RhoA/ROCK Signaling Regulates Sox9 Expression and Actin Organization during Chondrogenesis

Guoyan Wang,Anita Woods,Frank Beier

doi:10.1074/jbc.m409158200

Abstract

Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes (chondrogenesis). This process is characterized by a strong interdependence of cell shape, cytoskeletal organization, and the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. Here we investigated the role of the RhoA/ROCK pathway, a well characterized regulator of cytoskeletal organization, in chondrogenesis. We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects. Suppression of Sox9 expression by ROCK signaling was achieved through repression of Sox9 promoter activity. These molecular changes were accompanied by reorganization of the actin cytoskeleton, where RhoA/ROCK signaling suppressed cortical actin organization, a hallmark of differentiated chondrocytes. This led us to analyze the regulation of Sox9 expression by drugs affecting cytoskeletal dynamics. Both inhibition of actin polymerization by cytochalasin D and stabilization of existing actin filaments by jasplakinolide resulted in increased Sox9 mRNA levels, whereas inhibition of microtubule polymerization by colchicine completely blocked Sox9 expression. In conclusion, our data suggest that RhoA/ROCK signaling suppresses chondrogenesis through the control of Sox9 expression and actin organization.

Highlights

Chondrocytes fulfill two major roles in mammals
We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects
The number of Alcian blue-stained nodules at this time point was not affected by Y27632 (Fig. 2c). The size of these nodules does not appear to change between the control and treated cultures. These data suggest that ROCK inhibition does not affect cell condensation but results in increased chondrogenic differentiation and chondrocyte-specific extracellular matrix synthesis of mesenchymal precursor cells within nodules

Summary

Introduction

Chondrocytes fulfill two major roles in mammals. During development, most of our bones form through endochondral ossification in which bones are first laid down as cartilage precursors [1, 2]. We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects. Our data suggest that RhoA/ROCK signaling suppresses chondrogenesis through the control of Sox9 expression and actin organization.

Results

Conclusion