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Sample records for 25-dihydroxyvitamin d3-induced osteoclastogenesis

  1. Downregulation of Runx2 by 1,25-Dihydroxyvitamin D3 Induces the Transdifferentiation of Osteoblasts to Adipocytes

    PubMed Central

    Kim, Jung Ha; Seong, Semun; Kim, Kabsun; Kim, Inyoung; Jeong, Byung-Chul; Kim, Nacksung

    2016-01-01

    1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)2D3 enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)2D3 in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)2D3 enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)2D3 were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)2D3 induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts. PMID:27213351

  2. RUNX2 Mutation Impairs 1α,25-Dihydroxyvitamin D3 mediated Osteoclastogenesis in Dental Follicle Cells

    PubMed Central

    Wang, X. Z.; Sun, X. Y.; Zhang, C. Y.; Yang, X.; Yan, W. J.; Ge, L. H.; Zheng, S. G.

    2016-01-01

    Cleidocranial dysplasia (CCD), a skeletal disorder characterized by delayed permanent tooth eruption and other dental abnormalities, is caused by heterozygous RUNX2 mutations. As an osteoblast-specific transcription factor, RUNX2 plays a role in bone remodeling, tooth formation and tooth eruption. To investigate the crosstalk between RUNX2 and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) in human dental follicle cells (hDFCs) during osteoclast formation, we established a co-culture system of hDFCs from CCD patient and healthy donors with peripheral blood mononuclear cells (PBMCs). Expression of the osteoclast-associated genes and the number of TRAP+ cells were reduced in CCD hDFCs, indicating its suppressed osteoclast-inductive ability, which was reflected by the downregulated RANKL/OPG ratio. In addition, 1α,25-(OH)2D3-stimulation elevated the expression of osteoclast-related genes, as well as RANKL mRNA levels and RANKL/OPG ratios in control hDFCs. Conversely, RUNX2 mutation abolished this 1α,25-(OH)2D3-induced RANKL gene activation and osteoclast formation in CCD hDFCs. Therefore, RUNX2 haploinsufficiency impairs dental follicle-induced osteoclast formation capacity through RANKL/OPG signaling, which may be partially responsible for delayed permanent tooth eruption in CCD patients. Furthermore, this abnormality was not rescued by 1α,25-(OH)2D3 application because 1α,25-(OH)2D3-induced RANKL activation in hDFCs is mediated principally via the RUNX2-dependent pathway. PMID:27068678

  3. The impact of methylmercury on 1,25-dihydroxyvitamin D3-induced transcriptomic responses in dolphin skin cells.

    PubMed

    Ellis, Blake C; Gattoni-Celli, Sebastiano; Kindy, Mark S

    2010-01-01

    The Atlantic bottlenose dolphin has been the focus of much attention owing to the considerable impact of environmental stress on its health and the associated implications for human health. Here, we used skin cells from the dolphin to investigate the protective role of the vitamin D pathway against environmental stressors. We previously reported that dolphin skin cells respond to 1,25-dihydroxyvitamin D3 (1,25D3), the bioactive metabolite of vitamin D3, by upregulation of the vitamin D receptor (VDR) and expression of several genes. Methylmercury is a highly bioaccumulative environmental stressor of relevance to the dolphin. We currently report that in dolphin cells sublethal concentrations of methylmercury compromise the ability of 1,25D3 to upregulate VDR, to transactivate a vitamin D-sensitive promoter, and to express specific target genes. These results help elucidate the effects of vitamin D and methylmercury on innate immunity in dolphin skin and potentially in human skin as well, considering similarities in the vitamin D pathway between the two species.

  4. 1,25-Dihydroxyvitamin D3 Induces LL-37 and HBD-2 Production in Keratinocytes from Diabetic Foot Ulcers Promoting Wound Healing: An In Vitro Model

    PubMed Central

    Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A.; Rivas-Santiago, Bruno

    2014-01-01

    Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU. PMID:25337708

  5. 1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

    PubMed

    Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A; Rivas-Santiago, Bruno

    2014-01-01

    Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

  6. Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin.

    PubMed

    Yasuhara, Rika; Miyamoto, Yoichi; Takami, Masamichi; Imamura, Takahisa; Potempa, Jan; Yoshimura, Kentaro; Kamijo, Ryutaro

    2009-04-01

    Porphyromonas gingivalis is one of the major pathogens of periodontitis, a condition characterized by excessive alveolar bone resorption by osteoclasts. The bacterium produces cysteine proteases called gingipains, which are classified according to their cleavage-site specificity into Kgps (lysine-specific gingipains) and Rgps (arginine-specific gingipains). In the present study we examined the effects of gingipains on osteoclast differentiation. In co-cultures of mouse bone-marrow cells and osteoblasts, formation of multinucleated osteoclasts induced by 1alpha,25(OH)(2)D(3) (1alpha,25-dihydroxyvitamin D(3)) was augmented by Kgp but not by RgpB. A physiological concentration (0.1 nM) of 1alpha,25(OH)(2)D(3) induced the osteoclast formation in the presence of 100 nM Kgp to an extent comparable with that induced by 10 nM 1alpha,25(OH)(2)D(3). Kgp also enhanced osteoclastogenesis induced by various microbial components, including lipopolysaccharide. Combined use of Kgp and 1alpha,25(OH)(2)D(3) or lipopolysaccharide also increased the number of resorption pits developed on dentin slices, indicating that the osteoclasts formed in the presence of Kgp possess bone-resorbing activity. The enhanced osteoclastogenesis by Kgp was correlated with a depletion of osteoprotegerin in co-culture medium and was proteolytic-activity-dependent, since benzyloxycarbonyl-L-phenylalanyl-L-lysylacycloxyketone, an inhibitor of Kgp, completely abolished osteoclastogenesis induced by Kgp. Kgp digested osteoprotegerin, since its recombinant protein was susceptible to degradation by Kgp in the presence of serum. As a result, Kgp did not augment osteoclastogenesis in co-cultures of osteoprotegerin-deficient osteoblasts and bone-marrow cells. In addition, enhanced osteoclastogenesis by Kgp was abolished by an excess amount of recombinant osteoprotegerin. These findings suggest that degradation of osteoprotegerin is one of the mechanisms underlying promotion of osteoclastogenesis by Kgp.

  7. Serum Concentrations of 1,25-Dihydroxyvitamin D2 and 1,25-Dihydroxyvitamin D3 in Response to Vitamin D2 and Vitamin D3 Supplementation

    PubMed Central

    Biancuzzo, Rachael M.; Clarke, Nigel; Reitz, Richard E.; Travison, Thomas G.

    2013-01-01

    Objective: The purpose of this study was to determine 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 1,25-dihydroxyvitamin D2 [1,25(OH)2D2] levels in healthy adults consuming 1000 IU vitamin D2 or vitamin D3 per day for 11 weeks. Subjects and Design: Blood from 34 healthy male and female adults, aged 18 to 79 years, from a placebo-controlled, double-blind study who received a placebo, 1000 IU vitamin D3, or 1000 IU vitamin D2 daily for 11 weeks at end of winter was analyzed. Serum levels of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25(OH)2D2, and 1,25(OH)2D3 were determined by liquid chromatography–tandem mass spectroscopy. Results: Of the adults, 82% were vitamin D insufficient (serum 25-hydroxyvitamin D [25(OH)D <30 ng/mL]) at the start of the study. Administration of vitamin D2 and vitamin D3 induced similar increases in total 25(OH)D as well as in 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, respectively. Compared with placebo and adjusting for baseline levels, 1000 IU daily of vitamin D2 was associated with a mean increase of 7.4 pg/mL (95% confidence interval, 4.4–10.3) in 1,25(OH)2D2, which was accompanied by a mean decrease of 9.9 pg/mL (−15.8 to −4.0) in 1,25(OH)2D3. No such differences accompanied administration of 1000 IU daily of vitamin D3. Conclusion: Vitamin D2 and vitamin D3 were effective in raising and maintaining total serum concentrations of 25(OH)D. Ingestion of vitamin D2 also resulted in an increase in serum concentrations of 1,25(OH)2D2. This increase was accompanied by a comparable decrease in serum concentrations of 1,25(OH)2D3; therefore, the total 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations did not significantly change after 11 weeks compared with baseline levels. Ingestion of vitamin D3 did not alter serum concentrations of 1,25(OH)2D3 or total 1,25(OH)2D. Therefore, ingestion of 1000 IU vitamin D2 or vitamin D3 for 11 weeks was effective in raising total serum concentrations of 25(OH)D as well as sustaining serum

  8. 1,25-dihydroxyvitamin D sub 3 up-regulates the 1,25-dihydroxyvitamin D sub 3 receptor in vivo

    SciTech Connect

    Strom, M.; Sandgren, M.E.; Brown, T.A.; DeLuca, H.F. )

    1989-12-01

    The level of mRNA encoding the 1,25-dihydroxyvitamin D{sub 3} receptor in the intestine of vitamin D-deficient rats given 1,25-dihydroxyvitamin D{sub 3} was determined by Northern blot analysis using a {sup 32}P-labeled cDNA probe to the 1,25-dihydroxyvitamin D{sub 3} receptor. mRNA levels increased 10-fold above deficiency levels at 6 and 12 hr after an intravenous dose of 1,25-dihydroxyvitamin D{sub 3}, returning to predosing levels at 24 hr. Total receptor protein level determined by an immunoradiometric assay was increased 2-fold at 12 hr. No change in unoccupied receptor levels determined by ligand-binding assay was observed during this period. These results suggest that 1,25-dihydroxyvitamin D{sub 3} increases receptor mRNA and total receptor level to maintain constant levels of unoccupied receptor.

  9. Skeletal unloading decreases production of 1,25-dihydroxyvitamin D

    NASA Technical Reports Server (NTRS)

    Halloran, B. P.; Bikle, D. D.; Harris, J.; Foskett, H. C.; Morey-Holton, E.

    1993-01-01

    The plasma concentration of 1,25-dihydroxyvitamin D [1,25(OH)2D] decreases during skeletal unloading and increases when normal weight bearing is restored. To determine whether these changes in plasma 1,25(OH)2D reflect changes in production, metabolic clearance, or both we measured the kinetics of 1,25(OH)2D metabolism in rats whose skeletons were normally loaded, unloaded, or reloaded after a period of nonweight bearing. Skeletal unloading produced a transient but striking fall in the production (-73%) and plasma concentration (-72%) of 1,25(OH)2D without having a significant effect (< 20%) on metabolic clearance. Skeletal reloading returned production to normal. Bone formation predictably decreased during unloading and returned to normal after return to weight bearing. No consistent changes in blood ionized calcium, plasma immunoreactive parathyroid hormone (irPTH), or plasma phosphorus occurred. These data suggest that the changes in plasma 1,25-(OH)2D associated with changes in skeletal weight bearing primarily reflect changes in 1,25(OH)2D production. The data provide no evidence that the changes in 1,25(OH)2D production are a consequence of changes in blood ionized calcium, plasma irPTH, or phosphorus.

