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 Citations (Scopus)

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

Fingerprint

Parathyroid Hormone
Protein Kinase C
Modulators
Calcium
Protein C Inhibitor
Inositol 1,4,5-Trisphosphate
Protein Kinase Inhibitors
Cells
Coloring Agents
Osteoblasts
Stress Fibers
protein kinase modulator
Actins
Phorbol 12,13-Dibutyrate
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
Single-Cell Analysis
Fibers
Fura-2
Phase-Contrast Microscopy
Cell Shape

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells. / Babich, Michael; Foti, Lisa R P; Mathias, Kevin L.

In: Journal of Cellular Biochemistry, Vol. 65, No. 2, 05.1997, p. 276-285.

Research output: Contribution to journalArticle

@article{7ed5efcf66174963a63ed17b36b5dadc,
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 (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+).",
keywords = "calcium imaging, inositol 1,4,5-triphosphate, inositol phosphate, morphology, osteoblasts, thrombin",
author = "Michael Babich and Foti, {Lisa R P} and Mathias, {Kevin L.}",
year = "1997",
month = "5",
language = "English (US)",
volume = "65",
pages = "276--285",
journal = "Journal of Cellular Biochemistry",
issn = "0730-2312",
publisher = "Wiley-Liss Inc.",
number = "2",

}

TY - JOUR

T1 - Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells

AU - Babich, Michael

AU - Foti, Lisa R P

AU - Mathias, Kevin L.

PY - 1997/5

Y1 - 1997/5

N2 - 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+).

AB - 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+).

KW - calcium imaging

KW - inositol 1,4,5-triphosphate

KW - inositol phosphate

KW - morphology

KW - osteoblasts

KW - thrombin

UR - http://www.scopus.com/inward/record.url?scp=0030949861&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030949861&partnerID=8YFLogxK

M3 - Article

C2 - 9136085

AN - SCOPUS:0030949861

VL - 65

SP - 276

EP - 285

JO - Journal of Cellular Biochemistry

JF - Journal of Cellular Biochemistry

SN - 0730-2312

IS - 2

ER -