Expression and phorbol ester–induced down-regulation of protein kinase C isozymes in osteoblasts

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Abstract
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.
Funding Information
  • National Osteoporosis Foundation
  • Army Medical Research and Development Command Breast Cancer Research Program
  • NIH (AR 11262 (P.H.S.))

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