Disruption of PTH receptor 1 in T cells protects against PTH-induced bone loss

Abstract

Background: Hyperparathyroidism in humans and continuous parathyroid hormone (cPTH) treatment in mice cause bone loss by regulating the production of RANKL and OPG by stromal cells (SCs) and osteoblasts (OBs). Recently, it has been reported that T cells are required for cPTH to induce bone loss as the binding of the T cell costimulatory molecule CD40L to SC receptor CD40 augments SC sensitivity to cPTH. However it is unknown whether direct PTH stimulation of T cells is required for cPTH to induce bone loss, and whether T cells contribute to the bone catabolic activity of PTH with mechanisms other than induction of CD40 signaling in SCs.

Methodology/principal findings: Here we show that silencing of PTH receptor 1 (PPR) in T cells blocks the bone loss and the osteoclastic expansion induced by cPTH, thus demonstrating that PPR signaling in T cells is central for PTH-induced reduction of bone mass. Mechanistic studies revealed that PTH activation of the T cell PPR stimulates T cell production of the osteoclastogenic cytokine tumor necrosis factor alpha (TNF). Attesting to the relevance of this effect, disruption of T cell TNF production prevents PTH-induced bone loss. We also show that a novel mechanism by which TNF mediates PTH induced osteoclast formation is upregulation of CD40 expression in SCs, which increases their RANKL/OPG production ratio.

Conclusions/significance: These findings demonstrate that PPR signaling in T cells plays an essential role in PTH induced bone loss by promoting T cell production of TNF. A previously unknown effect of TNF is to increase SC expression of CD40, which in turn increases SC osteoclastogenic activity by upregulating their RANKL/OPG production ratio. PPR-dependent stimulation of TNF production by T cells and the resulting TNF regulation of CD40 signaling in SCs are potential new therapeutic targets for the bone loss of hyperparathyroidism.

Figure 1. Confirmation of PPR gene disruption in PPR^{T cells} −/− mice.

A Characterization of floxed and Cre recombined PPR alleles in BMMs, SCs and T cells from PPR^{T cells} −/− and control mice. B PPR mRNA levels (Mean ± SEM) in CD4+ and CD8+ T cells from PPR^{T cells} −/− and control mice. * = p<0.05 and ** = p<0.01 compared to the corresponding PPR^fl/fl group. C Effect (Mean ± SEM) of in vitro PTH treatment (50 nM) on cAMP accumulation by splenic CD4+ and CD8+ T cells from PPR^{T cells} −/− and control mice. * = p<0.05 and ** = p<0.01 compared to the corresponding vehicle group.

Figure 2. Effects of cPTH treatment on indices of bone structure and turnover in PPR^{T cells} −/− and control mice.

A. µCT analysis of femur cortical bone thickness (Co.Th). B. µCT analysis of femur cortical bone volume (Co.Vo). C. µCT analysis of trabecular bone volume (BV/TV). D Analysis of serum CTX levels by ELISA. E Analysis of serum OCN levels by ELISA. F–L Histomorphometric analysis femoral trabecular bone. F: percentage of bone surface covered by osteoclasts (Oc.S/BS). G: number of osteoclasts per mm bone surface (N.Oc/BS). H: Bone formation rate (BFR). I: Mineral Apposition rate (MAR), J: Percentage of bone surface covered by osteoblasts (Ob.S/BS). K: Number of osteoblasts per mm bone surface (N.Ob/BS). L: percentage of mineralized bone surface (MS/BS). * = p<0.05, ** = p<0.01 and *** = p<0.001 compared to the corresponding vehicle treated group. # = p<0.05 compared to the corresponding PPR^fl/fl mice. n = 18–22 mice per group for µCT and serum measurements. n = 10–12 mice per group for bone histomorphometry. Data are means ± SEM.

Figure 3. PTH effects on OC formation, SC osteoclastogenic activity and SC RANKL, OPG, and CD40 expression.

A. Images of in vitro PTH treated BM stained for TRAP to visualize OCs (top panel), and number of OCs per well (bottom panel). BM from PPR^Tcells−/− and control mice were stimulated with PTH (1 nM) for 7 days, stained for TRAP, and OCs counted. ** = p<0.01 and *** = p<0.001 compared to vehicle. # = p<0.05 compared to the other PTH treated groups. B WT BMMs were cocultured with SCs isolated from PPR^{T cells} −/− and control mice treated in vivo with cPTH or vehicle. Cells were cultured for 7 days in the presence of PTH (1 nM), stained for TRAP, and OCs counted. ** = p<0.01 and *** = p<0.001 compared to vehicle. # = p<0.05 compared to the other PTH treated groups. C. mRNA levels of RANKL and OPG in SCs from PPR^{T cells} −/− and control mice treated in vivo with vehicle or cPTH. * = p<0.05 and ** = p<0.01 compared to the corresponding vehicle treated group. D mRNA levels of CD40 in SCs from PPR^{T cells} −/− and control mice treated in vivo with vehicle or cPTH. * = p<0.05 compared to the corresponding vehicle treated group. Data are Means + SEM.

Figure 4. Regulation of SC CD40 expression and T cell TNF production.

A Effect of in vitro TNF treatment on CD40 mRNA expression by SCs. B Effect of in vivo cPTH treatment on CD40 mRNA levels in SCs from WT and TNF−/− mice. C Effect of in vivo cPTH treatment on TNF mRNA and TNF protein production by CD4+ cells from PPR^{T cells} −/− and control mice. D Effect of in vivo cPTH treatment on TNF mRNA and TNF protein production by CD8+ cells isolated from PPR^{T cells} −/− and control mice. All data are expressed as mean ± SEM. * = p<0.05 and * = p<0.01 compared to the corresponding vehicle group. n = 6 mice per group.

Figure 5. Effects of cPTH on bone structure and turnover in TNF −/− mice.

A µCT analysis of femoral Co.Th. B µCT analysis of femoral Co.Vo. C µCT analysis of BV/TV. D Serum CTX levels. E Serum OCN levels. F–J Histomorphometric analysis of femoral trabecular bone. F: percentage of bone surface covered by osteoclasts (Oc.S/BS). G: number of osteoclasts per mm bone surface (N.Oc/BS). H: Bone formation rate (BFR). I: Percentage of bone surface covered by osteoblasts (Ob.S/BS). J: Number of osteoblasts per mm bone surface (N.Ob/BS). K–L mRNA levels of RANKL and OPG in SCs from WT and TNF−/− mice treated in vivo with vehicle or cPTH. * = p<0.05 compared to the corresponding vehicle group. n = 9–15 mice per group. Data are Means + SEM.

Figure 6. Effects of cPTH treatment in mice lacking T cell TNF production.

A µCT analysis of femoral Co.Th. B µCT analysis of femoral Co.Vo. C µCT analysis of BV/TV. D Serum CTX levels. E Serum OCN levels. * = p<0.05, ** = p<0.01 and *** = p<0.001 compared to the corresponding vehicle treated group. n = 10–17 mice per group. Data are Means + SEM.

Figure 7. Schematic representation of the role of T cell PPR in cPTH stimulation of OC formation.

PTH binding to PPR in T cells stimulates the production of TNF. This cytokine increases CD40 expression by SCs. Binding of CD40 by T cell expressed CD40L increases SC sensitivity to PTH resulting in enhanced SC production of RANKL and diminished secretion of OPG in response to PTH. T cell produced TNF further stimulates OC formation through its direct effects on maturing OC precursors. The red arrows represent the main modifications induced by activation of PPR signaling in T cells.