Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism.
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 micrograms/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, (Bu2)cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 micrograms/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 [35S]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-IIA-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.
Paniccia
R
R
Department of Experimental Medicine, University of L'Aquila, Italy.
Riccioni
T
T
Zani
B M
BM
Zigrino
P
P
Scotlandi
K
K
Teti
A
A
D000818:Animals; D002116:Calcitonin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D016023:Integrins / Q000502:physiology*; D009994:Osmolar Concentration; D010010:Osteoclasts / Q000187:drug effects* / Q000502:physiology; D010446:Peptide Fragments / Q000494:pharmacology*; D012474:Salmon; D012795:Sialoglycoproteins / Q000737:chemistry / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D014407:Tumor Cells, Cultured
C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D016023:Integrins; D010446:Peptide Fragments; D012795:Sialoglycoproteins; D002116:Calcitonin; D002118:Calcium