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

J Bone Miner Res. 1996 Dec;11(12):1862-72. doi: 10.1002/jbmr.5650111206.

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 alpha and beta I isozymes, but not gamma, were present in each of the osteoblasts examined; PKC-beta II was detectable in all but the ROS 24/1 line. PKC-epsilon was expressed in all osteoblasts screened, but other novel PKCs, delta, eta, and theta, were detectable only in select lines. The atypical zeta and iota/lambda 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 microM 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 (alpha, beta I) and novel (delta, epsilon, eta) 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 delta and epsilon isozymes, however, showed more rapid and dramatic down-regulation than conventional isozymes. The observed down-regulation was dose-dependent (0.3-3 microM) and specific; 48 h treatment with the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate (4 alpha-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-eta, 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Bone Development / physiology*
  • Bone Resorption / enzymology*
  • Cells, Cultured
  • Down-Regulation
  • Humans
  • Isoenzymes / biosynthesis
  • Isoenzymes / physiology*
  • Mice
  • Osteoblasts / drug effects*
  • Osteoblasts / metabolism
  • Phorbol 12,13-Dibutyrate / pharmacology*
  • Protein Kinase C / biosynthesis
  • Protein Kinase C / physiology*
  • Rats
  • Tumor Cells, Cultured

Substances

  • Isoenzymes
  • Phorbol 12,13-Dibutyrate
  • Protein Kinase C