DESCRIPTION (provided by applicant): Endochondral ossification is the major process of embryonic skeletal development and longitudinal bone growth. During this process chondrocytes within each skeletal element become organized into growth plates and undergo maturation and hypertrophy. Following hypertrophy, the cells reach a final late-hypertrophic stage during which they facilitate cartilage matrix remodeling and mineralization and invasion of blood vessels, leading to replacement of calcified cartilage by bone. This sequence of phenotypic changes requires tight control by numerous systemic and local factors, and alterations of such regulatory mechanisms underlie congenital and acquired skeletal pathologies. The retinoic acid receptors α, β and γ (RARα, RARβ and RARγ) are nuclear hormone receptors that regulate fundamental biological processes. During the previous funding cycle, we studied the roles of RAR signaling in growth plate function and found that (1) RARγ is the dominant receptor in growth plate; (2) expression of RARγ enhances aggrecan production in cultured chondrocytes under retinoid-free condition; (3) RARγ mRNA is predominantly localized in proliferative and prehypertrophic zones but the active form of retinoic acid (atRA) is hardly detectable in these zones; and (4) RARγ deficiency induces skeletal growth retardation. These findings led us to conclude that RARγ exerts an essential role in cartilage matrix production as a ligand-less transcriptional repressor in growth plate. Surprisingly, immuno- histochemical analysis of RARγ revealed that RARγ protein was localized to the hypertrophic zone where atRA was present, indicating that ligand-bound RARγ acts on hypertrophic chondrocytes. We found also that selective RARγ agonists markedly stimulated gene expression levels linked to late-hypertrophic function in cultured primary chondrocytes and that administration of RARγ agonist accelerated chondrocyte hypertrophy and caused growth-plate closure in mouse. These and other findings lead to our central hypothesis that ligand-bound RARγ in hypertrophic zone stimulates late-hypertrophic differentiation of chondrocytes and endochondral ossification and that modulations of RARγ pathway could have therapeutic value. Our specific goals are (1) To define the role of RARγ in regulation of late-hypertrophic differentiation of growth plate chondrocytes and transition from cartilage to bone; (2) To determine the molecular mechanisms by which RARγ regulates late-hypertrophic differentiation of chondrocytes; and (3) To examine the role of RARγ in pathological growth-arrest of long bones and test if selective inhibition of RARγ ameliorates the condition. We will modify RARγ signaling in hypertrophic chondrocytes by use of genetically modified mouse systems and/or pharmacological manipulations and clarify physiological importance of RARγ in growth plate. The results could also provide a new avenue to therapy for precocious growth plate closure and...