Intact insulin-like growth factor binding protein-5 (IGFBP-5) associates with bone matrix and the soluble fragments of IGFBP-5 accumulated in culture medium of neonatal mouse calvariae by parathyroid hormone and prostaglandin E2-treatment.
We examined the distribution of insulin-like growth factor binding proteins (IGFBPs) in cultured neonatal mouse calvariae. IGFBP-3 and -4 were predominantly found in the conditioned medium. IGFBP-2 was partitioned between conditioned medium and bone and extracellular matrix (BECM), while intact (31-kDa) IGFBP-5 was most abundant in BECM extracts. After treatment with parathyroid hormone (PTH, 10(-8) M) or prostaglandin E2 (PGE2, 10(-6) M), immunoreactive IGFBP-5 accumulated in the conditioned medium in a 21-kDa form which did not bind IGF-I on Western ligand blots. PTH and PGE2 did not alter the level of steady-state IGFBP-5 mRNA, nor markedly stimulate IGFBP-5 synthesis in the calvariae, and thus accumulation of 21-kDa IGFBP-5 was largely due to release from BECM. This accumulation of truncated IGFBP-5 in the conditioned medium was not dependent on osteoclastic bone resorption, since it was not blocked by calcitonin or a bisphosphonate which inhibited PTH- and PGE2-stimulated 45Ca-release. The conditioned medium from PTH- or PGE2-treated cultures degraded recombinant human IGFBP-5 into lower molecular weight fragments. Addition of IGF-1 at 10(-8) M into the culture 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, responsive to PTH and PGE2. Aprotinin, leupeptin, cystatin, and bestatin did not inhibit the effects of PTH and PGE2 in the cultures. The localization of IGFBP-5 in BECM and its release and proteolysis induced by PTH and PGE2 could play a role in the local regulation of bone metabolism.
Hakeda
Y
Y
Division of Endocrinology and Metabolism, University of Connecticut Health Center, Farmington 06030, USA.
Kawaguchi
H
H
Hurley
M
M
Pilbeam
C C
CC
Abreu
C
C
Linkhart
T A
TA
Mohan
S
S
Kumegawa
M
M
Raisz
L G
LG
D000818:Animals; D000831:Animals, Newborn; D001857:Bone Matrix / Q000378:metabolism*; D001842:Bone and Bones / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000737:chemistry; D046508:Culture Techniques; D015232:Dinoprostone / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012886:Skull / Q000378:metabolism; D012995:Solubility
D017077:Culture Media, Conditioned; D018975:Insulin-Like Growth Factor Binding Protein 5; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D012333:RNA, Messenger; D010450:Endopeptidases; D015232:Dinoprostone