Uridine Triphosphate Inhibits Bone Growth in Rat Cells In Vitro

Abstract

Osteoblast responses to nucleotides increase during differentiation.

Accumulating evidence suggests that extracellular nucleotides, signaling through P2 receptors, play a role in modulating bone cell function. ATP and ADP stimulate osteoclastic resorption, while ATP and UTP are powerful inhibitors of bone formation by osteoblasts. We investigated changes in the expression of P2 receptors with cell differentiation in primary osteoblast cultures. Rat calvarial osteoblasts, cultured for up to 10 days, were loaded with the intracellular Ca(2+)-sensing fluorophore, Fluo-4 AM, and a fluorescence imaging plate reader was used to measure responses to nucleotide agonists. Peak responses occurred within 20 s and were evoked by ATP or UTP at concentrations as low as 2 microM. Osteoblast number doubled between day 4 and 10 of culture, but the peak intracellular Ca(2+) response to ATP or UTP increased up to 6-fold over the same period, indicating that osteoblast responsiveness to nucleotides increases as cell differentiation proceeds. The approximate order of potency for the most active nucleotide agonists at day 8 of culture was ATP > UTP and ATPgammaS > ADP > UDP, consistent with the expression of functional P2Y(2), P2X(2), P2Y(4), P2Y(1) and P2Y(6) receptors. Smaller responses were elicited by 2-MeSATP, Bz-ATP and alpha,beta-meATP, additionally suggesting the presence of functional P2X(1), P2X(3), P2X(5) and P2X(7) receptors. Expression of mRNA for the ATP- and UTP-selective P2Y(2) receptor increased strongly between day 6 and 15 in primary rat osteoblasts, whereas mRNAs for the P2Y(4) (also ATP/UTP selective) and P2Y(6) (UDP/UTP selective) receptors were highly expressed at intermediate time points. In contrast, mRNA for the cell-proliferation-associated P2X(5) receptor decreased to undetectable as osteoblasts matured, but mRNA for the cell-death-associated P2X(7) receptor was detected at all time points. Similar trends were evident using immunostaining and Western blotting for P2 receptors. Exposure to 10 muM ATP or UTP during days 10-14 of culture was sufficient to cause near-total blockade of the ‘trabecular’ bone nodules formed by osteoblasts; however, UDP and ADP were without effect. Our results show that there is a shift from P2X to P2Y expression during differentiation in culture, with mature osteoblasts preferentially expressing the P2Y(2) receptor and to a lesser extent P2Y(4) and P2Y(6) receptors. Taken together, these data suggest that the P2Y(2) receptor, and possibly the P2Y(4) receptor, could function as ‘off-switches’ for mineralized bone formation.

Orriss IR, Knight GE, Ranasinghe S, Burnstock G…
Bone Aug 2006
PMID: 16616882

Abstract

ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling.

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally-occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16-21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2-methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 microM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations >or= 1 microM. ATP was inhibitory at >or= 10 microM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low-dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the ‘universal’ agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states.

Hoebertz A, Mahendran S, Burnstock G, Arnett TR
J. Cell. Biochem. 2002
PMID: 12210747

Abstract

Regulation of the osteogenic and adipogenic differentiation of bone marrow-derived stromal cells by extracellular uridine triphosphate: The role of P2Y2 receptor and ERK1/2 signaling.

