Regulatory T cells are expanded by Teriparatide treatment in humans and mediate intermittent PTH‐induced bone anabolism in mice

Teriparatide is a bone anabolic treatment for osteoporosis, modeled in animals by intermittent PTH (iPTH) administration, but the cellular and molecular mechanisms of action of iPTH are largely unknown. Here, we show that Teriparatide and iPTH cause a ~two‐threefold increase in the number of regulatory T cells (Tregs) in humans and mice. Attesting in vivo relevance, blockade of the Treg increase in mice prevents the increase in bone formation and trabecular bone volume and structure induced by iPTH. Therefore, increasing the number of Tregs is a pivotal mechanism by which iPTH exerts its bone anabolic activity. Increasing Tregs pharmacologically may represent a novel bone anabolic therapy, while iPTH‐induced Treg increase may find applications in inflammatory conditions and transplant medicine.     

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including G_sα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of G_sα in the osteoblast lineage. Postnatal removal of G_sα in the osteoblast lineage (P-G_sα^OsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1–34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-G_sα^OsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-G_sα^OsxKO mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via G_sα but not phospholipase C via G_q/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of G_sα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond G_sα and G_q/11 act downstream of PTH on osteoblast differentiation.     

Cyclooxygenase-2 Suppresses the Anabolic Response to PTH Infusion in Mice

We previously reported that the ability of continuously elevated PTH to stimulate osteoblastic differentiation in bone marrow stromal cell cultures was abrogated by an osteoclastic factor secreted in response to cyclooxygenase-2 (Cox2)-produced prostaglandin E₂. We now examine the impact of Cox2 (Ptgs2) knockout (KO) on the anabolic response to continuously elevated PTH in vivo. PTH (40 μg/kg/d) or vehicle was infused for 12 or 21 days in 3-mo-old male wild type (WT) and KO mice in the outbred CD-1 background. Changes in bone phenotype were assessed by bone mineral density (BMD), μCT and histomorphometry. PTH infusion for both 12 and 21 days increased femoral BMD in Cox2 KO mice and decreased BMD in WT mice. Femoral and vertebral trabecular bone volume fractions were increased in KO mice, but not in WT mice, by PTH infusion. In the femoral diaphysis, PTH infusion increased cortical area in Cox2 KO, but not WT, femurs. PTH infusion markedly increased trabecular bone formation rate in the femur, serum markers of bone formation, and expression of bone formation-related genes, growth factors, and Wnt target genes in KO mice relative to WT mice, and decreased gene expression of Wnt antagonists only in KO mice. In contrast to the differential effects of PTH on anabolic factors in WT and KO mice, PTH infusion increased serum markers of resorption, expression of resorption-related genes, and the percent bone surface covered by osteoclasts similarly in both WT and KO mice. We conclude that Cox2 inhibits the anabolic, but not the catabolic, effects of continuous PTH. These data suggest that the bone loss with continuously infused PTH in mice is due largely to suppression of bone formation and that this suppression is mediated by Cox2.     

Physical training prevents oxidative stress in L‐NAME‐induced hypertension rats

The present study investigated the effects of a 6‐week swimming training on blood pressure, nitric oxide (NO) levels and oxidative stress parameters such as protein and lipid oxidation, antioxidant enzyme activity and endogenous non‐enzymatic antioxidant content in kidney and circulating fluids, as well as on serum biochemical parameters (cholesterol, triglycerides, urea and creatinine) from Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME)‐induced hypertension treated rats. Animals were divided into four groups (n = 10): Control, Exercise, L‐NAME and Exercise L‐NAME. Results showed that exercise prevented a decrease in NO levels in hypertensive rats (P < 0·05). An increase in protein and lipid oxidation observed in the L‐NAME‐treated group was reverted by physical training in serum from the Exercise L‐NAME group (P < 0·05). A decrease in the catalase (CAT) and superoxide dismutase (SOD) activities in the L‐NAME group was observed when compared with normotensive groups (P < 0·05). In kidney, exercise significantly augmented the CAT and SOD activities in the Exercise L‐NAME group when compared with the L‐NAME group (P < 0·05). There was a decrease in the non‐protein thiols (NPSH) levels in the L‐NAME‐treated group when compared with the normotensive groups (P < 0·05). In the Exercise L‐NAME group, there was an increase in NPSH levels when compared with the L‐NAME group (P < 0·05). The elevation in serum cholesterol, triglycerides, urea and creatinine levels observed in the L‐NAME group were reverted to levels close to normal by exercise in the Exercise L‐NAME group (P < 0·05). Exercise training had hypotensive effect, reducing blood pressure in the Exercise L‐NAME group (P < 0·05). These findings suggest that physical training could have a protector effect against oxidative damage and renal injury caused by hypertension. Copyright © 2012 John Wiley & Sons, Ltd.     

