Pyrroloquinoline quinone alleviates natural aging-related osteoporosis via a novel MCM3-Keap1-Nrf2 axis-mediated stress response and Fbn1 upregulation.

Age-related osteoporosis is associated with increased oxidative stress and cellular senescence. Pyrroloquinoline quinone (PQQ) is a water-soluble vitamin-like compound that has strong antioxidant capacity; however, the effect and underlying mechanism of PQQ on aging-related osteoporosis remain unclear. The purpose of this study was to investigate whether dietary PQQ supplementation can prevent osteoporosis caused by natural aging, and the potential mechanism underlying PQQ antioxidant activity. Here, we found that when 6-month-old or 12-month-old wild-type mice were supplemented with PQQ for 12 months or 6 months, respectively, PQQ could prevent age-related osteoporosis in mice by inhibiting osteoclastic bone resorption and stimulating osteoblastic bone formation. Mechanistically, pharmmapper screening and molecular docking studies revealed that PQQ appears to bind to MCM3 and reduces its ubiquitination-mediated degradation; stabilized MCM3 then competes with Nrf2 for binding to Keap1, thus activating Nrf2-antioxidant response element (ARE) signaling. PQQ-induced Nrf2 activation inhibited bone resorption through increasing stress response capacity and transcriptionally upregulating fibrillin-1 (Fbn1), thus reducing Rankl production in osteoblast-lineage cells and decreasing osteoclast activation; as well, bone formation was stimulated by inhibiting osteoblastic DNA damage and osteocyte senescence. Furthermore, Nrf2 knockout significantly blunted the inhibitory effects of PQQ on oxidative stress, on increased osteoclast activity and on the development of aging-related osteoporosis. This study reveals the underlying mechanism of PQQ's strong antioxidant capacity and provides evidence for PQQ as a potential agent for clinical prevention and treatment of natural aging-induced osteoporosis.

1,25-Dihydroxyvitamin D Deficiency Accelerates Aging-related Osteoarthritis via Downregulation of Sirt1 in Mice.

Emerging observational data suggest that vitamin D deficiency is associated with the onset and progression of knee osteoarthritis (OA). However, the relationship between vitamin D level and OA and the role of vitamin D supplementation in the prevention of knee OA are controversial. To address these issues, we analyzed the articular cartilage phenotype of 6- and 12-month-old wild-type and 1α(OH)ase-/- mice and found that 1,25(OH)2D deficiency accelerated the development of age-related spontaneous knee OA, including cartilage surface destruction, cartilage erosion, proteoglycan loss and cytopenia, increased OARSI score, collagen X and Mmp13 positive chondrocytes, and increased chondrocyte senescence with senescence-associated secretory phenotype (SASP). 1,25(OH)2D3 supplementation rescued all knee OA phenotypes of 1α(OH)ase-/- mice in vivo, and 1,25(OH)2D3 rescued IL-1ß-induced chondrocyte OA phenotypes in vitro, including decreased chondrocyte proliferation and cartilage matrix protein synthesis, and increased oxidative stress and cell senescence. We also demonstrated that VDR was expressed in mouse articular chondrocytes, and that VDR knockout mice exhibited knee OA phenotypes. Furthermore, we demonstrated that the down-regulation of Sirt1 in articular chondrocytes of 1α(OH)ase-/- mice was corrected by supplementing 1,25(OH)2D3 or overexpression of Sirt1 in mesenchymal stem cells (MSCs) and 1,25(OH)2D3 up-regulated Sirt1 through VDR mediated transcription. Finally, we demonstrated that overexpression of Sirt1 in MSCs rescued knee OA phenotypes in 1α(OH)ase-/- mice. Thus, we conclude that 1,25(OH)2D3, via VDR-mediated gene transcription, plays a key role in preventing the onset of aging-related knee OA in mouse models by up-regulating Sirt1, an aging-related gene that promotes articular chondrocyte proliferation and extracellular matrix protein synthesis, and inhibits senescence and SASP.

Mechanisms of action of vitamin D in delaying aging and preventing disease by inhibiting oxidative stress.