  10. 1,25-dihydroxyvitamin D and PTHrP mediated malignant hypercalcemia in a seminoma

    PubMed Central

    2014-01-01

    Background Seminomas have been rarely associated with malignant hypercalcemia. The responsible mechanism of hypercalcemia in this setting has been described to be secondary to 1,25-dihydroxyvitamin D secretion. The relationship with PTHrP has not been determined or studied. The aim of this study is to describe and discuss the case and the pathophysiological mechanisms involved in a malignant hypercalcemia mediated by 1,25-dihydroxyvitamin D and PTHrP cosecretion in a patient with seminoma. Case presentation A 35-year-old man was consulted for assessment and management of severe hypercalcemia related to an abdominal mass. Nausea, polyuria, polydipsia, lethargy and confusion led him to the emergency department. An abdominal and pelvic enhanced CT confirmed a calcified pelvic mass, along with multiple retroperitoneal lymphadenopathy. Chest x-ray revealed “cannon ball” pulmonary metastases. The histopathology result was consistent with a seminoma. Serum calcium was 14.7 mg/dl, PTH was undetectable, 25-dihydroxyvitamin D was within normal values and PTHrP and 1,25-dihydroxyvitamin were elevated (35.0 pg/ml, and 212 pg/ml, respectively). After the first cycle of chemotherapy with bleomycin, etoposide and cisplatin, normocalcemia was restored. Both PTHrP and 1,25-dihydroxyvitamin D, dropped dramatically to 9.0 pg/ml and 8.0 pg/ml, respectively. Conclusion The association of seminoma and malignant hypercalcemia is extremely rare. We describe a case of a patient with a seminoma and malignant hypercalcemia related to paraneoplastic cosecretion of 1,25-dihydroxyvitamin D and PTHrP. After successful chemotherapy, calcium, PTHrP and 1,25-Dihydroxyvitamin D returned to normal values. PMID:24721620

  11. Dynamics of 1α,25-dihydroxyvitamin D3-dependent chromatin accessibility of early vitamin D receptor target genes.

    PubMed

    Seuter, Sabine; Pehkonen, Petri; Heikkinen, Sami; Carlberg, Carsten

    2013-12-01

    The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes.

  12. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin.

    PubMed

    Yuk, Jae-Min; Shin, Dong-Min; Lee, Hye-Mi; Yang, Chul-Su; Jin, Hyo Sun; Kim, Kwang-Kyu; Lee, Zee-Won; Lee, Sang-Hee; Kim, Jin-Man; Jo, Eun-Kyeong

    2009-09-17

    Autophagy and vitamin D3-mediated innate immunity have been shown to confer protection against infection with intracellular Mycobacterium tuberculosis. Here, we show that these two antimycobacterial defenses are physiologically linked via a regulatory function of human cathelicidin (hCAP-18/LL-37), a member of the cathelicidin family of antimicrobial proteins. We show that 1,25-dihydroxyvitamin D3 (1,25D3), the active form of vitamin D, induced autophagy in human monocytes via cathelicidin, which activated transcription of the autophagy-related genes Beclin-1 and Atg5. 1,25D3 also induced the colocalization of mycobacterial phagosomes with autophagosomes in human macrophages in a cathelicidin-dependent manner. Furthermore, the antimycobacterial activity in human macrophages mediated by physiological levels of 1,25D3 required autophagy and cathelicidin. These results indicate that human cathelicidin, a protein that has direct antimicrobial activity, also serves as a mediator of vitamin D3-induced autophagy.

  13. Metabolism and pharmacokinetics of 24,25-dihydroxyvitamin D3 in the vitamin D3-replete rat

    SciTech Connect

    Jarnagin, K.; Zeng, S.Y.; Phelps, M.; DeLuca, H.F.

    1985-11-05

    The time course of in vivo metabolism of 24,25-dihydroxyvitamin D3 in rats has been examined. Several tissues were surveyed in an effort to discover new metabolites of 24,25-dihydroxyvitamin D3 and to estimate the concentrations of previously identified metabolites. Rapidly growing male rats were dosed with 24,25-dihydroxyvitamin D3 orally until plasma concentrations of 24,25-dihydroxyvitamin D3 were at steady state. 24,25-Dihydroxyvitamin (3-TH)D3 was then administered. At 10 min and 1, 6, 15, 24, 96, and 192 h after dosing, the animals were killed, and plasma, liver, intestine, and bones were analyzed with a newly developed gradient straight-phase high performance liquid chromatography system. The high performance liquid chromatography system is capable of base-line resolution of most of the major vitamin D metabolites. 24,25-Dihydroxyvitamin D3 clearance from plasma, liver, and kidney but not intestine followed a two-compartment model. 24,25-Dihydroxyvitamin D3 disappeared from plasma with a half-life of 0.55 h (fast phase) and 73.8 h (slow phase). Only two lipid-soluble metabolites of 24,25-dihydroxyvitamin D3 were detected: 24-oxo-25-hydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3. These compounds circulate at very low concentrations in the plasma (50 pg/ml of plasma).

  14. Genomic Determinants of Gene Regulation by 1,25-Dihydroxyvitamin D3 during Osteoblast-lineage Cell Differentiation*♦

    PubMed Central

    Meyer, Mark B.; Benkusky, Nancy A.; Lee, Chang-Hun; Pike, J. Wesley

    2014-01-01

    The biological effects of 1α,25-dihydroxyvitamin D3 (1,25 (OH)2D3) on osteoblast differentiation and function differ significantly depending upon the cellular state of maturation. To explore this phenomenon mechanistically, we examined the impact of 1,25(OH)2D3 on the transcriptomes of both pre-osteoblastic (POBs) and differentiated osteoblastic (OBs) MC3T3-E1 cells, and assessed localization of the vitamin D receptor (VDR) at sites of action on a genome-scale using ChIP sequence analysis. We observed that the 1,25(OH)2D3-induced transcriptomes of POBs and OBs were quantitatively and qualitatively different, supporting not only the altered biology observed but the potential for a change in VDR interaction at the genome as well. This idea was confirmed through discovery that VDR cistromes in POBs and OBs were also strikingly different. Depletion of VDR-binding sites in OBs, due in part to reduced VDR expression, was the likely cause of the loss of VDR-target gene interaction. Continued novel regulation by 1,25(OH)2D3, however, suggested that factors in addition to the VDR might also be involved. Accordingly, we show that transcriptomic modifications are also accompanied by changes in genome binding of the master osteoblast regulator RUNX2 and the chromatin remodeler CCAAT/enhancer-binding protein β. Importantly, genome occupancy was also highlighted by the presence of epigenetic enhancer signatures that were selectively changed in response to both differentiation and 1,25(OH)2D3. The impact of VDR, RUNX2, and C/EBPβ on osteoblast differentiation is exemplified by their actions at the Runx2 and Sp7 gene loci. We conclude that each of these mechanisms may contribute to the diverse actions of 1,25(OH)2D3 on differentiating osteoblasts. PMID:24891508

  15. The effect of microgravity on 1,25-dihydroxyvitamin d3 signalling in osteoblasts

    NASA Astrophysics Data System (ADS)

    Coenegrachts, Lieve; Stockmans, Ingrid; Segers, Ilse; Bouillon, Roger; Carmeliet, Geert

    2007-09-01

    Microgravity encountered during space flight induces bone loss, as seen in both humans and rats. This type of bone loss is mainly caused by decreased bone formation due to reduced osteoblast proliferation and differentiation. Yet, the molecular alterations induced by microgravity during osteoblast differentiation are still enigmatic. Therefore, the effect of microgravity on the intracellular signalling pathway of 1,25-dihydroxyvita-min D3 was investigated during the Odissea Mission. The ligand 1,25-dihydroxyvitamin D3 interacts with the vitamin D receptor (VDR) and this complex binds to vitamin D response elements (VDRE) in the promoter region of target genes to stimulate or suppress gene transcription. To investigate the interaction of liganded VDR with VDRE, the mouse osteoblastic cell line, MC3T3, was stable transfected with a construct containing multiple VDREs of the rat osteocalcin promoter fused to growth hormone as reporter gene. Treatment of these transfectants with 1,25-dihydroxyvitamin D3 resulted in a time- and dose-dependent release of growth hormone in the culture medium. Space flight cultures responded to 1,25-dihydroxyvitamin D3 treatment with increased growth hormone production that was comparable with the induction observed in ground cultures. No 1g centrifuge was available during the space flight. These data indicate that microgravity for 5 days did not alter the interaction of VDR with the osteocalcin VDRE or the subsequent gene transcription.

  16. Conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in renal slices from the rat

    SciTech Connect

    Armbrecht, H.J.; Zenser, T.V.; Davis, B.B.

    1981-07-01

    Isolated renal cortical slices were used to study the conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25)(OH2)D3) by the rat kidney. Production of 1,25-(OH)2D3 and 24,25-(OH)2D3 was linear with time (30-90 min) and tissue weight (40-250 mg). Production of 1,25-(OH)2D3 was greatest (134 +/- 17 pg/mg tissue.h) in animals fed a low calcium, vitamin D-deficient diet. The greatest 24,25-(OH)2D3 production (106 +/- 17 pg/mg tissue.h) was seen in animals fed a high calcium, vitamin D-replete diet, 1,25-(OH)2D3 production was reduced to 23% of maximum by the addition of 1.2% calcium or 0.8% strontium to the vitamin D-deficient, low calcium diet. Production of 1,25-(OH)2D3 and 24,25-(OH)2D3 was greatly reduced in renal cortical slices that had been heated before incubation. Slices of renal medulla produced only small amounts of 1,25-(OH)2D3 compared to slices of renal cortex. These studies provide direct evidence for the production of 1,25-(OH)2D3 and 24,25-(OH)2D3 by the mammalian renal cortex. They also demonstrate that this production may be modulated by dietary calcium, strontium, and vitamin D.

