PTH) Receptor 1 in PNAS PLUS T Cells Blunts the Bone Anabolic Activity of PTH

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

Brahmchetna Bedia,b,1, Jau-Yi Lia,1, Hesham Tawfeeka, Ki-Hyun Baeka, Jonathan Adamsa, Sameera S. Vangaraa, Ming-Kang Changc, Michaela Kneisselc, M. Neale Weitzmanna,b, and Roberto Paciﬁcia,d,2

aDivision of Endocrinology, Metabolism, and Lipids, Department of Medicine, Emory University, Atlanta, GA 30322; bAtlanta Veterans Affairs Medical Center, Decatur, GA 30033; cNovartis Institutes for Biomedical Research, CH4002 Basel, Switzerland; and dImmunology and Molecular Pathogenesis Program, Emory University, Atlanta, GA 30322

Edited by John T. Potts, Massachusetts General Hospital, Charlestown, MA, and approved February 6, 2012 (received for review December 28, 2011) Intermittent parathyroid hormone (iPTH) treatment stimulates T- been described in models of hyperparathyroidism characterized cell production of the osteogenic Wnt ligand Wnt10b, a factor by a continuous overproduction of PTH but not in mice treated required for iPTH to activate Wnt signaling in osteoblasts and with intermittent PTH. For example, continuous PTH treatment stimulate bone formation. However, it is unknown whether iPTH regulates the Wnt antagonist Dkk1 (34, 35) and the Wnt re- induces Wnt10b production and bone anabolism through direct ceptor LRP6 (28), whereas iPTH does not. activation of the parathyroid hormone (PTH)/PTH-related protein Although SCs, OBs, and osteocytes represent the major tar- receptor (PPR) in T cells. Here, we show that conditional silencing gets of PTH in bone, reports from our laboratory have disclosed of PPR in T cells blunts the capacity of iPTH to induce T-cell that T lymphocytes play an unexpected role in the mechanism of production of Wnt10b; activate Wnt signaling in osteoblasts; action of PTH (30, 36, 37). We have shown that treatment with expand the osteoblastic pool; and increase bone turnover, bone iPTH increases the T-cell production of Wnt10b, a Wnt ligand mineral density, and trabecular bone volume. These findings that stimulates osteoblastogenesis by activating Wnt signaling in demonstrate that direct PPR signaling in T cells plays an important SCs and OBs. As a result, the bone anabolic activity of iPTH is role in PTH-induced bone anabolism by promoting T-cell pro- markedly reduced in T cell-deficient mice and in mice with duction of Wnt10b and suggest that T cells may provide pharma- a specific disruption of Wnt10b production by T cells (30). cological targets for bone anabolism. Despite the evidence supporting a role for T cells in the actions of iPTH in bone, the mechanisms involved are only partly bone mass | T lymphocytes | bone cells understood because it remains unknown whether direct activation of PPR in T cells by PTH is required for iPTH treatment to arathyroid hormone (PTH) is a major regulator of calcium exert its full anabolic activity. Because SCs regulate T-cell Pmetabolism and defends against hypocalcemia, in part, by function (38, 39) and OBs express CD40 (40), a surface receptor stimulating bone resorption, and thereby the release of calcium that signals to its counterpart CD40L expressed in T cells, iPTH from the skeleton. However, when injected daily, a regimen could indeed affect T cells indirectly through osteoblastic cells. known as intermittent parathyroid hormone (iPTH) treatment, To address these issues, we have conditionally silenced PPR in the hormone markedly stimulates trabecular and cortical bone T cells and determined whether PTH activation of PPR in T cells formation. Although this bone-forming activity is antagonized is required for iPTH to exert its bone anabolic activity. We show by a stimulation of bone resorption, the net effect of iPTH that blocking PPR signaling in T cells blunts the capacity of iPTH treatment is an improvement in bone microarchitecture and treatment to induce the production of Wnt10b by T cells, stim- increased strength (1, 2). As a result, intermittent treatment ulate osteoblastogenesis and bone formation, and increase bone with the 1–34 fragment of PTH is a Food and Drug Adminis- mass. Therefore, direct targeting of T cells by PTH is required tration-approved treatment modality for postmenopausal os- for iPTH to induce maximal bone anabolism. teoporosis (3). The effects of PTH on bone result from its binding to the Results PTH/PTH-related protein receptor (PPR or PTHR1) expressed PPR Signaling in T Cells Is Required for iPTH to Increase Wnt10b on bone marrow (BM), stromal cells (SCs), osteoblasts (OBs), Production by T Cells and Activate Wnt Signaling in OBs. Intermittent and osteocytes (1, 4, 5). iPTH stimulates bone formation by in- PTH treatment stimulates the production of the osteogenic Wnt creasing the number of OBs (6–8), a phenomenon achieved ligand Wnt10b by BM T cells, leading to an overall increase in the through activation of quiescent lining cells (9), increased OB BM levels of Wnt10b (30). To determine whether this stimulatory