An imbalance in the osteogenesis and adipogenesis of bone marrow-derived stromal cells (BMSCs) is a crucial pathological factor in the development of osteoporosis. Growing evidence suggests that extracellular nucleotide signaling involving the P2 receptors plays a significant role in bone metabolism. The aim of the present study was to investigate the effects of uridine triphosphate (UTP) on the osteogenic and adipogenic differentiation of BMSCs, and to elucidate the underlying mechanisms. The differentiation of the BMSCs was determined by measuring the mRNA and protein expression levels of osteogenic- and adipogenic-related markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining. The effects of UTP on BMSC differentiation were assayed using selective P2Y receptor antagonists, small interfering RNA (siRNA) and an intracellular signaling inhibitor. The incubation of the BMSCs with UTP resulted in a dose-dependent decrease in osteogenesis and an increase in adipogenesis, without affecting cell proliferation. Significantly, siRNA targeting the P2Y2 receptor prevented the effects of UTP, whereas the P2Y6 receptor antagonist (MRS2578) and siRNA targeting the P2Y4 receptor had little effect. The activation of P2Y receptors by UTP transduced to the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. This transduction was prevented by the mitogen-activated protein kinase inhibitor (U0126) and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture, as opposed to 3 to 7 days of culture. Thus, our data suggest that UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process.

Li W, Wei S, Liu C, Song M…
Int. J. Mol. Med. Jan 2016
PMID: 26531757 | Free Full Text

High Salt Diet May be a Risk Factor for Osteoporosis in Korean Women

Abstract

Association between Urinary Sodium Excretion and Bone Health in Male and Female Adults.

High salt intake is a well-known risk factor for osteoporosis, but the association between bone mass and urinary sodium excretion has not been studied as yet. This study investigates the hypothesis that urinary sodium excretion is negatively associated with bone mass and the risk of osteoporosis.
This cross-sectional study was performed using data from the Korea National Health and Nutrition Examination Survey, 2008-2011. Participants (n = 16,279) were divided into age groups; men were categorized as younger than 50 years of age or 50 years or greater, women were categorized as pre- or post-menopausal. Multivariate linear regression analysis showed that urinary sodium excretion was negatively associated with bone mineral content (BMC) and bone mineral density (BMD) in premenopausal and postmenopausal women. Sodium excretion was negatively associated with BMC and BMD of the lumbar spine in women with normal bone health, osteopenia and osteoporosis, but there was no association in men. Increased sodium excretion was significantly associated with risk for osteoporosis/osteopenia in premenopausal women. This study demonstrates that urinary sodium excretion is negatively associated with bone health, suggesting that high salt intake could be a possible risk factor for osteoporosis in Korean women, but not in men.

Park Y, Kwon SJ, Ha YC
Ann. Nutr. Metab. 2016
PMID: 26967579

Gastrodin Inhibits Osteoclasts Multiple Ways and Stimulates Mesenchymal Stem Cells In Vitro

Abstract

Gastrodin inhibits osteoclastogenesis via down-regulating the NFATc1 signaling pathway and stimulates osseointegration in vitro.

Bone is a rigid yet dynamic organ, and this dynamism is mediated by the delicate balance between osteoclastic bone resorption and osteoblastic bone formation. However, excessive activation of osteoclasts is responsible for many bone diseases such as osteoporosis, Paget disease, and tumor bone metastasis. Agents that could inhibit osteoclast formation or function are regarded as promising alternatives to treat osteoclast-related diseases. Recently, traditional Chinese medicine has attracted attention because of its multiple activities in bone metabolism. Among them, gastrodin has been reported as an anti-osteoporosis agent that reduces reactive oxygen species. However, the direct action of gastrodin on osteoclast differentiation and bone resorption, and its underlying molecular mechanism, remain unknown. In this study, we investigated the effects of gastrodin on receptor activator NF-κB ligand (RANKL)-activated osteoclasts formation and bone resorption. Our results showed that gastrodin retarded RANKL-induced osteoclast differentiation efficiently by downregulating transcriptional and translational expression of nuclear factor of activated T cells cl (NFATc1), a major factor in RANKL-mediated osteoclastogenesis. Meanwhile, gastrodin prevented osteoclast maturation and migration by inhibiting the gene expression of dendrocyte expressed seven transmembrane protein (DC-STAMP), an osteoclastic-specific gene that controls cells fusion and movement. And gastrodin prevented RANKL-induced osteoclastic bone erosion in vitro. In addition, gastrodin also stimulated bone mesenchymal stem cell (BMSC) spreading and osseointegration in titanium plate. In summary, gastrodin could prevent osteoclasts formation and bone resorption via blockage of NFATc1 activity, and stimulate osseointegration in vitro. Gastrodin could be developed as a potent phytochemical candidate to treat osteolytic diseases.