  17. Epigenetic regulation of BMP2 by 1,25-dihydroxyvitamin D3 through DNA methylation and histone modification.

    PubMed

    Fu, Baisheng; Wang, Hongwei; Wang, Jinhua; Barouhas, Ivana; Liu, Wanqing; Shuboy, Adam; Bushinsky, David A; Zhou, Dongsheng; Favus, Murray J

    2013-01-01

    Genetic hypercalciuric stone-forming (GHS) rats have increased intestinal Ca absorption, decreased renal tubule Ca reabsorption and low bone mass, all of which are mediated at least in part by elevated tissue levels of the vitamin D receptor (VDR). Both 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and bone morphogenetic protein 2 (BMP2) are critical for normal maintenance of bone metabolism and bone formation, respectively. The complex nature of bone cell regulation suggests a potential interaction of these two important regulators in GHS rats. In the present study, BMP2 expression is suppressed by the VDR-1,25(OH)2D3 complex in Bone Marrow Stromal Cells (BMSCs) from GHS and SD rat and in UMR-106 cell line. We used chromatin immunoprecipitation (ChIP) assays to identify VDR binding to only one of several potential binding sites within the BMP2 promoter regions. This negative region also mediates suppressor reporter gene activity. The molecular mechanisms underlying the down-regulation of BMP2 by 1,25(OH)2D3 were studied in vitro in BMSCs and UMR-106 cells using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) and the histone deacetylase inhibitor trichostatin A (TSA). Both DAC and TSA activate BMP2 expression in combination with 1,25(OH)2D3. Bisulfite DNA pyrosequencing reveals 1,25(OH)2D3 to completely hypermethylate a single CpG site in the same BMP2 promoter region identified by the ChIP and reporter gene assays. ChIP assays also show that 1,25(OH)2D3 can increase the repressive histone mark H3K9me2 and reduce the acetylation of histone H3 at the same BMP2 promoter region. Taken together, our results indicate that 1,25(OH)2D3 binding to VDR down-regulates BMP2 gene expression in BMSCs and osteoblast-like UMR-106 cells by binding to the BMP2 promoter region. The mechanism of this 1,25(OH)2D3-induced transcriptional repression of BMP2 involves DNA methylation and histone modification. The study provides novel evidence that 1,25(OH)2D3 represses bone

  18. Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

    PubMed Central

    van der Meijden, Karen; Lips, Paul; van Driel, Marjolein; Heijboer, Annemieke C.; Schulten, Engelbert A. J. M.; den Heijer, Martin; Bravenboer, Nathalie

    2014-01-01

    The most biologically active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D3 (25(OH)D3) can affect osteoblast function via conversion to 1,25(OH)2D3, however, it is largely unknown whether 25(OH)D3 can affect primary osteoblast function on its own. Furthermore, 25(OH)D3 is not only converted to 1,25(OH)2D3, but also to 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D3 itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)2D3 can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D3 and 1,25(OH)2D3 by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)2D3 and 24R,25(OH)2D3 dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)2D3 synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D3. We demonstrated that 24R,25(OH)2D3 increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)2D3 strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D3, 1,25(OH)2D3 and 24R,25(OH)2D3 can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)2D3, but also to 24R,25(OH)2D3 by enhancing osteoblast differentiation. This suggests that 25(OH)D3 can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)2D3, but also via conversion to 24R,25(OH)2D3. Whether 25(OH)D3 has direct actions on osteoblast function needs further

  19. 1,25-dihydroxyvitamin D3 suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin gene promoter.

    PubMed

    Yuan, Weihua; Pan, Wei; Kong, Juan; Zheng, Wei; Szeto, Frances L; Wong, Kari E; Cohen, Ronald; Klopot, Anna; Zhang, Zhongyi; Li, Yan Chun

    2007-10-12

    We have shown that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from -2,725 to -2,647 (distal fragment) and from -117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)(2)D(3) inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)(2)D(3)-induced suppression. EMSA revealed that 1,25(OH)(2)D(3) markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)(2)D(3) suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex.

  20. Effect of simulated weightlessness and chronic 1,25-dihydroxyvitamin D administration on bone metabolism

    NASA Technical Reports Server (NTRS)

    Halloran, B. P.; Bikle, D. D.; Globus, R. K.; Levens, M. J.; Wronski, T. J.; Morey-Holton, E.

    1985-01-01

    Weightlessness, as experienced during space flight, and simulated weightlessness induce osteopenia. Using the suspended rat model to simulate weightlessness, a reduction in total tibia Ca and bone formation rate at the tibiofibular junction as well as an inhibition of Ca-45 and H-3-proline uptake by bone within 5-7 days of skeletal unloading was observed. Between days 7 and 15 of unloading, uptake of Ca-45 and H-3-proline, and bone formation rate return to normal, although total bone Ca remains abnormally low. To examine the relationship between these characteristic changes in bone metabolism induced by skeletal unloading and vitamin D metabolism, the serum concentrations of 25-hydroxyvitamin D (25-OH-D), 24, 25-dihydroxyvitamin D (24,25(OH)2D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) at various times after skeletal unloading were measured. The effect of chronic infusion of 1,25(OH)2D3 on the bone changes associated with unloading was also determined.

  1. Synthesis and evaluation of geometric analogs of 1α,25-dihydroxyvitamin D2 as potential therapeutics.

    PubMed

    Bolla, Narasimha Rao; Corcoran, Aoife; Yasuda, Kaori; Chodyński, Michał; Krajewski, Krzysztof; Cmoch, Piotr; Marcinkowska, Ewa; Brown, Geoffrey; Sakaki, Toshiyuki; Kutner, Andrzej

    2016-11-01

    An improved convergent strategy was developed for the synthesis of the previously obtained side-chain extended and rigidified analogs of 1α,25-dihydroxyvitamin D2, PRI-1906 and PRI-1907. New (24Z) geometric isomers of the analogs, PRI-1916 and PRI-1917, were also obtained and identified. These side-chain isomers were separable by flash chromatography, as C-25 alcohols, from the synthetic precursors of PRI-1906 and PRI-1907, respectively. The structures of new analogs were determined by advanced techniques of (1)H and (13)C NMR, including COSY, HSQC and HMBC sequences. Binding affinities of the geometric analogs PRI-1906 and PRI-1916 and their respective C-26, C-27 homologs PRI-1907 and PRI-1917 for the full-length human vitamin D receptor were determined by a fluorescence polarization competition assay. The binding affinity of (24Z) methyl analog PRI-1906 was much higher than that of (24E) analog PRI-1906, while the affinity of (24Z) ethyl analog PRI-1917 was lower than that of the respective PRI-1907. Investigation of the metabolism of these compounds by human CYP24A1 revealed they are much more resistant to CYP24A1 than 1α,25-dihydroxyvitamin D2, indicating they could have longer-term biological effects on target tissues.

  2. A humanized mouse model of hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia.

    PubMed

    Lee, Seong Min; Goellner, Joseph J; O'Brien, Charles A; Pike, J Wesley

    2014-11-01

    The syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a genetic disease of altered mineral homeostasis due to mutations in the vitamin D receptor (VDR) gene. It is frequently, but not always, accompanied by the presence of alopecia. Mouse models that recapitulate this syndrome have been prepared through genetic deletion of the Vdr gene and are characterized by the presence of rickets and alopecia. Subsequent studies have revealed that VDR expression in hair follicle keratinocytes protects against alopecia and that this activity is independent of the protein's ability to bind 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we introduced into VDR-null mice a human VDR (hVDR) bacterial artificial chromosome minigene containing a mutation that converts leucine to serine at amino acid 233 in the hVDR protein, which prevents 1,25(OH)2D3 binding. We then assessed whether this transgene recreated features of the HVDRR syndrome without alopecia. RT-PCR and Western blot analysis in one strain showed an appropriate level of mutant hVDR expression in all tissues examined including skin. The hVDR-L233S mutant failed to rescue the aberrant systemic and skeletal phenotype characteristic of the VDR null mouse due to the inability of the mutant receptor to activate transcription after treatment with 1,25(OH)2D3. Importantly, however, neither alopecia nor the dermal cysts characteristic of VDR-null mice were observed in the skin of these hVDR-L233S mutant mice. This study confirms that we have created a humanized mouse model of HVDRR without alopecia that will be useful in defining additional features of this syndrome and in identifying potential novel functions of the unoccupied VDR.

  3. Targeted delivery of 1,25-dihydroxyvitamin D3 to colon tissue and identification of a major 1,25-dihydroxyvitamin D3 glycoside from Solanumglaucophyllum plant leaves.

    PubMed

    Zimmerman, Duane R; Koszewski, Nicholas J; Hoy, Derrel A; Goff, Jesse P; Horst, Ronald L

    2015-04-01

    Leaves of the Solanum glaucophyllum (Sg) plant, indigenous to South America, have long been known for their calcinogenic toxicity in ruminant animals. It was determined the leaves contained glycosidic derivatives of 1,25-dihydroxyvitamin D3 (1,25D3) and liberation of the free hormone by rumen bacterial populations elicited a hypercalcemic response. Our interest in the leaves is predicated on the concept that the glycoside forms of 1,25D3 would target release of the active hormone in the lower gut of non-ruminant mammals. This would provide a means of delivering 1,25D3 directly to the colon, where the hormone has been shown to have beneficial effects in models of inflammatory bowel disease (IBD) and colon cancer. We fed mice for 10 days with variable amounts of Sg leaf. Feeding 7-333μg leaf/day produced no changes in plasma Ca(2+) and 1,25D3 concentrations, and only at ≥1000μg leaf/day did these values become significantly elevated compared to controls. Gene expression studies from colon tissue indicated a linear relationship between the amount of leaf consumed and expression of the Cyp24a1 gene. In contrast, Cyp24a1 gene expression in the duodenums and ileums of these mice was unchanged compared to controls. One of the major 1,25D3-glycosides was isolated from leaves following extraction and purification by Sep-Pak cartridges and HPLC fractionation. Ultraviolet absorbance was consistent with modification of the 1-hydroxyl group, and positive ion ESI mass spectrometry indicated a diglycoside of 1,25D3. 2-Dimensional NMR analyses were carried out and established the C1 proton of the A-ring was interacting with a C1' sugar proton, while the C3 proton of the A-ring was linked with a second C1' sugar proton. The structure of the isolated compound is therefore consistent with a β-linked 1,3-diglycoside of 1,25D3. Thus, Sg leaf administered to mice at up to 333 ug/day can elicit colon-specific enhancement of Cyp24a1 gene expression without inducing hypercalcemia, and

  4. Chondrocyte-specific modulation of Cyp27b1 expression supports a role for local synthesis of 1,25-dihydroxyvitamin D3 in growth plate development.

    PubMed

    Naja, Roy Pascal; Dardenne, Olivier; Arabian, Alice; St Arnaud, René

    2009-09-01

    The Cyp27b1 enzyme (25-hydroxyvitamin D-1alpha-hydroxylase) that converts 25-hydroxyvitamin D into the active metabolite, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is expressed in kidney but also in other cell types such as chondrocytes. This suggests that local production of 1,25(OH)(2)D(3) could play an important role in the differentiation of these cells. To test this hypothesis, we engineered mutant mice that do not express the Cyp27b1 gene in chondrocytes. Inactivation of both alleles of the Cyp27b1 gene led to decreased RANKL expression and reduced osteoclastogenesis, increased width of the hypertrophic zone of the growth plate at embryonic d 15.5, increased bone volume in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. The expression of the angiogenic marker VEGF was decreased, accompanied by decreased platelet/endothelial cell adhesion molecule-1 staining in the neonatal growth plate, suggesting a delay in vascularization. In parallel, we engineered strains of mice overexpressing a Cyp27b1 transgene in chondrocytes by coupling the Cyp27b1 cDNA to the collagen alpha(1)(II) promoter. The transgenic mice showed a mirror image phenotype when compared with the tissue-specific inactivation, i.e. a reduction in the width of the hypertrophic zone of the embryonic growth plate, decreased bone volume in neonatal long bones, and inverse expression patterns of chondrocytic differentiation markers. These results support an intracrine role of 1,25(OH)(2)D(3) in endochondral ossification and chondrocyte development in vivo.

  5. Effect of 1,25-dihydroxyvitamin D3 on the expression of mannose receptor, DC-SIGN and autophagy genes in pulmonary tuberculosis.