proliferation (10, 11) and differentiation (10, 12, 13), attenuation effect of PTH is attributable to direct PPR signaling in T cells, we MEDICAL SCIENCES − − of OB apoptosis (14–17), and signaling in osteocytes (18). made use of PPRT cells / mice, a strain with a silent PPR in all T − − fl fl However, the specific contribution of each of these effects of cells (37). Six-week-old female PPRT cells / and control PPR / iPTH remains controversial. The expansion of the osteoblastic mice were injected daily with vehicle or 80 μg/kg of human PTH 1– pool induced by iPTH is initiated by the release from the matrix 34 for 4 wk, a treatment modality referred to hereafter as iPTH. undergoing resorption of TGF-β, insulin-like growth factor 1, and other growth factors that recruit SCs to remodeling areas – (19 22). Subsequent events are driven primarily by the activation Author contributions: M.N.W., and R.P. designed research; B.B., J.-Y.L., H.T., K.-H.B., M.-K.C., of Wnt signaling in osteoblastic cells (23). Activation of Wnt J.A., and M.K. performed research; B.B., J.-Y.L., S.S.V., M.-K.C., M.K., and R.P. analyzed data; signaling induces OB proliferation (24) and differentiation (23, and M.N.W. and R.P. wrote the paper. 25), prevents OB apoptosis (16, 17, 26), and augments OB The authors declare no conflict of interest. production of osteoprotegerin (OPG) (27). This article is a PNAS Direct Submission. iPTH activates Wnt signaling in OBs through multiple mech- 1B.B. and J.-Y.L. contributed equally to this work. anisms that include Wnt ligand-independent activation of the 2To whom correspondence should be addressed. E-mail: roberto.pacifi[email protected]. Wnt coreceptor LRP6 (28), increased production of Wnt ligands See Author Summary on page 4355 (volume 109, number 12). by bone and BM cells (29, 30), and suppression of sclerostin This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. production (31–33). Additional effects on the Wnt system have 1073/pnas.1120735109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1120735109 PNAS | Published online March 5, 2012 | E725–E733 Downloaded by guest on September 24, 2021 Whole BM and BM T cells were harvested, and their expression of BM levels of Wnt10b, PTH increased the levels of Wnt10b Wnt10b mRNA was analyzed by real-time PCR (RT-PCR). This mRNA by approximately fourfold in unfractionated BM samples assay disclosed that iPTH increased Wnt10b mRNA levels by from control mice. However, PTH had no significant effect in BM − − approximately sixfold in T cells from control mice. By contrast, from PPRT cells / mice (Fig. 1B). − − PTH had no stimulatory effect in T cells from PPRT cells / mice To confirm these findings, BM T cells and whole BM from − − (Fig. 1A). Attesting to the contribution of T cells to the overall 6-wk-old control and PPRT cells / mice were treated in vitro with

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 − − − − − − fl fl genes, bone density, and BV/TV in PPRT cells / mice or Wnt10b / mice. (A) Effect of iPTH on BM T-cell levels of Wnt10b mRNA in PPRT cells / and control PPR / − − female mice at 6 wk of age. (B) Effect of iPTH on whole-BM levels of Wnt10b mRNA in PPRT 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 PPRT 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 PPRT 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 PPRT 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 fl fl compared with the corresponding PPR / group. n = 10 mice per group.

