Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Dr. Frank Beier


Longitudinal growth of endochondral bones is controlled by the cartilage growth plate. Chondrocyte proliferation and hypertrophy, vascular invasion, formation of ossification centers and cartilage replacement by bone tissue are all important processes required for normal growth. These biological processes have to be tightly regulated or disturbances will lead to skeletal diseases. A large number of genes, growth factors and hormones have been implicated in the regulation of growth plate biology, however, less is known about the intracellular signaling pathways involved. Nitric oxide (NO) has been identified as a regulator of cellular proliferation, differentiation, migration, survival and metabolism in multiple cell types. In bone biology, it has been implicated in bone remodeling and the pathogenesis of osteoarthritis, but the roles of specific nitric oxide synthase (NOS) enzymes in chondrocyte physiology and cartilage development are unclear. The goal of this thesis was to analyze the roles of NOS/NO signaling in specific stages of endochondral bone formation. We had shown recently that chondrocyte-specific deletion of the Rac1 gene results in severe dwarfism due to reduced chondrocyte proliferation in mice, but the molecular pathways involved remained unknown. Employing a Rac1-deficient monolayer chondrocyte culture, we showed that loss of Rac1 results in severely reduced levels of inducible nitric oxide synthase (iNOS) protein and NO production. Additionally, reduced iNOS expression was found in Rac1-decifient mice in vivo. Using a tibia organ culture system, we showed that NO donors rescued the antiproliferative effects of Rac1 inhibition. Examination of the growth plate of iNOS-deficient mice revealed reduced chondrocyte proliferation and decreased expression of cyclin D1, while ATF3, a suppressor of cyclin D1 transcription, showed increased expression. Thus, we identified iNOS/NO as a novel mediator of Rac1 signaling and ATF3 as a link between iNOS and chondrocyte cell cycle. Doe to the skeletal phenotypes we observed in iNOS-deficient mice, my next study investigated effects of inactivation of endothelial nitric oxide synthase (eNOS) on cartilage development in mice. eNOS-deficient mice showed increased lethality and reduced bone growth, delayed ossification and a marked reduction in the number of proliferating chondrocytes. The mechanisms leading to these bone phenotypes appear to be caused by decreased cyclin D1 and increased p57 expressions in mutants, resulting in slower cell cycle progression and earlier cell cycle exit. Additionally, expression of early chondrocyte markers such as Sox9 was reduced and prehypertrophic markers were upregulated in mutant mice. Because my studies had shown upregulation of nNOS in eNOS-null cartilage, next I analyzed the skeletal phenotype of nNOS-deficient mice. Transient growth retardation, reduced length of long bones, less trabecular bone and decreased mineralization were shown in nNOS KO mice. Reduced proliferating chondrocyte numbers in mutants may in part be due to premature cell cycle exit, shown by reduced cyclin D1 and upregulated p57 expressions. Similar to the other two mutant strains, ATF3 was a link between nNOS and reduced cyclin D1 expression. In addition, I demonstrated increased apoptosis, reduced early chondrocyte markers such as Sox genes and increased prehypertrophic markers RORα and c-Fos in mutant mice. Together, these data suggest that NOS/NO presents a core signaling pathway to regulate chondrocyte proliferation and differentiation through control of cell cycle protein.