    PubMed

    Afsal, K; Selvaraj, P

    2016-07-01

    1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a powerful immuno-modulator, which enhances expression of antimicrobial peptides and induces autophagy in monocytes/macrophages. Since 1,25(OH)2D3 increases the phagocytic potential of monocytes/macrophages, we have explored the effect of 1,25(OH)2D3 on the expression of receptors such as mannose receptor (CD206) and DC-SIGN (CD209) as well as autophagy genes such as ATG5 and Beclin-1 (BECN1) in monocytes/macrophages of healthy controls (HCs) and pulmonary tuberculosis (PTB) patients with and without cavitary disease. Peripheral blood mononuclear cells (PBMCs) were isolated from 40 HCs and 40 PTB patients and were cultured for 72 h with Mtb in the presence or absence of 1,25(OH)2D3 at 10(-7) M concentration. 1,25(OH)2D3 significantly upregulated the expression of mannose receptor, ATG5 and BECN1; whereas DC-SIGN expression was suppressed in Mtb infected cells of both study groups (p < 0.05). The 1,25(OH)2D3-induced expression of CD206, ATG5 and BECN1 genes was lower in PTB patients compared to HCs, whereas expression of these genes was impaired in PTB patients with cavitary disease. Moreover, the relative expression of ATG5 and BECN1 was positively correlated with monocyte/macrophage phagocytosis and cathelicidin antimicrobial peptide gene expression in HCs and PTB patients (p < 0.05). Our study results suggest that vitamin D supplementation in PTB patients without cavitary disease could enhance innate immune functions and may help to control intracellular growth of mycobacteria in macrophages.

  6. The Use of 1α,25-Dihydroxyvitamin D3 as an Anticancer Agent

    PubMed Central

    Marcinkowska, Ewa; Wallace, Graham R.; Brown, Geoffrey

    2016-01-01

    The notion that vitamin D can influence the incidence of cancer arose from epidemiological studies. The major source of vitamin D in the organism is skin production upon exposure to ultra violet-B. The very first observation of an inverse correlation between exposure of individuals to the sun and the likelihood of cancer was reported as early as 1941. In 1980, Garland and Garland hypothesised, from findings from epidemiological studies of patients in the US with colon cancer, that vitamin D produced in response to sun exposure is protective against cancer as opposed to sunlight per se. Later studies revealed inverse correlations between sun exposure and the occurrence of prostate and breast cancers. These observations prompted laboratory investigation of whether or not vitamin D had an effect on cancer cells. Vitamin D is not active against cancer cells, but the most active metabolite 1α,25-dihydroxyvitamin D3 (1,25D) has profound biological effects. Here, we review the anticancer action of 1,25D, clinical trials of 1,25D to date and the prospects of the future therapeutic use of new and low calcaemic analogues. PMID:27187375

  7. Fluorometric assay of 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 in plasma.

    PubMed

    Shimizu, M; Gao, Y; Aso, T; Nakatsu, K; Yamada, S

    1992-08-01

    The first practical fluorometric assay of plasma 25-hydroxyvitamin D3 (25-OH-D3) and 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is described. The method uses a highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1, 2,4- triazoline-3,5-dione (DMEQ-TAD), to fluorescence-label vitamin D. Vitamin D metabolites were roughly purified with a short cartridge column followed by HPLC, labeled with DMEQ-TAD, and the product was analyzed on HPLC. In the assay of 25-OH-D3 the new fluorometric method was compared with the HPLC-uv method and was confirmed to be as accurate and reliable (CV, 4-5%) as the HPLC-uv method. Plasma 24,25-(OH)2D3 was accurately assayed by the HPLC-FL method, where the standard addition method was successfully used to calculate the overall recovery.

  8. 1,25-Dihydroxyvitamin D{sub 3} regulates genes responsible for detoxification in intestine

    SciTech Connect

    Kutuzova, Galina D.; DeLuca, Hector F. . E-mail: deluca@biochem.wisc.edu

    2007-01-01

    1{alpha},25-Dihydroxyvitamin D{sub 3} (1,25-(OH){sub 2}D{sub 3}), the biologically active form of vitamin D{sub 3}, not only plays a major role in mammalian calcium and phosphorous homeostasis but also exerts pleiotropic effects on cell proliferation, differentiation and the immune system. Further, vitamin D is believed to play a significant role in the prevention of colon, prostate, and breast cancer and in reducing the risk of autoimmune diseases. To gain insight into the mechanism whereby vitamin D can have such diverse actions, we have employed microarray technology. We studied the effect of a single dose of 1,25-(OH){sub 2}D{sub 3} on gene expression in the intestine of vitamin D-deficient rats. Within 6 h, 1,25-(OH){sub 2}D{sub 3} stimulates the expression of several phase I and phase II biotransformation genes. There is also an increased expression of antioxidant genes. These results support the idea that vitamin D is a significant factor in detoxification and protection against environmental toxins.

  9. 1α,25-dihydroxyvitamin D₃ counteracts the effects of cigarette smoke in airway epithelial cells.

    PubMed

    Zhang, Ruhui; Zhao, Haijin; Dong, Hangming; Zou, Fei; Cai, Shaoxi

    2015-06-01

    Cigarette smoke extracts (CSE) alter calpain-1 expression via ERK signaling pathway in bronchial epithelial cells. 1α,25-dihydroxyvitamin D3 (1,25D3) inhibits cigarette smoke-induced epithelial barrier disruption. This study was aimed to explore whether the 1,25D3 counteracted the CSE effects in a human bronchial epithelial cell line (16HBE). In particular, transepithelial electrical resistance (TER) and permeability, expression and distribution of E-cadherin and β-catenin, calpain-1 expression, and ERK phosphorylation were assessed in the CSE-stimulated 16HBE cells. The CSE induced the ERK phosphorylation, improved the calpain-1 expression, increased the distribution anomalies and the cleaving of E-cadherin and β-catenin, and resulted in the TER reduction and the permeability increase. The 1,25D3 reduced these pathological changes. The 1,25D3 mediated effects were associated with a reduced ERK phosphorylation. In conclusion, the present study provides compelling evidences that the 1,25D3 may be considered a possible valid therapeutic option in controlling the cigarette smoke-induced epithelial barrier disruption.

  10. The effectiveness of cinacalcet as an adjunctive therapy for hereditary 1-25 dihydroxyvitamin -D3 resistant rickets.

    PubMed

    Akıncı, Ayşehan; Dündar, İsmail; Kıvılcım, Meltem

    2016-10-31

    High doses of oral calcium or long-term calcium infusions are recommended to correct the hypocalcemia and secondary hyperparathyroidism in patients with hereditary 1-25 dihydroxyvitamin D3 resistant rickets (HVDRR). Preliminary studies revealed that calcimimetics may be safe and effective therapeutic choise in children with secondary hyperparathyroidism. Our aim was to observe the efficacy of cinacalcet on the normalization of secondary hyperparathroidism and hypophosphatemia in two siblings with VDRR who did not respond to traditional therapy regimes.

  11. Plasma 1,25-Dihydroxyvitamin D and the Risk of Developing Hypertension: The Prevention of Renal and Vascular End-Stage Disease Study.

    PubMed

    van Ballegooijen, Adriana J; Gansevoort, Ron T; Lambers-Heerspink, Hiddo J; de Zeeuw, Dick; Visser, Marjolein; Brouwer, Ingeborg A; Kema, Ido P; de Borst, Martin H; Bakker, Stephan J L; Joosten, Michel M

    2015-09-01

    Previous observational studies on the vascular effects of vitamin D have predominantly relied on measurement of its inactive precursor, 25-hydroxyvitamin D, whereas the active metabolite 1,25-dihydroxyvitamin D may be of more physiological relevance. We prospectively studied the associations of 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D with hypertension risk (blood pressure ≥140/90 mm Hg or initiation of blood pressure-lowering drugs) in 5066 participants aged 28 to 75 years, free of hypertension at baseline from the Prevention of Renal and Vascular End-Stage Disease Study, a well-defined cohort with serial follow-up. We measured plasma 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D using liquid chromatography-tandem mass spectrometry. Mean±SD plasma concentration of 1,25-dihydroxyvitamin D was 145±47.0 pmol/L and 25-hydroxyvitamin D was 58.6±23.8 nmol/L. During a median follow-up of 6.4 years, 1036 participants (20.5%) developed hypertension. As expected, low 25-hydroxyvitamin D was associated with a higher hypertension risk; each 1-SD decrement in 25-hydroxyvitamin D was associated with a 8% higher hypertension risk (hazard ratio, 1.08; 95% confidence interval, 1.01-1.16) after adjustment for potential confounders. However, the association of 1,25-dihydroxyvitamin D was in the opposite direction; each 1-SD decrement of 1,25-dihydroxyvitamin D was associated with a 10% lower hypertension risk (hazard ratio, 0.90; 95% confidence interval, 0.84-0.96), independent of potential confounders. In contrast to the inverse association between 25-hydroxyvitamin D and hypertension risk, 1,25-dihydroxyvitamin D was positively associated with risk of hypertension. Thus, higher circulating concentrations of 1,25-dihydroxyvitamin D are associated with a higher risk of hypertension.

  12. Preparation and biological activity of 24-epi-26,26,26,27,27,27-hexafluoro- 1 alpha,25-dihydroxyvitamin D2.

    PubMed

    Iseki, K; Oishi, S; Namba, H; Taguchi, T; Kobayashi, Y

    1995-11-01

    A new fluorinated analog of vitamin D2, 24-epi-26,26,26,27,27,27-hexafluoro- 1 alpha,25-dihydroxyvitamin D2, was efficiently synthesized starting from (R)-4-isopropyl-3-propionyl-2- oxazolidinone with high stereochemical control. In all four physiological test systems, the fluorinate vitamin D2 analog was found to be slightly less active than 1 alpha,25-dihydroxyvitamin D3.

  13. Gravity affects the responsiveness of Runx2 to 1, 25-dihydroxyvitamin D3 (VD3)

    NASA Astrophysics Data System (ADS)

    Guo, Feima; Dai, Zhongquan; Wu, Feng; Liu, Zhaoxia; Tan, Yingjun; Wan, Yumin; Shang, Peng; Li, Yinghui

    2013-03-01

    Bone loss resulting from spaceflight is mainly caused by decreased bone formation, and decreased osteoblast proliferation and differentiation. Transcription factor Runx2 plays an important role in osteoblast differentiation and function by responding to microenvironment changes including cytokine and mechanical factors. The effects of 1, 25-dihydroxyvitamin D3 (VD3) on Runx2 in terms of mechanical competence is far less clear. This study describes how gravity affects the response of Runx2 to VD3. A MC3T3-6OSE2-Luc osteoblast model was constructed in which the activity of Runx2 was reflected by reporter luciferase activity identifed by bone-related cytokines. The results showed that luciferase activity in MC3T3-6OSE2-Luc cells transfected with Runx2 was twice that of the vacant vector. Alkaline phosphatase (ALP) activity was increased in MC3T3-6OSE2-Luc cells by different concentrations of IGF-I and BMP2. MC3T3-6OSE2-Luc cells were cultured under simulated microgravity or centrifuge with or without VD3. In simulated microgravity, luciferase activity was decreased after 48 h of clinorotation culture, but increased in the centrifuge culture. Luciferase activity was increased after VD3 treatment in normal conditions and simulated microgravity, the increase in luciferase activity in simulated microgravity was lower than that in the 1 g condition when simultaneously treated with VD3 and higher than that in the centrifuge condition. Co-immunoprecipitation showed that the interaction between the VD3 receptor (VDR) and Runx2 was decreased by simulated microgravity, but increased by centrifugation. From these results, we conclude that gravity affects the response of Runx2 to VD3 which results from an alteration in the interaction between VDR and Runx2 under different gravity conditions.

