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10% Fluoro-Ruby 溶液是将 10 mg Fluoro-Ruby 干粉溶解在100 µL 蒸馏水。然后使用 1 uL Hamilton 微注射器通过颅内立体定向注射来压力注射示踪剂。注射体积通常为 0.02-0.1 µL，并在 10-15 分钟的间隔内逐渐注射。让动物恢复并通常在 4-14 天后处死。
然后用中性缓冲甲醛（10% 福尔马林）灌注动物。n 或 4% 多聚甲醛溶于 0.1 M 中性磷酸盐缓冲液中。）取出大脑并在相同的固定液中后固定至少过夜，并添加 20% 蔗糖进行冷冻保护。然后在冷冻滑动切片机或低温恒温器上将大脑切成厚度通常在 20 至 40 微米之间的切片。
将组织切片安装在涂有明胶的载玻片上，并在 50-50 ℃的载玻片加热器中风干。 60摄氏度至少半小时。然后将干燥的载玻片转移到二甲苯透明溶液中至少一分钟，然后用 DPX 封固剂盖上盖玻片。然后可以使用适合可视化 TRITC（绿光激发）的滤光片在落射荧光显微镜下检查载玻片。
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Silencing of parathyroid hormone (PTH) receptor 1 in T cells blunts the bone anabolic activity of PTH | PNAS Edited by John T. Potts, Massachusetts General Hospital, Charlestown, MA, and approved February 6, 2012 (received for review December 28, 2011) AbstractIntermittent 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.bone massT lymphocytesbone cellsParathyroid hormone (PTH) is a major regulator of calcium metabolism and defends against hypocalcemia, in part, by stimulating bone resorption, and thereby the release of calcium from the skeleton. However, when injected daily, a regimen known as intermittent parathyroid hormone (iPTH) treatment, the hormone markedly stimulates trabecular and cortical bone formation. Although this bone-forming activity is antagonized by a stimulation of bone resorption, the net effect of iPTH treatment is an improvement in bone microarchitecture and increased strength (1, 2). As a result, intermittent treatment with the 1–34 fragment of PTH is a Food and Drug Administration-approved treatment modality for postmenopausal osteoporosis (3).The effects of PTH on bone result from its binding to the PTH/PTH-related protein receptor (PPR or PTHR1) expressed on bone marrow (BM), stromal cells (SCs), osteoblasts (OBs), and osteocytes (1, 4, 5). iPTH stimulates bone formation by increasing the number of OBs (6⇓–8), a phenomenon achieved through activation of quiescent lining cells (9), increased OB proliferation (10, 11) and differentiation (10, 12, 13), attenuation of OB apoptosis (14⇓⇓–17), and signaling in osteocytes (18). However, the specific contribution of each of these effects of iPTH remains controversial. The expansion of the osteoblastic pool induced by iPTH is initiated by the release from the matrix 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 of Wnt signaling in osteoblastic cells (23). Activation of Wnt signaling induces OB proliferation (24) and differentiation (23, 25), prevents OB apoptosis (16, 17, 26), and augments OB production of osteoprotegerin (OPG) (27).iPTH activates Wnt signaling in OBs through multiple mechanisms that include Wnt ligand-independent activation of the Wnt coreceptor LRP6 (28), increased production of Wnt ligands by bone and BM cells (29, 30), and suppression of sclerostin production (31⇓–33). Additional effects on the Wnt system have been described in models of hyperparathyroidism characterized by a continuous overproduction of PTH but not in mice treated with intermittent PTH. For example, continuous PTH treatment regulates the Wnt antagonist Dkk1 (34, 35) and the Wnt receptor LRP6 (28), whereas iPTH does not.Although SCs, OBs, and osteocytes represent the major targets of PTH in bone, reports from our laboratory have disclosed that T lymphocytes play an unexpected role in the mechanism of action of PTH (30, 36, 37). We have shown that treatment with iPTH increases the T-cell production of Wnt10b, a Wnt ligand that stimulates osteoblastogenesis by activating Wnt signaling in SCs and OBs. As a result, the bone anabolic activity of iPTH is markedly reduced in T cell-deficient mice and in mice with a specific disruption of Wnt10b production by T cells (30).Despite the evidence supporting a role for T cells in the actions of iPTH in bone, the mechanisms involved are only partly understood because it remains unknown whether direct activation of PPR in T cells by PTH is required for iPTH treatment to exert its full anabolic activity. Because SCs regulate T-cell function (38, 39) and OBs