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 intensively conserved across species, has been identified as a secretory product of several different cell types, including keratinocytes and squamous carcinomas. As recent data suggest that a midregion PTHrP fragment may be biologically active, we hypothesized that midregion PTHrPs interact with unique cell surface receptors that mediate autocrine or paracrine action. Dose-dependent transient elevations in intracellular calcium ([Ca2-]i) were observed in fura-2-loaded SqCC/Y1 squamous carcinoma cells exposed to human (h) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 microM. The effects of maximal stimulatory concentrations of [Tyr36]PTHrP-(1-36)NH2 and PTHrP-(67-86)NH2 on [Ca2+]i were additive. The inhibitory PTH analog, [D-Trp12,Tyr34]bovine PTH-(7-34)NH2, attenuated the [Ca2+]i response to [Tyr36]hPTHrP-(1-36)NH2, but not that to PTHrP-(67-86)NH2. These data suggest that PTHrP-(67-86)NH2 activates a different receptor pathway in SqCC/Y1 cells from the one activated by [Tyr36]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 125I-labeled [Tyr36]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 min and measuring the accumulation of inositol trisphosphates. PTHrP-(67-86)NH2 treatment (100 nM) resulted in maximal stimulation of inositol trisphosphates of 3.1 +/- 0.1-fold over the control value, with an EC50 of 1.5 +/- 1.2 nm. In contrast, PTHrP-(67-86)NH2 (0.1 nM to 1 microM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation of cAMP formation by isoproterenol (1 microM) to 66-fold over 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 expression studies in Xenopus oocytes. Fluo-3 fluorescence in Xenopus oocytes expressing SqCC/Y1 mRNA was visualized by confocal video microscopy after exposure to 1 microM PTHrP-(67-86)NH2. Clear increases in [Ca2+]i were detected in mRNA-injected, but not in sham-injected, oocytes. Finally, we examined the effect of PTHrP-(67-86)NH2 treatment on fibronectin secretion from SqCC/YN1 cells. A significant 3.5-fold increase in fibronectin secretion into conditioned medium was observed when SqCC/Y1 cells were exposed to 100 nM PTHrP-(67-86)NH2, and this effect was dose dependent, with an EC50 of 0.1 nM. We conclude that PTHrP-(67-86)NH2 activates phospholipase C-dependent pathways in SqCC/Y1 cells through a receptor distinct from that activated by PTHrP-(1-36) in the same cells. As a midregion secretory fragment of PTHrP has been partially purified from several different cell types, this receptor may have broad biological significance.
Orloff
J J
JJ
Division of Endocrinology and Metabolism, Veterans Affairs Connecticut Healthcare System, West Haven 06516, USA.
Ganz
M B
MB
Nathanson
M H
MH
Moyer
M S
MS
Kats
Y
Y
Mitnick
M
M
Behal
A
A
Gasalla-Herraiz
J
J
Isales
C M
CM
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis*; D009865:Oocytes / Q000378:metabolism; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology*; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014981:Xenopus
D005353:Fibronectins; C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D002118:Calcium