  14. Identification of Human UDP-glucuronosyltransferases Catalyzing Hepatic 1α,25-Dihydroxyvitamin D3 Conjugation

    PubMed Central

    Hashizume, Takanori; Xu, Yang; Mohutsky, Michael A.; Alberts, Jeffrey; Hadden, Chad; Kalhorn, Thomas F.; Isoherranen, Nina; Shuhart, Margaret C.; Thummel, Kenneth E.

    2009-01-01

    The biological effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH)2D3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D3. Among twelve different UGT isozymes tested, only UGT1A4 ≫ 2B4 and 2B7 supported the reaction. Two different 1,25(OH)2D3 monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25-O-glucuronide isomer. The formation of 25-O-glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective Km and Vmax values of 7.3 and 11.2 μM, and 33.7 ± 1.4 and 32.9 ± 1.9 pmol/min/mg protein. The calculated intrinsic 25-O-glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (4-fold) inter-liver variability in the 25-O-glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited > 80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH)2D3 represents a significant component of intestinal and systemic 1,25(OH)2D3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step. PMID:18177842

  15. Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation.

    PubMed

    Bollag, W B; Ducote, J; Harmon, C S

    1995-05-01

    1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has been proposed as a physiologic regulator of keratinocyte growth and differentiation. Utilizing a proliferative serum-free culture system, we have found that a physiologic (picomolar) concentrations this hormone stimulated proliferation of primary mouse epidermal keratinocytes; at higher (nanomolar to micromolar) doses, growth was inhibited by 1,25(OH)2D3. We investigated the nature of the signal transduction mechanism underlying the response to 1,25(OH)2D3 and observed little or no effect of either low or high concentrations of the hormone on cytosolic calcium levels or Fos expression. Furthermore, the protein kinase C inhibitor, Ro 31-7549, had very little effect on the growth inhibition induced by a high dose (1 microM) of 1,25(OH)2D3. This lack of rapid signal transduction events was consistent with the inability of a short (4-hour) exposure to 1,25(OH)2D3 to initiate a complete growth-inhibitory response as measured using [3H]thymidine incorporation. Our results indicate that physiologic concentrations of 1,25(OH)2D3 are required for optimal keratinocyte growth. Furthermore, we found no evidence of rapid effects of 1,25(OH)2D3 and suggest that in mouse epidermal keratinocytes, the response to this hormone is mediated by a slow transduction pathway, such as that activated by the intracellular 1,25(OH)2D3 receptor (VDR).

  16. Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D sub 3 receptor and 1,25-dihydroxyvitamin D sub 3 enhancement of mouse secreted phosphoprotein 1 (Spp-1 or osteopontin) gene expression

    SciTech Connect

    Noda, Masaki; Vogel, R.L. ); Craig, A.M. ); Prahl, J.; DeLuca, H.F. ); Denhardt, D.T. )

    1990-12-01

    Secreted phosphoprotein 1 (Spp-1; osteopontin) is one of the abundant noncollagenous proteins in bone matrix and is produced by osteoblasts. The authors examined the promoter region of the mouse Spp-1 gene and identified a sequence responsible for 1,25-dihydroxyvitamin D{sub 3} enhancement of the Spp-1 gene expression. This 24-base-pair (bp) sequence (vitamin D response element) is located 761 bp upstream of the transcription start site and consists of two direct repeats of a unique 9-bp motif, AGGTTCACG. The vitamin D response element confers responsiveness of a heterologous promoter to 1,25-dihydroxyvitamin D{sub 3} in a position-and orientation-independent and copy-number-dependent manner. The basal level of expression of the reporter constructs containing this sequence and its response to 1,25-dihydroxyvitamin D{sub 3} were not affected by cotreatment with transforming growth factor {beta} or the tumor promoter phorbol 12-myristate 13-acetate or by cotransfection with a JUN expression vector. The vitamin D response element forms DNA-protein complexes, as indicated by gel-retardation assays. The addition of a monoclonal antibody raised against the vitamin D receptor further retarded the mobility of the DNA-protein complex. Another antibody that recognized the DNA binding region of the vitamin D receptor attenuated its binding to the sequence. These results indicate that this 24-bp sequence containing two 9-bp motifs binds to the vitamin D receptor and mediates the vitamin D{sub 3} enhancement of murine Spp-1 gene expression.

  17. Tolerance to 1,25 dihydroxyvitamin D3 glycosides from Solanum glaucophyllum by the growing pig.

    PubMed

    Schlegel, P; Guggisberg, D; Gutzwiller, A

    2017-02-16

    Solanum glaucophyllum leaves contain high levels of glycosidically bound 1,25 dihydroxyvitamin D3, the most important vitamin D metabolite. The tolerance to this source was evaluated during six weeks with fifty weaned pigs fed increasing levels (0, 2.5, 5, 10 and 20μg 1,25(OH)2D3/kg diet). The diet contained, per kg, 9.7g Ca, 3.5g digestible P and 2000IU cholecalciferol. Ten additional pigs were fed a diet containing 1000IU cholecalciferol/kg, without 1,25(OH)2D3. Weekly plasma and final kidney, bone and urinary mineral contents, bone density and breaking strength served as indicators for possible adverse effects of the supplement. All animals grew well and remained clinically healthy. The measured parameters remained unchanged when 1000 replaced 2000IU cholecalciferol/kg and when 1,25(OH)2D3 was fed up to 10μg/kg. Twenty μg 1,25(OH)2D3 increased plasma Ca and decreased plasma P from the 2(nd) and the 4(th) experimental week onwards, respectively. Twenty μg 1,25(OH)2D3 increased final plasma Ca and 1,25(OH)2D3 and reduced final plasma P by respectively 19, 56 and 13%. Twenty μg 1,25(OH)2D3 also increased kidney Ca and urinary Ca by 43 and 69%, respectively, reduced bone breaking strength by 12% and tended to decrease bone ash by 3%. To conclude, 2000IU D3 was not beneficial compared to 1000IU cholecalciferol; up to 10μg 1,25(OH)2D3 per kg diet did not lead to observed adverse effects; 20μg 1,25(OH)2D3 altered the homeostatic regulation of Ca and P thus, may lead to first signs of possible adverse effects, such as soft tissue calcification.

  18. 1,25-Dihydroxyvitamin D(3) Inhibits Podocyte uPAR Expression and Reduces Proteinuria

    PubMed Central

    Liu, Shuangxin; Xie, Shaoting; Yang, Yun; Ma, Juan; Deng, Yujun; Wang, Wenjian; Xu, Lixia; Li, Ruizhao; Zhang, Li; Yu, Chunping; Shi, Wei

    2013-01-01

    Background Accumulating studies have demonstrated that 1,25-Dihydroxyvitamin D(3) (1,25(OH)2D3) reduces proteinuria and protects podocytes from injury. Recently, urokinase receptor (uPAR) and its soluble form have been shown to cause podocyte injury and focal segmental glomerulosclerosis (FSGS). Here, our findings showed that 1,25(OH)2D3 did inhibit podocyte uPAR expression and attenuate proteinuria and podocyte injury. Methodology/Principal Findings In this study, the antiproteinuric effect of 1,25(OH)2D3 was examined in the lipopolysaccharide mice model of transient proteinuria (LPS mice) and in the 5/6 nephrectomy rat FSGS model(NTX rats). uPAR protein expression were tested by flow cytometry, immune cytochemistry and western blot analysis, and uPAR mRNA expression by real-time quantitative PCR in cultured podocytes and kidney glomeruli isolated from mice and rats. Podocyte motility was observed by transwell migration assay and wound healing assay. Podocyte foot processes effacement was identified by transmission electron microscopy. We found that 1,25(OH)2D3 inhibited podocyte uPAR mRNA and protein synthesis in LPS-treated podocytes, LPS mice and NTX rats, along with 1,25(OH)2D3 reducing proteinuria in NTX rats and LPS mice.1,25(OH)2D3 reduced glomerulosclerosis in NTX rats and alleviated podocyte foot processes effacement in LPS mice. Transwell migration assay and wound healing assay showed that LPS-induced podocyte motility, irrespective of random or directed motility, were substantially reduced by 1,25(OH)2D3. Conclusions/Significance Our results demonstrated that 1,25(OH)2D3 inhibited podocyte uPAR expression in vitro and in vivo, which may be an unanticipated off target effect of 1,25(OH)2D3 and explain its antiproteinuric effect in the 5/6 nephrectomy rat FSGS model and the LPS mouse model of transient proteinuria. PMID:23741418

  19. 1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation.

    PubMed Central

    Khare, S; Bolt, M J; Wali, R K; Skarosi, S F; Roy, H K; Niedziela, S; Scaglione-Sewell, B; Aquino, B; Abraham, C; Sitrin, M D; Brasitus, T A; Bissonnette, M

    1997-01-01

    Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time

  20. The role of 1,25-dihydroxyvitamin D in the inhibition of bone formation induced by skeletal unloading

    NASA Technical Reports Server (NTRS)

    Halloran, B. P.; Bikle, D. D.; Wronski, T. J.; GLOBUS. R.; Levens, M. J.; Morey-Holton, E.

    1983-01-01

    Skeletal unloading results in osteopenia. To examine the involvement of vitamin D in this process, the rear limbs of growing rats were unloaded and alterations in bone calcium and bone histology were related to changes in serum calcium (Ca), inorganic phosphorus (P sub i), 25-hydroxyvitamin D (25-OH-D), 24,25-dihydroxyvitamin D (24,25(OH)2D and 1,25-dihydroxyvitamin D (1,25(OH)2D. Acute skeletal unloading induced a transitory inhibition of Ca accumulation in unloaded bones. This was accompanied by a transitory rise in serum Ca, a 21% decrease in longitudinal bone growth (P 0.01), a 32% decrease in bone surface lined with osteoblasts (P .05), no change in bone surface lined with osteoclasts and a decrease in circulating (1,25(OH)2D. No significant changes in the serum concentrations of P sub i, 25-OH-D or 24,25(OH)2D were observed. After 2 weeks of unloading, bone Ca stabilized at approximately 70% of control and serum Ca and 1,25(OH)2D returned to control values. Maintenance of a constant serum 1,25(OH)2D concentration by chronic infusion of 1,25(OH)2D (Alza osmotic minipump) throughout the study period did not prevent the bone changes induced by acute unloading. These results suggest that acute skeletal unloading in the growing rat produces a transitory inhibition of bone formation which in turn produces a transitory hypercalcemia.

  1. Control of macrophage cell differentiation in human promyelocytic HL-60 leukemia cells by 1,25-dihydroxyvitamin D/sub 3/ and phorbol-12-myristate-13-acetate

    SciTech Connect

    Murao, S.; Gemmell, M.A.; Callaham, M.F.; Anderson, N.L.; Huberman, E.