Although several recent studies have shown that vitamin D supplementation beneficially decreases oxidative stress parameters, there is no consensus on this subject in humans. Thus the role of vitamin D supplementation has recently become a controversial topic because large intervention studies in humans have not shown significant benefits. These studies have indicated that supplementation with precursor forms of active vitamin D has no effect on all-cause mortality, cannot reduce the fracture risk of the elderly, cannot reduce the incidence of cancer or cardiovascular disease in the elderly, and cannot significantly reduce the incidence risk of diabetes in the elderly. However, a link between several age-related diseases and enhanced oxidative stress has been found in mice with insufficient or deficient 1,25-dihydroxyvitamin D (1,25(OH)2D), the active form of vitamin D, which indicates that reduced active vitamin D accelerates aging and age-related diseases by increasing oxidative stress. Furthermore, supplementation of exogenous 1,25(OH)2D3, or antioxidants, could dramatically postpone aging, prevent osteoporosis and spontaneous tumor development induced by 1,25(OH)2D insufficiency or deficiency, by inhibiting oxidative stress. Mechanistically, the antioxidative effects of 1,25(OH)2D3 are carried out via the vitamin D receptor (VDR) by activation of the Nrf2 oxidative stress response pathway though transcriptional or posttranscriptional activation of Nrf2 or transcriptional upregulation of Sirt1 and Bmi1 expression. Whether discrepancies between studies in humans and in mice reflect the different forms of vitamin D examined remains to be determined.

Variation in bone mineral density and fractures over 20 years among Canadians: a comparison of the Canadian Multicenter Osteoporosis Study and the Canadian Longitudinal Study on Aging.

International variations in osteoporosis and fracture rates have been reported, with temporal trends differing between populations. We observed higher BMD and lower fracture prevalence in a recently recruited cohort compared to that of a cohort recruited 20 years ago, even after adjusting for multiple covariates. PURPOSE: We explored sex-specific differences in femoral neck bone mineral density (FN-BMD) and in prevalent major osteoporotic fractures (MOF) using two Canadian cohorts recruited 20 years apart. METHODS: We included men and women aged 50-85 years from the Canadian Multicentre Osteoporosis Study (CaMos, N = 6,479; 1995-1997) and the Canadian Longitudinal Study on Aging (CLSA, N = 19,534; 2012-2015). We created regression models to compare FN-BMD and fracture risk between cohorts, adjusting for important covariates. Among participants with prevalent MOF, we compared anti-osteoporosis medication use. RESULTS: Mean (SD) age in CaMos (65.4 years [8.6]) was higher than in CLSA (63.8 years [9.1]). CaMos participants had lower mean body mass index and higher prevalence of smoking (p < 0.001). Adjusted linear regression models (estimates [95%CI]) demonstrated lower FN-BMD in CaMos women (- 0.017 g/cm2 [- 0.021; - 0.014]) and men (- 0.006 g/cm2 [- 0.011; 0.000]), while adjusted odds ratios (95%CI) for prevalent MOF were higher in CaMos women (1.99 [1.71; 2.30]) and men (2.33 [1.82; 3.00]) compared to CLSA. In women with prevalent MOF, menopausal hormone therapy use was similar in both cohorts (43.3% vs 37.9%, p = 0.076), but supplements (32.0% vs 48.3%, p < 0.001) and bisphosphonate use (5.8% vs 17.3%, p < 0.001) were lower in CaMos. The proportion of men with MOF who received bisphosphonates was below 10% in both cohorts. CONCLUSION: Higher BMD and lower fracture prevalence were noted in the more recently recruited CLSA cohort compared to CaMos, even after adjusting for multiple covariates. We noted an increase in bisphosphonate use in the recent cohort, but it remained very low in men.

Inhibition of Nrf2 degradation alleviates age-related osteoporosis induced by 1,25-Dihydroxyvitamin D deficiency.

Previous studies have shown that 1,25(OH)2D plays an anti-osteoporosis role by an anti-aging mechanism. Oxidative stress is a key mediator of aging and bone loss; however, whether 1,25(OH)2D can exert its anti-osteoporosis effect by inhibiting oxidative stress is unclear. In this study, osteoporosis and the bone aging phenotype induced by 1,25(OH)2D deficiency in male mice were significantly rescued in vivo upon the supplementation of oltipraz, an inhibitor of Nrf2 degradation. Increased oxidative stress, cellular senescence and reduced osteogenesis of BM-MSCs from VDR knockout mice were also significantly rescued when the cells were pre-treated with oltipraz. We found that 1,25(OH)2D3 promoted Nrf2 accumulation by inhibiting its ubiquitin-proteasome degradation, thus facilitating Nrf2 activation of its transcriptional targets. Mechanistically, 1,25(OH)2D3 enhances VDR-mediated recruitment of Ezh2 and facilitation of H3K27me3 action at the promoter region of Keap1, thus transcriptionally repressing Keap1. To further validate that the Nrf2-Keap1 pathway serves as the key mediator in the anabolic effect of 1,25(OH)2D3 on bone, Nrf2-/- mice, or hBM-MSCs with shRNA-mediated Nrf2-knockdown, were treated with 1,25(OH)2D3; we found that Nrf2 knockout largely blocked the bone anabolic effect of 1,25(OH)2D3 in vivo and ex vivo, and Nrf2 knockdown in hBM-MSCs markedly blocked the role of 1,25(OH)2D3 in inhibiting oxidative stress and promoting osteogenic differentiation and bone formation. This study provides insight into the mechanism whereby 1,25(OH)2D3 postpones age-related osteoporosis via VDR-mediated activation of Nrf2-antioxidant signaling and inhibition of oxidative stress, and thus provides evidence for oltipraz as a potential reagent for clinical prevention and treatment of age-related osteoporosis.