Parathyroid Hormone (PTH)/PTH-related Peptide Type 1 Receptor (PPR) Signaling in Osteocytes Regulates Anabolic and Catabolic Skeletal Responses to PTH

Parathyroid hormone (PTH) is the only Food and Drug Administration-approved anabolic agent to treat osteoporosis; however, the cellular targets of PTH action in bone remain controversial. PTH modulates bone turnover by binding to the PTH/PTH-related peptide (PTHrP) type 1 receptor (PPR), a G-protein-coupled receptor highly expressed in bone and kidneys. Osteocytes, the most abundant cells in adult bone, also express PPR. However, the physiological relevance of PPR signaling in osteocytes remains to be elucidated. Toward this goal, we generated mice with PPR deletion in osteocytes (Ocy-PPRKO). Skeletal analysis of these mice revealed a significant increase in bone mineral density and trabecular and cortical bone parameters. Osteoblast activities were reduced in these animals, as demonstrated by decreased collagen type I α1 mRNA and receptor activator of NF-κB ligand (RANKL) expression. Importantly, when subjected to an anabolic or catabolic PTH regimen, Ocy-PPRKO animals demonstrated blunted skeletal responses. PTH failed to suppress SOST/Sclerostin or induce RANKL expression in Ocy-PPRKO animals compared with controls. In vitro, osteoclastogenesis was significantly impaired in Ocy-PPRKO upon PTH administration, indicating that osteocytes control osteoclast formation through a PPR-mediated mechanism. Taken together, these data indicate that PPR signaling in osteocytes is required for bone remodeling, and receptor signaling in osteocytes is needed for anabolic and catabolic skeletal responses.     

Amelioration of type I diabetes-induced osteoporosis by parathyroid hormone is associated with improved osteoblast survival

Type 1 diabetic osteoporosis results from impaired osteoblast activity and death. Therefore, anti-resorptive treatments may not effectively treat bone loss in this patient population. Intermittent parathyroid hormone (PTH) treatment stimulates bone remodeling and increases bone density in healthy subjects. However, PTH effects may be limited in patients with diseases that interfere with its signaling. Here, we examined the ability of 8 and 40 μg/kg intermittent PTH to counteract diabetic bone loss. PTH treatment reduced fat pad mass and blood glucose levels in non-diabetic PTH-treated mice, consistent with PTH-affecting glucose homeostasis. However, PTH treatment did not significantly affect general body parameters, including the blood glucose levels, of type 1 diabetic mice. We found that the high dose of PTH significantly increased tibial trabecular bone density parameters in control and diabetic mice, and the lower dose elevated trabecular bone parameters in diabetic mice. The increased bone density was due to increased mineral apposition and osteoblast surface, all of which are defective in type 1 diabetes. PTH treatment suppressed osteoblast apoptosis in diabetic bone, which could further contribute to the bone-enhancing effects. In addition, PTH treatment (40 μg/kg) reversed preexisting bone loss from diabetes. We conclude that intermittent PTH may increase type 1 diabetic trabecular bone volume through its anabolic effects on osteoblasts.     

Alterations in plasma biochemical composition in NO deficiency induced by L‐NAME in mice analysed by Fourier Transform Infrared Spectroscopy

Mouse model of nitric oxide deficiency, induced by prolonged treatment with N^G‐nitro‐L‐arginine methyl ester (L‐NAME) was used for infrared spectroscopy (FTIR) analysis of plasma. L‐NAME leads to increased peripheral resistance and systemic hypertension. Classification of spectral response was by principal component analysis (PCA) and linear discriminant analysis (LDA). PCA allowed to separate each animal group showing that FTIR spectra are sensitive to development of NO‐deficiency on contrary to blood pressure values indicating hypertension. Globally, the most pronounced spectral alternations were observed in the second and third week of L‐NAME treatment indicating that infrared signature of blood plasma can serve as indicator of early and late stages of the disease. The PLS‐DA method provided >95% classification accuracy. Spectral features characteristic for L‐NAME treatment were mainly associated with an elevated level of proteins accompanied by a decrease of a tyrosine content and changes in lipids/phospholipid concentration. In our work we discuss these changes for which statistically significant differences (p < 0.05 – 0.005) were observed between spectra collected for each time‐point of the L‐NAME treatment versus control subjects. We demonstrated for the first time that NO‐deficiency and hypertension resulted in changes in biochemical profile of plasma that was detected by FTIR spectroscopy.