    1983-10-01

    Human promyelocytic leukemia cells (HL-60) were induced to differentiate into macrophage-like cells in a dose (3 x 10/sup -10/ to 10/sup -7/ M) and time (1 to 6 days)-dependent manner by 1,25-dihydroxyvitamin D/sub 3/ and the tumor promoter, phorbol-12-myristate-13-acetate. Differentiation was determined by an increase in the percentage of morphologically mature cells, in lysozyme and nonspecific esterase activities, and in reactivity with the murine OKM1 monoclonal antibody. Two HL-60 cell variants, designated as R-80 and B-II, were also examined. R-80 cells, which are resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate, also exhibited resistance, although to a lesser degree, to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/. Te resistance to the action of the two compounds is presumably not due to similar binding sites for the two inducers, since 1,25-dihydroxyvitamin D/sub 3/ was unable to compete for the phorbol diester binding sites as measured by (/sup 3/H)phorbol-12,13-dibutyrate binding. B-II cells were resistant to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide. Two-dimensional electrophoretic analysis of HL-60 cell protein patterns indicated that treatment of the HL-60 cells with 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide caused the cells to express various monocyte-macrophage and granulocyte marker proteins. These results indicate that 1,25-dihydroxyvitamin D/sub 3/ induces in the HL-60 cells a phenotype that resembles, but is not identical to, that of peripheral monocytes-macrophages. 40 references, 3 figures, 1 table.

  2. Downregulation of Runx2 by 1,25-Dihydroxyvitamin D₃ Induces the Transdifferentiation of Osteoblasts to Adipocytes.

    PubMed

    Kim, Jung Ha; Seong, Semun; Kim, Kabsun; Kim, Inyoung; Jeong, Byung-Chul; Kim, Nacksung

    2016-05-19

    1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)₂D₃ enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)₂D₃ in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)₂D₃ enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)₂D₃ were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)₂D₃ induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts.

  3. The serum 24,25-dihydroxyvitamin D concentration, a marker of vitamin D catabolism, is reduced in chronic kidney disease

    PubMed Central

    Bosworth, Cortney; Levin, Gregory; Robinson-Cohen, Cassianne; Hoofnagle, Andrew N.; Ruzinski, John; Young, Bessie; Schwartz, Stephen; Himmelfarb, Jonathan; Kestenbaum, Bryan; de Boer, Ian H.

    2012-01-01

    Chronic kidney disease is characterized, in part, as a state of decreased production of 1,25-dihydroxyvitamin D (1,25(OH)2D); however, this paradigm overlooks the role of vitamin D catabolism. We developed a mass spectrometric assay to quantify serum concentration of 24,25-dihydroxyvitamin D (24,25(OH)2D), the first metabolic product of 25-hydroxyvitamin D (25(OH)D) by CYP24A1, and determined its clinical correlates and associated outcomes among 278 participants with chronic kidney disease in the Seattle Kidney Study. For eGFRs of 60 or more, 45–59, 30–44, 15–29, and under 15 ml/min/1.73m2, the mean serum 24,25(OH)2D concentrations significantly trended lower from 3.6, 3.2, 2.6, 2.6, to 1.7 ng/ml, respectively. Non-Hispanic Black race, diabetes, albuminuria, and lower serum bicarbonate were also independently and significantly associated with lower 24,25(OH)2D concentrations. The 24,25(OH)2D concentration was more strongly correlated with that of parathyroid hormone than was 25(OH)D or 1,25(OH)2D. A 24,25(OH)2D concentration below the median was associated with increased risk of mortality in unadjusted analysis, but this was attenuated with adjustment for potential confounding variables. Thus, chronic kidney disease is a state of stagnant vitamin D metabolism characterized by decreases in both 1,25(OH)2D production and vitamin D catabolism. PMID:22648296

  4. Antiproliferative Activity of Double Point Modified Analogs of 1,25-Dihydroxyvitamin D2 Against Human Malignant Melanoma Cell Lines

    PubMed Central

    Piotrowska, Anna; Wierzbicka, Justyna; Nadkarni, Sharmin; Brown, Geoffrey; Kutner, Andrzej; Żmijewski, Michał A.

    2016-01-01

    Vitamin D is a lipid soluble steroid hormone with pleiotropic biological properties, including regulation of cell proliferation, differentiation and apoptosis. As to these desirable anticancer actions, 1,25-dihydroxyvitamins D and analogs have been reported to inhibit the proliferation and to induce differentiation of a wide variety of cancer cell types, including human malignant melanoma. However, there is a need for novel and more efficacious vitamin D analogs, and how best to design such is still an open issue. A series of double point modified (DPM) analogs of 1,25-dihydroxyvitamin D2 (1,25(OH)2D2) induced differentiation of the vitamin D receptor (VDR) positive A375 and VDR negative SK-MEL 188b human malignant melanoma cell lines. Surprisingly, the dose of 1,25(OH)2D2 required to inhibit the proliferation of the A375 melanoma cell line by was several fold lower than that required in the case of 1,25(OH)2D3. To evaluate the impact of the modification in the side chain (additional 22-hydroxyl) and in the A-ring (5,6-trans modification), the regular side-chain of vitamin D2 or D3 was retained in the structure of our analogs. As expected, 5,6-trans modification was advantageous to enhancing the anti-proliferative activity of analogs, but not as a single point modification (SPM). Very unexpectedly, the additional 22-hydroxyl in the side-chain reduced significantly the anti-proliferative activity of both the natural and 5,6-trans series analogs. Finally, an induction of pigmentation in melanoma SK-MEL 188b cells was observed to sensitized cells to the effect of vitamin D analogs. PMID:26760999

  5. Inhibition of RelB by 1,25-dihydroxyvitamin D3 promotes sensitivity of breast cancer cells to radiation.

    PubMed

    Mineva, Nora D; Wang, Xiaobo; Yang, Sanghwa; Ying, Haoqiang; Xiao, Zhi-Xiong J; Holick, Michael F; Sonenshein, Gail E

    2009-09-01

    Aberrant constitutive expression of the NF-kappaB c-Rel and RelA subunits in breast cancer cells was shown to promote their survival. Recently, we demonstrated that aggressive breast cancers constitutively express high levels of the RelB subunit, which promotes their more invasive phenotype via induction of the BCL2 gene. As these cancers are frequently resistant to therapy, here we tested the hypothesis that RelB promotes their survival. High RelB expressing Hs578T and MDA-MB-231 breast cancer cells were more resistant to gamma-radiation than MCF7 and ZR-75 cells, which express lower RelB levels. Knockdown of RelB in Hs578T led to decreased survival in response to gamma-irradiation, while conversely ectopic expression of RelB in MCF7 cells protected these cells from radiation. Similar data were obtained upon treatment of Hs578T or MCF7 cells with the chemotherapeutic agent doxorubicin. High serum levels of 25-hydroxyvitamin D are associated with decreased breast cancer risk and mortality, although, the mechanisms of its protective actions have not been fully elucidated. Treatment of Hs578T and Her-2/neu-driven NF639 cells with 1,25-dihydroxyvitamin D3 decreased RelB/RELB gene expression and levels of pro-survival targets Survivin, MnSOD and Bcl-2, while increasing their sensitivity to gamma-irradiation. Thus, RelB, which promotes survival and a more highly invasive phenotype of breast cancer cells, is a target of 1,25-dihydroxyvitamin D3, providing one mechanism for the observed protective role of 25-hydroxyvitamin D in patients with breast cancer.

  6. Inhibition of RelB by 1,25-Dihydroxyvitamin D3 Promotes Sensitivity of Breast Cancer Cells to Radiation

    PubMed Central

    MINEVA, NORA D.; WANG, XIAOBO; YANG, SANGHWA; YING, HAOQIANG; XIAO, ZHI-XIONG J.; HOLICK, MICHAEL F.; SONENSHEIN, GAIL E.

    2010-01-01

    Aberrant constitutive expression of the NF-κB c-Rel and RelA subunits in breast cancer cells was shown to promote their survival. Recently, we demonstrated that aggressive breast cancers constitutively express high levels of the RelB subunit, which promotes their more invasive phenotype via induction of the BCL2 gene. As these cancers are frequently resistant to therapy, here we tested the hypothesis that RelB promotes their survival. High RelB expressing Hs578T and MDA-MB-231 breast cancer cells were more resistant to γ-radiation than MCF7 and ZR-75 cells, which express lower RelB levels. Knockdown of RelB in Hs578T led to decreased survival in response to γ-irradiation, while conversely ectopic expression of RelB in MCF7 cells protected these cells from radiation. Similar data were obtained upon treatment of Hs578T or MCF7 cells with the chemotherapeutic agent doxorubicin. High serum levels of 25-hydroxyvitamin D are associated with decreased breast cancer risk and mortality, although, the mechanism of its protective action has not been elucidated. Treatment of Hs578T and Her-2/neu-driven NF639 cells with 1,25-dihydroxyvitamin D3 decreased RelB/RELB gene expression and levels of pro-survival targets Survivin, MnSOD and Bcl-2, while increasing their sensitivity to γ-irradiation. Thus, RelB, which promotes survival and a more highly invasive phenotype of breast cancer cells, is a target of 1,25-dihydroxyvitamin D3, providing one mechanism for the observed protective role of 25-hydroxyvitamin D in patients with breast cancer. PMID:19373868

  7. Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1alpha,25-dihydroxyvitamin D3.

    PubMed

    Sicinski, R R; DeLuca, H F

    1999-12-01

    Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1alpha-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 and 22-iodo-1alpha,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1alpha,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.

  8. 1α,25-Dihydroxyvitamin D3 Regulates Mitochondrial Oxygen Consumption and Dynamics in Human Skeletal Muscle Cells*

    PubMed Central

    Ryan, Zachary C.; Craig, Theodore A.; Folmes, Clifford D.; Wang, Xuewei; Lanza, Ian R.; Schaible, Niccole S.; Salisbury, Jeffrey L.; Nair, K. Sreekumaran; Terzic, Andre; Sieck, Gary C.; Kumar, Rajiv

    2016-01-01

    Muscle weakness and myopathy are observed in vitamin D deficiency and chronic renal failure, where concentrations of the active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), are low. To evaluate the mechanism of action of 1α,25(OH)2D3 in skeletal muscle, we examined mitochondrial oxygen consumption, dynamics, and biogenesis and changes in expression of nuclear genes encoding mitochondrial proteins in human skeletal muscle cells following treatment with 1α,25(OH)2D3. The mitochondrial oxygen consumption rate (OCR) increased in 1α,25(OH)2D3-treated cells. Vitamin D3 metabolites lacking a 1α-hydroxyl group (vitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) decreased or failed to increase OCR. 1α-Hydroxyvitamin D3 did not increase OCR. In 1α,25(OH)2D3-treated cells, mitochondrial volume and branching and expression of the pro-fusion protein OPA1 (optic atrophy 1) increased, whereas expression of the pro-fission proteins Fis1 (fission 1) and Drp1 (dynamin 1-like) decreased. Phosphorylated pyruvate dehydrogenase (PDH) (Ser-293) and PDH kinase 4 (PDK4) decreased in 1α,25(OH)2D3-treated cells. There was a trend to increased PDH activity in 1α,25(OH)2D3-treated cells (p = 0.09). 83 nuclear mRNAs encoding mitochondrial proteins were changed following 1α,25(OH)2D3 treatment; notably, PDK4 mRNA decreased, and PDP2 mRNA increased. MYC, MAPK13, and EPAS1 mRNAs, which encode proteins that regulate mitochondrial biogenesis, were increased following 1α,25(OH)2D3 treatment. Vitamin D receptor-dependent changes in the expression of 1947 mRNAs encoding proteins involved in muscle contraction, focal adhesion, integrin, JAK/STAT, MAPK, growth factor, and p53 signaling pathways were observed following 1α,25(OH)2D3 treatment. Five micro-RNAs were induced or repressed by 1α,25(OH)2D3. 1α,25(OH)2D3 regulates mitochondrial function, dynamics, and enzyme function, which are likely to influence muscle strength. PMID:26601949

