Coordination of cell cycle and cell differentiation by receptor activator of NF-kappa-B ligand during osteoclast differentiation

Osteoclasts are bone resorbing multinuclear cells formed by the fusion of hematopoietic mononuclear precursor cells. Microphthalmia transcription factor (MITF) is a basic helix-loop-helix leucine zipper transcription factor important for the differentiation osteoclasts. MITF regulates the expression of osteoclast-differentiation marker genes, Tartrate-resistant acid phosphatase (TRAP) and cathepsin K. Deletion in arginine 215 in the basic domain of MITF results in severe osteopetrosis in Mitfmi/mi mice. A substitution of arginine 216, in the basic domain, with lysine results in age resolving osteopetrosis in Mitfor/or mice. Mitfwh/wh mice with a substitution of isoleucine to asparagine in the basic domain of Mitf do not exhibit any osteopetrosis. We identified several novel genes regulated by Mitf with potential roles in osteoclast differentiation via microarray analysis of cDNA from WT and Mitfmi/mi osteoclasts. In particular, Eos, HOX11L2, Hematopoietic cell phosphatase (HCP) and p9 were confirmed to be expressed in lower levels in Mitfmi/mi osteoclasts. We also observed that while TRAP mRNA levels were unregulated in Mitfor/or, similar to the levels in WT, Cathepsin K levels were lower in both Mitfmi/mi and Mitfor/or osteoclasts.; Osteoclast progenitors quit proliferating prior to fusion to become multinuclear osteoclasts. We observed that the cytokine, receptor activator of NF-kB ligand (RANKL), induces wild type (WT) osteoclast progenitors to withdraw from cell cycle within 24 hours of its application. This event coincides with elevation in p27KIP1 and p21CIP1 and with decreased CDK2 activity. Also, p27KIP1 is required by osteoclast progenitors to exit from the cell cycle in response to RANKL and p27KIP1 −/− osteoclasts express lower levels of TRAP mRNA. Osteoclast progenitors from p27KIP1−/− p21CIP1−/− double knockout mice do not withdraw from cell cycle in response to RANKL and express significantly lower levels of TRAP and cathepsin K mRNA. p27KIP1−/−p21 CIP1−/− mice exhibit age resolving osteopetrosis. Also, precursors from the double mutant mice form fewer multinuclear functional osteoclasts in vitro. These data suggest that both p21 CIP1 and p27KIP1 might have redundant roles during osteoclast differentiation.