INTACT INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-5 (ICFBP-5) ASSOCIATES WITH BONE-MATRIX AND THE SOLUBLE FRAGMENTS OF ICFBP-5 ACCUMULATED IN CULTURE-MEDIUM OF NEONATAL MOUSE CALVARIAE BY PARATHYROID-HORMONE AND PROSTAGLANDIN E(2)-TREATMENT

We examined the distribution of insulin-like growth factor binding pro teins (IGFBPs) in cultured neonatal mouse calvariae. IGFBP-3 and -4 we re predominantly found in the conditioned medium. IGFBP-2 was partitio ned between conditioned medium and bone and extracellular matrix (BECM ), while intact(31-kDa) IGFBP-5 was most abundant in BECM extracts. Af ter treatment with parathyroid hormone (PTH, 10(-8) M) or prostaglandi n E(2) (PGE(2), 10(-6) M), immunoreactive IGFBP-5 accumulated in the c onditioned medium in a 21-kDa form which did not bind IGF-1 on Western ligand blots. PTH and PGE, did not alter the level of steady-state IG FBP-5 mRNA, nor markedly stimulate IGFBP-5 synthesis in the calvariae, and thus accumulation of 21-kDa IGFBP-5 was largely due to release fr om BECM. This accumulation of truncated IGFBP-5 in the conditioned med ium was not dependent on osteoclastic bone resorption, since it was no t blocked by calcitonin or a bisphosphonate which inhibited PTH- and P GE(2)-stimulated Ca-45-release. The conditioned medium from PTH- or PG E(2)-treated cultures degraded recombinant human IGFBP-5 into lower mo lecular weight fragments. Addition of IGF-1 at 10(-8) M into the cultu re resulted in accumulation of native 31-kDa IGFBP-5. However, even in the presence of IGF-1, the native IGFBP-5 was degraded and the 21-kDa product accumulated in the culture medium. These results suggested a possible proteolytic mechanism for 21-kDa IGFBP-5 accumulation, respon sive to PTH and PGE(2). Aprotinin, leupeptin, cystatin, and bestatin d id not inhibit the effects of PTH and PGE(2) in the cultures. The loca lization of IGFBP-5 in BECM and its release and proteolysis induced by PTH and PGE(2) could play a role in the local regulation of bone meta bolism. (C) 1996 Wiley-Liss, Inc.