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

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, PPARγ 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 PPARγ, 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 fluidflow-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.

Geometric cues for directing the differentiation of mesenchymal stem cells

“…In both cases of normal media and mixed media, microarray analysis reveals differential gene expression for transcripts associated with MAP kinase pathways and Wnt signaling suggesting a role for secreted factors. In particular, we find that cells cultured on star shapes in growth media promote expression of noncanonical Wnt/Fzd signaling molecules (including downstream effectors RhoA and ROCK previously shown to be involved in osteogenesis (23,38)). Moreover, when exposed to mixed media the gene expression of tension specific MAP kinases (p38, ERK1/2, JNK) and both canonical and noncanonical Wnt transcripts is increased for cells on star shapes relative to those on flower shapes and on unpatterned surfaces.…”

“…Previous work has demonstrated the importance of Wnt signaling in regulating actomyosin contractility (37). Furthermore, high levels of the noncanonical Wnt downstream effector ROCK has been shown to trigger osteogenesis through a myosin-generated tension feedback loop (23,38). It is therefore reasonable to suggest that contractile cells may be poised in a state of higher susceptibility to soluble factors including autocrine/paracrine Wnt signals as well as osteogenic media supplements.…”

Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

“…111,[136][137][138][139] The effect of actin cytoskeletal tension on commitment to a stem cell lineage has been best characterized for MSCs in many studies, 140 including those using the high-content image-based approach for the prediction of a lineage fate of stem cells. 141,142 As summarized in Table 1, osteogenic lineage commitment and inhibition of differentiation into other lineage types require actin cytoskeletal tension mediated by RhoA and ROCK in human, 61,106,[143][144][145] mouse, 146 and rat 132 MSCs. In line with this observation, human MSCs on grooves or pits 300-500 nm deep and 5-50 mm wide (Fig.…”

Influence of Shear Stress in Perfusion Bioreactor Cultures for the Development of Three-Dimensional Bone Tissue Constructs: A Review

“…A wealth of literature exists for nonhuman 70 70 and peroxisome proliferator activated receptor gamma (PPARg) 70 showed upregulation of gene or protein expression as a consequence of shear stress application, whereas its influence was negative or even suppressive for expression of BMP4, 77 COX1, 90 and transforming growth factor-b 1 . 77 The variation in cell types used, culture conditions, and experimental platforms has increased the complexity of prizing out the importance of the role of fluid shear stress; nevertheless, it appears that a range of 0.1-0.5 Pa is most successful, and in some instances, values as high as 2 Pa have yielded positive responses, showing a good correlation with the levels of shear stress expected to occur in vivo (0.8-3 Pa).…”