Apoptosis Mediated by Activation of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH)/ PTH-Related Protein (PTHrP)

Abstract

The present studies were carried out to evaluate the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences cell viability. In 293 cells expressing recombinant PTHRs, PTH treatment markedly reduced the number of viable cells. This effect was associated with a marked apoptotic response including DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition of tumor necrosis factor (TNFalpha). Addition of caspase inhibitors or overexpression of bcl-2 partially abrogated apoptosis induced by serum withdrawal. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apoptosis were neither mimicked by activators of the cAMP pathway (forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However, elevation of Ca(i)2+ by addition of thapsigargin induced rapid apoptosis, and suppression of Ca(i)2+ by overexpression of the calcium- binding protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109203X partially inhibited PTH-induced apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of the activity of the alpha-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the beta(gamma)-subunits of G proteins) was without effect. Chemical mutagenesis allowed selection of a series of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNFalpha. These results suggest that 1) apoptosis produced by PTHR and TNF receptor signaling involve converging pathways; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediated cAMP signaling, is required for the apoptotic effects of PTHR activation.

Parathyroid Hormone and Parathyroid Hormone-related Protein Exert Both Pro- and Anti-apoptotic Effects in Mesenchymal Cells

“…PTH is antiapoptotic in osteoblasts (5) and chick embryo hypertrophic chondrocytes (56), whereas it promotes apoptosis in 293 cells, a transformed primary embryonal kidney cell line (29). Interestingly, our findings of a bi-directional effect of PTH on cells of differing maturity stage are similar to those reported for another important factor in skeletal development, fibroblast growth factor (57)(58)(59).…”

“…The underlying mechanisms of this dual effect are not totally clear at present but appear to be dependent on the PKA pathway. This is in contrast to the proapoptotic effects of PTH reported in kidney cells that were found to be dependent on the PKC pathway (29).…”

“…The PKA pathway is mediated by the G s protein that activates adenylyl cyclase and leads to cAMP production and protein kinase A activation. The PKC pathway is mediated by the G q protein that activates phospholipase C, resulting in an increase in intracellular Ca 2ϩ levels and activation of protein kinase C. In previous work, PTH induced apoptosis in 293 cells through PKC pathway rather than PKA pathway (29). However, other studies indicate that cAMP suppresses apoptosis in rat periosteal cells (60) and human osteoblasts (61).…”

“…To determine the effects of PTH/PTHrP on apoptosis, caspase inhibitors 2 ϫ 10 Ϫ7 M YVAD-cmk (inhibits caspase-1) and DEVD-fmk (inhibits caspase-3 and caspase-8) (CLONTECH) were used to inhibit apoptosis (29) and dexamethasone (10 Ϫ7 M) was used to induce apoptosis (5) as described previously. Flow Cytometry Assay-During apoptosis, the cell membrane phospholipid phosphatidylserine is translocated from the inner to the outer leaflet of the plasma membrane.…”

Glucagon-like Peptide-2 Receptor Activation Engages Bad and Glycogen Synthase Kinase-3 in a Protein Kinase A-dependent Manner and Prevents Apoptosis following Inhibition of Phosphatidylinositol 3-Kinase

“…Parathyroid hormone-related hormone, bradykinin, corticotropin-releasing hormone, vasoactive intestinal peptide, opiates, pituitary adenylate cyclase-activating polypeptide, endothelins, adenosine, somatostatin, and lysophosphatidic acid all modulate cell death through GPCRs in a diverse number of cell types (45)(46)(47)(48)(49)(50)(51)(52). Although activation of PKA is essential for prevention of cell death in some cell types, regulators of G protein signaling may also modulate GPCR-regulated apoptosis (53) independently of PKA (46). GPCR activation leads to diverse and often opposing effects on cell survival via increasingly complex signaling mechanisms, even among highly related members of the same receptor superfamily.…”