EXPRESSION AND PHORBOL ESTER-INDUCED DOWN-REGULATION OF PROTEIN-KINASE-C ISOZYMES IN OSTEOBLASTS

The protein kinase C (PKC) enzyme family consists of at least 11 isozy mes in three classes, with characteristic tissue distributions. Phorbo l esters activate and ultimately down-regulate phorbol-sensitive isozy mes. PKC is a signal transducer in bane, and phorbol esters influence bone resorption. Little is known about specific PKC isozymes in this t issue, however. We describe here the expression and phorbol ester-indu ced down-regulation of PKC isozymes in osteoblasts. Normal mouse osteo blasts and seven osteoblastic cell lines (rat UMR-106, ROS 17/2.8, ROS 24/1, and human MG-63, G-292, SaOS-2, HOS-TE85) were screened for iso zyme expression by Western immunoblotting using isozyme-specific anti- PKC antibodies. The conventional alpha and beta(1) isozymes, but not g amma, were present in each of the osteoblasts examined; PKC-beta(II) w as detectable in all but the ROS 24/1 line. PKC-epsilon was expressed in all osteoblasts screened, but other novel PKCs, delta, eta, and the ta, were detectable only in select lines. The atypical zeta and i/lamb da PKCs were in all osteoblasts examined. To determine the sensitivity of the isozymes to prolonged phorbol ester treatment, normal osteobla sts and the UMR-106 cell line were treated with vehicle or 1 mu M phor bol 12, 13-dibutyrate (PDB) for 1, 3, 6, 12, 24, or 48 h, and Western blot analysis was performed. Normal and UMR-106 cells showed similar p horbol sensitivities; conventional (alpha, beta(I)) and novel (delta, epsilon, eta) isozymes were down-regulated by prolonged phorbol treatm ent but atypical isozymes were not. Down-regulation of all sensitive P KCs was detectable within 6 h of phorbol treatment; the novel delta an d epsilon isozymes, however, showed more rapid and dramatic down-regul ation than conventional isozymes. The observed down-regulation was dos e-dependent (0.3-3 mu M) and specific; 48 h treatment with the inactiv e phorbol, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), failed to down-regulate PDB-sensitive isozymes. The phorbol-induced down-regulat ion was also reversible; 24 h after withdrawing PDB, all phorbol-sensi tive isozymes, except PKC-eta, had recovered at least partially. These studies, the first to characterize thoroughly PKC isozyme expression in osteoblastic cells from several species, demonstrate that osteoblas ts have a characteristic PKC isozyme profile, including both phorbol e ster-sensitive and -insensitive isozymes. The time course of down-regu lation and the presence of phorbol-insensitive PKCs must be considered in interpreting the effects of phorbol esters on bone remodeling.