Changes in the insulin-like growth factor-system may contribute to in vitro age-related impaired osteoblast functions

Age-related bone loss is thought to be due to impaired osteoblast functions . Insulin-like growth factors (IGFs) have been shown to be important stimul ators of bone formation and osteoblast activities in vitro and in vivo. We tested the hypothesis that in vitro osteoblast senescence is associated wit h changes in components of the IGF-system including IGF-I, IGF-II, IGF-bind ing proteins (IGFBPs) and IGFBP-specific proteases. We employed a human dip loid osteoblast cell line obtained from trabecular bone explants and that e xhibit typical characteristics of in vitro senescence during serial subcult uring. Using a non-competitive reverse-transcriptase polymerase-chain react ion (RT-PCR) assay, we found that the constitutive level of IGF-I mRNA decr eased progressively to 49.9 +/- 4.9% in old osteoblasts as compared to the levels found in the young cells. No age-related change was found in IGF-II steady-state mRNA levels. Changes in IGFBPs gene expression and protein pro duction were assessed using Northern blot analysis and Western ligand blott ing (WLB), respectively. IGFBP-3 mRNA levels decreased to 30% and protein p roduction to 16% in aged osteoblasts as compared to levels found in young c ells. We also found age-related decreases in mRNA levels of both IGFBP-4 an d IGFBP-5 to 70% and 60% in aged osteoblasts, respectively, compared to you ng cells. While IGFBP-5 protein was not detected by WLB, IGFBP-4 protein pr oduction showed a biphasic change with 50% decrease in middle-aged cells an d a subsequent increase in aged osteoblasts to levels similar to those in y oung osteoblasts. We found an age-related increase in the immunoreactive le vels of IGFBP-4 protease, however, no detectable IGFBP-4 or IGFBP-3 proteas e activities in conditioned media from osteoblast cultures were observed. O ur findings demonstrate that osteoblast aging is associated with impaired p roduction of the stimulatory components of the IGF-system, that may be a me chanism contributing to age-related decline in osteoblast functions. (C) 20 00 Elsevier Science Inc. All rights reserved.