Estrogen Stimulation of Pleiotrophin Enhances Osteoblast Differentiation and Maintains Bone Mass in IGFBP-2 Null Mice

Abstract

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.

Keywords: bone mineral density; insulin-like growth factor-1; osteocalcin; receptor tyrosine phosphatase beta; sexual dimophism.

Figure 1.

Sexual dimorphisms in bone mass and expression of pleiotrophin (PTN) and receptor tyrosine phosphatase beta (RPTPβ) in male and female Igfbp2^–/– mice at 6 months of age. (A) Femoral areal bone mineral density (aBMD). (B) Distal femur trabecular bone volume/total volume (BV/TV) in Igfbp2^–/– and Igfbp2^+/+ male and female mice (n = 10/sex/genotype). Femoral mRNA expression of (C) PTN (D) RPTPβ (n = 6/sex/genotype). In vitro osteoclast expression of Ptn (E) Rptpβ (F) (n = 6/sex/genotype). For all analysis *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 2.

Pleiotrophin (PTN) expression is enhanced in calvarial osteoblasts isolated from insulin-like growth factor binding protein-2 (IGFBP-2) knockout mice or in IGFBP-2 knockdown MC3T3 cells. (A, B, C) Lysates were prepared from calvarial osteoblasts isolated from female IGFBP-2 knockout (–) mice or female wild type (+) mice following the instructions described in methods (A) or from IGFBP-2 knockdown (IGFBP-2 Si) or control knockdown (LacZ Si) MC3T3 cells (B, C). Cell lysates were harvested at the indicated time points and were immunoblotted with an anti-PTN (PTN) (A, C), anti-IGFBP-2 (B), or anti-β-actin antibody.

Figure 3.

Pleiotrophin (PTN) expression is upregulated at the early stage of osteoblasts differentiation but downregulated at the late stage of differentiation. (A, B) Lysates were prepared from the CL4 cells (A) or calvarial osteoblasts isolated from the wild type mice (both sexes) (B).The cultures were exposed to differentiation medium at day 0 and were analyzed at the indicated time points. The lysates were immunoblotted with an anti-PTN antibody or anti-β-actin antibody.

Figure 4.

Knockdown of pleiotrophin (PTN) inhibits osteocalcin expression which is restored by adding PTN exogenously. (A) Lysates were harvested from the CL4 cells transfected with a control siRNA (Ctrl Si) or a siRNA targeting PTN (PTN Si). The lysates were immunoblotted with an anti-PTN antibody or anti-β-actin antibody. (B) CL4 cells were treated with a control siRNA or a siRNA targeting PTN in the presence or absence of PTN. Cell lysates were immunoblotted with an anti-osteocalcin (OCN), anti-collagen 1A (Col 1A) or anti-β-actin antibody.

Figure 5.

Pleiotrophin (PTN)-stimulated PTEN tyrosine phosphorylation, AKT activation and osteocalcin expression are attenuated by an anti-fibronectin antibody. (A) Lysates were prepared from the CL4 cells treated with exogenous PTN in the presence or absence of an anti-fibronectin-3 (FN3) antibody. Cell lysates were immunoprecipitated with an anti-phospho-tyrosine (pY99) antibody and immunoblotted with an anti-PTEN antibody. The same amount of lysate was immunoblotted with an anti-PTEN antibody. (B, C) Lysates were prepared from the CL4 cells treated with exogenous PTN in the presence or absence of an anti-FN3 antibody. Cell lysates were immunoblotted with an anti-pAKT antibody (B) or anti-osteocalcin (OCN), anti-collagen 1A (Col 1A) (C) or anti-β-actin antibody.

Figure 6.

Estradiol-stimulated osteoblasts differentiation requires the presence of pleiotrophin (PTN). (A) Cell lysates were prepared from the CL4 cells treated with the different amounts of estradiol. The lysates were immunoblotted with an anti-osteocalcin (OCN) antibody or anti-β-actin antibody. (B) Lysates were prepared using CL4 cells transfected with a control siRNA (Ctrl si) or a siRNA targeting PTN (PTN si) in the presence or absence of estradiol. The lysates were immunoblotted with an anti- PTN antibody or anti-β-actin antibody. (C) CL4 cells were treated with a control siRNA or a siRNA targeting PTN in the presence or absence of PTN or estradiol. Cell lysates were immunoblotted with an anti-osteocalcin (OCN) or anti-collagen 1A (Col 1A) or anti-β-actin antibody.

Figure 7.

Pleiotrophin (PTN) or estradiol stimulate osteoblast differentiation which is attenuated by an anti-fibronectin-3 antibody in IGFBP-2 knockdown cells. (A) Cell lysates were prepared from the LacZ-silenced or IGFBP-2 knockdown CL4 cells in the absence or presence of PTN or anti-fibronectin (FN3) antibody. Cell lysates were immunoblotted with an anti-collagen 1A (Col 1A), or anti-β-actin antibody. (B) Lysates were prepared from the LacZ-silenced or IGFBP-2 knockdown CL4 cells that had been cultured in the absence or presence of estradiol. The lysates were immunoblotted with an anti- PTN or anti-β-actin antibody. (C, D, E) Lysates were prepared from the LacZ-silenced or IGFBP-2 knockdown CL4 cells cultured in the presence or absence of estradiol or anti-fibronectin (FN3) antibody. The lysates were immunoblotted with an anti-pAKT or anti-β-actin antibody (C). Lysates were immunoprecipitated with an anti-phospho-tyrosine (pY99) antibody and immunoblotted with an anti-PTEN antibody. The same amount of cell lysate was immunoblotted with an anti-PTEN antibody (D). Cell lysates were immunoblotted with an anti-collagen 1A (Col 1A) or anti-β-actin antibody (E).

Figure 8.

Estradiol treatment significantly stimulates bone mass in Igfbp2^–/– and Igfbp2^+/+ male mice and RPTPβ expression in null males. (A) Whole body areal bone mineral density (aBMD) changes during estrogen treatment between 8 and 16 weeks of age (n = 10/treatment/genotype). (B) Distal femur trabecular bone volume/total volume (BV/TV) in Igfbp2^+/+ and Igfbp2^–/– after 8 weeks of estradiol treatment. Femoral mRNA expression of PTN (C) and RPTPβ (D) (n = 6/ treatment /genotype). For all analysis *P < .05; **P < .01; ***P < .001; ****P < .0001.