Apoptosis mediated by activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP)

The present studies were carried out to evaluate the mechanisms by which PT H/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 i ncluding DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition o f tumor necrosis factor (TNF alpha). Addition of caspase inhibitors or over expression of bcl-2 partially abrogated apoptosis induced by serum withdraw al. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apo ptosis 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 D 28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109 203X partially inhibited PTH-induced apoptosis. Regulator of G protein sign aling 4 (RGS4) tan inhibitor of the activity of the alpha-subunit of G(q)) 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 prot eins) was without effect. Chemical mutagenesis allowed selection of a serie s of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNF alpha. These results suggest that 1) apopt osis produced by PTHR and TNF receptor signaling involve converging pathway s; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediat ed cAMP signaling, is required for the apoptotic effects of PTHR activation .