The roles of the Rho GTPase protein Rac-1 in cell morphology and function during chondrocyte maturation
The objective of this dissertation was to determine the roles of Rac-1 in the regulation of chondrocyte morphology, the remodeling of the extracellular matrix and the expression of a mature phenotype. We established a replication competent, retroviral system to modulate Rac-1 activity in primary chick chondrocytes. The results of Specific Aim 1 indicated that Rac-1 activation induced cell enlargement and development of actin filamentation, which are associated with chondrocyte hypertrophy. Conversely, cells expressing inactive Rac-1 had a rounded morphology with a less developed actin architecture, comparable to immature chondrocytes. In Specific Aim 2, we determined that Rac-1 inactivation maintained an immature phenotype as shown by high proteoglycan accumulation and increased expression of aggrecan and collagen type IX. Conversely, Rac-1 activation upregulated the expression of hypertrophic markers: alkaline phosphatase activity and matrix metalloproteinase expression. Thus, the activation status of Rac-1 correlated with the maturation status of chondrocytes. The data presented in Specific Aim 3 described the activity and localization of Rac-1 during chondrocyte maturation. We demonstrated that Rac-1 activity increased in mature chondrocytes when compared to immature cells. In addition, Rac-1 tagged with GFP co-localized with F-actin at the cortical membrane. Further, these proteins identically altered their distribution in response to treatment with Wnt3A, a factor known to induce maturation in chondrocytes. In addition, cooperation between Rac-1 activation and Wnt signaling was suggested by the findings that Wnt3A stimulated Rac-1 activation and that morphological changes induced by Wnt3A required Rac-1 activation. In conclusion, the results of this investigation indicated that Rac-1 activation induces the expression of a mature, hypertrophic-like phenotype, while chondrocytes under Rac-1 inactivation retain an immature phenotype. Further, our data implies that Rac-1 controls actin organization and cell morphology in chondrocytes. Hence, the data presented in this dissertation reveal that Rac-1 has important roles in the regulation of the hypertrophic phenotype and the cytoskeletal organization of chondrocytes during endochondral ossification.
0541: Biomedical engineering