Zhou F, Shen Y, Liu B, Chen X…
Biochem. Biophys. Res. Commun. Mar 2017
PMID: 28161640

Protein Intake Above the Current RDA May Help Prevent Fractures

Abstract

Dietary Protein Intake above the Current RDA and Bone Health: A Systematic Review and Meta-Analysis.

Dietary intake of protein is fundamental for optimal acquisition and maintenance of bone across all life stages; however, it has been hypothesized that intakes above the current recommended dietary allowance (RDA) might be beneficial for bone health. We utilized the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines when preparing and reporting this systematic review and meta-analysis. A literature search strategy through April 11, 2017, was developed for the following 3 databases: PubMed, Ovid Medline, and Agricola. Included studies were those randomized controlled trials and prospective cohort studies among healthy adults ages 18 and older that examined the relationships between varying doses of protein intake at or above the current U.S. RDA (0.8 g/kg/d or 10%-15% of total caloric intake) from any source on fracture, bone mineral density (BMD)/bone mineral content (BMC), and/or markers of bone turnover. Twenty-nine articles were included for data extraction (16 randomized controlled trials [RCTs] and 13 prospective cohort studies). Meta-analysis of the prospective cohort studies showed high vs low protein intakes resulted in a statistically significant 16% decrease in hip fractures (standardized mean difference [SMD] = 0.84, 95% confidence interval [CI], 0.73, 0.95; I(2) = 36.8%). Data from studies included in these analyses collectively lean toward the hypothesis that protein intake above the current RDA is beneficial to BMD at several sites. This systematic review supports that protein intakes above the current RDA may have some beneficial role in preventing hip fractures and BMD loss. There were no differences between animal or plant proteins, although data in this area were scarce. Larger, long-term, and more well-controlled clinical trials measuring fracture outcomes and BMD are needed to adequately assess whether protein intake above the current RDA is beneficial as a preventative measure and/or intervention strategy for osteoporosis. Key teaching points: Bone health is a multifactorial musculoskeletal issue, and optimal protein intakes are key in developing and maintaining bone throughout the life span. Dietary protein at levels above the current RDA may be beneficial in preventing hip fractures and BMD loss. Plant vs animal proteins do not seem to differ in their ability to prevent bone loss; however, data in this area are scarce. Larger, long-term RCTs using women not using hormone replacement therapy (HRT) are needed to adequately assess the magnitude of impact that protein intakes above the RDA have on preventing bone loss.

Wallace TC, Frankenfeld CL
J Am Coll Nutr Aug 2017
PMID: 28686536

SIRT1 May Grow Bone by Downregulating PPAR Gamma

Abstract

Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells.

Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ-induced activity and expression of adipocyte-specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1-PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of osteoporosis.

Qu B, Ma Y, Yan M, Gong K…
Biochem. Biophys. Res. Commun. Sep 2016
PMID: 27378422

Can Leaky Gut Cause Bone Loss?

Abstract

Epithelial Barrier Function in Gut-Bone Signaling.

The intestinal epithelial barrier plays an essential role in maintaining host homeostasis. The barrier regulates nutrient absorption as well as prevents the invasion of pathogenic bacteria in the host. It is composed of epithelial cells, tight junctions, and a mucus layer. Several factors, such as cytokines, diet, and diseases, can affect this barrier. These factors have been shown to increase intestinal permeability, inflammation, and translocation of pathogenic bacteria. In addition, dysregulation of the epithelial barrier can result in inflammatory diseases such as inflammatory bowel disease. Our lab and others have also shown that barrier disruption can have systemic effects including bone loss. In this chapter, we will discuss the current literature to understand the link between intestinal barrier and bone. We will discuss how inflammation, aging, dysbiosis, and metabolic diseases can affect intestinal barrier-bone link. In addition, we will highlight the current suggested mechanism between intestinal barrier and bone.