express CD40 (40), a surface receptor that signals to its counterpart CD40L expressed in T cells, iPTH could indeed affect T cells indirectly through osteoblastic cells.To address these issues, we have conditionally silenced PPR in T cells and determined whether PTH activation of PPR in T cells is required for iPTH to exert its bone anabolic activity. We show that blocking PPR signaling in T cells blunts the capacity of iPTH treatment to induce the production of Wnt10b by T cells, stimulate osteoblastogenesis and bone formation, and increase bone mass. Therefore, direct targeting of T cells by PTH is required for iPTH to induce maximal bone anabolism.ResultsPPR Signaling in T Cells Is Required for iPTH to Increase Wnt10b Production by T Cells and Activate Wnt Signaling in OBs.Intermittent PTH treatment stimulates the production of the osteogenic Wnt ligand Wnt10b by BM T cells, leading to an overall increase in the BM levels of Wnt10b (30). To determine whether this stimulatory effect of PTH is attributable to direct PPR signaling in T cells, we made use of PPRT cells−/− mice, a strain with a silent PPR in all T cells (37). Six-week-old female PPRT cells−/− and control PPRfl/fl mice were injected daily with vehicle or 80 μg/kg of human PTH 1–34 for 4 wk, a treatment modality referred to hereafter as iPTH. Whole BM and BM T cells were harvested, and their expression of Wnt10b mRNA was analyzed by real-time PCR (RT-PCR). This assay disclosed that iPTH increased Wnt10b mRNA levels by approximately sixfold in T cells from control mice. By contrast, PTH had no stimulatory effect in T cells from PPRT cells−/− mice (Fig. 1A). Attesting to the contribution of T cells to the overall BM levels of Wnt10b, PTH increased the levels of Wnt10b mRNA by approximately fourfold in unfractionated BM samples from control mice. However, PTH had no significant effect in BM from PPRT cells−/− mice (Fig. 1B).Download figure Open in new tab Download powerpoint 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 PPRT cells−/− mice or Wnt10b−/− mice. (A) Effect of iPTH on BM T-cell levels of Wnt10b mRNA in PPRT cells−/− and control PPRfl/fl 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 compared with the corresponding PPRfl/fl group. n = 10 mice per group.To confirm these findings, BM T cells and whole BM from 6-wk-old control and PPRT cells−/− mice were treated in vitro with PTH for 24 h. Analysis by real-time RT-PCR disclosed that PTH increased Wnt10b mRNA levels in purified T cells and whole BM from control mice by approximately eightfold and approximately fourfold, respectively. By contrast, PTH had no stimulatory effect in T cells and whole BM from PPRT cells−/− mice (Fig. 1 C and D). Thus, PTH stimulates production of Wnt10b by directly targeting T cell-expressed PPR.Next, we investigated the role of PPR signaling in T cells for the activation of Wnt signaling in osteoblastic cells induced by iPTH. To this end, we assessed by RT-PCR the mRNA expression of genes up-regulated by Wnt signaling in SCs purified from PPRT cells−/− and control mice treated with vehicle or iPTH for 4 wk and killed at 10 wk of age. The analyzed genes were chosen based on known expression patterns during differentiation of primitive mesenchymal cells to the OB phenotype. Some of the selected genes are known to play a direct role in regulating OB differentiation (41), whereas others are not involved in OB differentiation but are sensitive Markers of Wnt activation (42). These analyses revealed (Fig. 1E) that iPTH activates Wnt signaling in SCs through PPR signaling in T cells. In fact, SC levels of mRNA for the nine tested genes were all increased by iPTH in SCs from control mice but not in those from PPRT cells−/− mice. The genes that were stimulated by iPTH 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 (Twist1), and Wnt1 inducible signaling pathway protein 1 (Wisp1).To determine the relevance of Wnt10b for the anabolic activity of iPTH, 6-wk-old male WT and Wnt10b−/− mice were injected daily with vehicle or iPTH for 