Parathyroid hormone stimulates translocation of protein kinase C isozymes in UMR-106 osteoblastic osteosarcoma cells.

Studies with antagonists have provided evidence that protein kinase C (PKC) is involved in several of the actions of parathyroid hormone (PTH) on bone. PTH increases total PKC activity in bone and bone cells. The current studies investigated whether PTH can activate specific PKC isozymes, as demonstrated by translocation of these isozymes from cytosolic to membrane fractions. The isozymes selected for study, alpha, betaI, delta, epsilon, and zeta, were shown previously by us to be present in normal osteoblasts and several osteosarcoma-derived osteoblastic cells. UMR-106 cells, a widely used osteoblastic cell line, were selected for the current study. PKC isozymes in whole cell lysates and cell fractions were visualized by western blotting; isozyme distribution was also visualized by immunofluorescence. The total amounts of the isozymes and their relative distribution between membrane and cytosolic fractions in untreated cells were stable over a range of passages (5-20 from initial plating). In untreated cells, the concentrations of PKC alpha, betaI, and zeta were higher in the cytosol, and PKC delta and epsilon were higher in the membrane fraction. Treatment with 1 or 10 nmol/L PTH for 1 or 5 min stimulated translocation of PKC alpha and betaI, with variable effects on the other isozymes. Treatment with phorbol-12,13-dibutyrate (PDBu), 1 micromol/L for 5 min, elicited similar effects to those of PTH on PKC alpha and betaI. Treatment with PDBu for 48 h resulted in a downregulation of PKC alpha, whereas a 48 h treatment with PTH did not cause downregulation. The results indicate that PTH can affect specific PKC isozymes, providing a mechanism for differential regulation of cellular actions through this pathway.

Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption.

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-beta, LY379196, was used to determine the role of the PKC-beta isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) induced translocation of PKC-alpha and -beta(I) to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-beta(I) translocation by PTH, TNF-alpha or IL-1 beta was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-alpha-, or IL-1 beta-stimulated translocation of PKC-alpha. PTH, TNF-alpha, and IL-1 beta increased luciferase expression in UMR-106 cells transiently transfected with a -224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-beta(I) is a component of the signaling pathway that mediates PTH-, TNF-alpha-, and IL-1 beta-stimulated IL-6 expression and PTH-stimulated bone resorption.

Stimulation of interleukin-6 promoter by parathyroid hormone, tumor necrosis factor alpha, and interleukin-1beta in UMR-106 osteoblastic cells is inhibited by protein kinase C antagonists.

To investigate the level at which protein kinase C (PKC) regulates expression of interleukin-6 (IL-6) in osteoblastic cells, effects of several PKC antagonists and PKC down-regulation by phorbol ester were studied in UMR-106 osteoblastic cells that had been transiently transfected with a -224/+11-base pair (bp) IL-6 promoter coupled to a luciferase reporter. Parathyroid hormone (PTH) elicited a dose-dependent stimulation of the IL-6 promoter expression, with significant increases produced by 5 h of treatment with concentrations of PTH as low as 10(-14) M. The increase in IL-6 promoter expression was inhibited by the PKC antagonists GF109203X, 30 nM to 1 microM, and calphostin C, 250 nM. Prior down-regulation of PKC with 100 nM phorbol-12,13-dibutyrate (PDBU) for 48 h inhibited the PTH effect as well as the smaller stimulatory effects elicited by tumor necrosis factor alpha (TNF-alpha), 10(-9)-10(-8) M, and by IL-1beta, 1-10 ng/ml. In contrast to these findings, the stimulatory effects of PTH, TNF-alpha, and IL-1beta on the IL-6 promoter expression were enhanced by staurosporine. Treatment with GF109203X or down-regulation of PKC with PDBU prevented the stimulatory effects of staurosporine. PKC activity was increased by staurosporine. The findings with staurosporine are consistent with our earlier observations that this agent enhances the calcium signaling and bone resorption elicited by PTH. The studies support the role of PKC in the stimulatory effects of PTH, TNF-alpha, and IL-1beta on IL-6 expression.

Phosphatidylcholine-specific phospholipase C inhibitor, tricyclodecan-9-yl xanthogenate (D609), increases phospholipase D-mediated phosphatidylcholine hydrolysis in UMR-106 osteoblastic osteosarcoma cells.

Our previous studies have shown that parathyroid hormone (PTH) stimulates phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD) and transphosphatidylation in UMR-106 osteoblastic cells. To determine whether phospholipase C (PLC) is also involved in the PTH-mediated PC hydrolysis, we used the inhibitor, tricyclodecan-9-yl xanthogenate (D609), a putatively selective antagonist of this pathway. Consistent with this proposed mechanism, D609 decreased (3)H-phosphocholine in extracts from UMR-106 cells prelabeled with (3)H-choline. Unexpectedly, D609 enhanced PC hydrolysis and transphosphatidylation, suggesting that either there was a compensatory increase in PLD activity when PLC was inhibited, or that D609 directly increased PLD activity. The D609-stimulated increase in PC hydrolysis was rapid, being seen as early as 2 min. The effect of D609 was temperature-sensitive, consistent with an enzymatic mechanism. The D609-stimulated increase in PC hydrolysis was PKC-independent, based upon the lack of effect of down-regulation of PKC by phorbol 12,13-dibutyrate on the response. The studies reveal a novel action of this inhibitor on signaling in osteoblastic cells which might influence downstream responses.

Modulation of [3H]Dopamine release from rat nucleus accumbens by neuropeptide Y may involve a sigma1-like receptor.

Sigma receptors are located in limbic areas, including the nucleus accumbens, where increased dopamine levels have been linked to psychosis and reinforcement. Neuropeptide Y (NPY) has been named as a possible endogenous ligand for a subpopulation of sigma receptors on the basis of its ability to compete for sigma receptor binding. Using a superfusion system, we found that NPY enhanced N-methyl-D-asparate-stimulated [3H]dopamine release in rat nucleus accumbens, whereas the prototypical sigma agonist (+)pentazocine inhibited release. However, four sigma antagonists, one of which is sigma1 selective, as well as a Y receptor antagonist, all reversed the enhancement by NPY and the inhibition by (+)pentazocine. A sigma2-selective antagonist had no effect on either NPY-mediated enhancement or (+)pentazocine-mediated inhibition. [Leu31,Pro34]NPY and NPY13-36 also enhanced release, but the effects were not reversed by sigma antagonists. Peptide YY did not mimic the effect of NPY. Our findings are consistent with the potential role of NPY as an endogenous ligand for a subtype of sigma receptor with characteristics different from Y1, Y2 and Y3 receptors but sensitive to Ac-[3-(2,6-dichlorobenzyl)Tyr27,D-Thr32NPY-(27-36)amide. Our findings suggest a role for NPY, via sigma receptors, in the regulation of dopamine levels in areas of brain critical to psychosis and reinforcement.