Expression and phorbol ester sensitivity of protein kinase c (pkc) isozymes in osteoblasts

The PKC family consists of at least 12 isozymes in 3 classes, with characteristic tissue distributions. Phorbol esters activate and down-regulate sensitive isozymes. PKC is a signal transducer in bone, and phorbol esters influence bone résorption. Little is known about specific PKC isozytnes in this tissue, however. We examined the expression and phorbol ester sensitivity of PKC isozymes in osteoblasts. Normal mouse osteoblasts and 7 osteoblastic cell lines (rat UHR-106, ROS 17/2-8, ROS 24/1, and human MG-63, G292, SaOS-2, HOS-TE85) were screened by Western blotting using isozyme-specific anti-PKC antibodies. The conventional α, β_I and β^II isozymes, but not y are present in the osteoblasts examined. PKC-e is also expressed in all osteoblasts screened, but other novel PKCs, 6, TJ, and 6, are detectable only in select lines. The atypical C and i PKCs are in all cell types. To determine the sensitivity of isozymes to long-term phorbol ester treatment, normal osteoblasts and the UMR-106 line were treated with 1 pM phorbol 12,13-dibutyrate (PDB) for 1, 3, 6, 12, 24 or 48 hr. Normal and UMR-106 cells show similar phorbol sensitivities; conventional (a, ,) and novel isozymes are down-regulated by long-term treatment but atypical isozymes are not. Novel isozymes show more rapid and dramatic down-regulation than conventional isozymes. These studies demonstrate that osteoblasts have a characteristic PKC isozyme profile, including both phorbol eater-senaitive and insensitive isozymes. Down-regulation of sensitive PKCs is detectable within 6 hr of phorbol treatment. This time course and the presence of phorbol-insensitive isozymes in osteoblastic cells must be considered in interpreting the effects of phorbol esters on bone remodeling.