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.
Department of Medicine, University of North Carolina, Chapel Hill, NC, USA.
Gang
G
Xi
Christine
C
Wai
Victoria
V
DeMambro
Clifford J
CJ
Rosen
David R
DR
Clemmons
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D015675:Osteocalcin / Q000096:biosynthesis; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5 / Q000235:genetics / Q000378:metabolism
D018971:Insulin-Like Growth Factor Binding Protein 2; D015675:Osteocalcin; D051057:Proto-Oncogene Proteins c-akt; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5
IGFBP-2; OSTEOBLAST DIFFERENTIATION; PTEN; RPTPβ; pAKT