Apoptosis or programmed cell death is a process fundamental to normal growth and development, immune response, and tissue remodeling. The mammalian signal transduction pathways which mediate apoptosis, although under intense scrutiny, remain poorly understood. Parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) are peptides which initiate signals critical for regulation of cell growth and differentiation, as well as bone and mineral metabolism (). The responses of different cell types to these hormones are varied and include cell cycle arrest, differentiation, proliferation, and apoptotic cell death (). These two peptide hormones activate a single G-protein coupled receptor (GPCR), the parathyroid hormone receptor (PTHR), which signals via two G-protein coupled pathways: 1) Gq mediated activation of phospholipase C (PLC), which results in intracellular free calcium ([Ca2+]i) mobilization and activation of protein kinase C, and 2) Gs mediated activation of adenylate cyclase which leads to increases in cAMP and protein kinase A activation - the PTHR is also capable of activating Gi (). It is not known how activation of these signaling pathways is linked to changes in proliferation or apoptosis. Preliminary studies showed that activation of the PTHR induced apoptosis in HEK 293 cells. This apoptosis was abrogated by inhibition of caspase proteases, or by the expression of a calcium binding protein, calbindin. Signaling studies showed that Gs mediated activation of adenylate cyclase did not mediate PTHR-induced apoptosis, rather apoptosis appeared to occur as a result of PLC/Ca2+ signaling. In this proposal the roles of upstream signaling pathway components in PTH-mediated apoptosis will be evaluated 1) To evaluate the role of G-protein subunits in PTHR mediated apoptosis, the proposed studies will establish if G-protein alpha and betagamma subunits are necessary or sufficient to mediate apoptosis resulting from activation of the PTHR: a) The activity of G-protein a or betagamma subunits will be selectively blocked by expression of proteins known to inhibit their activity, b) Constitutively active G-protein subunits will be introduced and effects on cell growth and apoptosis will be determined. 2) To evaluate the role of [Ca2+]i in PTHR-mediated apoptosis, studies will establish if calcium changes are necessary or sufficient to mediate apoptosis. a) [Ca2+]i changes in response to PTHR activation will be documented. b) Establish if [Ca2+]i changes are sufficient to induce apoptosis: [Ca2+]i will be elevated and/or intracellular calcium stores will be depleted in HEK 293 cells, and the effects of these manipulations on cell growth and apoptosis examined. 3) Block PTH-induced mobilization of calcium, either by the use of intracellular calcium chelators; or binding proteins to buffer [Ca2+]i changes, or by use of calcium channel blockers to inhibit calcium influx. Blockers of PLC activation will also be used. The effects of PTHR activation on cell growth and apoptosis under these conditions will then be examined. 4) Demonstrate how changes in [Ca2+]i might result in apoptosis: caspase protease and calpain protease activities in cell lysates in response to buffered changes in [Ca+]i will be assayed. A long term goal of this research is to identify signaling components of this PTHR-activated apoptosis pathway, and to examine consequences of activation or blockage of PTHR-mediated apoptosis pathways in vivo.