     

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone

Summary Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm² show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.     

Studies on the mechanisms of the skeletal anabolic action of endogenous and exogenous parathyroid hormone

Parathyroid hormone (PTH) has been viewed as catabolic for bone. Nevertheless, exogenous PTH is anabolic when administered intermittently, at a frequency that permits complete clearance between doses. In the fetus and neonate, endogenous PTH is required for normal trabecular bone formation. In older animals PTH produces net bone loss in fulfilling its calcium homeostatic role, whereas PTH-related peptide (PTHrP), acting in a paracrine/autocrine mode, is anabolic. The proliferative, differentiating, and anti-apoptotic effects of PTH on cells of the osteoblast lineage leading to anabolism can be direct, or indirect via release of local growth factors. The anabolic effect of PTH is also influenced by osteoclastic activity such that suppression of osteoclasts with anti-resorptive agents, concomitant to administering PTH, may enhance the anabolic effect by delaying a reactive osteoclastic response. In contrast, prolonged suppression of osteoclast activity prior to administering PTH appears to diminish molecular signals that increase the osteoblast pool and thereby reduces the anabolic efficacy of PTH. These observations may define the proper timing of the use of PTH as a therapeutic in diseases of bone loss. Finally, the capacity of exogenous PTH to modulate extra-osseous factors such as 1,25 dihydroxyvitamin D may also modulate its potency as an anabolic agent.     

Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt‐NOS signaling

Ischemia/reperfusion (I/R) injury in cardiomyocytes is related to excess reactive oxygen species (ROS) generation and can be modulated by nitric oxide (NO). We have previously shown that grape seed proanthocyanidin extract (GSPE), a naturally occurring antioxidant, decreased ROS and may potentially stimulate NO production. In this study, we investigated whether GSPE administration at reperfusion was associated with cardioprotection and enhanced NO production in a cardiomyocyte I/R model. GSPE attenuated I/R‐induced cell death [18.0 ± 1.8% (GSPE, 50 µg/ml) vs. 42.3 ± 3.0% (I/R control), P < 0.001], restored contractility (6/6 vs. 0/6, respectively), and increased NO release. The NO synthase (NOS) inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 200 µM) significantly reduced GSPE‐induced NO release and its associated cardioprotection [32.7 ± 2.7% (GSPE + L‐NAME) vs. 18.0 ± 1.8% (GSPE alone), P < 0.01]. To determine whether GSPE induced NO production was mediated by the Akt‐eNOS pathway, we utilized the Akt inhibitor API‐2. API‐2 (10 µM) abrogated GSPE‐induced protection [44.3% ± 2.2% (GSPE + API‐2) vs. 27.0% ± 4.3% (GSPE alone), P < 0.01], attenuated the enhanced phosphorylation of Akt at Ser473 in GSPE‐treated cells and attenuated GSPE‐induced NO increases. Simultaneously blocking NOS activation (L‐NAME) and Akt (API‐2) resulted in decreased NO levels similar to using each inhibitor independently. These data suggest that in the context of GSPE stimulation, Akt may help activate eNOS, leading to protective levels of NO. GSPE offers an alternative approach to therapeutic cardioprotection against I/R injury and may offer unique opportunities to improve cardiovascular health by enhancing NO production and increasing Akt‐eNOS signaling. J. Cell. Biochem. 107: 697–705, 2009. © 2009 Wiley‐Liss, Inc.     

Rapamycin impairs trabecular bone acquisition from high‐dose but not low‐dose intermittent parathyroid hormone treatment

