An Estrogen Stimulated Alternative Signaling Pathway Responsible for the Sexual Dimorphisms Observed in Igfbp2-/- Mice

Presentation Number: SAT 351
Date of Presentation: April 1st, 2017

Victoria DeMambro*1, Lindsey Gower1, Carolina Figueroa1, Susan D'Costa2, David Clemmons2 and Clifford Rosen1
1Maine Medical Center Research Institute, Scarborough, ME, 2University of North Carolina, Chapel Hill, NC

Abstract

We previously reported a sexual dimorphism in the Igfbp2-/- mice in regards to bone, where males have reduced bone mass while females have normal bone relative to controls. Ovariectomized Igfbp2-/- female mice exhibited greater bone loss than controls, becoming a skeletal phenocopy of the Igfbp2-/- males. IGFBP-2 has been shown to bind to the PTPRZ1 receptor, which also binds pleotrophin (PTN), a molecule produced by bone cells and stimulated by estrogen. We hypothesized that PTN/PTPRZ1 signaling could be an alternative pathway for female Igfbp2-/- mice to maintain bone mass in the absence of IGFBP-2 and that the gender differences in bone in the absence of IGFBP-2 are related to the estrogen stimulation of PTN expression. To test this hypothesis in vitro we silenced IGFBP-2 in a MC3T3L1 cell line and treated with estrogen (200pg/ml) in the presence and absence of an anti-fibronectin3 antibody (α-FN3), which disrupts ligand binding to the PTPRZ1 receptor. In vivo we implanted either 17B estradiol (0.18mg) or control pellets in 8 week old +/+ and Igfbp2-/- male mice for 8 weeks to determine if estrogen stimulation of PTN/PTPRZ1 signaling was sufficient to rescue the bone phenotype in the male null mice or if the presence of IGFBP-2 was also required for full rescue. Mice were assessed for body composition and aBMD by DEXA at 8 and 16 weeks of age. At harvest bones were isolated for gene expression and MicroCT. In vitro, in the absence of IGFBP-2 estrogen stimulated PTN secretion, which increased differentiation, while an inhibitor of ligand binding to the PTPRZ1 receptor repressed the effect of PTN. Likewise PTN knockdown in the absence of IGFBP-2 inhibited differentiation. In vivo at 8 weeks of age Igfbp2-/- mice had significant decreases in whole body aBMD (p=0.01) and aBMC (p=0.003) as expected. At 16 weeks of age estrogen treatment increased aBMD and aBMC compared to controls in both genotypes (P<0.0001). Strikingly Igfbp2-/- mice had a 26% increase in aBMD vs. 21% increase in +/+ mice (p=0.04), eliminating the low aBMD present in the 16 week Igfbp2-/- controls. MicroCT analysis of the femur confirmed that the Igfbp2-/- mice were more responsive to estrogen treatment with greater increases in trabecular BV/TV (+/+ = 129% vs. -/- 221%; p= 0.007), due to increased number and thickness of the trabeculae. Igfbp2-/- cortical bone was also more sensitive to estrogen stimulation with a 30% increase in Ct. A/TA vs. a 22% increase in the +/+ mice (p= 0.007). Gene expression analysis of the femur revealed a two-fold increase in Ptn and Ptprz1 in the mice treated with estrogen confirming the upregulation of this pathway observed in vitro. In summary, an estrogen stimulated alternate signaling pathway through PTN/PTPRZ1 in the absence of IGFBP-2 can completely rescue the low bone mass observed in males and appears to be responsible for the gender differences observed in the bones of the Igfbp2-/- mice.

 

Nothing to Disclose: VD, LG, CF, SD, DC, CR