Hyväri Laura, Vanhatupa Sari, Halonen Heidi T, Kääriäinen Minna, Miettinen Susanna
Adult Stem Cell Group, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.
Research, Development and Innovation Centre, Tampere University Hospital, Tampere, Finland.
Stem Cells Int. 2020 Sep 22;2020:8853541. doi: 10.1155/2020/8853541. eCollection 2020.
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 -smooth muscle actin (-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.
先前的研究表明，心肌相关转录因子A（MRTF-A）与其共调节因子血清反应因子（SRF）共同建立了肌动蛋白细胞骨架动力学与基因表达之间的联系。MRTF-A也被认为是干细胞分化的调节因子。然而，MRTF-A在人骨髓间充质干细胞分化中的作用仍未得到充分研究。我们旨在阐明MRTF-A是否是人类脂肪干细胞（hASC）向脂肪生成和成骨谱系分化的潜在调节因子。为了研究MRTF-A活性在分化过程中的作用，将hASC在添加了抑制剂分子CCG-1423或CCG-100602的脂肪生成和成骨培养基中培养，这些抑制剂已被证明可阻断MRTF-A/SRF激活基因的表达。我们基于图像对油红O染色的脂滴和 perilipin 1染色进行定量分析的结果表明，抑制MRTF-A可增强脂肪生成分化。相反，抑制MRTF-A会导致早期成骨标志物碱性磷酸酶的活性降低，以及细胞外基质（ECM）蛋白I型胶原蛋白和骨桥蛋白的分泌减少。此外，在抑制MRTF-A的情况下，代表ECM矿化的茜素红定量染色显著降低。基于图像的鬼笔环肽染色分析表明，抑制MRTF-A可减少F-肌动蛋白的形成以及肌动蛋白丝的平行排列。此外，抑制MRTF-A会影响α-平滑肌肌动蛋白（α-SMA）、肌球蛋白轻链（MLC）和磷酸化MLC的蛋白量，这表明MRTF-A可能通过SRF活性调节分化。我们的结果强烈表明，MRTF-A通过介导细胞骨架动力学，是hASC成骨和脂肪生成平衡的重要调节因子。这些结果为MRTF-A作为再生医学组织工程应用中指导干细胞分化的一个新的有趣靶点提供了依据。