SIRT1/FOXO3a axis plays an important role in the prevention of mandibular bone loss induced by 1,25(OH)2D deficiency.

It has been reported that 1,25 dihydroxyvitamin D [1,25(OH)2D] deficiency leads to the loss of mandibular bone, however the mechanism is unclear. We investigated whether the Sirt1/FOXO3a signaling pathway is involved in this process. Using a 1,25(OH)2D deficiency model induced by genetic deletion in mice of 25-hydroxyvitamin D-1α hydroxylase [1α(OH)ase-/- mice]. We first documented a sharp reduction of expression levels of Sirt1 in the 1α(OH)ase-/- mice in vivo. Next, we demonstrated dose-dependent upregulation of Sirt1 by treatment with exogenous 1,25(OH)2D3in vitro. We then identified a functional VDR binding site in the Sirt1 promoter. By crossing Prx1-Sirt1 transgenic mice with 1α(OH)ase-/- mice we demonstrated that the overexpression of Sirt1 in mesenchymal stem cells (MSCs) greatly improved the 1α(OH)ase-/- mandibular bone loss phenotype by increasing osteoblastic bone formation and reducing osteoclastic bone resorption. In mechanistic studies, we showed, in 1α(OH)ase-/- mice, decreases of Sirt1 and FoxO3a, an increase in oxidative stress as reflected by a reduction of the antioxidant enzymes peroxiredoxin1 (Prdx1), SOD1 and SOD2 expression, and an increase of markers for osteocyte senescence and senescence associated secretory phenotypes (SASP), including ß-galactosidase (ß-gal), p16, p53 and p21. The targeted overexpression of Sirt1 in the 1α(OH)ase-/- mice restored the expression levels of these molecules. Finally, we demonstrated that a Sirt1 agonist can upregulate FOXO3a activity by increasing deacetylation and nuclear translocation. Overall, results from this study support the concept that targeted increases in Sirt1/FOXO3a signaling levels can greatly improve the bone loss caused by 1,25(OH)2D deficiency.

1,25-Dihydroxyvitamin D protects against age-related osteoporosis by a novel VDR-Ezh2-p16 signal axis.

To determine whether 1,25-dihydroxyvitamin D (1,25(OH)2 D) can exert an anti-osteoporosis role through anti-aging mechanisms, we analyzed the bone phenotype of mice with 1,25(OH)2 D deficiency due to deletion of the enzyme, 25-hydroxyvitamin D 1α-hydroxylase, while on a rescue diet. 1,25(OH)2 D deficiency accelerated age-related bone loss by activating the p16/p19 senescence signaling pathway, inhibiting osteoblastic bone formation, and stimulating osteoclastic bone resorption, osteocyte senescence, and senescence-associated secretory phenotype (SASP). Supplementation of exogenous 1,25(OH)2 D3 corrected the osteoporotic phenotype caused by 1,25(OH)2 D deficiency or natural aging by inhibiting the p16/p19 pathway. The proliferation, osteogenic differentiation, and ectopic bone formation of bone marrow mesenchymal stem cells derived from mice with genetically induced deficiency of the vitamin D receptor (VDR) were significantly reduced by mechanisms including increased oxidative stress, DNA damage, and cellular senescence. We also demonstrated that p16 deletion largely rescued the osteoporotic phenotype caused by 1,25(OH)2 D3 deficiency, whereas 1,25(OH)2 D3 could up-regulate the enzyme Ezh2 via VDR-mediated transcription thereby enriching H3K27me3 and repressing p16/p19 transcription. Finally, we demonstrated that treatment with 1,25(OH)2 D3 improved the osteogenic defects of human BM-MSCs caused by repeated passages by stimulating their proliferation and inhibiting their senescence via the VDR-Ezh2-p16 axis. The results of this study therefore indicate that 1,25(OH)2 D3 plays a role in preventing age-related osteoporosis by up-regulating Ezh2 via VDR-mediated transcription, increasing H3K27me3 and repressing p16 transcription, thus promoting the proliferation and osteogenesis of BM-MSCs and inhibiting their senescence, while also stimulating osteoblastic bone formation, and inhibiting osteocyte senescence, SASP, and osteoclastic bone resorption.

