Rac1/Cdc42 and RhoA GTPases Antagonistically Regulate Chondrocyte Proliferation, Hypertrophy, and Apoptosis

Abstract

The intracellular signaling pathways controlling chondrocyte physiology are largely unknown. Here we show that the small GTPases, Rac1 and Cdc42, accelerate the rate of chondrocyte differentiation and apoptosis, thereby antagonizing the activity of RhoA. These results identify Rac1 and Cdc42 pathways as novel regulators of cartilage development.Introduction: Proliferation, hypertrophic differentiation, and ultimate apoptosis of chondrocytes regulate endochondral bone growth and development, but the intracellular signaling pathways controlling chondrocyte biology are incompletely understood. In this study, we investigated the role of the small GTPases Rac1 and Cdc42 in chondrocytes. Materials and Methods: Rac1 and Cdc42 expression during chondrogenic differentiation was assessed by RT-PCR and Western blotting. Effects of Rac1 and Cdc42 on parameters of chondrocyte biology were studied using transient transfections into primary mouse chondrocytes and stable transfections of the chondrogenic cell line ATDC5. Luciferase assays, RT-PCR, cell proliferation, alkaline phosphatases assays, staining procedures, TUNEL assays, and caspase activity assays were performed to study the chondrocyte response to overexpression of Rac1 and Cdc42 proteins. Activation of the p38 pathway was analyzed using Western blotting with phospho-specific antibodies, and mitogen-activated protein (MAP) kinase pathways were inhibited using pharmacological approaches. Results and Conclusions: Rac1 and Cdc42 activities are required for maximal activity of the collagen X promoter, a hypertrophic marker, in primary chondrocytes, suggesting essential roles of these GTPases in chondrocyte hypertrophy. Overexpression of Rac1 or Cdc42 in chondrogenic ATDC5 cells results in reductions in cell numbers and marked acceleration of hypertrophic differentiation, thus opposing the effects of the related GTPase RhoA. Rac1 and Cdc42 also induce accelerated chondrocyte apoptosis, as shown by TUNEL and caspase activity assays and changes in cell morphology and actin organization. Rac1 and Cdc42 overexpression results in activation of the p38 MAP kinase pathway in ATDC5 cells, and pharmacological inhibition of p38 signaling blocks the effects of Rac1 and Cdc42 overexpression on hypertrophy and apoptosis. Our results therefore suggest that Rac1 and Cdc42 signaling accelerates progression through the chondrocyte life cycle in a p38-dependent fashion and antagonizes RhoA signaling pathways in chondrocyte proliferation, hypertrophy, and apoptosis.

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

“…[20,27] Thus, we first determined the ability of Rac-1 to modulate cell attachment and spreading. The infected chondrocytes were plated on collagen type I, fibronectin, gelatin, or collagen type II substrates and cell attachment was quantified by computer-assisted analysis one hour after plating.…”

“…These findings are consistent with the previous observation that Rac-1 activation stimulates a hypertrophic phenotype in ATCD5 cells, a chondrogenic cell line. [20] On the contrary, inactivation of Rac-1 increases proteoglycan synthesis, while it decreases alkaline phosphatase activity and gene expression of hypertrophic markers. This is also in line with the findings that deficiency of Rac-1 in cartilage induces a delay of chondrocyte maturation and endochondral ossification.…”

“…[19] In contrast, Rac-1 null limb micromass cultures have lowered proteoglycan content and levels of collagen type II and aggrecan mRNA. [20,21] However, the experiment using limb micromass cultures monitors chondrogenic induction, while our experimental system recapitulates chondrocyte maturation. Thus, these two findings indicate that Rac-1 diversely regulates chondrocyte matrix metabolism at the initiation versus late maturation stages.…”

“…Unlike previous reports, a loss of Rac-1 function in our cultures does not strongly inhibit cell spreading, which could be due to the different levels of Rac-1 activity in the chondrocytes used for the experiments. [19][20][21] We use immature chondrocytes, whereas the cultures in the previous study likely contained immature and mature chondrocytes. Since we use immature chondrocytes, which have low Rac-1 activity, a further decrease in Rac-1 function might not induce a strong effect.…”

“…[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.…”