AJ18, a member of the growing family of KRAB/C2H2 zinc finger genes, was originally identified by its responsiveness to BMP-7 in fetal rat calvarial cells. Subsequent studies have shown that AJ18 is expressed in the same tissues as BMP-7, is differentially expressed during osteoblastic differentiation, and has the ability to abrogate Runx2-mediated transactivation through competitive binding of the osteoblast-specific element (OSE2). Taken together, AJ18 is believed to play an important role in bone development downstream of BMP-7. Objectives: To better understand the role of AJ18 during bone differentiation. Methods: AJ18 cDNA in the sense (S – expression of exogenous AJ18) and antisense (AS – inhibition of endogenous AJ18) orientations were over-expressed in a clonal rat bone marrow cell (RBMC) line under the control of two separate promoters: 1) the constitutively-activated cytomegalovirus (CMV) promoter and 2) the collagen type I (COLI) promoter, which is upregulated during osteogenic differentiation. Stably-transfected RBMCs were isolated, grown in the presence of ascorbic acid and
b-glycerophosphate, and the formation of bone-nodules measured by von Kossa staining. Results: COLI-driven AJ18 (S and AS) and CMV-driven AJ18 (AS) RBMCs showed a decrease in bone formation, whereas the CMV-driven AJ18 (S) cells showed a significant increase in bone formation in comparison to their respective empty vector controls. Moreover, whereas the COLI-driven population showed little difference in cell proliferation, the CMV-driven AJ18 (S) and AJ18 (AS) cells demonstrated higher and lower rates of cell proliferation, respectively, relative to the empty vector and wild type controls. Conclusions: Collectively these studies suggest that forced expression of AJ18 during the proliferative phase in osteogenic cells increases the rate of cell proliferation, and thereby increases bone formation, whereas de-regulation of AJ18 during osteogenic differentiation results in decreased bone formation.