  9. Parathyroid hormone suppression by intravenous 1,25-dihydroxyvitamin D. A role for increased sensitivity to calcium.

    PubMed Central

    Delmez, J A; Tindira, C; Grooms, P; Dusso, A; Windus, D W; Slatopolsky, E

    1989-01-01

    Numerous in vitro studies in experimental animals have demonstrated a direct suppressive effect of 1,25-dihydroxyvitamin D (1,25(OH)2D) on parathyroid hormone (PTH) synthesis. We therefore sought to determine whether such an effect could be demonstrated in uremic patients undergoing maneuvers designed to avoid changes in serum calcium concentrations. In addition, the response of the parathyroid gland in patients undergoing hypercalcemic suppression (protocol I) and hypocalcemic stimulation (protocol II) before and after 2 wk of intravenous 1,25(OH)2D was evaluated. In those enlisted in protocol I, PTH values fell from 375 +/- 66 to 294 +/- 50 pg (P less than 0.01) after 1,25(OH)2D administration. During hypercalcemic suppression, the "set point" (PTH max + PTH min/2) for PTH suppression by calcium fell from 5.24 +/- 0.14 to 5.06 +/- 0.15 mg/dl (P less than 0.05) with 1,25(OH)2D. A similar decline in PTH levels after giving intravenous 1,25(OH)2D was noted in protocol II patients. During hypocalcemic stimulation, the parathyroid response was attenuated by 1,25(OH)2D. We conclude that intravenous 1,25(OH)2D directly suppresses PTH secretion in uremic patients. This suppression, in part, appears to be due to increased sensitivity of the gland to ambient calcium levels. PMID:2703535

  10. Elevated 1,25-dihydroxyvitamin D levels in patients with chronic obstructive pulmonary disease treated with prednisone

    NASA Technical Reports Server (NTRS)

    Bikle, D. D.; Halloran, B.; Fong, L.; Steinbach, L.; Shellito, J.

    1993-01-01

    Glucocorticoid administration is a well established cause of osteopenia. Mechanisms underlying the deleterious effect of glucocorticoids on bone may include direct inhibition of bone formation as well as indirect effects through changes in intestinal calcium absorption, renal calcium excretion, and/or levels of the calciotropic hormones. To further examine the potential role of the calciotropic hormones we measured serum levels of PTH and 1,25 dihydroxyvitamin D [1,25(OH)2D], as well as serum and urine levels of calcium and vertebral bone density in patients with chronic obstructive pulmonary disease being managed with or without prednisone. Patients treated with prednisone had lower spinal bone density (53 vs. 106 mg/cm3) and higher serum calcium (2.40 vs. 2.33 mmol/l), urine calcium (6.9 vs. 2.7 mmol/24h), and 1,25(OH)2D levels (147 vs. 95 pmol/L). Compared to the patients not treated with glucocorticoids. PTH levels also tended to be higher (33 vs. 26 microliters-eq/ml), but the difference was not significant. Serum and urine calcium levels correlated positively with 1,25(OH)2D levels, but none of these measurements correlated with PTH levels. Our results suggest that prednisone treatment alters the regulation of 1,25(OH)2D production, and this may contribute to the loss of bone mineral induced by prednisone.

  11. CYP24 inhibition preserves 1α,25-dihydroxyvitamin D3 anti-proliferative signaling in lung cancer cells

    PubMed Central

    Zhang, Qiuhong; Kanterewicz, Beatriz; Buch, Shama; Petkovich, Martin; Parise, Robert; Beumer, Jan; Lin, Yan; Diergaarde, Brenda; Hershberger, Pamela A.

    2012-01-01

    Human lung tumors aberrantly express the 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3)-catabolizing enzyme, CYP24. We hypothesized that CYP24 reduces 1,25(OH)2D3-mediated transcription and allows lung cancer cells to escape its growth-inhibitory action. To test this, H292 lung cancer cells and the CYP24-selective inhibitor CTA091 were utilized. In H292 cells, CTA091 reduces 1,25(OH)2D3 catabolism, significantly increases 1,25(OH)2D3-mediated growth inhibition, and increases 1,25(OH)2D3 effects on induced and repressed genesin gene expression profiling studies. Pathway mapping of repressed genes uncovered cell cycle as a predominant 1,25(OH)2D3 target. In H292 cells, 1,25(OH)2D3 significantly decreases cyclin E2 levels and induces G0/G1 arrest. A broader set of cyclins is down-regulated when 1,25(OH)2D3 is combined with CTA091, and cell cycle arrest further increases. Effects of CTA091 on 1,25(OH)2D3 signaling are vitamin D receptor-dependent. These data provide evidence that CYP24 limits 1,25(OH)2D3 anti-proliferative signaling in cancer cells, and suggest that CTA091 may be beneficial in preserving 1,25(OH)2D3 action in lung cancer. PMID:22386975

  12. Dexamethasone and 1,25-dihydroxyvitamin D3 reduce oxidative stress-related DNA damage in differentiating osteoblasts.

    PubMed

    Pawlowska, Elzbieta; Wysokiński, Daniel; Tokarz, Paulina; Piastowska-Ciesielska, Agnieszka; Szczepanska, Joanna; Blasiak, Janusz

    2014-09-19

    The process of osteoblast differentiation is regulated by several factors, including RUNX2. Recent reports suggest an involvement of RUNX2 in DNA damage response (DDR), which is important due to association of differentiation with oxidative stress. In the present work we explore the influence of two RUNX2 modifiers, dexamethasone (DEX) and 1,25-dihydroxyvitamin D3 (1,25-D3), in DDR in differentiating MC3T3-E1 preosteoblasts challenged by oxidative stress. The process of differentiation was associated with reactive oxygen species (ROS) production and tert-butyl hydroperoxide (TBH) reduced the rate of differentiation. The activity of alkaline phosphatase (ALP), a marker of the process of osteoblasts differentiation, increased in a time-dependent manner and TBH further increased this activity. This may indicate that additional oxidative stress, induced by TBH, may accelerate the differentiation process. The cells displayed changes in the sensitivity to TBH in the course of differentiation. DEX increased ALP activity, but 1,25-D3 had no effect on it. These results suggest that DEX might stimulate the process of preosteoblasts differentiation. Finally, we observed a protective effect of DEX and 1,25-D3 against DNA damage induced by TBH, except the day 24 of differentiation, when DEX increased the extent of TBH-induced DNA damage. We conclude that oxidative stress is associated with osteoblasts differentiation and induce DDR, which may be modulated by RUNX2-modifiers, DEX and 1,25-D3.

  13. Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein

    NASA Technical Reports Server (NTRS)

    Kutner, A.; Link, R. P.; Schnoes, H. K.; DeLuca, H. F.

    1986-01-01

    3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

  14. An ochre mutation in the vitamin D receptor gene causes hereditary 1,25-dihydroxyvitamin D sub 3 -resistant rickets in three families

    SciTech Connect

    Ritchie, H.H.; Hughes, M.R.; Thompson, E.T.; Pike, J.W.; O'Malley, B.W. ); Malloy, P.J.; Feldman, D. ); Hochberg, Z. )

    1989-12-01

    Hereditary 1,25-dihydroxyvitamin D{sub 3}-resistant rickets is a rare autosomal-recessive disease resulting from target-organ resistance to the action of the active hormonal form of vitamin D. Four affected children from three related families with the classical syndrome of hereditary 1,25-dihydroxyvitamin D{sub 3}-resistant rickets and the absence of detectable binding to the vitamin D receptor (VDR) in cultured fibroblasts or lymphoblasts were examined for genetic abnormalities in the VDR gene. Genomic DNA from Epstein-Barr virus-transformed lymphoblasts of eight family members was isolated and amplified by polymerase chain reaction techniques. Amplified fragments containing the eight structural exons encoding the VDR protein were sequenced. The DNA from all affected children exhibited a single C {yields} A base substitution within exon 7 at nucleotide 970. Although the affected children were all homozygotic for the mutation, the four parents tested all exhibited both wild-type and mutant alleles, indicating a heterozygous state. Recreated mutant receptor exhibited no specific 1,25-({sup 3}H)dihydroxyvitamin D{sub 3} binding and failed to activate a cotransfected VDR promoter-reporter gene construct. Thus these findings identify an ochre mutation in a human steroid hormone receptor in patients with hereditary 1,25-dihydroxyvitamin D{sub 3}-resistant rickets.

  15. Vitamin D3 supports osteoclastogenesis via functional vitamin D response element of human RANKL gene promoter.

    PubMed

    Kitazawa, Sohei; Kajimoto, Kazuyoshi; Kondo, Takeshi; Kitazawa, Riko

    2003-07-01

    Receptor activator of NF-kappaB ligand (RANKL) has been identified as requisite for osteoclastogenesis. To elucidate the molecular mechanism that conducts its catabolic action on bone, the effect of 1alpha,25 dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) on osteoclastogenesis and RANKL mRNA expression was examined by coculture, RT-PCR and nuclear run-on studies. By accelerating the transcription rate of the RANKL gene in SaOS2 osteoblastic cells, 1alpha,25(OH)(2)D(3) enhanced in vitro osteoclast formation from peripheral monocytes. Cloning and characterization of the 5'-flanking region of the human RANKL gene revealed that the basic promoter comprises inverted TATA- and CAAT-boxes flanked by RUNX2 binding sites. Both electrophoresis mobility shift assay (EMSA) and transfection studies demonstrated that 1alpha,25(OH)(2)D(3) activated human RANKL promoter through vitamin D responsive elements (VDRE) located at -1584/-1570 by binding VDR and RXRalpha heterodimers in a ligand-dependent manner. The results provide direct evidence that 1alpha,25(OH)(2)D(3) augments osteoclastogenesis by transactivating the human RANKL gene in osteoblastic cells through VDRE.

  16. Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.