Endocrine aryl hydrocarbon receptor signaling is induced by moderate cutaneous exposure to ultraviolet light.

Links between solar UV exposure and immunity date back to the ancient Greeks with the development of heliotherapy. Skin contains several UV-sensitive chromophores and exposure to sunlight can produce molecules, such as vitamin D3, that act in an endocrine manner. We investigated the role of the aryl hydrocarbon receptor (AHR), an environmental sensor and ligand-regulated transcription factor activated by numerous planar compounds of endogenous, dietary or environmental origin. 15- to 30-minute exposure of cells to a minimal erythemal dose of UVB irradiation in vitro induced translocation of the AHR to the nucleus, rapidly inducing site-specific DNA binding and target gene regulation. Importantly, ex vivo studies with Ahr wild-type or null fibroblasts showed that serum from mice whose skin was exposed to a 15 min UVB dose, but not control serum, contained agonist activity within 30 min of UV irradiation, inducing AHR-dependent gene expression. Moreover, a 15-min cutaneous UVB exposure induced AHR site-specific DNA binding and target gene regulation in vivo within 3-6 hr post-irradiation in blood and in peripheral tissues, including intestine. These results show that cutaneous exposure of mice to a single minimal erythemic dose of UVB induces rapid AHR signaling in multiple peripheral organs, providing compelling evidence that moderate sun exposure can exert endocrine control of immunity through the AHR.

1,25-Dihydroxyvitamin D exerts an antiaging role by activation of Nrf2-antioxidant signaling and inactivation of p16/p53-senescence signaling.

We tested the hypothesis that 1,25-dihydroxyvitamin D3 [1α,25(OH)2 D3 ] has antiaging effects via upregulating nuclear factor (erythroid-derived 2)-like 2 (Nrf2), reducing reactive oxygen species (ROS), decreasing DNA damage, reducing p16/Rb and p53/p21 signaling, increasing cell proliferation, and reducing cellular senescence and the senescence-associated secretory phenotype (SASP). We demonstrated that 1,25(OH)2 D3 -deficient [1α(OH)ase-/- ] mice survived on average for only 3 months. Increased tissue oxidative stress and DNA damage, downregulated Bmi1 and upregulated p16, p53 and p21 expression levels, reduced cell proliferation, and induced cell senescence and the senescence-associated secretory phenotype (SASP) were observed. Supplementation of 1α(OH)ase-/- mice with dietary calcium and phosphate, which normalized serum calcium and phosphorus, prolonged their average lifespan to more than 8 months with reduced oxidative stress and cellular senescence and SASP. However, supplementation with exogenous 1,25(OH)2 D3 or with combined calcium/phosphate and the antioxidant N-acetyl-l-cysteine prolonged their average lifespan to more than 16 months and nearly 14 months, respectively, largely rescuing the aging phenotypes. We demonstrated that 1,25(OH)2 D3 exerted an antioxidant role by transcriptional regulation of Nrf2 via the vitamin D receptor (VDR). Homozygous ablation of p16 or heterozygous ablation of p53 prolonged the average lifespan of 1α(OH)ase-/- mice on the normal diet from 3 to 6 months by enhancing cell proliferative ability and reducing cell senescence or apoptosis. This study suggests that 1,25(OH)2 D3 plays a role in delaying aging by upregulating Nrf2, inhibiting oxidative stress and DNA damage，inactivating p53-p21 and p16-Rb signaling pathways, and inhibiting cell senescence and SASP.

1,25-dihydroxyvitamin D deficiency accelerates alveolar bone loss independent of aging and extracellular calcium and phosphorus.

