Insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins-2 (IGFBP-2) function coordinately to stimulate osteoblast differentiation. Induction of AMP-activated protein kinase (AMPK) is required for differentiation and is stimulated by these two factors. These studies were undertaken to determine how these two peptides lead to activation of AMPK. Enzymatic inhibitors and small interfering RNA were utilized to attenuate calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) activity in osteoblasts, and both manipulations resulted in failure to activate AMPK, thereby resulting in inhibition of osteoblast differentiation. IGFBP-2 and IGF-I stimulated an increase in CaMKK2, and inhibition of IGFBP-2 binding its receptor resulted in failure to induce CaMKK2 and AMPK activation. Injection of a peptide that contained the IGFBP-2 receptor-binding domain into IGFBP-2 −/− mice activated CaMKK2 and injection of a CaMKK2 inhibitor into normal mice inhibited both CamKK2 and AMPK activation in osteoblasts. We conclude that induction of CaMKK2 by IGFBP-2 and IGF-I in osteoblasts is an important signaling event that occurs early in differentiation and is responsible for activation of AMPK, which is required for optimal osteoblast differentiation.
Insulin-like growth factor binding protein-2 (IGFBP-2) stimulates osteoblast differentiation but only male Igfbp2 null mice have a skeletal phenotype. The trophic actions of IGFBP-2 in bone are mediated through its binding to receptor tyrosine phosphatase beta (RPTPβ). Another important ligand for RPTPβ is pleiotrophin (PTN), which also stimulates osteoblast differentiation. We determined the change in PTN and RPTPβ in Igfbp2–/– mice. Analysis of whole bone mRNA in wild-type and knockout mice revealed increased expression of Ptn. Rptpβ increased in gene-deleted animals with females having greater expression than males. Knockdown of PTN expression in osteoblasts in vitro inhibited differentiation, and addition of PTN to the incubation medium rescued the response. Estradiol stimulated PTN secretion and PTN knockdown blocked estradiol-stimulated differentiation. PTN addition to IGFBP-2 silenced osteoblast stimulated differentiation, and an anti-fibronectin-3 antibody, which inhibits PTN binding to RPTPβ, inhibited this response. Estrogen stimulated PTN secretion and downstream signaling in the IGFBP-2 silenced osteoblasts and these effects were inhibited with anti-fibronectin-3. Administration of estrogen to wild-type and Igfbp2–/– male mice stimulated an increase in both areal bone mineral density and trabecular bone volume fraction but the increase was significantly greater in the Igfbp2–/– animals. Estrogen also stimulated RPTPβ expression in the null mice. We conclude that loss of IGFBP-2 expression is accompanied by upregulation of PTN and RPTPβ expression in osteoblasts, that the degree of increase is greater in females due to estrogen secretion, and that this compensatory change may account for some component of the maintenance of normal bone mass in female mice.
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