T cell-expressed CD40L potentiates the bone anabolic activity of intermittent PTH treatment

Abstract

T cells are known to potentiate the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. One of the involved mechanisms is increased T cell secretion of Wnt10b, a potent osteogenic Wnt ligand that activates Wnt signaling in stromal cells (SCs). However, additional mechanisms might play a role, including direct interactions between surface receptors expressed by T cells and SCs. Here we show that iPTH failed to promote SC proliferation and differentiation into osteoblasts (OBs) and activate Wnt signaling in SCs of mice with a global or T cell-specific deletion of the T cell costimulatory molecule CD40 ligand (CD40L). Attesting to the relevance of T cell-expressed CD40L, iPTH induced a blunted increase in bone formation and failed to increase trabecular bone volume in CD40L(-/-) mice and mice with a T cell-specific deletion of CD40L. CD40L null mice exhibited a blunted increase in T cell production of Wnt10b and abrogated CD40 signaling in SCs in response to iPTH treatment. Therefore, expression of the T cell surface receptor CD40L enables iPTH to exert its bone anabolic activity by activating CD40 signaling in SCs and maximally stimulating T cell production of Wnt10b.

Keywords: BM; BONE MARROW; OB; OSTEOBLAST; PARATHYROID HORMONE; PPR; PTH; PTH/PTHRP RECEPTOR; SC; STROMAL CELL.

Figure 1

Effects (mean ± SEM) of iPTH treatment on femoral bone structure indices in WT and CD40L−/− mice as assessed by μCT. (A) Trabecular bone volume fraction (BV/TV). (B) Trabecular thickness (Tb.Th), (C) Trabecular number (Tb.N), (D), Trabecular spacing (Tb.Sp), (E) Cortical volume (Ct.Vo) and (F) Cortical thickness (Ct.Th). n = 12 mice per group. * = p<0.05, **= p<0.01, and **** = p<0.0001 versus the respective vehicle treated group.

Figure 2

Effects (mean ± SEM) of iPTH treatment on histomorphometric and biochemical indices of bone turnover and RANKL and OPG mRNA levels in WT and CD40L−/− mice. (A) Number of OCs per mm bone surface (N.Oc/BS), (B) Percentage of bone surface covered by OCs (Oc.S/BS). (C) Mineral Apposition rate (MAR) and bone formation rate per μm² bone surface (BFR/BS) as measured from the interlabel period of calcein injections at 7 and 2 days before sacrifice. (D) Serum levels of CTX, a marker of resorption. (E) Serum levels of P1NP, a marker of formation. (F) SC RANKL mRNA levels. (G) Whole BM OPG mRNA levels. n = 10–12 per group for A–E. n = 4 per group for F,G. * = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001 versus the respective vehicle treated group. # = p<0.05 versus WT vehicle treated mice.

Figure 3

Effects (mean ± SEM) of iPTH treatment on Wnt10b mRNA expression by BM T cells (A), and whole BM (B), and on the SC expression of the NFkB subunits p100 and p52, where lanes 1 and 2 are from representative vehicle treated mice and lanes 3 and 4 are from representative iPTH treated mice, with GAPDH as a loading control. (C), Pim-1 mRNA levels (D), and Survivin mRNA levels (E). n = 4 per group. * = p<0.05, and *** = p<0.001 compared to the respective vehicle treated group.

Figure 4

Effects (mean ± SEM) of iPTH treatment on SC differentiation, proliferation, apoptosis and osteoblastic gene expression in WT and CD40L−/− mice. (A) Analysis of BM alkaline phosphatase (ALP) positive colony forming-unit fibroblast (CFU-ALP), (B) SC proliferation, (C) SC apoptosis, (D) Alkaline Phosphatase (ALP) mRNA levels in SCs. (E) Analysis of osteoblastic gene expression in SCs, (F) SC cell expression of Wnt dependent genes. n = 10 per group. * = p<0.05, ** = p<0.01 and *** = p<0.001 compared to the respective vehicle treated group.

Figure 5

Effects (mean ± SEM) of iPTH treatment on indices of bone volume, structure and turnover in TCRβ−/− mice and TCRβ−/− mice reconstituted with either WT or CD40L−/− T cells. (A) Trabecular bone volume fraction (BV/TV), (B) Trabecular thickness (Tb.Th), (C) Trabecular number (Tb.N), (D) Trabecular spacing (Tb.Sp), (E) Cortical volume (Ct.Vo), (F) Cortical thickness (Ct.Th), (G) Serum CTX levels, (H) Serum P1NP levels. n = 8–10 mice per group. * = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001 versus the respective vehicle treated group. # = p<0.05 versus TCRβ−/− vehicle treated mice.

Figure 6

Effects (mean ± SEM) of iPTH treatment on SC proliferation, apoptosis and differentiation in TCRβ−/− mice and TCRβ−/− mice reconstituted with either WT or CD40L−/− T cells. (A) SC proliferation. (B) SC apoptosis. (C) Analysis of osteoblastic gene expression in SCs. (D) SC expression of Wnt dependent genes. n = 8 per group. * = p<0.05, ** = p<0.01, and *** = p<0.001 versus the respective vehicle treated group.

Figure 7

Schematic representation of the role of T cells in the anabolic activity of iPTH in trabecular bone. When CD40L is expressed on T cells, iPTH stimulates T cells to express Wnt10b, a Wnt ligand loosely associated with cell membranes required to activate Wnt signaling in osteoblastic cells. The Wnt10b promoter contains two essential CREB binding sites. Wnt10b gene expression is activated by the PPR/ GαS/cAMP/PKA pathway, which phosphorylates CREB proteins leading to their binding to the CREB sites in the Wnt10b promoter. Activation of CD40L potentiates CREB phosphorylation, further inducing Wnt10b gene expression. T cell produced Wnt10b activates Wnt signaling in SCs and OBs in the trabecular compartment of the skeleton. Wnt signaling stimulates OB proliferation, differentiation and survival. In addition, CD40L directly activates Wnt signaling in osteoblastic cells and provides proliferative and survival cues to SCs and OBs by activating CD40 signaling. Since T cells do not reside on the periosteal surface and have limited communications with osteocytes (OCY), T cell expressed CD40L and Wnt10b do not contribute to the activation of Wnt signaling in osteocytes. Therefore the anabolic effects of iPTH in cortical bone are T cell independent and result primarily from direct inhibition of osteocytic sclerostin production. It is possible that increased production of Wnt10b or other Wnt ligands by OCYs may contribute to the T cell and CD40L–independent effects of iPTH on cortical bone.