BACKGROUND: Vitamin D is critical for bone homeostasis and immunomodulation. We therefore assessed whether 1,25-dihydroxyvitamin D (1,25(OH)2 D) deficiency in mice with targeted deletion of the gene encoding 25-hydroxyvitamin D-1α-hydroxylase (1α(OH)ase [1αOH)ase-/- mice]) results in alveolar bone loss and periodontal inflammation in vivo. METHODS: Ten-week-old and 12-month-old 1α(OH)ase-/- mice and wild-type littermates were fed a normal diet or a rescue diet, and the phenotype of the periodontium was then analyzed using microcomputed tomography, histology, immunohistochemistry, and real-time Reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Alveolar bone loss was increased and maxillary bone mineral density (BMD), osteoblast numbers, and the number of osterix-positive cells were decreased significantly in 1α(OH)ase-/- mice compared with wild-type mice. Although aging from 10 weeks to 12 months accentuated these changes, and a rescue diet reduced them, the alterations in the 1α(OH)ase-/- mice exceeded the effects of aging and diet change. Nuclear factor kappa light-chain-enhancer of activated B cells (NF-кB) p65 and CD3 positive cells, and the gene expression levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, matrix metalloproteinase (MMP)-3 and -8 were all increased significantly in periodontal tissues of 1α(OH)ase-/- mice compared with wild-type mice. Aging from 10 weeks to 12 months also accentuated these changes, and a rescue diet reduced them, however, the alterations in the 1α(OH)ase-/- mice exceeded the effects of aging and diet change. CONCLUSION: 1,25(OH)2 D deficiency in the 1α(OH)ase-/- mice accelerated alveolar bone loss by inhibiting osteoblastic bone formation and enhancing periodontal tissue degeneration in a calcium- and phosphorus- as well as an age-independent manner.

1,25-Dihydroxy vitamin D prevents tumorigenesis by inhibiting oxidative stress and inducing tumor cellular senescence in mice.

Human epidemiological studies suggest that 1,25(OH)2 D3 deficiency might increase cancer incidence, but no spontaneous tumors have been reported in mice lacking 1,25(OH)2 D3 or deficient in its receptor. In our study, we detected, for the first time, diverse types of spontaneous tumors in l,25(OH)2 D3 deficient mice more than 1 year of age. This was associated with increased oxidative stress, cellular senescence and senescence-associated secretory phenotype molecules, such as hepatocyte growth factor, mediated via its receptor c-Met. Furthermore, 1,25(OH)2 D3 prevented spontaneous tumor development. We also demonstrated that l,25(OH)2 D3 deficiency accelerates allograft tumor initiation and growth by increasing oxidative stress and DNA damage, activating oncogenes, inactivating tumor suppressor genes, stimulating malignant cell proliferation and inhibiting their senescence; in contrast, supplementation with exogenous l,25(OH)2 D3 or antioxidant, or knock-down of the Bmi1 or c-Met oncogene, largely rescued the phenotypes of allograft tumors. Results from our study suggest that 1,25(OH)2 D3 deficiency enhances tumorigenesis by increasing malignant cell oxidative stress and DNA damage, stimulating microenvironmental cell senescence and a senescence-associated secretory phenotype, and activating oncogenes and inactivating tumor suppressor genes, thus increasing malignant cell proliferation. Our study provides direct evidence supporting the role of vitamin D deficiency in increasing cancer incidence. Conversely, 1,25(OH)2 D3 prevented spontaneous tumor development, suggesting that this inhibitory effect prevents the initiation and progression of tumorigenesis, thus provides a mechanistic basis for 1,25(OH)2 D3 to prevent tumorigenesis in an aging organism.

Feasibility of a clinical trial to assess the effect of dietary calcium v. supplemental calcium on vascular and bone markers in healthy postmenopausal women.

Whether supplemental Ca has similar effects to dietary Ca on vascular and bone markers is unknown. The present trial investigated the feasibility of applying dietary and supplemental interventions in a randomised-controlled trial (RCT) aiming to estimate the effect of supplemental Ca as compared with dietary Ca on vascular and bone markers in postmenopausal women. In total, thirteen participants were randomised to a Ca supplement group (CaSuppl) (750 mg Ca from CaCO3+450 mg Ca from food+20 µg vitamin D supplement) or a Ca diet group (CaDiet) (1200 mg Ca from food+10 µg vitamin D supplement). Participants were instructed on Ca consumption targets at baseline. Monthly telephone follow-ups were conducted to assess adherence to interventions (±20 % of target total Ca) using the multiple-pass 24-h recall method and reported pill count. Measurements of arterial stiffness, peripheral blood pressure and body composition were performed at baseline and after 6 and 12 months in all participants who completed the trial (n 9). Blood and serum biomarkers were measured at baseline and at 12 months. Both groups were compliant to trial interventions (±20 % of target total Ca intake; pill count ≥80 %). CaSuppl participants maintained a significantly lower average dietary Ca intake compared with CaDiet participants throughout the trial (453 (sd 187) mg/d v. 1241 (sd 319) mg/d; P<0·001). There were no significant differences in selected vascular outcomes between intervention groups over time. Our pilot trial demonstrated the feasibility of conducting a large-scale RCT to estimate the differential effects of supplemental and dietary Ca on vascular and bone health markers in healthy postmenopausal women.

