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 stimulators of bone formation and osteoblast activities in vitro and in vivo. We tested the hypothesis that in vitro osteoblast senescence is associated with changes in components of the IGF-system including IGF-I, IGF-II, IGF-binding proteins (IGFBPs) and IGFBP-specific proteases. We employed a human diploid osteoblast cell line obtained from trabecular bone explants and that exhibit typical characteristics of in vitro senescence during serial subculturing. Using a non-competitive reverse-transcriptase polymerase-chain reaction (RT-PCR) assay, we found that the constitutive level of IGF-I mRNA decreased 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 production were assessed using Northern blot analysis and Western ligand blotting (WLB), respectively. IGFBP-3 mRNA levels decreased to 30% and protein production to 16% in aged osteoblasts as compared to levels found in young cells. We also found age-related decreases in mRNA levels of both IGFBP-4 and IGFBP-5 to 70% and 60% in aged osteoblasts, respectively, compared to young cells. While IGFBP-5 protein was not detected by WLB, IGFBP-4 protein production showed a biphasic change with 50% decrease in middle-aged cells and a subsequent increase in aged osteoblasts to levels similar to those in young osteoblasts. We found an age-related increase in the immunoreactive levels of IGFBP-4 protease, however, no detectable IGFBP-4 or IGFBP-3 protease activities in conditioned media from osteoblast cultures were observed. Our findings demonstrate that osteoblast aging is associated with impaired production of the stimulatory components of the IGF-system, that may be a mechanism contributing to age-related decline in osteoblast functions.