The sclerostin-independent bone anabolic activity of intermittent PTH treatment is mediated by T-cell-produced Wnt10b

Abstract

Both blunted osteocytic production of the Wnt inhibitor sclerostin (Scl) and increased T-cell production of the Wnt ligand Wnt10b contribute to the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. However, the relative contribution of these mechanisms is unknown. In this study, we modeled the repressive effects of iPTH on Scl production in mice by treatment with a neutralizing anti-Scl antibody (Scl-Ab) to determine the contribution of T-cell-produced Wnt10b to the Scl-independent modalities of action of iPTH. We report that combined treatment with Scl-Ab and iPTH was more potent than either iPTH or Scl-Ab alone in increasing stromal cell production of OPG, osteoblastogenesis, osteoblast life span, bone turnover, bone mineral density, and trabecular bone volume and structure in mice with T cells capable of producing Wnt10b. In T-cell-null mice and mice lacking T-cell production of Wnt10b, combined treatment increased bone turnover significantly more than iPTH or Scl-Ab alone. However, in these mice, combined treatment with Scl-Ab and iPTH was equally effective as Scl-Ab alone in increasing the osteoblastic pool, bone volume, density, and structure. These findings demonstrate that the Scl-independent activity of iPTH on osteoblasts and bone mass is mediated by T-cell-produced Wnt10b. The data provide a proof of concept of a more potent therapeutic effect of combined treatment with iPTH and Scl-Ab than either alone.

Keywords: ANTIBODY; BONE; PTH; SCL; T CELLS; WNT10B.

Fig. 1

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on spine BMD in WT mice (A), TCRβ^−/− mice (B), TCRβ^−/− mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b T cells (D) and global Wnt10b^−/− mice (E). In vivo spinal BMD was measured by DXA at 2 and 4 weeks of treatment (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle +Irr.Ig-treated group or the indicated group. #p < 0.05 compared with the corresponding group at 2 weeks.

Fig. 2

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on spine trabecular bone volume (BV/TV) as measured by μCT scanning in WT mice (A), TCRβ^−/− mice (B), TCRβ^−/− mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b^−/− T cells (D) and global Wnt10b^−/− mice (E) (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the indicated group.

Fig. 3

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on femoral cortical volume (Co.Vo) as measured by μCT scanning in WT mice (A), TCRβ^−/− mice (B), TCRβ^−/− mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b^−/− T cells (D) and global Wnt10b^−/− mice (E) (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the indicated group.

Fig. 4

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on histomorphometric indices of bone formation and resorption. (A) Number of osteoblasts per mm bone surface (N.Ob/BS). (B) Percentage of bone surface covered by osteoblasts (Ob.S/BS). (C) Number of osteoclasts per mm bone surface (N.Oc/BS). (D) Percentage of bone surface covered by osteoclasts (Oc.S/BS). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle + Irr.Ig-treated group. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the Scl-Ab–treated group.

Fig. 5

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on serum P1NP levels in WT mice (A), TCRβ^−/− mice (B), TCRβ^−/− mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b^−/− T cells (D) and global Wnt10b^−/− mice (E) (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the indicated group.

Fig. 6

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on serum CTX levels in WT mice (A), TCRβ^−/− mice (B), TCRβ^−/− mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b^−/− T cells (D) and global Wnt10b^−/− mice (E) (n = 10 mice per group). *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the indicated group.

Fig. 7

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on CFU-ALP formation, SC number, SC proliferation, and SC apoptosis in WT mice, TCRβ^−/− mice, TCRβ^−/− mice previously subjected to adoptive transfer of WT T cells or Wnt10b^−/− T cells, and global Wnt10b^−/− mice. (A) Whole BM was cultured for 7 days to assess the formation CFU-ALP. (B) BM harvested at death was cultured for 1 week and SCs purified and counted. (C) SCs were purified from BM cultured for 1 week, seeded in equal number and pulsed with ^3H-thymidine for 18 hours to assess their proliferation. Data are expressed in CPM. (D) SCs were purified from BM cultured for 1 week and the rate of apoptosis quantified by determinations of caspase3 activity (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle or to the indicated group. #p < 0.05 compared with Scl-Ab treatment.

Fig. 8

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on OB differentiation in WT mice (A), TCRβ^−/− mice (B), TCRβ^−/− mice previously subjected to adoptive transfer of WT T cells (C), or Wnt10b^−/− T cells (D) and global Wnt10b^−/− mice (E). SCs were purified from BM cultured for 1 week and the level of OB marker gene mRNAs, bone sialoprotein (BSP), type I collagen (Col1), osteocalcin (Ocn), osterix (Osx), and runt related transcription factor 2 (Runx2) analyzed by RT-PCR (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle. #p < 0.05 compared with Scl-Ab treatment.

Fig. 9

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on SC expression of OPG (A) and RANKL (B) mRNA in WT mice, TCRβ^−/− mice, TCRβ^−/− mice previously subjected to adoptive transfer of WT T cells or Wnt10b^−/− T cells and global Wnt10b^−/− mice. SCs were purified from BM cultured for 1 week and the level of OPG and RANKL mRNA analyzed by RT-PCR (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle or to the indicated group. #p < 0.05 compared with Scl-Ab treatment.

Fig. 10

Schematic representation of the mechanism of action of iPTH. In baseline conditions, T cells produce low levels of Wnt10b, whereas osteocytes secrete high levels of Scl. The presence of a low Wnt10b/Scl ration prevents the activation of Wnt signaling in OBs leading to low osteoblastogenesis and high OB apoptosis. Treatment with iPTH increases the T-cell production of Wnt10b and blunts the osteocytic secretion of Scl, thus increasing the Wnt10b/Scl ration. Wnt10b is now capable of activating Wnt signaling in OBs leading to increased osteoblastogenesis and decreased OB apoptosis.