CYP24 inhibition as a therapeutic target in FGF23-mediated renal phosphate wasting disorders.

CYP24A1 (hereafter referred to as CYP24) enzymatic activity is pivotal in the inactivation of vitamin D metabolites. Basal renal and extrarenal CYP24 is usually low but is highly induced by its substrate 1,25-dihydroxyvitamin D. Unbalanced high and/or long-lasting CYP24 expression has been proposed to underlie diseases like chronic kidney disease, cancers, and psoriasis that otherwise should favorably respond to supplemental vitamin D. Using genetically modified mice, we have shown that renal phosphate wasting hypophosphatemic states arising from high levels of fibroblast growth factor 23 (FGF23) are also associated with increased renal Cyp24 expression, suggesting that elevated CYP24 activity is pivotal to the pathophysiology of these disorders. We therefore crossed 2 mouse strains, each with distinct etiology for high levels of circulating FGF23, onto a Cyp24-null background. Specifically, we evaluated Cyp24 deficiency in Hyp mice, the murine homolog of X-linked dominant hypophosphatemic rickets, and transgenic mice that overexpress a mutant FGF23 (FGF23R176Q) that is associated with the autosomal dominant form of hypophosphatemic rickets. Loss of Cyp24 in these murine models of human disease resulted in near-complete recovery of rachitic/osteomalacic bony abnormalities in the absence of any improvement in the serum biochemical profile. Moreover, treatment of Hyp and FGF23R1760-transgenic mice with the CYP24 inhibitor CTA102 also ameliorated their rachitic bones. Our results link CYP24 activity to the pathophysiology of FGF23-dependent renal phosphate wasting states and implicate pharmacologic CYP24 inhibition as a therapeutic adjunct for their treatment.

Administration of exogenous 1,25(OH)2D3 normalizes overactivation of the central renin-angiotensin system in 1α(OH)ase knockout mice.

Previously, we reported that active vitamin D deficiency in mice causes secondary hypertension and cardiac dysfunction, but the underlying mechanism remains largely unknown. To clarify whether exogenous active vitamin D rescues hypertension by normalizing the altered central renin-angiotensin system (RAS) via an antioxidative stress mechanism, 1-alpha-hydroxylase [1α(OH)ase] knockout mice [1α(OH)ase(-/-)] and their wild-type littermates were fed a normal diet alone or with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or a high-calcium, high-phosphorus "rescue" diet with or without antioxidant N-acetyl-l-cysteine (NAC) supplementation for 4 weeks. Compared with their wild-type littermates, 1α(OH)ase(-/-)mice had high mean arterial pressure, increased levels of renin, angiotensin II (Ang II), and Ang II type 1 receptor, and increased malondialdehyde levels, but decreased anti-peroxiredoxin I and IV proteins and the antioxidative genes glutathione reductase (Gsr) and glutathione peroxidase 4 (Gpx4) in the brain samples. Except Ang II type 1 receptor, these pathophysiological changes were rescued by exogenous 1,25(OH)2D3 or NAC plus rescue diet, but not by rescue diet alone. We conclude that 1,25(OH)2D3 normalizes the altered central RAS in 1α(OH)ase(-/-)mice, at least partially, through a central antioxidative mechanism.

Active vitamin D deficiency mediated by extracellular calcium and phosphorus results in male infertility in young mice.

