IGFBP-2 directly stimulates osteoblast differentiation

Abstract

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.

Keywords: IGFBP-2; OSTEOBLAST DIFFERENTIATION; PTEN; RPTPβ; pAKT.

Figure 1. IGFBP-2 stimulates osteoblast differentiation

(A) Equal amounts of cell lysate from MC-3T3 cells expressing a shRNA sequence targeting LacZ (Ctrl Si) or IGFBP-2 (IGFBP-2 Si) were immunoblotted with indicated antibody. β-actin was immunoblotted as a loading control. (B) Cell lysate from Ctrl Si or IGFBP-2 Si cells on the indicated day after differentiation medium (DM) exposure were immunoblotted with the indicated antibody. (C) Ctrl Si or IGFBP-2 Si expressing cells were stained by Alizarin Red following the procedure described in “Materials and Methods” on indicated day after DM exposure. (D) Cell lysates from IGFBP-2−/− or IGFBP-2 +/+ derived calvarial osteoblasts prepared on indicated day after DM exposure were immunoblotted with indicated antibody. The bar graph shows the ratio of scanning densitometry units of osteocalcin/β-actin obtained from three individual experiments. (E) Calvarial osteoblasts isolated from IGFBP-2 +/+ or IGFBP-2 −/− mice were exposed to DM alone or DM plus IGFBP-2 (1 ug/ml) and stained with Alizarin Red on day 21. (F) Lysates from cells expressing LacZ or IGFBP-2 were immunoblotted with indicated antibody. (G) Lysates from cells expressing LacZ or IGFBP-2 on indicated day after DM exposure were immunoblotted with indicated antibody. β-actin was immunoblotted as a loading control. The bar graphs show the ratio of scanning densitometry units of osteocalcin/β-actin obtained from three individual experiments. (H) Cells expressing LacZ or IGFBP-2 were stained with Alizarin Red on indicated day after DM exposure.

Figure 2. AKT activation is required for osteoblast differentiation

(A) Lysates obtained from MC-3T3 cells, clone 24 (CL24) or clone 4 (CL4) on day 6 after DM exposure were immunoblotted with indicated antibody. Lysates from quiescent cells expressing LacZ or IGFBP-2 on day 6 after DM exposure were immunoblotted with the indicated antibody. Wild type MC-3T3 cells (B) or IGFBP-2 overexpressing cells (C) were stained by Alizarin Red on day 21 after differentiation medium (DM) alone or DM plus LY294002 which was added on the indicated day. The medium was changed every 72hr. (D) Lysates obtained from cells overexpressing IGFBP-2 or LacZ after indicated day of DM exposure were immunblotted with indicated antibody. The bar graph shows the ratio of scanning densitometry units of pAKT/β-actin obtained from three individual experiments. (E) Lysates from IGFBP-2−/− or IGFBP-2 +/+ derived calvarial osteoblasts obtained on indicated day after DM exposure were immunoblotted with indicated antibody. The bar graph shows the ratio of scanning densitometry units of pAKT/β-actin obtained from three individual experiments.

Figure 3. IGFBP-2 enhances AKT activation via suppressing RPTPβ dephosphorylation of PTEN

(A) Cell lysates from quiescent LacZ or IGFBP-2 overexpressing cells were immunoprecipitated with an anti-IGFBP-2 antibody and immunoblotted with an anti-RPTPβ antibody. β-actin was immunoblotted as a loading control. (B) Lysates from LacZ or IGFBP-2 overexpressing cells on indicated day after differentiation medium (DM) exposure were immunoprecipated with an anti-PY99 antibody and immunoblotted with an anti-PTEN antibody. PTEN was immunoblotted as an input control. (C, D) Lysates from MT-3C3 cells expressing shRNA sequence targeting LacZ (Ctrl Si) or RPTPβ (RPTPβ Si) were immunoblotted with the indicated antibodies. (E) Cells expressing Ctrl Si or RPTPβ Si were stained by Alizarin Red on indicated day after DM exposure. (F) Lysates from LacZ or RPTPβ overexpressing cells were immunoblotted with anti-HA and β-actin antibodies. The same cell lysates were immunoprecipitated with an anti-PY99 antibody and immuoblotted with an anti-PTEN antibody. PTEN was immunoblotted as an input control. (G) Cells expressing LacZ or RPTPβ were stained by Alizarin Red on indicated day after DM exposure. (H) Cells expressing RPTPβ were stained by Alizarin Red on day 21 after DM alone or DM plus IGFBP-2 exposure. Lysates from the same RPTPβ overexpressing cultures were immunoblotted with anti-osteocalcin and β-actin antibodies. (I) Lysates from quiescent RPTPβ overexpressing cells obtained on day 6 after IGF-I alone or IGFBP-2 alone (1ug/ml) or IGF-I plus IGFBP-2 (1 ug/ml) were immunoblotted with anti-pAKT and β-actin antibodies.

Figure 4. Disruption of IGFBP-2/RPTPβ interaction impairs IGF-I-stimulated AKT activation and osteoblast differentiation

(A) Lysates from quiescent LacZ or IGFBP-2 overexpressing cells obtained on day 6 after differentiation medium (DM) exposure treated with or without IGF-I alone (10 min) or IGF-I following a 4 hr exposure to anti-fibronectin domain (FN3) antibody were immunoprecipitated using an anti-IGFBP-2 antibody and immunoblotted with an anti-RPTPβ antibody (B) Lysates from quiescent IGFBP-2 overexpressing cells that received the same treatments as in panel A were obtained on Day 6 after DM exposure and were immunoblotted with indicated antibody. (C) Lysates from LacZ overexpressing cells obtained on day 21 after DM exposure following incubation with the indicated concentration of anti-fibronectin antibody (FN3) were immunoblotted with anti-osteocalcin and anti-β-actin antibodies. (D) Lysates from IGFBP-2 overexpressing cells obtained on day 9 and 12 after DM exposure following incubation with PQ401 (10 uM) were immunoblotted with anti-osteocalcin and anti-β-actin antibodies. (E) Cells expressing IGFBP-2 that had been incubated with or without PQ401 were stained with Alizarin Red on day 18 after DM exposure.

Figure 5. The heparin binding domain-1 (HBD-1) mediates the IGFBP-2 effect on osteoblast differentiation

(A) Calvarial osteoblasts isolated from IGFBP-2 +/+ or IGFBP-2 −/− mice were exposed to differentiation medium (DM) alone, DM plus IGFBP-2 (1 ug/ml), DM plus control peptide (Ctrl Pep, 1 ug/ml), DM plus HBD-1 (1 ug/ml) or DM plus the HBD-1 IGFBP-2 mutant protein (IGFBP-2 MP, 1 ug/ml) then stained with Alizarin Red on day 21. The bar graph shows the percentage of stained area that was quantified using NIH Image J (1.47n). (B, C) Calvarial osteoblasts isolated from IGFBP-2 −/− mice were exposed to DM alone (Ctrl) or DM plus the indicated concentration of HBD-1 peptide for 21 days and stained with Alizarin Red (B). Cell lysates were immunoblotted with anti-osteocalcin and β-actin antibodies (C).