The osteo‐anabolic effects of intermittent parathyroid hormone (PTH) treatment require insulin‐like growth factor (IGF) signaling through the IGF‐I receptor. A major downstream target of the IGF‐I receptor (via Akt) is the mammalian target of rapamycin (mTOR), a kinase involved in protein synthesis. We investigated whether the bone‐building effects of intermittent PTH require functional mTOR signaling. Mice were treated with daily PTH 1–34 (0, 10, 30, or 90 µg/kg) for 6 weeks in the presence or absence of rapamycin, a selective inhibitor of mTOR. We found that all PTH doses were effective in enhancing bone mass, whether rapamycin was present or not. Rapamycin had little to no effect on the anabolic response at low (10 µg) PTH doses, small effects in a minority of anabolic measures at moderate doses (30 µg), but the anabolic effects of high‐dose PTH (90 µg) were consistently and significantly suppressed by rapamycin (～4–36% reduction). Serum levels of Trap5b, a marker of resorption, were significantly enhanced by rapamycin, but these effects were observed whether PTH was absent or present. Our data suggest that intermittent PTH, particularly at lower doses, is effective in building bone mass in the presence of rapamycin. However, the full anabolic effects of higher doses of PTH are significantly suppressed by rapamycin, suggesting that PTH might normally activate additional pathways (including mTOR) for its enhanced high‐dose anabolic effects. Clinical doses of intermittent PTH could be an effective treatment for maintaining or increasing bone mass among patients taking rapamycin analogs for unrelated health issues. J. Cell. Physiol. 221: 579–585, 2009. © 2009 Wiley‐Liss, Inc.     

两种骨丢失动物模型骨质,甲状旁腺激素和降钙素的变化比较

本文观察了模拟失重１４天、２１天大鼠和卵巢切除（ＯＶＸ）３０天、６０天大鼠第６胸椎（Ｔ６）、第３腰椎（Ｌ３）和股骨干骨量、骨生物力学特性,血ＰＴＨ和ＣＴ变化特点。实验结果显示,悬吊１４天大鼠Ｔ６和Ｌ３矿盐密度显著增加,２１天Ｔ６矿盐密度显著增加,但Ｌ３矿盐密度显著降低。ＯＶＸ大鼠Ｔ６矿盐密度无变化,但Ｌ３矿盐密度显著降低。悬吊１４天、２１天大鼠股骨干近侧１／３段矿盐含量显著降低,ＯＶＸ６０天大鼠股骨干近侧１／３段矿盐含量显著降低。悬吊大鼠骨力学特性降低较ＯＶＸ大鼠严重。悬吊大鼠血ＰＴＨ无变化,但ＣＴ显著增加,ＯＶＸ大鼠ＰＴＨ、ＣＴ显著降低。实验结果提示,模拟失重大鼠有骨矿盐再分布现象发生,ＯＶＸ大鼠无此现象。悬吊模拟失重大鼠骨质量的降低较ＯＶＸ大鼠严重。在两个动物模型中,皮质骨和松质骨均受影响。     

Bone mineral density in chimpanzees,humans, and Japanese macaques

We performed a comparative study of bone mechanical properties in the radii of chimpanzees (Pan troglodytes), humans (Homo sapiens), and Japanese macaques (Macaca fuscata) using peripheral quantitative computed tomography. We investigated: (1)cortical bone area relative to the total periosteal area (PrA); (2) trabecular bone area relative to PrA; (3) cortical bone density; and (4) trabecular bone density. The cortical bone area index for chimpanzees was almost the same as that of Japanese macaques, whereas the equivalent value in humans was about the two-fifths that of the others. Values for the other three properties were constant among these three catarrhine species. Chimpanzees do not particularly resemble humans, but are more similar to digitigrade macaques in terms of bone properties. The constant trabecular bone area index and trabecular density value in these species may suggest that a certain amount of trabecular bone (20–30% of total bone area at the distal 4% level of the forearm) is necessary to achieve normal bone turnover. The physiological metabolism of bone, including cortical bone density, might be conserved in these catarrhines. Electronic Publication     

PIXE study on the effects of parathyroid hormone on elemental content in rat bones

Parathyroid hormone (PTH) has attracted considerable interest as a bone anabolic agent. PTH plays a central role in regulating calcium phosphate metabolism and its increases in production in response to low serum calcium levels. A continuous hypersecretion of PTH, as occurs in primary hyperparathyroidism, leads to bone resorption. In this study, the effect of different doses of parathyroid hormone (PTH) on bone mineral content (BMC) in rats was investigated by particle-induced X-ray emission (PIXE). This study will help in investigating further the toxicity of extremely high doses of PTH on BMC. For this study, PTH at doses of 15, 45, or 135 μg/kg/day were applied to 9-month-old male and female Sprague Dawley (SD) rats. The concentrations of calcium (Ca), phosphorus (P), strontium (Sr), and zinc (Zn) were measured for bone treatment of PTH. From the results of the research, it was revealed that the biomechanical characteristics of the bone as well as the bone mass were enhanced after the treatment. It was further found that the concentrations of other elements also increased, excluding Zn. This research proved that PTH assists in the treatment of osteoporosis as revealed by the characteristics of different elements. PIXE can be used to determine the concentrations of bone mineral content.