We used mice with targeted deletion of 25-hydroxyvitamin D-1 α-hydroxylase [1α(OH)ase(-/-)] to investigate whether 1,25(OH)2D3 deficiency results in male infertility mediated by 1,25(OH)2D3 or extracellular calcium and phosphorus. Male 1α(OH)ase(-/-) and their wild-type littermates fed either a normal diet or a rescue diet from weaning were mated at 6-14 wk of age with female wild-type mice on the same diet. The fertility efficiency of females was analyzed, and the reproductive phenotypes of males were evaluated by histopathological and molecular techniques. Hypocalcemic and hypophosphatemic male 1α(OH)ase(-/-) mice on a normal diet developed infertility characterized by hypergonadotropic hypogonadism, with downregulation of testicular calcium channels, lower intracellular calcium levels, decreased sperm count and motility, and histological abnormalities of the testes. The proliferation of spermatogenic cells was decreased with downregulation of cyclin E and CDK2 and upregulation of p53 and p21 expression, whereas apoptosis of spermatogenic cells was increased with upregulation of Bax and p-caspase 3 expression and downregulation of Bcl-xl expression. When serum calcium and phosphorus were normalized by the rescue diet, the defective reproductive phenotype in the male 1α(OH)ase(-/-) mice, including the hypergonadotropic hypogonadism, decreased sperm count and motility, histological abnormalities of testis, and defective spermatogenesis, was reversed. These results indicate that the infertility seen in male 1,25(OH)2D3-deficient mice is not a direct effect of active vitamin D deficiency on the reproductive system but is an indirect effect mediated by extracellular calcium and phosphorus.

Calcium and vitamin D intake and mortality: results from the Canadian Multicentre Osteoporosis Study (CaMos).

CONTEXT: Calcium and vitamin D are recommended for bone health, but there are concerns about adverse risks. Some clinical studies suggest that calcium intake may be cardioprotective, whereas others report increased risk associated with calcium supplements. Both low and high serum levels of 25-hydroxyvitamin D have been associated with increased mortality. OBJECTIVE: The purpose of this study was to determine the association between total calcium and vitamin D intake and mortality and heterogeneity by source of intake. DESIGN: The Canadian Multicentre Osteoporosis Study cohort is a population-based longitudinal cohort with a 10-year follow-up (1995-2007). SETTING: This study included randomly selected community-dwelling men and women. PARTICIPANTS: A total of 9033 participants with nonmissing calcium and vitamin D intake data and follow-up were studied. EXPOSURE: Total calcium intake (dairy, nondairy food, and supplements) and total vitamin D intake (milk, yogurt, and supplements) were recorded. OUTCOME: The outcome variable was all-cause mortality. RESULTS: There were 1160 deaths during the 10-year period. For women only, we found a possible benefit of higher total calcium intake, with a hazard ratio of 0.95 (95% confidence interval, 0.89-1.01) per 500-mg increase in daily calcium intake and no evidence of heterogeneity by source; use of calcium supplements was also associated with reduced mortality, with hazard ratio of 0.78 (95% confidence interval, 0.66-0.92) for users vs nonusers with statistically significant reductions remaining among those with doses up to 1000 mg/d. These associations were not modified by levels of concurrent vitamin D intake. No definitive associations were found among men. CONCLUSIONS: Calcium supplements, up to 1000 mg/d, and increased dietary intake of calcium may be associated with reduced risk of mortality in women. We found no evidence of mortality benefit or harm associated with vitamin D intake.

Meta-analysis of genome-wide studies identifies MEF2C SNPs associated with bone mineral density at forearm.

BACKGROUND: Forearm fractures affect 1.7 million individuals worldwide each year and most occur earlier in life than hip fractures. While the heritability of forearm bone mineral density (BMD) and fracture is high, their genetic determinants are largely unknown. AIM: To identify genetic variants associated with forearm BMD and forearm fractures. METHODS: BMD at distal radius, measured by dual-energy x-ray absorptiometry, was tested for association with common genetic variants. We conducted a meta-analysis of genome-wide association studies for BMD in 5866 subjects of European descent and then selected the variants for replication in 715 Mexican American samples. Gene-based association was carried out to supplement the single-nucleotide polymorphism (SNP) association test. We then tested the BMD-associated SNPs for association with forearm fracture in 2023 cases and 3740 controls. RESULTS: We found that five SNPs in the introns of MEF2C were associated with forearm BMD at a genome-wide significance level (p<5×10(-8)) in meta-analysis (lead SNP, rs11951031[T] -0.20 SDs per allele, p=9.01×10(-9)). The gene-based association test suggested an association between MEF2C and forearm BMD (p=0.003). The association between MEF2C variants and risk of fracture did not achieve statistical significance (SNP rs12521522[A]: OR=1.14 (95% CI 0.92 to 1.35), p=0.14). Meta-analysis also revealed two genome-wide suggestive loci at CTNNA2 and 6q23.2. CONCLUSIONS: These findings demonstrate that variants at MEF2C were associated with forearm BMD, implicating this gene in the determination of BMD at forearm.