E726 | www.pnas.org/cgi/doi/10.1073/pnas.1120735109 Bedi et al. Downloaded by guest on September 24, 2021 PTH for 24 h. Analysis by real-time RT-PCR disclosed that PTH of BV/TV revealed a less pronounced anabolic response to PTH PNAS PLUS − − increased Wnt10b mRNA levels in purified T cells and whole in PPRT cells / mice than in control mice. In fact, iPTH in- fl fl BM from control mice by approximately eightfold and approxi- creased BV/TV by ∼234% in PPR / mice and by ∼33% in − − mately fourfold, respectively. By contrast, PTH had no stimula- PPRT cells / mice (Fig. 2B). μCT analysis confirmed that acti- − − tory effect in T cells and whole BM from PPRT cells / mice (Fig. vation of PPR signaling in T cells by iPTH is required for iPTH- 1 C and D). Thus, PTH stimulates production of Wnt10b by induced anabolism, because iPTH induced a greater increase in directly targeting T cell-expressed PPR. BV/TV in control mice than in those with a silent PPR receptor Next, we investigated the role of PPR signaling in T cells for in T cells (Fig. 2C). Tb.N, Conn.D, and Tb.Th, three indices of fl fl the activation of Wnt signaling in osteoblastic cells induced by trabecular structure, were more substantially improved in PPR / − − iPTH. To this end, we assessed by RT-PCR the mRNA expres- mice than in PPRT cells / mice (Fig. 2 D–F). sion of genes up-regulated by Wnt signaling in SCs purified from By contrast, μCT analysis of cortical bone showed that iPTH − − PPRT cells / and control mice treated with vehicle or iPTH for 4 induced similar increases in cortical thickness (Co.Th) and cor- wk and killed at 10 wk of age. The analyzed genes were chosen tical volume (Co.Vo.) in all groups of mice (Fig. 2 G and H), thus based on known expression patterns during differentiation of demonstrating that PPR signaling in T cells specifically augments primitive mesenchymal cells to the OB phenotype. Some of the the capacity of iPTH to improve architecture in trabecular bone. selected genes are known to play a direct role in regulating OB Representative μCT images of trabecular and cortical bone are differentiation (41), whereas others are not involved in OB dif- shown in Fig. S3. ferentiation but are sensitive markers of Wnt activation (42). Analysis of the secondary spongiosa by bone histomorphometry These analyses revealed (Fig. 1E) that iPTH activates Wnt sig- revealed that iPTH increased two static indices of bone forma- naling in SCs through PPR signaling in T cells. In fact, SC levels tion, the number of OBs per bone surface (N.Ob/BS) (Fig. 3A) of mRNA for the nine tested genes were all increased by iPTH in and the percentage of surfaces covered by OBs (Ob.S/BS) (Fig. − − − − fl fl SCs from control mice but not in those from PPRT cells / mice. 3B), to a lower extent in PPRT cells / mice than in PPR / mice. The genes that were stimulated by iPTH were aryl-hydrocarbon Analysis of dynamic indices of formation in iPTH-treated control receptor (Ahr), axin2, cysteine-rich protein 61 (Cyr61), naked mice revealed massive and diffuse fluorochrome marker labeling cuticle 2 homolog (Nkd2), transgelin (tagln), TGF-β3, thrombo- that prevented an accurate measurement of mineral apposition spondin 1 (Thbs1), Twist gene homolog 1 (Twist1), and Wnt1 rate, mineralizing surface, and thus bone formation rate. Meas- inducible signaling pathway protein 1 (Wisp1). urements of serum osteocalcin (OCN), a marker of bone for- To determine the relevance of Wnt10b for the anabolic activity mation, confirmed that iPTH induced a larger increase in bone − − fl fl − − of iPTH, 6-wk-old male WT and Wnt10b / mice were injected turnover in PPR / mice than in PPRT cells / mice (Fig. 3C). daily with vehicle or iPTH for 4 wk. Dual X-ray absorptiometry Histomorphometric analysis showed no significant effects of (DXA) was used to measure in vivo total body bone mineral iPTH on two indices of trabecular bone resorption, the number density (BMD), and micro-computerized tomography (μCT) of osteoclasts (OCs) per bone surface (Fig. 3D) and the per- was used to assess the trabecular compartment of femurs har- centage of surfaces covered by OCs (Fig. 3E), whereas meas- vested at