Protein kinase C modulates parathyroid hormone- but not prostaglandin E2-mediated stimulation of cyclic AMP production via the inhibitory guanine nucleotide binding protein in UMR-106 osteosarcoma cells

Abstract

In UMR-106 osteosarcoma cells we found that PTH activated both the cAMP/protein kinase A and the Ca(2+)-dependent phosphoinositide/protein kinase C (PKC) pathways, but prostaglandin E2 (PGE2) activated only the cAMP pathway. Activation of PKC by the phorbol ester PMA had no effect on cAMP production but enhanced PTH-stimulated cAMP production by 50% or more; the effect on PGE2-induced cAMP was negligible. Inhibition of the alpha-subunit of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin pretreatment also enhanced PTH-mediated cAMP production but had no effect on PGE2-induced cAMP production. These results suggest that although PTH-mediated adenylate cyclase activity is regulated via both the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, only Gs regulates PGE2-mediated adenylate cyclase activity in UMR-106 cells. Costimulation with pertussis toxin and PMA did not increase PTH-stimulated cAMP production above that obtained with PMA alone. This implies a similar target of action for pertussis toxin and PMA, that is, the alpha-subunit of Gi. The alpha-subunit of Gi was found to be a substrate for in vitro PKC phosphorylation of membrane fractions from UMR-106 cells, seen as a +/- 40 kD band on SDS-PAGE. Stimulation of in situ 32P-labeled cells with either PMA or PTH also enhanced incorporation of 32P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin-labeled subunits of both Gi1 alpha/Gi2 alpha and Gi3 alpha could be immunoprecipitated, respectively, but immunoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only Gi2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

A2B adenosine receptor activation and modulation by protein kinase C

“…A PKC-induced increase or decrease of receptor function has previously been suggested to be via receptors, G proteins, and adenylyl cyclase. 2 , 7 , 11 , 12 , 26 , 27 , 28 Here, we first tested if PMA treatment directly affects adenylyl cyclase activity in H9C2 cells. Figure 3 A shows that PMA pretreatment did not inhibit or enhance the forskolin-stimulated cAMP accumulation in H9C2 cells, suggesting the action of PKC is probably at the receptor or G protein level, but not at the level of adenylyl cyclase.…”

“… 26 The α-subunit of G i 2α in the membrane fractions from UMR-106 osteosarcoma cells was proposed as a PKC phosphorylation substrate. 27 It remains to be examined which G i isoform(s) are involved in the A 2B AR modulation by PKC.…”

“… 37 Koch et al. 27 showed that PKC activation by PMA in UMR-106 cells had no effect on basal cAMP accumulation but enhanced parathyroid hormone (PTH)-stimulated cAMP accumulation. PTX-treatment also enhanced PTH-mediated cAMP production.…”

Protein Kinase C Involvement in Interleukin-6 Production by Parathyroid Hormone and Tumor Necrosis Factor-α in UMR-106 Osteoblastic Cells

“…Because there also is evidence that cAMP is important for PTH‐stimulated IL‐6 production in certain osteoblastic cells, the role of the PKA pathway in the current observations must be considered (13,15) . Cross‐talk between PTH stimulation of phospholipase C/PKC and cAMP/PKA pathways has been shown in osteoblasts (30–35) . Acute pretreatment or cotreatment (≤1 h) with a phorbol ester has been found to enhance PTH‐stimulated adenylate cyclase (AC) activity and cAMP production in UMR‐106 and other osteoblastic cells, whereas longer phorbol pretreatments (2–16 h) were found to decrease PTH‐stimulated AC activity, PTH receptor number, or PTH binding in osteoblasts (30–35) .…”

“…Cross‐talk between PTH stimulation of phospholipase C/PKC and cAMP/PKA pathways has been shown in osteoblasts (30–35) . Acute pretreatment or cotreatment (≤1 h) with a phorbol ester has been found to enhance PTH‐stimulated adenylate cyclase (AC) activity and cAMP production in UMR‐106 and other osteoblastic cells, whereas longer phorbol pretreatments (2–16 h) were found to decrease PTH‐stimulated AC activity, PTH receptor number, or PTH binding in osteoblasts (30–35) . In view of these previously reported interactions, we examined the effects of our PDB regimen on cAMP production.…”