RhoA/ROCK Signaling Suppresses Hypertrophic Chondrocyte Differentiation

Abstract

Coordinated proliferation and differentiation of growth plate chondrocytes is required for normal growth and development of the endochondral skeleton, but little is known about the intracellular signal transduction pathways regulating these processes. We have investigated the roles of the GTPase RhoA and its effector kinases ROCK1/2 in hypertrophic chondrocyte differentiation. RhoA, ROCK1, and ROCK2 are expressed throughout chondrogenic differentiation. RhoA overexpression in chondrogenic ATDC5 cells results in increased proliferation and a marked delay of hypertrophic differentiation, as shown by decreased induction of alkaline phosphatase activity, mineralization, and expression of the hypertrophic markers collagen X, bone sialoprotein, and matrix metalloproteinase 13. These effects are accompanied by activation of cyclin D1 transcription and repression of the collagen X promoter by RhoA. In contrast, inhibition of Rho/ROCK signaling by the pharmacological inhibitor Y27632 inhibits chondrocyte proliferation and accelerates hypertrophic differentiation. Dominant-negative RhoA also inhibits induction of the cyclin D1 promoter by parathyroid hormone-related peptide. Finally, Y27632 treatment partially rescues the effects of RhoA overexpression. In summary, we identify the RhoA/ROCK signaling pathway as a novel and important regulator of chondrocyte proliferation and differentiation.The development and growth of endochondral bones (such as ribs, vertebrae, and the long bones of vertebrate limbs) are regulated through the highly controlled rates of proliferation and hypertrophic differentiation of growth plate chondrocytes (1-3). In the growth plate, chondrocytes first undergo a series of cell divisions along the longitudinal axis of the growing bone, thereby forming characteristic columns of clonal cells. Chondrocytes then withdraw from the cell cycle and begin to increase their cell volume until reaching the fully differentiated state of hypertrophic chondrocytes. Transition from a proliferating to a hypertrophic phenotype involves numerous changes in gene expression, for example the induction of the collagen X (4, 5), matrix metalloproteinase 13 (6, 7), and bone sialoprotein (BSP) 1 (8 -10) genes. The latter two genes are also expressed by osteoblasts, suggesting similar biological properties of hypertrophic chondrocytes and osteoblasts. The fate of hypertrophic chondrocytes is still debated, but it appears that the majority of these cells undergo apoptosis and are replaced by bone tissue (11). Longitudinal growth of endochondral bones therefore requires both proliferation and differentiation-associated hypertrophy of growth plate chondrocytes.Disruption of chondrocyte proliferation and/or differentiation by gene mutations commonly results in chondrodysplasias that are characterized by skeletal deformities and reduced growth (12)(13)(14). Mutations in genes encoding extracellular matrix molecules, growth factors, receptors, and transcription factors have been identified as causes of several chondrodysplasias. Fo...

Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

“…Overexpression of RhoA inhibits Sox9 expression, stimulates cyclin D1 activation, and suppresses hypertrophy in ATDC5 cells. [16,17,34] However, in micromass limb bud cells, RhoA induces Sox9 expression and early chondrocyte differentiation. We do not see an increase in RhoA activity with maturation.…”

“…In gain-of-function experiments using the chondrogenic cell line ATDC5, RhoA over-expression was found to induce actin filament organization and stress fibers, to increase proliferation and proteoglycan production, and to suppress maturation and hypertrophy. [16,17] Conversely, overexpression of Rac-1 and Cdc42 in ATDC5 cells induces collagen type X expression, alkaline phosphatase activity, and matrix mineralization, which are associated with mature chondrocytes. [18] A recent study has indicated that genetic ablation of Rac-1 in developing cartilage leads to growth retardation with irregular growth plate organization, suppression of chondrocyte proliferation and a decreased area of collagen type X expression, indicating a delay in chondrocyte differentiation.…”

“…[16,17,[19][20][21] In this study, we monitor the status of Rho GTPases in primary chondrocytes by a biochemical pull down assay and live cell imaging analysis. We find that Rac-1 and Cdc42, but not RhoA, activation increases with chondrocyte maturation, and that Rac-1 distribution corresponds to changes in chondrocyte morphology.…”

Cell‐cycle control and the cartilage growth plate

“…Furthermore, the connections between extracellular signals and intracellular mediators in the control of cyclin D1 expression is poorly understood in most cases. While protein kinase A, RhoA, and the transcription factor CREB have been shown to be required for induction of cyclin D1 transcription by PTHrP (Beier et al, 2001;Ionescu et al, 2001;Beier and LuValle, 2002;Wang et al, 2004), and ATF-2 has been implicated in activation of the cyclin D1 promoter by TGFb (Beier et al, 2001), much less is known about the signaling molecules and transcription factors utilized by the other pathways listed above. Moreover, the regulation of cyclin D2 and D3 genes by these (or other) pathways has not been investigated.…”

“…However, Wnt5a deficiency also results in reduced cyclin D1 expression, but this effect might be secondary to a loss of Indian hedgehog expression in the bones of Wnt5a-deficient mice . Similarly, intracellular signaling molecules such as integrin-linked kinase (Grashoff et al, 2003;Terpstra et al, 2003), the small GTPase RhoA (Wang et al, 2004) and the transcription factor c-Fos (Sunters et al, 1998) also stimulate cyclin D1 expression in cartilage.…”