sacrifice. In response to iPTH treatment, WT mice urements of serum C-terminal telopeptide of collagen (CTX), displayed a significant increase in BMD and trabecular bone a biochemical marker of resorption, indicated that iPTH induced volume (BV/TV) (Fig. 1 F and G). By contrast, iPTH had no a significant increase in bone resorption in control but not in − − − − anabolic effects in Wnt10b / mice, thus confirming that Wnt10b PPRT cells / mice (Fig. 3F). The discrepancy between histo- is required for iPTH to exert its anabolic activity. Parameters of morphometric and biochemical indices of resorption is likely trabecular structure were also differentially affected in WT and explained by the fact that iPTH increased bone surfaces as ex- − − Wnt10b / mice, because trabecular number (Tb.N), connectivity tensively as OC number and OC surfaces. density (Conn.D), and trabecular thickness (Tb.Th) were im- One mechanism by which iPTH stimulates bone resorption is − − proved in WT mice but not in Wnt10b / mice (Fig. S1). by increasing the CD8+ cell production of receptor activator of nuclear factor-κB ligand (RANKL) (30). Therefore, CD8+ cells Mice Lacking PPR Signaling in T Cells Exhibit a Blunted Increase in were purified from the BM of vehicle and iPTH-treated mice and Bone Mass and Bone Turnover Response to iPTH. To determine assayed for RANKL mRNA levels. These experiments revealed whether PPR signaling in T cells is required for iPTH to exert its fi − − that iPTH increased the levels of RANKL mRNA signi cantly in effects on bone, we again made use of PPRT cells / mice (37). At + fl/fl T cells−/− − − CD8 cells from PPR mice but not in those from PPR 6 wk of age PPRT cells / mice have a normal bone phenotype mice (Fig. 3G). and normal serum calcium, phosphate, and PTH levels (37), To confirm the relevance of direct PPR signaling in T cells in − − indicating that PPR signaling in T cells does not play an essential mature mice, 17-wk-old male PPRT cells / mice and littermate role in bone modeling and baseline remodeling. Moreover, controls were treated with iPTH for 4 wk. Analysis of trabecular T cells−/− PPR mice have a normal number of T cells, which exhibit bone by μCT revealed that iPTH increased BV/TV in control MEDICAL SCIENCES a degree of activation and proliferation similar to control T cells fi − − mice, although it had no signi cant effects in mice with a silent (37). PPRT cells / and control mice also have a similar number of PPR receptor in T cells (Fig. 4A). Similarly, iPTH increased Tb. B cells and myeloid cells (Fig. S2). fl/fl T cells−/− − − Th and Conn.D in PPR mice but not in PPR mice In a first experiment, 6-wk-old female PPRT cells / mice and (Fig. 4 B and D). Tb.N was unaffected by iPTH in both genotypes littermate controls were treated with iPTH for 4 wk, and their (Fig. 4C). Confirming the data obtained in younger mice, iPTH BMD was measured in vivo by DXA, a technique that provides equally increased indices of cortical structures in all groups of a combined measure of cortical and trabecular bone. As a result mice (Fig. 4 E and F). Measurements of serum OCN and CTX fl fl of physiological growth, BMD increased in all mice treated with levels confirmed that iPTH increased bone turnover in PPR / − − vehicle. Treatment with iPTH induced a further increase in mice but not in PPRT cells / mice (Fig. 4 G and H). BMD in all groups. However, the bone anabolic effect of iPTH − − was significantly smaller in PPRT cells / mice than in control Mice Lacking PPR Signaling in T Cells Exhibit a Blunted Osteoblastogenic − − mice, as demonstrated by the fact that at 4 wk, iPTH induced Response to iPTH. BM from female PPRT cells / and control mice − − a 26% increase in the BMD of PPRT cells / mice compared with was used to assess the formation of alkaline phosphatase-positive fl fl a 41% increase in that of PPR / mice (Fig. 2A). colony-forming unit fibroblast (herein defined as CFU-ALP), an Cancellous bone was analyzed by histology and μCT using index of SC commitment to the osteoblastic lineage. iPTH femurs harvested at sacrifice. Histomorphometric measurements treatment for 4 wk in mice from 6 to 10 wk of age increased

