A MIDREGION PARATHYROID HORMONE-RELATED PEPTIDE MOBILIZES CYTOSOLIC CALCIUM AND STIMULATES FORMATION OF INOSITOL TRISPHOSPHATE IN A SQUAMOUS CARCINOMA CELL-LINE

A midregion fragment of PTH-related protein (PTHrP), which is intensiv ely conserved across species, has been identified as a secretory produ ct of several different cell types, including keratinocytes and squamo us carcinomas. As recent data suggest that a midregion PTHrP fragment may be biologically active, we hypothesized that midregion PTHrPs inte ract with unique cell surface receptors that mediate autocrine or para crine action. Dose-dependent transient elevations in intracellular cal cium ([Ca2+](i)) were observed in fura-2-loaded SqCC/Y1 squamous carci noma cells exposed to human (h) PTHrP-(67-86)NH2, [Tyr(36)]hPTHrP-(1-3 6)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 mu M. The effects of maximal stimulatory concentrations of[Tyr(36)]PTHrP- (1-36)NH2 and PTHrP-(67-86)NH2 on [Ca2+](i) were additive. The inhibit ory PTH analog, [D-Trp(12),Tyr(34)]bovine PTH-(7-34)NH2, attenuated th e [Ca2+](i) response to [Tyr(36)]hPTHrP-(1-36)NH2, but not that to PTH rP-(67-86)NH2. These data suggest that PTHrP-(67-86)NH2 activates a di fferent receptor pathway in SqCC/Y1 cells from the one activated by [T yr(36)]hPTHrP-(1-36)NH2. Radiolabeled PTHrP-(67-86)NH2 did not bind to SqCC/Y1 cells, and PTHrP-(67-86)NH2 did not compete for binding of I- 125-labeled [Tyr(36)]PTHrP-(1-36)NH2 to PTH/PTHrP receptors on SaOS-2 osteosarcoma cells. Activation of the phospholipase C pathway by PTHrP -(67-86)NH2 was confirmed by exposing SqCC/Y1 cells to peptide for 1 m in and measuring the ac cumulation of inositol trisphosphates. PTHrP-( 67-86)NH2 treatment (100 nM) resulted in maximal stimulation of inosit ol trisphosphates of 3.1+/-0.1-fold over the control value, with an EC (50) of 1.5+/-1.2 nM. In contrast, PTHrP-(67-86)NH2 (0.1 nM to 1 mu M) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation of cAMP formation by isoproterenol (1 mu M) to 66-fold ove r the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation of a receptor protein that mediated a [Ca2+](i) response to PTHrP-(67-86)NH2, we performed e xpression studies in Xenopus oocytes. Fluo-3 fluorescence in Xenopus o ocytes expressing SqCC/Y1 mRNA was visualized by confocal video micros copy after exposure to 1 mu M I PTHrP-(67-86)NH2. Clear increases in [ Ca2+](i) were detected in mRNA-injected, but not in sham-injected, ooc ytes. Finally, we examined the effect of PTHrP-(67-86)NH2 treatment on fibronectin secretion from SqCC/Y1 cells. A significant 3.5-fold incr ease in fibronectin secretion into conditioned medium was observed whe n SqCC/Y1 cells were exposed to 100 nM PTHrP-(67-86)NH2, and this effe ct was dose dependent, with an EC(50) of 0.1 nM. We conclude that PTHr P-(67-86)NH2 activates phospholipase C-dependent pathways in SqCC/Y1 c ells through a receptor distinct from that activated by PTHrP-(1-36) i n the same cells. As a midregion secretory fragment of PTHrP has been partially purified from several different cell types, this receptor ma y have broad biological significance.