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

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.

PKC‐ζ expression is lower in osteoblasts from arthritic patients: IL1‐β and TNF‐α induce a similar decrease in non‐arthritic human osteoblasts

“…We did not observe the presence of -g and -h isoforms. Sanders and Stern [1996] also reported the absence of -g isoform in normal mouse osteoblasts and in osteoblastic cell lines (rat and human), and the presence of -h isoform in only selected cell lines. Furthermore, these two isoforms were also not detected in normal human osteoblasts obtained from bone explants retrieved during oral surgery [Lampasso et al, 2001].…”

“…Several reports have described the basal expression of some PKC isoforms in osteoblast cell lines (animal and human origin) [Sanders and Stern, 1996;Lampasso et al, 2006] and in primary human osteoblast cultures [Lampasso et al, 2001]. PKC isoforms have also been demonstrated to be involved in the early phase of mechanotransduction of osteoblastic cell lines through the activation of cytoskeletal molecules [Geng et al, 2001].…”

Effects of phospholipase D during cultured osteoblast mineralization and bone formation

“…On the contrary, pharmacological inhibition allows shutting down PLD1 activity for short time, so that compensatory mechanisms may not be established in cultured cells. [40][41][42][43] We thus investigated whether PLD1 role in mineralization is linked to PKC pathway. 19 The observation that the manipulation of PLD2 level and activity did not have significant effects on the mineralization process is in agreement with the finding that highlighted PLD1 as the main isoform involved in other physiological processes of differentiation.…”

Insulin-Like Growth Factor I Production Is Essential for Anabolic Effects of Thyroid Hormone in Osteoblasts

“…Primary osteoblast cultures were prepared from calvarial bones obtained from 6–7‐day‐old neonatal mice (30) . Mice were housed and killed in accordance with policy at the Northwestern University Animal Care and Use Facility.…”