Bedi et al. PNAS | Published online March 5, 2012 | E727 Downloaded by guest on September 24, 2021 − − Fig. 2. Analysis of the effects (mean ± SEM) of iPTH treatment in PPRT 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. ﬂ ﬂ #P < 0.05 compared with corresponding PPR / mice. n =10–20 mice per group.

fl fl − − CFU-ALP formation in the BM of PPR / mice by approxi- from PPRT cells / mice (Fig. 5C). Moreover, iPTH decreased − − − − mately twofold, whereas it had no effect on that of PPRT cells / the rate of SC apoptosis in control mice but not in PPRT cells / mice (Fig. 5A), thus indicating that PPR signaling in T cells mice (Fig. 5D). Analysis of the expression levels of osteoblastic potentiates the capacity of iPTH to increase the number of SCs genes in SCs revealed that iPTH treatment for 4 wk increased with osteogenic potential. the expression of type 1 collagen, Runx2, osterix, bone sialo- To determine whether PPR signaling in T cells is required for protein, and OCN mRNAs by approximately two- to threefold in − − fl fl iPTH-induced osteoblastogenesis, BM from PPRT cells / and SCs from PPR / mice, whereas it had no effects on that from − − control female mice harvested after 4 wk of iPTH treatment PPRT cells / mice (Fig. 5E). These findings demonstrate that (when the mice were 10 wk old) was cultured for 1 wk to allow direct PPR signaling in T cells is indispensable for the expansion SCs to proliferate. SCs were then purified and counted. This of the osteoblastic pool and the differentiation along the oste- analysis revealed (Fig. 5B) that in vivo iPTH treatment increases oblastic lineage induced by iPTH. fl fl the number of SCs in samples from PPR / mice. By contrast, − − iPTH had no effects on the number of SCs from PPRT cells / Discussion mice. To investigate the mechanism involved, BM was cultured We report that mice lacking PPR signaling in T cells exhibit for 1 wk and SCs were purified and used to determine their rate a blunted bone anabolic response to iPTH because of impaired T- of proliferation and apoptosis. These experiments revealed that cell production of Wnt10b, an osteogenic factor that promotes iPTH increases the proliferation of SCs from control mice sig- Wnt signaling in OBs. Therefore, T cells are direct targets of PTH nificantly, whereas it had no effect on the proliferation of SCs that play a pivotal role in the osteoblastogenic response to iPTH.

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Fig. 3. Analysis of the effects (mean ± SEM) of iPTH treatment on histomorphometric and biochemical indices of bone turnover in PPRT 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 PPRT 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 < fl fl 0.001 compared with the corresponding vehicle-treated group. #P < 0.05 compared with the corresponding PPR / mice. n =10–20 mice per group for A–F and n = 4 mice per group for G.

Based on a previous report (30), we speculated that ligand- cluding Wnt7a and Wnt3b, resulting from PPR signaling in bone dependent activation of PPR signaling in T cells is required cells (43, 44). A second mechanism might be the inhibition of the

for iPTH to induce the production of Wnt10b and promote osteocytic production of the Wnt antagonist sclerostin (31, 32). MEDICAL SCIENCES bone anabolism. To investigate this hypothesis, we made use of A less likely possibility is that iPTH may signal in T cells through − − PPRT cells / mice, a strain with a silent PPR in all T cells (37). an alternative PTH receptor. One such receptor is PTHR2, Demonstrating a requirement for direct PTH signaling in T cells, a protein expressed primarily in the central nervous system and we found that the silencing of PPR in T cells blocks the capacity of the cardiovascular system. However, PTHR2 is not known to iPTH to induce the production of Wnt10b by T cells, the activation play a role in bone (45–47). Recently, a receptor specific for the of Wnt signaling in SCs, and trabecular bone accretion. Moreover, carboxyl terminal PTH fragments has been proposed and is yet − − analysis of the effect of iPTH in Wnt10b / mice confirmed that to be cloned (48). The function of this receptor in bone is un- Wnt10b is indispensable for the anabolic activity of iPTH. known but not relevant for the effects of PTH 1–34. We found that the capacity of iPTH to increase BV/TV was Although silencing of PPR in T cells resulted in a blunted − − decreased but not completely abolished in young PPRT cells / response of the trabecular compartment to iPTH, the hormone mice. By contrast, the anabolic activity of iPTH was completely had similar anabolic effects in the cortical bone of all groups of − − blocked in mature PPRT cells / mice. These findings suggest that mice. These findings are consistent with our earlier report that T the contribution of T cells to the activity of iPTH increases with cells specifically potentiate the anabolic effect of iPTH in can- age. A mechanism likely to account for the T cell-independent cellous bone (30) and indicate that iPTH stimulates cortical bone anabolic activity of iPTH observed in young mice is the osteo- formation through a mechanism unrelated to PPR signaling blastic production of several bone anabolic Wnt ligands, in- in T cells.