Rios-Arce ND, Collins FL, Schepper JD, Steury MD…
Adv. Exp. Med. Biol. 2017
PMID: 29101655

CoQ10 Increases Osteoblasts and Decreases Resorption in Rats

Abstract

Coenzyme Q10 promotes osteoblast proliferation and differentiation and protects against ovariectomy-induced osteoporosis.

Coenzyme Q10 (CoQ10) is a fat‑soluble vitamin‑like substance used for the treatment of a variety of disorders, including osteoporosis. The exact mechanism underlying CoQ10‑mediated protection against osteoporosis remains to be elucidated. The present study aimed to evaluate the effect of CoQ10 on osteoblastic cell proliferation and differentiation, and therapeutic effects on a rat model of osteoporosis. Following treatment with different concentrations of CoQ10, cell proliferation and differentiation of rat bone marrow stromal cells (BMSCs), and expression of osteoblastogenic markers, were measured. Rats with osteoporosis subjected to ovariectomy (OVX) were treated with different concentrations of CoQ10. Serum levels of estrogen and bone metabolism markers were measured. Micro computed tomography scans were used to analyze morphological changes in bones. In addition, mRNA and protein levels of phosphatidylinositol 3,4,5‑trisphosphate 3‑phosphatase and dual‑specificity protein phosphatase PTEN (PTEN)/phosphatidylinositol 4,5‑bisphosphate 3‑kinase (PI3K)/RAC‑alpha serine/threonine‑protein kinase(AKT), were determined. CoQ10 significantly increased the proliferation and osteogenic differentiation of BMSCs in a dose‑dependent manner, with an increased expression of osteogenic markers. CoQ10 significantly decreased bone resorption but exhibited no effect on serum E2 levels in vivo. CoQ10 markedly enhanced bone formation. Furthermore, the abundance of p‑PI3K and p‑AKT increased while PTEN levels decreased in a dose‑dependent manner following administration of CoQ10. CoQ10 stimulates the proliferation and differentiation of BMSCs and is effective for the treatment of OVX‑induced osteoporosis in rats. The above effects of CoQ10 may be mediated by activation of the PTEN/PI3K/AKT pathway.

Zheng D, Cui C, Yu M, Li X…
Mol Med Rep Oct 2017
PMID: 29115467

Green Tea Extract is Bad for Growing Bones in Rats

Abstract

Long-Term Intake of Green Tea Extract Causes Mal-Conformation of Trabecular Bone Microarchitecture in Growing Rats.

The purpose of this study was to examine the effects of green tea extract (GTE) intake on bone structural and physiological properties, such as bone mass, trabecular bone microarchitecture, cortical bone geometry, and bone mechanical strength, in growing rats. Four-week-old male Wistar rats were divided into the following four eenoups: standard diet feeding for 85 days (S-CON) or 170 days (L-CON), and GTE diet feeding for 85 days (S-GTE) or 170 days (L-GTE). At the end of the experiment, in addition to measurement of circulating bone formation/resorption markers, bone mass, trabecular bone microarchitecture, and cortical bone geometry were analyzed in the left femur, and bone mechanical strength of the right femur was measured. There was no difference in all bone parameters between the S-CON and S-GTE groups. On the other hand, the L-GTE group showed the decrease in some trabecular bone mass/microarchitecture parameters and no change in cortical bone mass/geometry parameters compared with the L-CON group, and consequently the reduction in bone weight corrected by body weight. There was no difference in bone formation/resorption markers and bone mechanical strength between the S-CON and S-GTE groups and also between the L-CON and L-GTE groups. However, serum leptin levels were significantly lower in the L-GTE group than in the L-CON group. Thus, the long-term GTE intake had negative effects on bone, especially trabecular bone loss and microarchitecture mal-conformation, in growing rats.