CaSR-mediated interactions between calcium and magnesium homeostasis in mice.

Calcium (Ca) and magnesium (Mg) homeostasis are interrelated and share common regulatory hormones, including parathyroid hormone (PTH) and vitamin D. However, the role of the calcium-sensing receptor (CaSR) in Mg homeostasis in vivo is not well understood. We sought to investigate the interactions between Mg and Ca homeostasis using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR) (double knockout, DKO). Serum Mg is lower in PTH KO and DKO mice than in WT mice on standard chow, whereas supplemental dietary Ca leads to equivalent Mg levels for all three genotypes. Mg loading increases serum Mg in all genotypes; however, the increase in serum Mg is most pronounced in the DKO mice. Serum Ca is increased with Mg loading in the PTH KO and DKO mice but not in the WT mice. Here, too, the hypercalcemia is much greater in the DKO mice. Serum and especially urinary phosphate are reduced during Mg loading, which is likely due to intestinal chelation of phosphate by Mg. Mg loading decreases serum PTH in WT mice and increases serum calcitonin in both WT and PTH KO mice but not DKO mice. Furthermore, Mg loading elevates serum 1,25-dihydroxyvitamin D in all genotypes, with greater effects in PTH KO and DKO mice, possibly due to reduced levels of serum phosphorus and FGF23. These hormonal responses to Mg loading and the CaSR's role in regulating renal function may help to explain changes in serum Mg and Ca found during Mg loading.

Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo.

Calcium and phosphorus homeostasis are highly interrelated and share common regulatory hormones, including FGF23. However, little is known about calcium's role in the regulation of FGF23. We sought to investigate the regulatory roles of calcium and phosphorus in FGF23 production using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR; PTH-CaSR DKO). In wild-type, PTH KO, and PTH-CaSR DKO mice, elevation of either serum calcium or phosphorus by intraperitoneal injection increased serum FGF23 levels. In PTH KO and PTH-CaSR DKO mice, however, increases in serum phosphorus by dietary manipulation were accompanied by severe hypocalcemia, which appeared to blunt stimulation of FGF23 release. Increases in dietary phosphorus in PTH-CaSR DKO mice markedly decreased serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] despite no change in FGF23, suggesting direct regulation of 1,25(OH)(2)D(3) synthesis by serum phosphorus. Calcium-mediated increases in serum FGF23 required a threshold level of serum phosphorus of about 5 mg/dl. Analogously, phosphorus-elicited increases in FGF23 were markedly blunted if serum calcium was less than 8 mg/dl. The best correlation between calcium and phosphorus and serum FGF23 was found between FGF23 and the calcium × phosphorus product. Since calcium stimulated FGF23 production in the PTH-CaSR DKO mice, this effect cannot be mediated by the full-length CaSR. Thus the regulation of FGF23 by both calcium and phosphorus appears to be fundamentally important in coordinating the serum levels of both mineral ions and ensuring that the calcium × phosphorus product remains within a physiological range.

Bone marrow ablation demonstrates that excess endogenous parathyroid hormone plays distinct roles in trabecular and cortical bone.

Mice null for Cyp27b1, which encodes the 25-hydroxyvitamin D-1α-hydroxylase [1α(OH)ase(-/-) mice], lack 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and have hypocalcemia and high parathyroid hormone (PTH) secretion. Intermittent, exogenous PTH is anabolic for bone. To determine the effect of the chronic excess endogenous PTH on osteogenesis and bone turnover, bone marrow ablations (BMX) were performed in tibiae and femurs of 6-week-old 1α(OH)ase(-/-) mice and in wild-type (WT) controls. Newly formed bone tissue was analyzed at 1, 2, and 3 weeks after BMX. BMX did not alter the higher levels of PTH in 1α(OH)ase(-/-) mice. In the marrow cavity, trabecular volume, osteoblast number, alkaline phosphatase-positive areas, type I collagen-positive areas, bone formation-related genes, and protein expression levels all increased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. Osteoclast numbers and surface and ratio of RANKL/OPG-relative mRNA levels decreased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. In the cortex, alkaline phosphatase-positive osteoblasts and osteoclast numbers increased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. These results demonstrate that chronic excess endogenous PTH exerts an anabolic role in trabecular bone by stimulating osteogenic cells and reducing bone resorption, but plays a catabolic role in cortical bone by enhancing bone turnover with an increase in resorption.