4 wk. Dual X-ray absorptiometry (DXA) was used to measure in vivo total body bone mineral density (BMD), and micro-computerized tomography (μCT) was used to assess the trabecular compartment of femurs harvested at sacrifice. In response to iPTH treatment, WT mice displayed a significant increase in BMD and trabecular bone volume (BV/TV) (Fig. 1 F and G). By contrast, iPTH had no anabolic effects in Wnt10b−/− mice, thus confirming that Wnt10b is required for iPTH to exert its anabolic activity. Parameters of trabecular structure were also differentially affected in WT and Wnt10b−/− mice, because trabecular number (Tb.N), connectivity density (Conn.D), and trabecular thickness (Tb.Th) were improved in WT mice but not in Wnt10b−/− mice (Fig. S1).Mice Lacking PPR Signaling in T Cells Exhibit a Blunted Increase in Bone Mass and Bone Turnover Response to iPTH.To determine whether PPR signaling in T cells is required for iPTH to exert its effects on bone, we again made use of PPRT cells−/− mice (37). At 6 wk of age PPRT cells−/− mice have a normal bone phenotype and normal serum calcium, phosphate, and PTH levels (37), indicating that PPR signaling in T cells does not play an essential role in bone modeling and baseline remodeling. Moreover, PPRT cells−/− mice have a normal number of T cells, which exhibit a degree of activation and proliferation similar to control T cells (37). PPRT cells−/− and control mice also have a similar number of B cells and myeloid cells (Fig. S2).In a first experiment, 6-wk-old female PPRT cells−/− mice and littermate controls were treated with iPTH for 4 wk, and their BMD was measured in vivo by DXA, a technique that provides a combined measure of cortical and trabecular bone. As a result of physiological growth, BMD increased in all mice treated with vehicle. Treatment with iPTH induced a further increase in BMD in all groups. However, the bone anabolic effect of iPTH was significantly smaller in PPRT cells−/− mice than in control mice, as demonstrated by the fact that at 4 wk, iPTH induced a 26% increase in the BMD of PPRT cells−/− mice compared with a 41% increase in that of PPRfl/fl mice (Fig. 2A).Download figure Open in new tab Download powerpoint 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. #P 0.05 compared with corresponding PPRfl/fl mice. n = 10–20 mice per group.Cancellous bone was analyzed by histology and μCT using femurs harvested at sacrifice. Histomorphometric measurements of BV/TV revealed a less pronounced anabolic response to PTH in PPRT cells−/− mice than in control mice. In fact, iPTH increased BV/TV by ∼234% in PPRfl/fl mice and by ∼33% in PPRT cells−/− mice (Fig. 2B). μCT analysis confirmed that activation of PPR signaling in T cells by iPTH is required for iPTH-induced anabolism, because iPTH induced a greater increase in BV/TV in control mice than in those with a silent PPR receptor in T cells (Fig. 2C). Tb.N, Conn.D, and Tb.Th, three indices of trabecular structure, were more substantially improved in PPRfl/fl mice than in PPRT cells−/− mice (Fig. 2 D–F).By contrast, μCT analysis of cortical bone showed that iPTH induced similar increases in cortical thickness (Co.Th) and cortical volume (Co.Vo.) in all groups of mice (Fig. 2 G and H), thus demonstrating that PPR signaling in T cells specifically augments the capacity of iPTH to improve architecture in trabecular bone. Representative μCT images of trabecular and cortical bone are shown in Fig. S3.Analysis of the secondary spongiosa by bone histomorphometry revealed that iPTH increased two static indices of bone formation, the number of OBs per bone surface (N.Ob/BS) (Fig. 3A) and the percentage of surfaces covered by OBs (Ob.S/BS) (Fig. 3B), to a lower extent in PPRT cells−/− mice than in PPRfl/fl mice. Analysis of dynamic indices of formation in iPTH-treated control mice revealed massive and diffuse fluorochrome marker labeling that prevented an accurate measurement of mineral apposition rate, mineralizing surface, and thus bone formation rate. Measurements of serum osteocalcin (OCN), a marker of bone formation, confirmed that iPTH induced a larger increase in bone turnover in PPRfl/fl mice than in PPRT cells−/− mice (Fig. 3C).Download figure Open in new tab Download powerpoint 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 0.001 compared with the corresponding vehicle-treated group. #P 0.05 compared with the corresponding PPRfl/fl mice. n = 10–20 mice per group for A–F and n = 4 mice per group for G.Histomorphometric analysis showed no significant effects of iPTH on two indices of trabecular bone resorption, the number of osteoclasts (OCs) per bone surface (Fig. 3D) and the percentage of surfaces covered by OCs (Fig. 3E), whereas measurements of serum C-terminal telopeptide of collagen (CTX), a biochemical marker of resorption, indicated that iPTH induced a significant increase in bone resorption in control but not in PPRT cells−/− mice (Fig. 3F). The discrepancy between histomorphometric and biochemical indices of resorption is likely explained by the fact that iPTH increased bone surfaces as extensively as OC number and OC surfaces.One mechanism by which iPTH stimulates bone resorption is by increasing the CD8+ cell production of receptor activator of nuclear factor-κB ligand (RANKL) (30). Therefore, CD8+ cells were purified from the BM of vehicle and iPTH-treated mice and assayed for RANKL mRNA levels. These experiments revealed that iPTH increased the levels of RANKL mRNA significantly in CD8+ cells from PPRfl/fl mice but not in those from PPRT cells−/− mice (Fig. 3G).To confirm the relevance of direct PPR signaling in T cells in mature mice, 17-wk-old male PPRT cells−/− mice and littermate controls were treated with iPTH for 4 wk. Analysis of trabecular bone by μCT revealed that iPTH increased BV/TV in control mice, although it had no significant effects in mice with a silent PPR receptor in T cells (Fig. 4A). Similarly, iPTH increased Tb.Th and Conn.D in PPRfl/fl mice but not in PPRT cells−/− mice (Fig. 4 B and D). Tb.N was unaffected by iPTH in both genotypes (Fig. 4C). Confirming the data obtained in younger mice, iPTH equally increased indices of cortical structures in all groups of mice (Fig. 4 E and F). Measurements of serum OCN and CTX levels confirmed that iPTH increased bone turnover in PPRfl/fl mice but not in PPRT cells−/− mice (Fig. 4 G and H).Download figure Open in new tab Download powerpoint 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 group. #P 0.05 compared with the corresponding PPRfl/fl mice. n = 8–10 mice per group.Mice Lacking PPR Signaling in T Cells Exhibit a Blunted Osteoblastogenic Response to iPTH.BM from female PPRT cells−/− and control mice was used to assess the formation of alkaline phosphatase-positive colony-forming unit fibroblast (herein defined as CFU-ALP), an index of SC commitment to the osteoblastic lineage. iPTH treatment for 4 wk in mice from 6 to 10 wk of age increased CFU-ALP formation in the BM of PPRfl/fl mice by approximately twofold, whereas it had no effect on that of PPRT cells−/− mice (Fig. 5A), thus indicating that PPR signaling in T cells potentiates the capacity of iPTH to increase the number of SCs with osteogenic potential.Download figure Open in new tab Download powerpoint 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.To determine whether PPR signaling in T cells is required for iPTH-induced osteoblastogenesis, BM from PPRT cells−/− and control female mice harvested after 4 wk of iPTH treatment (when the mice were 10 wk old) was cultured for 1 wk to allow SCs to proliferate. SCs were then purified and counted. This analysis revealed (Fig. 5B) that in vivo iPTH treatment increases the number of SCs in samples from PPRfl/fl mice. By contrast, iPTH had no effects on the number of SCs from PPRT cells−/− mice. To investigate the mechanism involved, BM was cultured for 1 wk and SCs were purified and used to determine their rate of proliferation and apoptosis. These experiments revealed that iPTH increases the proliferation of SCs from control mice significantly, whereas it had no effect