Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism

  • Kawase, Tomoyuki
  • Okuda, Kazuhiro
  • Burns, Douglas M.

  • aDepartment of Signal Transduction Research, Division of Cellular Pharmacology, Graduate School of Medical and Dental Sciences, Niigata University, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan

    bDepartment of Oral Biological Science, Division of Periodontology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8514, Japan

    cMedical Research Service, Kansas City Department of Veterans Affairs Medical Center and Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, MO 64128, USA

*Corresponding author. Tel.: +81-25-227-2927; fax: +81-25-227-2927.

E-mail address:[email protected]

Received April 10, 2003; accepted April 23, 2003.

European Journal of Pharmacology 470(3):p 125-137, June 2003. | DOI: 10.1016/S0014-2999(03)01763-1

Abstract

Although accumulated data suggest that calcitonin gene-related peptide (CGRP) produces anabolic effects in skeletal tissue by directly acting on osteogenic cells, neither the distribution of CGRP receptor subtypes nor the associated cellular signaling pathways are well understood. In this study, we have pharmacologically and biochemically characterized CGRP-binding sites in immature human osteoblastic MG63 cells. In a [125I]CGRP whole-cell-binding assay, nonlinear regression curve-fitting analysis demonstrated a single binding site (KD=405±29 pM; 13,100±223 sites per cell). Immunocytochemical and Western blot analyses demonstrated that 48-, 52-, and 120-kDa forms of the calcitonin receptor-like receptor (CRLR) and a 15-kDa form of the receptor-activity-modifying protein-1 (RAMP-1) was expressed on the plasma membrane. CGRP strongly stimulated cellular cAMP production and this effect was antagonized not only by an antagonist of the subtype-1 CGRP (CGRP1) receptor, CGRP-(8-37), but by an agonist of the putative subtype-2 CGRP (CGRP2) receptor, [Cys(Acm)2,7]-CGRP, that also itself acted as a weak agonist. In contrast to published data, CGRP dose- and time-dependently dephosphorylated and inactivated extracellular signal response kinase (ERK). This action was blocked by CGRP-(8-37), by an inhibitor of cAMP-dependent protein kinase (H-89), or by an inhibitor of protein phosphatases (vanadate). Prolonged CGRP treatments significantly suppressed DNA synthesis at 27 h, but up-regulated type I collagen. Both these actions were blocked by CGRP-(8-37) and mimicked by a specific inhibitor of ERK (PD98059). In summary, our data suggest that the CGRP receptors in MG63 cells meet many, but not all, of the classical criteria used to define CGRP1 receptors. These receptors that functioned in a pharmacologically distinct manner could inhibit cell proliferation, and were substantially more sensitive to a CGRP2 receptor agonist than are typical CGRP1 receptors. These receptor proteins were not exactly matched with the known components of a CGRP1 receptor that have been reported. Therefore, it is possible that the CGRP receptors expressed in immature osteoblastic human MG63 cells represent a variation of the known CGRP1 receptor.

Copyright © 2003Elsevier, Inc.