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IGFBP‐2 Directly Stimulates Osteoblast Differentiation

Insulin‐like growth factor binding protein 2 (IGFBP‐2) is important for acquisition of normal bone mass in mice; however, the mechanism by which IGFBP‐2 functions is not defined. These studies investigated the role of IGFBP‐2 in stimulating osteoblast differentiation. MC‐3T3 preosteoblasts expressed IGFBP‐2, and IGFBP‐2 knockdown resulted in a substantial delay in osteoblast differentiation, reduced osteocalcin expression and Alizarin red staining. These findings were replicated in primary calvarial osteoblasts obtained from IGFBP‐2−/− mice, and addition of IGFBP‐2 rescued the differentiation program. In contrast, overexpression of IGFBP‐2 accelerated the time course of differentiation as well as increasing the total number of differentiating cells. By day 6, IGFBP‐2–overexpressing cells expressed twice as much osteocalcin as control cultures and this difference persisted. To determine the mechanism by which IGFBP‐2 functions, the interaction between IGFBP‐2 and receptor tyrosine phosphatase β (RPTPβ) was examined. Disruption of this interaction inhibited the ability of IGFBP‐2 to stimulate AKT activation and osteoblast differentiation. Knockdown of RPTPβ enhanced osteoblast differentiation, whereas overexpression of RPTPβ was inhibitory. Adding back IGFBP‐2 to RPTPβ‐overexpressing cells was able to rescue cell differentiation via enhancement of AKT activation. To determine the region of IGFBP‐2 that mediated this effect, an IGFBP‐2 mutant that contained substitutions of key amino acids in the heparin‐binding domain‐1 (HBD‐1) was prepared. This mutant had a major reduction in its ability to stimulate differentiation of calvarial osteoblasts from IGFBP‐2−/− mice. Addition of a synthetic peptide that contained the HBD‐1 sequence to calvarial osteoblasts from IGFBP‐2−/− mice rescued differentiation and osteocalcin expression. In summary, the results clearly demonstrate that IGFBP‐2 stimulates osteoblast differentiation and that this effect is mediated through its heparin‐binding domain‐1 interacting with RPTPβ. The results suggest that stimulation of differentiation is an important mechanism by which IGFBP‐2 regulates the acquisition of normal bone mass in mice. © 2014 American Society for Bone and Mineral Research.