Involvement ofc-fos gene in the regulation of osteoblast proliferation and osteoclast differentiation by parathyroid hormone and parathyroid hormone-related protein

Abstract

Although there is a recent evidence that PTH induces c-fos gene expression in osteoblasts, the physiological role of this expression remains unknown. We, therefore, employed c-fos antisense oligodeoxynucleotide (as-ODN) and sought to clarify the role of c-fos gene in the regulation of osteoblast proliferation and differentiation as well as osteoclast differentiation and bone-resorbing activity in the presence of osteoblasts by PTH and PTH-related protein (PTHrP). We employed osteoclastlike cell formation from mouse bone cells for the evaluation of osseoclast differentiation and the pit formation assay on the dentin slice in mouse bone cells for the evaluation of bone-resorbing activity by mature osteoclasts. Northern blot analysis revealed that both human (h)PTH-(1-34) and hPTHrP-(1-34) (10⁻⁸M) induced a transient c-fos gene expression to a similar degree in osteoblastic UMR-106 cells. Sp-cAMPS (10⁻⁴M), an activator of cAMP-dependent protein kinase (PKA), as well as dbcAMP induced a weak c-fos gene expression and Rp-cAMPS (10⁻⁴M), an inhibitor of PKA, almost completely antagonized these expressions. However, Rp-cAMPS only slightly blocked c-fos gene expression by PTH and PTHrP. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC) (10⁻⁷ to 10⁻⁶M), but not 4 alpha-phorbol 12, 13-didecanoate, incapable of activating PKC induced an intense expression of c-fos gene. Calcium ionophores (A23187 and ionomycin, 10⁻⁷ to 10⁻⁶M) did not induce the expression of c-fos gene. An inhibitor of PKC (H-7, 50µM) almost completely blocked the c-fos gene expression by PTH and PTHrP as well as PMA. Pretreatment with 1µM as-ODN significantly antagonized the inhibition of [³H] thymidine incorporation into UMR-106 cells and the stimulation of osteoclast-like cell formation by PTH and PTHrP, compared to pretreatment with the control oligodeoxynucleotide consisting of same nucleotides as as-ODN but with a random sequence. On the other hand, as-ODN did not affect an increase in alkaline phosphatase activity of UMR-106 cells and pit formation by PTH and PTHrP. In all experiments so far, the effects of PTHrP were virtually the same as those of PTH. The present study indicates, first, the direct involvement of PKC as well as PKA in PTH- and PTHrP-induced c-fos gene expression and, second, the participation of its expression in the regulation of osteoblast proliferation and osteoclast differentiation by PTH as well as PTHrP.