Bedi et al. PNAS | Published online March 5, 2012 | E729 Downloaded by guest on September 24, 2021 Fig. 4. Analysis of the effects (mean ± SEM) of iPTH treatment in PPRT 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 ﬂ ﬂ group. #P < 0.05 compared with the corresponding PPR / mice. n =8–10 mice per group.

− − The resistance to iPTH-induced bone anabolism in PPRTcells / In WT mice, the enhancement of bone formation induced by mice is explained by the reduced capacity of iPTH to induce iPTH is accompanied by a stimulation of bone resorption that is Wnt10b production, activate Wnt signaling in osteoblastic cells, driven by increased production of RANKL by OBs (52) and T expand the osteoblastic pool, and thus stimulate bone formation. cells (30). These effects are mitigated, in part, by iPTH-induced − − Differences between control and PPRT cells / mice with respect activation of β-catenin in OBs, because this transcriptional reg- to iPTH-induced SC proliferation, differentiation, and life span ulator stimulates their production of OPG (27) and represses were demonstrated using purified SCs cultured for 1 wk, sug- that of RANKL (53). The latter is one of the mechanisms that gesting that in vivo activation of PPR signaling in T cells regu- prevents bone resorption from offsetting the anabolic activity lates the selection and differentiation of SCs, leading to the of iPTH. emergence of highly osteoblastogenic SC lineages and/or line- In agreement with earlier studies by us (30) and others (54), in ages of SCs that are highly responsive to the direct effects of this investigation, the stimulation of bone resorption induced by PTH on SCs. These steps are not reversed by the absence of T iPTH was not reflected by measurements of the number of OCs cells in vitro. This model is consistent with the capacity of Wnt and OC surfaces per unit of trabecular bone surface. This was signaling to guide cell fate determination (49). A similar para- mainly because iPTH increased bone surfaces in control mice digm has been described in our earlier studies on the effects of more markedly than OC number and OC surfaces. However, we iPTH in T cell-deficient mice (30) and in ovariectomized mice, found that iPTH increases serum levels of CTX and CD8+ cell − − a model in which estrogen withdrawal leads to the formation of production of RANKL in control mice but not in PPRT cells / SCs that exhibit increased osteoclastogenic activity, which per- mice. This suggests that iPTH regulates bone resorption by in- sists in vitro for 4 wk (50, 51). ducing T-cell production of RANKL through activation of PPR

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− − Fig. 5. Analysis of the effects (mean ± SEM) of iPTH treatment on osteoblastogenesis and on SC apoptosis in PPRT 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 PPRfl/fl mice. n =4–5 per group.

