Abstract
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.
Highlights
The actin cytoskeleton of a cell is continuously modified to allow dynamic cell functions such as stem cell differentiation [1, 2]
Our study aimed to investigate the role of myocardin-related transcription factor A (MRTF-A) coregulator in guiding the differentiation commitment of human adipose stem cells
We discovered that MRTF-A inhibition significantly decreased the coherency of actin orientation of human adipose stem cell (hASC) in basic culture medium (BM) condition, and a similar trend was seen in the intensity of Phalloidin staining representing F-actin
Summary
The actin cytoskeleton of a cell is continuously modified to allow dynamic cell functions such as stem cell differentiation [1, 2]. Actin dynamics are accomplished by continuous actin turnover and treadmilling regulated by Rho GTPase RhoARho-associated coiled-coil kinase (Rho-ROCK) pathway [3, 4]. RhoA-ROCK pathway has been reported to regulate the fate decision of mesenchymal stem cells (MSCs) [1, 2, 5]. The role of ROCK downstream effector myocardin-related transcription factor A (MRTF-A) in the regulation of MSC differentiation remains less studied. MRTF-A, known as megakaryocyte acute leukemia protein (MAL) and megakaryoblastic leukemia (MKL1), belongs to the myocardin family [6, 7] and is found in numerous embryonic and adult tissues [8]. MRTF-A generates a unique link between actin dynamics and gene expression because the activity of MRTF-A is controlled by the balance between
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