title: Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells.
abstract: The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP3) and intracellular free calcium (Cai2+) in osteoblasts are variable, whereas adenylate cyclase activity is consistently stimulated. Cyclic AMP is considered a mediator in the contractile effects of PTH on osteoblasts, but the regulation and role of Cai2+ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Cai2+ increases in osteoblastic cells. Therefore, the objectives of this study were to determine the effects of PKC modulators and PTH on UMR 106-H5 rat osteoblastic cell morphology, and the relationship between cell shape and PTH-induced Cai2+ changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM x 1 h) and H-7 (30 microM x 18 h) potentiated the effects of 1 microgram/ml bPTH (1-84) on Cai2+ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 micrograms/ml) was without significant effect on adherent cell Cai2+ as measured by single-cell image analysis, although another in vitro bone resorbing agent, thrombin (10 U/ml), produced an acute 3-fold increase in the ratio (R) of emission (approximately lambda 510 nm) detected and optimized at lambda 348/374 nm (i.e., Ca-bound dye/free dye) in control cells. Phase-contrast microscopy revealed PKC inhibitor-treated cells changed from a spread configuration to a stellate form with retracting processes or cell rounding and a collapse of actin stress fibers. Within 1 h of PTH addition, PKC inhibitor-treated cells continually became extended/respread up to 3 h with an associated increase in actin stress fibers that was preceded by an acute 1.6-fold Cai2+ increase. In contrast, control or PKC activator-treated cells (phorbol 12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate; TPA) contracted/retracted within 5 min in response to PTH. A role for Cai2+ in PTH-induced cell spreading was further indicated by a contractile response to PTH when PKC-inhibitor-treated cells were loaded with the intracellular calcium chelator dimethyl BAPTA (3 microM x 30 min). PTH-induced Cai2+ increases in adherent PKC inhibitor-treated cells were also associated with a 1.8-fold 1,4,5-IP3 increase as measured by mass assay. The data suggest PKC contributes to UMR 106-H5 cell morphology and selectively regulates signal pathways activated by PTH to promote either cell contraction (cAMP) or extension (1,4,5-IP3/Cai2+).
authors.affiliation: University of Illinois College of Medicine, Rockford, Illinois 61107-1897, USA. michaelb@uicvm.uic.edu
authors.forename: M
authors.initials: M
authors.lastname: Babich
authors.forename: L R
authors.initials: LR
authors.lastname: Foti
authors.forename: K L
authors.initials: KL
authors.lastname: Mathias
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D012516:Osteosarcoma; D010281:Parathyroid Hormone / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009281:Naphthalenes; D010281:Parathyroid Hormone; C061465:calphostin complex; D004533:Egtazic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D016257:Fura-2