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 isozymes in three classes, with characteristic tissue distributions. Phorbol esters activate and ultimately down‐regulate phorbol‐sensitive isozymes. PKC is a signal transducer in bone, and phorbol esters influence bone resorption. Little is known about specific PKC isozymes in this tissue, however. We describe here the expression and phorbol ester‐induced down‐regulation of PKC isozymes in osteoblasts. Normal mouse osteoblasts 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 isozyme expression by Western immunoblotting using isozyme‐specific anti‐PKC antibodies. The conventional α and βI, isozymes, but not γ, were present in each of the osteoblasts examined; PKC‐βII was detectable in all but the ROS 24/1 line. PKC‐ε was expressed in all osteoblasts screened, but other novel PKCs, δ, η, and θ, were detectable only in select lines. The atypical ζ and ι/λ PKCs were in all osteoblasts examined. To determine the sensitivity of the isozymes to prolonged phorbol ester treatment, normal osteoblasts and the UMR‐106 cell line were treated with vehicle or 1 μM phorbol 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 phorbol sensitivities; conventional (α, βI) and novel (δ, ε, η) isozymes were down‐regulated by prolonged phorbol treatment but atypical isozymes were not. Down‐regulation of all sensitive PKCs was detectable within 6 h of phorbol treatment; the novel δ and ε isozymes, however, showed more rapid and dramatic down‐regulation than conventional isozymes. The observed down‐regulation was dose‐dependent (0.3–3 μM) and specific; 48 h treatment with the inactive phorbol, 4α‐phorbol 12,13‐didecanoate (4α‐PDD), failed to down‐regulate PDB‐sensitive isozymes. The phorbol‐induced down‐regulation was also reversible; 24 h after withdrawing PDB, all phorbol‐sensitive isozymes, except PKC‐η, had recovered at least partially. These studies, the first to characterize thoroughly PKC isozyme expression in osteoblastic cells from several species, demonstrate that osteoblasts have a characteristic PKC isozyme profile, including both phorbol ester–sensitive and –insensitive isozymes. The time course of down‐regulation and the presence of phorbol‐insensitive PKCs must be considered in interpreting the effects of phorbol esters on bone remodeling.