Mechanically induced osteogenic differentiation – the role of RhoA, ROCKII and cytoskeletal dynamics

TLDR

The role of the local mechanical environment on stem cell fate has only recently been investigated, whereas biochemical factors have been extensively studied. The study examined whether oscillatory fluid flow regulates lineage differentiation of C3H10T1/2 mesenchymal stem cells and hypothesized that RhoA and actin tension are essential for flow‑induced differentiation. They measured Runx2, PPARγ, and SOX9 gene expression to assess osteogenic, adipogenic, and chondrogenic differentiation under oscillatory fluid flow. Oscillatory fluid flow upregulated Runx2, Sox9, and PPARγ, and RhoA/ROCKII activity promoted osteogenic differentiation while RhoA activation and actin tension suppressed adipogenic and chondrogenic pathways, with an intact dynamic actin cytoskeleton required for flow‑induced gene expression.

Abstract

Many biochemical factors regulating progenitor cell differentiation have been examined in detail; however, the role of the local mechanical environment on stem cell fate has only recently been investigated. In this study, we examined whether oscillatory fluid flow, an exogenous mechanical signal within bone, regulates osteogenic, adipogenic or chondrogenic differentiation of C3H10T1/2 murine mesenchymal stem cells by measuring Runx2, PPARgamma and SOX9 gene expression, respectively. Furthermore, we hypothesized that the small GTPase RhoA and isometric tension within the actin cytoskeleton are essential in flow-induced differentiation. We found that oscillatory fluid flow induces the upregulation of Runx2, Sox9 and PPARgamma, indicating that it has the potential to regulate transcription factors involved in multiple unique lineage pathways. Furthermore, we demonstrate that the small GTPase RhoA and its effector protein ROCKII regulate fluid-flow-induced osteogenic differentiation. Additionally, activated RhoA and fluid flow have an additive effect on Runx2 expression. Finally, we show RhoA activation and actin tension are negative regulators of both adipogenic and chondrogenic differentiation. However, an intact, dynamic actin cytoskeleton under tension is necessary for flow-induced gene expression.