的定量茜素红染色显着降低。基于鬼笔环肽染色的图像分析表明，MRTF-A抑制作用可降低肌动蛋白丝的F-肌动蛋白形成和平行方向。此外，MRTF-A抑制作用影响了蛋白质的含量 相反，MRTF-A抑制导致早期成骨标记碱性磷酸酶的活性降低，并输出I型胶原和骨桥蛋白的细胞外基质（ECM）蛋白。此外，在MRTF-A抑制下，代表ECM矿化的定量茜素红染色显着降低。基于鬼笔环肽染色的图像分析表明，MRTF-A抑制作用可降低肌动蛋白丝的F-肌动蛋白形成和平行方向。此外，MRTF-A抑制作用影响了蛋白质的含量 相反，MRTF-A抑制导致早期成骨标记碱性磷酸酶的活性降低，并输出I型胶原和骨桥蛋白的细胞外基质（ECM）蛋白。此外，在MRTF-A抑制下，代表ECM矿化的定量茜素红染色显着降低。基于鬼笔环肽染色的图像分析表明，MRTF-A抑制作用可降低肌动蛋白丝的F-肌动蛋白形成和平行方向。此外，MRTF-A抑制作用影响了 基于鬼笔环肽染色的图像分析表明，MRTF-A抑制作用可降低肌动蛋白丝的F-肌动蛋白形成和平行方向。此外，MRTF-A抑制作用影响了蛋白质的含量 基于鬼笔环肽染色的图像分析表明，MRTF-A抑制作用可减少F-肌动蛋白的形成和肌动蛋白丝的平行方向。此外，MRTF-A抑制作用影响了蛋白质的含量α-平滑肌肌动蛋白（α- SMA），肌球蛋白轻链（MLC）和磷酸化的MLC提示MRTF-A将通过SRF活性调节分化。我们的研究结果强烈表明，MRTF-A通过介导细胞骨架动力学，是hASC的成骨与脂肪形成之间平衡的重要调节剂。这些结果提供了MRTF-A作为新的有趣靶标，用于指导再生医学组织工程应用中的干细胞分化。