signaling in T lymphocytes. Another likely contributory mecha- Materials and Methods nism is the capacity of iPTH-stimulated T cells to lead to the Animals. All animal procedures were approved by the Institutional Animal generation of BM SCs that respond to iPTH by producing higher Care and Use Committee of Emory University. C57BL6 WT mice were purchased − − levels of RANKL and lower levels of OPG compared with the from the Jackson Laboratory. C57BL6 Wnt10b / mice were generated as de- SCs, which differentiate in the BM of T cell-deficient mice (36). scribed (55) and provided by T. F. Lane (University of California, Los Angeles, Tcells−/− In a previous report, we have shown that continuous activation CA). C57BL6 mice with T cell-specific PPR gene disruption (PPR mice) fl/fl of PPR in T cells induced by continuous PTH treatment is re- were generated by crossing homozygous PPR mice with Cre transgenic mice expressing Cre under the T cell-specific promoter Lck as previously quired for the hormone to stimulate bone resorption but not fl fl described (37). Experiments were conducted using 6-wk-old female PPR / bone formation (37). The changes in bone turnover induced by T cells−/− fl/fl T cells−/− and PPR mice and 17-wk-old male PPR and PPR mice. Ad- MEDICAL SCIENCES fl − − continuous PTH re ect the capacity of continuous activation of ditional studies were conducted using 6-wk-old male WT and Wnt10b / PPR in T cells to increase the production of TNF but not RANKL mice. All mice were maintained under specific pathogen-free conditions and and Wnt10b (37). By contrast, we show herein that intermittent fed sterilized food and autoclaved water ad libitum. activation of T cell-expressed PPR by iPTH is required for the hormone to stimulate bone turnover fully. Moreover, intermittent Intermittent Administration of PTH. In all the “in vivo” experiments, 80 activation of T cell-expressed PPR induces T-cell production of μg·kg·d of human PTH (1–34; Bachem California, Inc.) or vehicle was injected Wnt10b and RANKL but not of TNF. Although the molecular daily s.c. for 4 wk as described (30). mechanism of these phenomena remains to be determined, the data demonstrate that cell-autonomous effects of PTH in T cells In Vivo BMD Measurements. Total-body BMD was measured in anesthetized resulting from intermittent or continuous PPR signaling play mice using a PIXImus2 bone densitometer (GE Medical Systems) as described (56). pivotal roles in the overall mechanism of action of PTH in bone. μ μ fi CT Measurements. CT scanning and analysis were performed as reported Our ndings provide insight into the signaling integration be- previously (30, 37) using a Scanco μCT-40 scanner (Scanco Medical). tween cells of the immune system and bone cells and the impact of such cellular interaction on bone homeostasis. Understanding the Quantitative Bone Histomorphometry. The left femur was fixed in 10% (vol/ PPR signaling in T cells may therefore yield novel therapeutic vol) neutral-buffered formalin for 48 h, dehydrated, defattened at 4 8C, and strategies for potentiating bone anabolic agents. embedded in methyl methacrylate resin. In brief, 5-μm, nonconsecutive,

Bedi et al. PNAS | Published online March 5, 2012 | E731 Downloaded by guest on September 24, 2021 longitudinal sections in the frontal midbody plane (RM2155 microtome; Thymidine Incorporation Assay. SC proliferation was measured by [3H]-thy- Leica Microsystems) were cut and analyzed. OB and OC number and surface midine incorporation assay. SCs were pulsed with [3H]-thymidine (0.5 μCi per were determined on toluidine blue-stained sections using a Merz grid 10,000 cells) for 18 h and were harvested using a Cell Harvestor (Skatron, (magnification of 400×). The measurements, terminology, and units used for Inc.). [3H]-thymidine incorporation was determined by means of an LS 6000 histomorphometric analysis were those recommended by the Nomenclature IC Liquid Scintillation Counter (Beckman Coulter, Inc.). Committee of the American Society of Bone and Mineral Research (57). Apoptosis Assay. The activity of caspase-3, the key protease in the induction of SC Purification. SCs were purified as previously described (30, 36, 58). Addi- apoptosis, was measured in SCs using a CaspACE Assay System (Promega tional information is provided in SI Materials and Methods. Corporation) according to the manufacturer’s protocol.

Markers of Bone Turnover. Serum CTX was measured by a rodent-specific Real-Time RT-PCR and Primers. Assays were carried out as previously described ELISA (Immunodiagnostic Systems). Serum OCN was measured using a Rat- (30). Additional information is provided in SI Materials and Methods. MID Osteocalcin ELISA kit (Immunodiagnostic Systems). These methods have been previously described (30, 36, 58). Statistical Analysis. Analysis was carried out as previously described (30). Additional information is provided in SI Materials and Methods. CFU-ALP Assay. Colony-forming assays were carried out as described (30, 36, 58). ACKNOWLEDGMENTS. This study was supported by the National Institutes of Health Grants AR54625 and AR49659. M.N.W. acknowledges financial support from the Biomedical Laboratory Research and Development Service T-Cell Purification and Culture. T cells were purified from the BM by positive of the Veterans Affairs Office of Research and Development through Grant immunoselection using MACS Microbeads (Miltenyi Biotech) coupled to anti- 5I01BX000105; National Institute of Arthritis and Musculoskeletal and Skin CD90 or anti-CD8 antibody, as described (30, 36). Cell purity was verified to Diseases through Grants AR059364, AR056090, and AR053607; National Institute be >90% by flow cytometry. on Aging Grant AG040013; and the Georgia Research Alliance.

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