on the proliferation of SCs from PPRT cells−/− mice (Fig. 5C). Moreover, iPTH decreased the rate of SC apoptosis in control mice but not in PPRT cells−/− mice (Fig. 5D). Analysis of the expression levels of osteoblastic genes in SCs revealed that iPTH treatment for 4 wk increased the expression of type 1 collagen, Runx2, osterix, bone sialoprotein, and OCN mRNAs by approximately two- to threefold in SCs from PPRfl/fl mice, whereas it had no effects on that from PPRT cells−/− mice (Fig. 5E). These findings demonstrate that direct PPR signaling in T cells is indispensable for the expansion of the osteoblastic pool and the differentiation along the osteoblastic lineage induced by iPTH.DiscussionWe report that mice lacking PPR signaling in T cells exhibit a blunted bone anabolic response to iPTH because of impaired T-cell production of Wnt10b, an osteogenic factor that promotes Wnt signaling in OBs. Therefore, T cells are direct targets of PTH that play a pivotal role in the osteoblastogenic response to iPTH.Based on a previous report (30), we speculated that ligand-dependent activation of PPR signaling in T cells is required for iPTH to induce the production of Wnt10b and promote bone anabolism. To investigate this hypothesis, we made use of PPRT cells−/− mice, a strain with a silent PPR in all T cells (37). Demonstrating a requirement for direct PTH signaling in T cells, we found that the silencing of PPR in T cells blocks the capacity of iPTH to induce the production of Wnt10b by T cells, the activation of Wnt signaling in SCs, and trabecular bone accretion. Moreover, analysis of the effect of iPTH in Wnt10b−/− mice confirmed that Wnt10b is indispensable for the anabolic activity of iPTH.We found that the capacity of iPTH to increase BV/TV was decreased but not completely abolished in young PPRT cells−/− mice. By contrast, the anabolic activity of iPTH was completely blocked in mature PPRT cells−/− mice. These findings suggest that the contribution of T cells to the activity of iPTH increases with age. A mechanism likely to account for the T cell-independent anabolic activity of iPTH observed in young mice is the osteoblastic production of several bone anabolic Wnt ligands, including Wnt7a and Wnt3b, resulting from PPR signaling in bone cells (43, 44). A second mechanism might be the inhibition of the osteocytic production of the Wnt antagonist sclerostin (31, 32). A less likely possibility is that iPTH may signal in T cells through an alternative PTH receptor. One such receptor is PTHR2, a protein expressed primarily in the central nervous system and the cardiovascular system. However, PTHR2 is not known to play a role in bone (45⇓–47). Recently, a receptor specific for the carboxyl terminal PTH fragments has been proposed and is yet to be cloned (48). The function of this receptor in bone is unknown but not relevant for the effects of PTH 1–34.Although silencing of PPR in T cells resulted in a blunted response of the trabecular compartment to iPTH, the hormone had similar anabolic effects in the cortical bone of all groups of mice. These findings are consistent with our earlier report that T cells specifically potentiate the anabolic effect of iPTH in cancellous bone (30) and indicate that iPTH stimulates cortical bone formation through a mechanism unrelated to PPR signaling in T cells.The resistance to iPTH-induced bone anabolism in PPRT cells−/− mice is explained by the reduced capacity of iPTH to induce Wnt10b production, activate Wnt signaling in osteoblastic cells, expand the osteoblastic pool, and thus stimulate bone formation. Differences between control and PPRT cells−/− mice with respect to iPTH-induced SC proliferation, differentiation, and life span were demonstrated using purified SCs cultured for 1 wk, suggesting that in vivo activation of PPR signaling in T cells regulates the selection and differentiation of SCs, leading to the emergence of highly osteoblastogenic SC lineages and/or lineages of SCs that are highly responsive to the direct effects of PTH on SCs. These