Calcitonin (CT) is a peptide hormone that interacts with the cAMP- and phospholipase C-associated CT receptor subtypes. We investigated whether CT modulates the interaction of human tumoral osteoclast-like (GCT23) cells with a protein of the bone matrix, bone sialoprotein-II (BSP-II). Single GCT23 cells loaded with the intracellular Ca2+ indicator fura-2 were treated with the maximal active dose (300 mu g/ml) of the 18-mer Arg-Gly-Asp (RGD)-containing BSP-IIA fragment, and the cytosolic free Ca2+ concentration ([Ca2(+)](i)) was measured by dual wavelength microfluorometry. BSP-IIA stimulated an elevation in [Ca2+](i), consisting mainly of a peak, followed by a rapid return toward baseline. Pretreatment with CT induced a modest elevation of[Ca2+](i). However, CT significantly inhibited the response to BSP-IIA in a dose-dependent manner. Maximal inhibition (90% vs. untreated) was observed in the micromolar range. The intracellular mechanisms leading to this effect were investigated by pretreatment of GCT23 cells with the cAMP permeant analog, (Bu(2))cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 mu g/ml BSP-IIA. The effect induced by CT was specific, because an increase in the extracellular Ca2+ concentration, which is also known to inhibit bone resorption, failed to modify the ability of BSP-IIA to alter [Ca2+](i) in GCT23 cells. To investigate whether the CT-induced alteration of BSP-IIA-dependent cell signals was due to a modification in the synthesis of cell surface receptors (integrins) for the extracellular matrix macromolecules, 1-h CT-treated [S-35]methionine metabolically labeled GCT23 cell lysates were immunoprecipitated with anti-alpha(3)-, -alpha(v)-, beta(1)-, and -beta(3)-integrin subunit antibodies. Autoradiography demonstrated that 10(-7)-10(-6) M CT did not alter new synthesis of the alpha(v) beta(3) and the alpha(3) beta(1) receptors. Similarly, CT did not affect surface expression of these receptors, assessed by enzyme-linked immunosorbent assay. Finally, no alteration of the adhesion rate and spreading of GCT23 cells onto BSP-ILA-coated substrates was observed. This indicates that CT-induced down-regulation of immediate cell signals prompted by BSP-IIA in GCT23 cells is a postintegrin receptor event.

CALCITONIN DOWN-REGULATES IMMEDIATE CELL SIGNALS INDUCED IN HUMAN OSTEOCLAST-LIKE CELLS BY THE BONE SIALOPROTEIN-IIA FRAGMENT THROUGH A POSTINTEGRIN RECEPTOR MECHANISM

ZANI, Bianca Maria;TETI, ANNA MARIA
1995

Abstract

Calcitonin (CT) is a peptide hormone that interacts with the cAMP- and phospholipase C-associated CT receptor subtypes. We investigated whether CT modulates the interaction of human tumoral osteoclast-like (GCT23) cells with a protein of the bone matrix, bone sialoprotein-II (BSP-II). Single GCT23 cells loaded with the intracellular Ca2+ indicator fura-2 were treated with the maximal active dose (300 mu g/ml) of the 18-mer Arg-Gly-Asp (RGD)-containing BSP-IIA fragment, and the cytosolic free Ca2+ concentration ([Ca2(+)](i)) was measured by dual wavelength microfluorometry. BSP-IIA stimulated an elevation in [Ca2+](i), consisting mainly of a peak, followed by a rapid return toward baseline. Pretreatment with CT induced a modest elevation of[Ca2+](i). However, CT significantly inhibited the response to BSP-IIA in a dose-dependent manner. Maximal inhibition (90% vs. untreated) was observed in the micromolar range. The intracellular mechanisms leading to this effect were investigated by pretreatment of GCT23 cells with the cAMP permeant analog, (Bu(2))cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 mu g/ml BSP-IIA. The effect induced by CT was specific, because an increase in the extracellular Ca2+ concentration, which is also known to inhibit bone resorption, failed to modify the ability of BSP-IIA to alter [Ca2+](i) in GCT23 cells. To investigate whether the CT-induced alteration of BSP-IIA-dependent cell signals was due to a modification in the synthesis of cell surface receptors (integrins) for the extracellular matrix macromolecules, 1-h CT-treated [S-35]methionine metabolically labeled GCT23 cell lysates were immunoprecipitated with anti-alpha(3)-, -alpha(v)-, beta(1)-, and -beta(3)-integrin subunit antibodies. Autoradiography demonstrated that 10(-7)-10(-6) M CT did not alter new synthesis of the alpha(v) beta(3) and the alpha(3) beta(1) receptors. Similarly, CT did not affect surface expression of these receptors, assessed by enzyme-linked immunosorbent assay. Finally, no alteration of the adhesion rate and spreading of GCT23 cells onto BSP-ILA-coated substrates was observed. This indicates that CT-induced down-regulation of immediate cell signals prompted by BSP-IIA in GCT23 cells is a postintegrin receptor event.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11697/11273
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