A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium and stimulates formation of inositol trisphosphate in a squamous carcinoma cell line

John J. Orloff, Michael B. Ganz, Michael H. Nathanson, M. Susan Moyer, Yuliya Kats, Maryann Mitnick, Amita Behal, Jose Gasalla-Herraiz, Carlos M Isales

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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 (b) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 μM. 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 μM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation el cAMP formation by isoproterenol (1 μM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation era 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 μM 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-(6786)NH2 treatment on fibronectin secretion from SqCC/Y1 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.

Original languageEnglish (US)
Pages (from-to)5376-5385
Number of pages10
JournalEndocrinology
Volume137
Issue number12
DOIs
StatePublished - Jan 1 1996

Fingerprint

Parathyroid Hormone-Related Protein
Inositol
Squamous Cell Carcinoma
Calcium
Cell Line
Proteins
Oocytes
Type C Phospholipases
Xenopus
Fibronectins
Messenger RNA
Parathyroid Hormone Receptor Type 1
Video Microscopy

ASJC Scopus subject areas

  • Endocrinology

Cite this

A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium and stimulates formation of inositol trisphosphate in a squamous carcinoma cell line. / Orloff, John J.; Ganz, Michael B.; Nathanson, Michael H.; Moyer, M. Susan; Kats, Yuliya; Mitnick, Maryann; Behal, Amita; Gasalla-Herraiz, Jose; Isales, Carlos M.

In: Endocrinology, Vol. 137, No. 12, 01.01.1996, p. 5376-5385.

Research output: Contribution to journalArticle

Orloff, John J. ; Ganz, Michael B. ; Nathanson, Michael H. ; Moyer, M. Susan ; Kats, Yuliya ; Mitnick, Maryann ; Behal, Amita ; Gasalla-Herraiz, Jose ; Isales, Carlos M. / A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium and stimulates formation of inositol trisphosphate in a squamous carcinoma cell line. In: Endocrinology. 1996 ; Vol. 137, No. 12. pp. 5376-5385.
@article{a5c152981da648fe928dad4acce287d3,
title = "A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium and stimulates formation of inositol trisphosphate in a squamous carcinoma cell line",
abstract = "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 (b) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 μM. 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 μM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation el cAMP formation by isoproterenol (1 μM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation era 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 μM 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-(6786)NH2 treatment on fibronectin secretion from SqCC/Y1 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.",
author = "Orloff, {John J.} and Ganz, {Michael B.} and Nathanson, {Michael H.} and Moyer, {M. Susan} and Yuliya Kats and Maryann Mitnick and Amita Behal and Jose Gasalla-Herraiz and Isales, {Carlos M}",
year = "1996",
month = "1",
day = "1",
doi = "10.1210/endo.137.12.8940360",
language = "English (US)",
volume = "137",
pages = "5376--5385",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "12",

}

TY - JOUR

T1 - A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium and stimulates formation of inositol trisphosphate in a squamous carcinoma cell line

AU - Orloff, John J.

AU - Ganz, Michael B.

AU - Nathanson, Michael H.

AU - Moyer, M. Susan

AU - Kats, Yuliya

AU - Mitnick, Maryann

AU - Behal, Amita

AU - Gasalla-Herraiz, Jose

AU - Isales, Carlos M

PY - 1996/1/1

Y1 - 1996/1/1

N2 - 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 (b) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 μM. 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 μM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation el cAMP formation by isoproterenol (1 μM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation era 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 μM 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-(6786)NH2 treatment on fibronectin secretion from SqCC/Y1 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.

AB - 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 (b) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 μM. 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 μM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation el cAMP formation by isoproterenol (1 μM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation era 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 μM 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-(6786)NH2 treatment on fibronectin secretion from SqCC/Y1 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.

UR - http://www.scopus.com/inward/record.url?scp=0030444856&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030444856&partnerID=8YFLogxK

U2 - 10.1210/endo.137.12.8940360

DO - 10.1210/endo.137.12.8940360

M3 - Article

C2 - 8940360

AN - SCOPUS:0030444856

VL - 137

SP - 5376

EP - 5385

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 12

ER -