Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells

Michael Babich, Lisa R P Foti, Kevin L. Mathias

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

The effects of parathyroid hormone (PTH) on 1,4,5 inositol triphosphate (1,4,5-IP3) and intracellular free calcium (Ca(i)/2+) 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 Ca(i)/2+ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Ca(i)/2+ 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 Ca(i)2+ 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 μM x 18 h) potentiated the effects of 1 μg/ml bPTH(1-84) on Ca(i)2+ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 μg/ml) was without significant effect on adherent cell Ca(i)2+ 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 (~λ510 nm) detected and optimized at λ348/374 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 Ca(i)2+ 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 Ca(i)2+ 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 μM x 30 min). PTH-induced Ca(i)2+ 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/Ca(i)2+).

Original languageEnglish (US)
Pages (from-to)276-285
Number of pages10
JournalJournal of Cellular Biochemistry
Volume65
Issue number2
StatePublished - May 1997
Externally publishedYes

Keywords

  • calcium imaging
  • inositol 1,4,5-triphosphate
  • inositol phosphate
  • morphology
  • osteoblasts
  • thrombin

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

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