Differentiation Potential of the Human Adipose Stem Cells in Response to Regulation of ROCK, FAK and MEK-ERK Signaling Pathways

Abstract

Background and aims: Adipose stem cells (ASCs), obtained from adipose tissue, are a promising cell source for bone tissue engineering applications. ASCs exhibit stable growth and proliferation in vitro and they possess multi-lineage differentiation capacity into various cell lineages including adipocytes, osteoblasts and chondrocytes. The differentiation process of mesenchymal stem cells (MSC) is known to be regulated through cell attachment and morphology, however, the intracellular details of the regulation remain unidentified. The aim of this study was to enlighten the signaling events in cell attachment or morphology -mediated differentiation in hASCs. The significance of Rho-kinase ROCK), Focal adhesion kinase (FAK) and Mitogen-activated protein kinase/Extracellular signal-regulated kinase (MEK-ERK) signaling to hASC differentiation potential towards osteoblasts and adipocytes were analyzed.

Methods: To assess the significance of ROCK, FAK and MEK-ERK signaling pathways to hASC differentiation, specific inhibitor molecules targeted to these cascades were used: ROCK1 inhibitor Y-27632 2HCl, FAK inhibitor PF-562271 and MEK inhibitor PD98059. The inhibitor effect on cell viability and proliferation were assessed with LIVE/DEAD® assay and CyQUANT® assay, respectively. The hASC adipogenic differentiation was determined by Oil Red O staining, and the osteogenic differentiation was assessed by quantitative alkaline phosphatase assay (qALP), and qualitative and quantitative Alizarin Red staining. The intracellular protein activation was evaluated with Western Blot analysis method.

Results: The results indicated that cell attachment to the culture platform and to the extracellular matrix (ECM) is critical for cell viability, proliferation and induction of the osteogenic differentiation of hASCs. Disruption of the cell adhesion with FAK or ROCK inhibition suppressed the osteogenic differentiation. Cell morphology guided hASC lineage commitment: spread cytoskeleton induced osteogenesis while rounded shape favoured commitment to adipogenesis. ROCK was found to be a positive regulator of osteogenesis and the inhibition of ROCK enhanced adipogenesis in the hASC lines studied. Also functional MEK-ERK pathway was required for various intracellular processes regulating cell proliferation and differentiation in hASCs.

Conclusions: Taken together, the cell adhesion is an essential part and a prerequisite of many intracellular functions in hASC differentiation. Based on this study, ROCK, FAK and MEK-ERK signaling cascades were involved in the osteogenic differentiation of hASCs. An active ROCK protein worked as a negative regulator of adipogenesis, representing the influence of cell morphology to the differentiation potential of hASCs. The results indicate, that the differentiation mechanisms of hASCs could be supported by enhancing the cell adhesion and regulating the morphology of the cells.