Minematsu A, Nishii Y, Imagita H, Sakata S
Calcif. Tissue Int. Nov 2017
PMID: 29103160

PQQ Stimulates Osteoblastic Bone Formation in Testosterone-Deficient Mice

Abstract

Pyrroloquinoline quinone prevents testosterone deficiency-induced osteoporosis by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption.

Accumulating evidences suggest that oxidative stress caused and deteriorated the aging related osteoporosis and pyrroloquinoline quinone (PQQ) is a powerful antioxidant. However, it is unclear whether PQQ can prevent testosterone deficiency-induced osteoporosis. In this study, the orchidectomized (ORX) mice were supplemented in diet with/without PQQ for 48 weeks, and compared with each other and with sham mice. Results showed that bone mineral density, trabecular bone volume, collagen deposition and osteoblast number were decreased significantly in ORX mice compared with shame mice, whereas PQQ supplementation largely prevented these alterations. In contrast, osteoclast surface and ratio of RANKL and OPG mRNA relative expression levels were increased significantly in ORX mice compared with shame mice, but were decreased significantly by PQQ supplementation. Furthermore, we found that CFU-f and ALP positive CFU-f forming efficiency and the proliferation of mesenchymal stem cells were reduced significantly in ORX mice compared with shame mice, but were increased significantly by PQQ supplementation. Reactive oxygen species (ROS) levels in thymus were increased, antioxidant enzymes SOD-1, SOD-2, Prdx I and Prdx IV protein expression levels in bony tissue were down-regulated, whereas the protein expression levels of DNA damage response related molecules including γ-H2AX, p53, Chk2 and NFκB-p65 in bony tissue were up-regulated significantly in ORX mice compared with shame mice, whereas PQQ supplementation largely rescued these alterations observed in ORX mice. Our results indicate that PQQ supplementation can prevent testosterone deficiency-induced osteoporosis by inhibiting oxidative stress and DNA damage, stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption.

Wu X, Li J, Zhang H, Wang H…
Am J Transl Res 2017
PMID: 28386349 | Free Full Text

PQQ Increases Osteoblasts in Bmi-1 Knockout Mice

Abstract

Effect and mechanism of pyrroloquinoline quinone on anti-osteoporosis in Bmi-1 knockout mice-Anti-oxidant effect of pyrroloquinoline quinone.

Pyrroloquinoline quinone (PQQ), considered as an ROS scavenger,could protect mitochondrial activity from damage of oxidative stress. To determine the role of PQQ supplement in rescuing long bone osteoporosis in Bmi-1(-/-) mice. We fed Bmi-1 knockout mice a diet supplemented with PQQ (BKO+PQQ), BKO mice with normal diet (BKO) and wild type mice with normal diet (WT) as controls. We compared the differences of skeletal phenotype by means of imaging, histopathological and molecular biology methods in three groups of animals. Results showed that BKO+PQQ mice increased morphology of tibia, decreased X-ray transmittance, and increased bone density, thickness of cortical bone, width of growth plate and trabecular bone mass compared with BKO mice. Our study also investigated that, compared mice BKO, PCNA positive cells percentage of tibial growth plate areas significantly increased in BKO+PQQ mice, and TUNEL positive cells percentage was significantly decreased. To detect the effect of PQQ on osteoblast formation of tibiae. Our results showed, compared with BKO mice, osteogenic cell, osteoblast number areas, ALP, Col I and OCN positive areas significantly increased in tibia of BKO+PQQ mice. Further studies showed that supplemental PQQ played a role in anti-osteoporosis by up-regulating antioxidant capacity, inhibiting oxidative stress and reducing DNA damage, down-regulating CDKI proteins levels, and decreasing cell apoptosis. This study not only reveals the mechanism of PQQ supplementation in anti-osteoporosis, but also provides the experimental and theoretical basis for the clinical application of PQQ in osteoporosis.

Huang Y, Chen N, Miao D
Am J Transl Res 2017
PMID: 29118900 | Free Full Text