steps are not reversed by the absence of T cells in vitro. This model is consistent with the capacity of Wnt signaling to guide cell fate determination (49). A similar paradigm has been described in our earlier studies on the effects of iPTH in T cell-deficient mice (30) and in ovariectomized mice, a model in which estrogen withdrawal leads to the formation of SCs that exhibit increased osteoclastogenic activity, which persists in vitro for 4 wk (50, 51).In WT mice, the enhancement of bone formation induced by iPTH is accompanied by a stimulation of bone resorption that is driven by increased production of RANKL by OBs (52) and T cells (30). These effects are mitigated, in part, by iPTH-induced activation of β-catenin in OBs, because this transcriptional regulator stimulates their production of OPG (27) and represses that of RANKL (53). The latter is one of the mechanisms that prevents bone resorption from offsetting the anabolic activity of iPTH.In agreement with earlier studies by us (30) and others (54), in this investigation, the stimulation of bone resorption induced by iPTH was not reflected by measurements of the number of OCs and OC surfaces per unit of trabecular bone surface. This was mainly because iPTH increased bone surfaces in control mice more markedly than OC number and OC surfaces. However, we found that iPTH increases serum levels of CTX and CD8+ cell production of RANKL in control mice but not in PPRT cells−/− mice. This suggests that iPTH regulates bone resorption by inducing T-cell production of RANKL through activation of PPR signaling in T lymphocytes. Another likely contributory mechanism is the capacity of iPTH-stimulated T cells to lead to the generation of BM SCs that respond to iPTH by producing higher levels of RANKL and lower levels of OPG compared with the SCs, which differentiate in the BM of T cell-deficient mice (36).In a previous report, we have shown that continuous activation of PPR in T cells induced by continuous PTH treatment is required for the hormone to stimulate bone resorption but not bone formation (37). The changes in bone turnover induced by continuous PTH reflect the capacity of continuous activation of PPR in T cells to increase the production of TNF but not RANKL and Wnt10b (37). By contrast, we show herein that intermittent activation of T cell-expressed PPR by iPTH is required for the hormone to stimulate bone turnover fully. Moreover, intermittent activation of T cell-expressed PPR induces T-cell production of Wnt10b and RANKL but not of TNF. Although the molecular mechanism of these phenomena remains to be determined, the data demonstrate that cell-autonomous effects of PTH in T cells resulting from intermittent or continuous PPR signaling play pivotal roles in the overall mechanism of action of PTH in bone.Our findings provide insight into the signaling integration between cells of the immune system and bone cells and the impact of such cellular interaction on bone homeostasis. Understanding the PPR signaling in T cells may therefore yield novel therapeutic strategies for potentiating bone anabolic agents.Materials and MethodsAnimals.All animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University. C57BL6 WT mice were purchased from the Jackson Laboratory. C57BL6 Wnt10b−/− mice were generated as described (55) and provided by T. F. Lane (University of California, Los Angeles, CA). C57BL6 mice with T cell-specific PPR gene disruption (PPRT cells−/− mice) were generated by crossing homozygous PPRfl/fl mice with Cre transgenic mice expressing Cre under the T cell-specific promoter Lck as previously described (37). Experiments were conducted using 6-wk-old female PPRfl/fl and PPRT cells−/− mice and 17-wk-old male PPRfl/fl and PPRT cells−/− mice. Additional studies were conducted using 6-wk-old male WT and Wnt10b−/− mice. All mice were maintained under specific pathogen-free conditions and fed sterilized food and autoclaved water ad libitum.Intermittent Administration of PTH.In all the \"in vivo” experiments, 80 μg⋅kg⋅d of human PTH (1–34; Bachem California, Inc.) or vehicle was injected daily s.c. for 4 wk as described (30).In Vivo BMD Measurements.Total-body BMD was measured in anesthetized mice using a PIXImus2 bone densitometer (GE Medical Systems) as described (56).