Silencing of parathyroid hormone (PTH) receptor 1 in T cells blunts the bone anabolic activity of PTH

Abstract

Intermittent parathyroid hormone (iPTH) treatment stimulates T-cell production of the osteogenic Wnt ligand Wnt10b, a factor required for iPTH to activate Wnt signaling in osteoblasts and stimulate bone formation. However, it is unknown whether iPTH induces Wnt10b production and bone anabolism through direct activation of the parathyroid hormone (PTH)/PTH-related protein receptor (PPR) in T cells. Here, we show that conditional silencing of PPR in T cells blunts the capacity of iPTH to induce T-cell production of Wnt10b; activate Wnt signaling in osteoblasts; expand the osteoblastic pool; and increase bone turnover, bone mineral density, and trabecular bone volume. These findings demonstrate that direct PPR signaling in T cells plays an important role in PTH-induced bone anabolism by promoting T-cell production of Wnt10b and suggest that T cells may provide pharmacological targets for bone anabolism.

Fig. 1.

Effect (mean ± SEM) of in vivo iPTH treatment and in vitro PTH treatment on the T-cell expression of Wnt10b mRNA, SC expression of Wnt-dependent genes, bone density, and BV/TV in PPR^{T cells−/−} mice or Wnt10b^−/− mice. (A) Effect of iPTH on BM T-cell levels of Wnt10b mRNA in PPR^{T cells−/−} and control PPR^fl/fl female mice at 6 wk of age. (B) Effect of iPTH on whole-BM levels of Wnt10b mRNA in PPR^{T cells−/−} and control female mice at 6 wk of age. (C) Effect of in vitro PTH treatment on Wnt10b mRNA levels in BM T cells from PPR^{T cells−/−} and control female mice at 6 wk of age. T cells were stimulated with plate-bound anti-CD3 plus anti-CD28 mAbs for 24 h and were cultured with vehicle (Veh) or PTH (50 nM) for 24 h. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 100 μM) or vehicle was added 1 h before PTH. (D) Effect of in vitro PTH treatment on whole-BM levels of Wnt10b mRNA in PPR^{T cells−/−} and control female mice at 6 wk of age. Whole-BM samples were stimulated with plate-bound anti-CD3 plus anti-CD28 mAbs for 24 h and cultured with vehicle or PTH (50 nM) for 24 h. IBMX (100 μM) or vehicle was added 1 h before PTH. (E) Effect of iPTH on the SC expression of mRNA of genes known to be up-regulated by Wnt signaling in PPR^{T cells−/−} and control female mice at 6 wk of age. The genes analyzed were aryl-hydrocarbon receptor (Ahr), axin2, cysteine-rich protein 61 (Cyr61), naked cuticle 2 homolog (Nkd2), transgelin (tagln), TGF-β3, thrombospondin 1 (Thbs1), Twist gene homolog 1 (Twst1), and Wnt1-inducible signaling pathway protein 1 (Wisp1). BM harvested at sacrifice was cultured for 1 wk. SCs were purified, and mRNA levels were determined by RT-PCR (n = 3 mice per group). (F) Effect of iPTH on total-body BMD as measured by DXA at 2 and 4 wk of treatment in WT and Wnt10b^−/− male mice at 6 wk of age. (G) Effect of iPTH on BV/TV in WT and Wnt10b^−/− male mice of 6 wk of age. *P < 0.05; ***P < 0.001 compared with the corresponding vehicle-treated group. ^#P < 0.01 compared with the corresponding PPR^fl/fl group. n = 10 mice per group.

Fig. 2.

Analysis of the effects (mean ± SEM) of iPTH treatment in PPR^{T cells−/−} and control female mice at 6 wk of age. (A) In vivo total-body BMD measurements by DXA at 2 and 4 wk of treatment. (B) Measurement of BV/TV by quantitative bone histomorphometry. (C) BV/TV as measured by μCT. (D–F) Measurements of trabecular structural indices by μCT. Tb.Th, Tb.N, and Conn.D are shown. (G and H) Measurements of cortical indices by μCT. Cortical thickness (Co.Th) and cortical volume (Co.V) are shown. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding vehicle (Veh)-treated group. ^#P < 0.05 compared with corresponding PPR^fl/fl mice. n = 10–20 mice per group.

Fig. 3.

Analysis of the effects (mean ± SEM) of iPTH treatment on histomorphometric and biochemical indices of bone turnover in PPR^{T cells−/−} and control female mice at 6 wk of age. (A) Number of OBs per millimeter of bone surface (N.Ob/BS). (B) Percentage of bone surface covered by OBs (Ob.S/BS). (C) Serum levels of OCN, a marker of formation. (D) Number of OCs per millimeter of bone surface (N.Oc/BS). (E) Percentage of bone surface covered by OCs (Oc.S/BS). (F) Serum levels of CTX, a marker of resorption. (G) RANKL mRNA levels in BM T cells. BM was harvested at sacrifice from PPR^{T cells−/−} and control mice treated with vehicle (Veh) of iPTH. CD8⁺ cells were purified by positive immunomagnetic selection and assayed for RANKL mRNA levels. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding vehicle-treated group. ^#P < 0.05 compared with the corresponding PPR^fl/fl mice. n = 10–20 mice per group for A–F and n = 4 mice per group for G.

Fig. 4.

Analysis of the effects (mean ± SEM) of iPTH treatment in PPR^{T cells−/−} and control male mice at 17 wk of age. (A–F) Trabecular and cortical structural indices as measured by μCT. (G and H) Serum OCN and CTX levels. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding vehicle (Veh)-treated group. ^#P < 0.05 compared with the corresponding PPR^fl/fl mice. n = 8–10 mice per group.

Fig. 5.

Analysis of the effects (mean ± SEM) of iPTH treatment on osteoblastogenesis and on SC apoptosis in PPR^{T cells−/−} and control female mice at 6 wk of age. (A) Whole BM was cultured for 7 d to assess the formation of CFU-ALP. The average of the colonies counted in six wells is shown. (B) BM harvested at sacrifice was cultured for 1 wk, and SCs were purified and counted. (C) SCs were purified from BM cultured for 1 wk, seeded in equal number, and pulsed with [³H]-thymidine for 18 h to assess their proliferation. Data are expressed in counts per minute. (D) SCs were purified from BM cultured for 1 wk, and the rate of apoptosis was quantified by determination of caspase3 activity. (E) SCs were purified from BM cultured for 1 wk, and the levels of OB marker gene mRNAs, bone sialoprotein (BSP), type I collagen (Col1a1), OCN (Ocn), osterix (Osx), and runt-related transcription factor 2 (Runx2) were analyzed by RT-PCR. *P < 0.05; **P < 0.01 compared with the corresponding vehicle (Veh)-treated group. ^#P < 0.05 compared with the corresponding PPR^fl/fl mice. n = 4–5 per group.

Fig. P1.

Schematic representation of the role of direct T-cell PPR signaling in the mechanism by which intermittent PTH treatment stimulates bone formation. In this schema, PPR signaling stimulates T cells to secrete Wnt10b, a Wnt ligand required to activate Wnt signaling in stromal cells and osteoblasts. In the presence of T cell-produced Wnt10b, stimulation of osteoblastic cells by PTH activates the Wnt signaling pathway. This, in turn, leads to increased commitment of mesenchymal stem cells to maturing into osteoblasts, increased osteoblast proliferation and differentiation, and decreased osteoblast apoptosis or cell death. The resultant increased bone formation is followed by an increase in bone mass. OB, osteoblast.