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 language | English (US) |
|---|---|
| Pages (from-to) | 276-285 |
| Number of pages | 10 |
| Journal | Journal of Cellular Biochemistry |
| Volume | 65 |
| Issue number | 2 |
| State | Published - May 1997 |
| Externally published | Yes |
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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 journal › Article
}
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 -