μCT Measurements.μCT scanning and analysis were performed as reported previously (30, 37) using a Scanco μCT-40 scanner (Scanco Medical).Quantitative Bone Histomorphometry.The left femur was fixed in 10% (vol/vol) neutral-buffered formalin for 48 h, dehydrated, defattened at 4 °C, and embedded in methyl methacrylate resin. In brief, 5-μm, nonconsecutive, longitudinal sections in the frontal midbody plane (RM2155 microtome; Leica Microsystems) were cut and analyzed. OB and OC number and surface were determined on toluidine blue-stained sections using a Merz grid (magnification of 400×). The measurements, terminology, and units used for histomorphometric analysis were those recommended by the Nomenclature Committee of the American Society of Bone and Mineral Research (57).SC Purification.SCs were purified as previously described (30, 36, 58). Additional information is provided in SI Materials and Methods.Markers of Bone Turnover.Serum CTX was measured by a rodent-specific ELISA (Immunodiagnostic Systems). Serum OCN was measured using a Rat-MID Osteocalcin ELISA kit (Immunodiagnostic Systems). These methods have been previously described (30, 36, 58).CFU-ALP Assay.Colony-forming assays were carried out as described (30, 36, 58).T-Cell Purification and Culture.T cells were purified from the BM by positive immunoselection using MACS Microbeads (Miltenyi Biotech) coupled to anti-CD90 or anti-CD8 antibody, as described (30, 36). Cell purity was verified to be 90% by flow cytometry.Thymidine Incorporation Assay.SC proliferation was measured by [3H]-thymidine incorporation assay. SCs were pulsed with [3H]-thymidine (0.5 μCi per 10,000 cells) for 18 h and were harvested using a Cell Harvestor (Skatron, Inc.). [3H]-thymidine incorporation was determined by means of an LS 6000 IC Liquid Scintillation Counter (Beckman Coulter, Inc.).Apoptosis Assay.The activity of caspase-3, the key protease in the induction of apoptosis, was measured in SCs using a CaspACE Assay System (Promega Corporation) according to the manufacturer\'s protocol.Real-Time RT-PCR and Primers.Assays were carried out as previously described (30). Additional information is provided in SI Materials and Methods.Statistical Analysis.Analysis was carried out as previously described (30). Additional information is provided in SI Materials and Methods.AcknowledgmentsThis 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 of the Veterans Affairs Office of Research and Development through Grant 5I01BX000105; National Institute of Arthritis and Musculoskeletal and Skin Diseases through Grants AR059364, AR056090, and AR053607; National Institute on Aging Grant AG040013; and the Georgia Research Alliance.Footnotes↵1B.B. and J.-Y.L. contributed equally to this work.↵2To whom correspondence should be addressed. 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Vangara, Ming-Kang Chang, Michaela Kneissel, M. Neale Weitzmann, Roberto Pacifici Proceedings of the National Academy of Sciences Mar 2012, 109 (12) E725-E733; DOI: 10.1073/pnas.1120735109 T-cell PTH receptor in PTH-induced bone anabolism Brahmchetna Bedi, Jau-Yi Li, Hesham Tawfeek, Ki-Hyun Baek, Jonathan Adams, Sameera S. Vangara, Ming-Kang Chang, Michaela Kneissel, M. Neale Weitzmann, Roberto Pacifici Proceedings of the National Academy of Sciences Mar 2012, 109 (12) E725-E733; DOI: 10.1073/pnas.1120735109 Sign up for the PNAS Highlights newsletter to get in-depth stories of science sent to your inbox twice a month: Relatively clean snow and ice in the Indus River Basin during the COVID-19 pandemic may have reduced meltwater in 2020, compared with the 20-year average. Atmospheric and climate conditions could have created a cloud greenhouse effect to warm Mars and support liquid surface water. 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