    PubMed Central

    Merke, J; Milde, P; Lewicka, S; Hügel, U; Klaus, G; Mangelsdorf, D J; Haussler, M R; Rauterberg, E W; Ritz, E

    1989-01-01

    Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population. Images PMID:2542376

  17. Mapping Variation in Cellular and Transcriptional Response to 1,25-Dihydroxyvitamin D3 in Peripheral Blood Mononuclear Cells

    PubMed Central

    Kariuki, Silvia N.; Maranville, Joseph C.; Baxter, Shaneen S.; Jeong, Choongwon; Nakagome, Shigeki; Hrusch, Cara L.; Witonsky, David B.; Sperling, Anne I.; Di Rienzo, Anna

    2016-01-01

    The active hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is an important modulator of the immune system, inhibiting cellular proliferation and regulating transcription of immune response genes. In order to characterize the genetic basis of variation in the immunomodulatory effects of 1,25D, we mapped quantitative traits of 1,25D response at both the cellular and the transcriptional level. We carried out a genome-wide association scan of percent inhibition of cell proliferation (Imax) induced by 1,25D treatment of peripheral blood mononuclear cells from 88 healthy African-American individuals. Two genome-wide significant variants were identified: rs1893662 in a gene desert on chromosome 18 (p = 2.32 x 10−8) and rs6451692 on chromosome 5 (p = 2.55 x 10−8), which may influence the anti-proliferative activity of 1,25D by regulating the expression of nearby genes such as the chemokine gene, CCL28, and the translation initiation gene, PAIP1. We also identified 8 expression quantitative trait loci at a FDR<0.10 for transcriptional response to 1,25D treatment, which include the transcriptional regulator ets variant 3-like (ETV3L) and EH-domain containing 4 (EHD4). In addition, we identified response eQTLs in vitamin D receptor binding sites near genes differentially expressed in response to 1,25D, such as FERM Domain Containing 6 (FRMD6), which plays a critical role in regulating both cell proliferation and apoptosis. Combining information from the GWAS of Imax and the response eQTL mapping enabled identification of putative Imax-associated candidate genes such as PAIP1 and the transcriptional repressor gene ZNF649. Overall, the variants identified in this study are strong candidates for immune traits and diseases linked to vitamin D, such as multiple sclerosis. PMID:27454520

  18. 1α, 25-Dihydroxyvitamin D₃ and the vitamin D receptor regulates ΔNp63α levels and keratinocyte proliferation.

    PubMed

    Hill, N T; Zhang, J; Leonard, M K; Lee, M; Shamma, H N; Kadakia, M

    2015-06-11

    1α, 25-dihydroxyvitamin D3 (VD3), a secosteriod that has been explored as an anti-cancer agent, was also shown to promote cell survival. Its receptor, the Vitamin D Receptor (VDR), is a direct target of the proto-oncogene ΔNp63α, which is overexpressed in non-melanoma skin cancers. The interconnection between VDR/VD3 signaling and ΔNp63α, led us to examine whether VDR/VD3 signaling promotes keratinocyte proliferation by regulating ΔNp63α levels. Our data demonstrate that VDR regulates ΔNp63α expression at both the transcript and protein level. Interestingly, although low doses of VD3 led to an increase in ΔNp63α protein levels and keratinocyte proliferation, high doses of VD3 failed to increase ΔNp63α protein levels and resulted in reduced proliferation. Increased expression of ΔNp63α by low dose VD3 was shown to be dependent on VDR and critical for the proliferative effects of VD3. VD3-mediated increases in ΔNp63α protein levels occur via activation of both p38 MAPK and Akt kinases. Finally, analysis of samples from patients with squamous cell carcinoma (SCC), basal cell carcinoma and precursors to invasive SCC demonstrated a significant correlation between p63 and VDR levels when compared with healthy normal skin control samples. Delineation of the mechanisms by which VD3 exerts its effect on ΔNp63α and cell proliferation is critical for determining the future of VD3 in cancer therapies.

  19. Biological Evaluation of Double Point Modified Analogues of 1,25-Dihydroxyvitamin D₂ as Potential Anti-Leukemic Agents.

    PubMed

    Corcoran, Aoife; Nadkarni, Sharmin; Yasuda, Kaori; Sakaki, Toshiyuki; Brown, Geoffrey; Kutner, Andrzej; Marcinkowska, Ewa

    2016-02-01

    Structurally similar double-point modified analogues of 1,25-dihydroxyvitamin D₂ (1,25D₂) were screened in vitro for their pro-differentiating activity against the promyeloid cell line HL60. Their affinities towards human full length vitamin D receptor (VDR) and metabolic stability against human vitamin D 24-hydroxylase (CYP24A1) were also tested. The analogues (PRI-1730, PRI-1731, PRI-1732, PRI-1733 and PRI-1734) contained 5,6-trans modification of the A-ring and of the triene system, additional hydroxyl or unsaturation at C-22 in the side chain and reversed absolute configuration (24-epi) at C-24 of 1,25D₂. As presented in this paper, introduction of selected structural modifications simultaneously in two distinct parts of the vitamin D molecule resulted in a divergent group of analogues. Analogues showed lower VDR affinity in comparison to that of the parent hormones, 1,25D₂ and 1,25D₃, and they caused effective HL60 cell differentiation only at high concentrations of 100 nM and above. Unexpectedly, introducing of a 5,6-trans modification combined with C-22 hydroxyl and 24-epi configuration switched off entirely the cell differentiation activity of the analogue (PRI-1734). However, this analogue remained a moderate substrate for CYP24A1, as it was metabolized at 22%, compared to 35% for 1,25D₂. Other analogues from this series were either less (12% for PRI-1731 and PRI-1733) or more (52% for PRI-1732) resistant to the enzymatic deactivation. Although the inactive analogue PRI-1734 failed to show VDR antagonism, when tested in HL60 cells, its structure might be a good starting point for our design of a vitamin D antagonist.

  20. Therapeutic effects of 1,25-dihydroxyvitamin D3 on diabetes-induced liver complications in a rat model

    PubMed Central

    LIU, LINA; LV, GUODONG; NING, CONGHUA; YANG, YE; ZHU, JUN

    2016-01-01

    It has been suggested that 1,25-dihydroxyvitamin D3 (vitamin D) plays a protective role against inflammation and insulin resistance (IR) in type 2 diabetes mellitus (T2DM). The present study investigate the hypothesis that vitamin D may exert beneficial effects on the liver in a rat model of T2DM by regulating the expression of inflammation-related cytokines and ameliorating IR induced by inflammation. Normal control group rats were fed a basic diet (NC). Experimental rats received a high-fat diet for 8 weeks and were then injected with streptozotocin (STZ) to induce T2DM. Half of the T2DM model rats received vitamin D (0.03 µg/kg/day) for 8 weeks (vitamin D-treated group; VD; n=11), while the other (T2DM group; DM; n=10) and NC group received an equivalent quantity of peanut oil. Following sacrifice, fasting plasma glucose (FPG) and fasting insulin (FINS) were recorded and homeostasis model assessment of IR (HOMA-IR) was calculated. Liver histopathology was examined using hematoxylin and eosin staining. The levels of the inflammatory cytokines C-Jun N-terminal kinase, C-Jun, tumor necrosis factor-α and interleukin-1β were measured using immunohistology, quantitative polymerase chain reaction and western blot analyses. The results revealed that treatment with vitamin D markedly alleviated the pathological alterations of liver and reduced the expression of inflammatory cytokines at the protein and mRNA levels. Furthermore, decreased levels of FPG, HOMA-IR and increased FINS were detected. In conclusion, the results of the present study indicate that vitamin D has therapeutic effects on diabetes-induced liver complications in T2DM model rats, which may involve the modulation of the inflammatory response, attenuating the crosstalk’ between inflammation and IR and ameliorating hyperglycemic state. PMID:27284312

  1. Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells.

    PubMed

    Ji, Shuhan; Doumit, Matthew E; Hill, Rodney A

    2015-01-01

    The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)2D3) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100 nM) of 1, 25-(OH)2D3 were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)2D3 were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)2D3. In response to 1, 25-(OH)2D3 (1, 10, and 100 nM), lipid accumulation and the expression of PPARγ, C/EBPα, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)2D3 tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBPα promoter activity response to 1, 25-(OH)2D3 was also tested, with no effect detected. These results indicate that 1, 25-(OH)2D3 inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPARγ activation or immediately up-stream thereof. Gene expression down-stream of PPARγ especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)2D3 in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity.

  2. Plasma 24,25-dihydroxyvitamin D concentration of Dahl salt-sensitive rats decreases during high salt intake

    NASA Technical Reports Server (NTRS)

    Thierry-Palmer, Myrtle; Tewolde, Teclemicael K.; Forte, Camille; Wang, Min; Bayorh, Mohamed A.; Emmett, Nerimiah L.; White, Jolanda; Griffin, Keri

    2002-01-01

    Dahl salt-sensitive rats, but not salt-resistant rats, develop hypertension in response to high salt intake. We have previously shown an inverse relationship between plasma 25-hydroxyvitamin D (25-OHD) concentration and blood pressure of Dahl salt-sensitive rats during high salt intake. In this study, we report on the relationship between high salt intake and plasma 24,25-dihydroxyvitamin D (24,25-(OH)(2)D) concentration of Dahl salt-sensitive and salt-resistant rats. Rats were fed a high salt diet (8%) and sacrificed at day 2, 7, 14, 21, and 28. Plasma 24,25-(OH)(2)D concentrations of salt-sensitive rats were reduced to 50% of that at baseline at day 2-when blood pressure and plasma 25-OHD concentration were unchanged, but 25-OHD content in the kidney was 81% of that at baseline. Plasma 24,25-(OH)(2)D concentration was reduced further to 10% of that at baseline from day 7 to 14 of high salt intake, a reduction that was prevented in rats switched to a low salt (0.3%) diet at day 7. Exogenous 24,25-dihydroxycholecalciferol (24,25-(OH)(2)D(3)), administered at a level that increased plasma 24,25-(OH)(2)D concentration to five times normal, did not attenuate the salt-induced hypertension of salt-sensitive rats. Plasma 24,25-(OH)(2)D concentration of salt-resistant rats was gradually reduced to 50% of that at baseline at day 14 and returned to baseline value at day 28 of high salt intake. We conclude that the decrease in plasma 24,25-(OH)(2)D concentration in salt-sensitive rats during high salt intake is caused by decreased 25-OHD content in the kidney and also by another unidentified mechanism.

  3. In vitro vitamin K(2) and 1α,25-dihydroxyvitamin D(3) combination enhances osteoblasts anabolism of diabetic mice.

    PubMed

    Poon, Christina C W; Li, Rachel W S; Seto, Sai Wang; Kong, Siu Kai; Ho, Ho Pui; Hoi, Maggie P M; Lee, Simon M Y; Ngai, Sai Ming; Chan, Shun Wan; Leung, George P H; Kwan, Yiu Wa

    2015-11-15

    In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K2 (10 nM) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (+/+) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K2 (10 nM) and 1,25(OH)2D3 (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K2 and 1,25(OH)2D3 co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K2 plus 1,25(OH)2D3 treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K2 plus D3 combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis.

  4. Low-calcium diets increase both production and clearance of 1,25-dihydroxyvitamin D3 in rats

    SciTech Connect

    Fox, J.; Bunker, J.E.; Kamimura, M.; Wong, P.F. )

    1990-02-01

    Administration of large doses of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to animals induces 1,25(OH)2D3 side-chain oxidative pathways. This study determined if the elevated plasma 1,25(OH)2D3 seen in rats fed low-Ca diets is associated not only with an increased production rate (PR) but also with an increased metabolic clearance rate (MCR) of the hormone. In vitamin D-replete rats fed a Ca-deficient diet for 3-4 wk, the PR increased 21-fold, plasma levels 15-fold, and the MCR by 37%. The increased MCR in Ca-deficient rats was associated with a 48% increase in hepatic microsomal UDP glucuronyl transferase enzyme activity, whereas 1,25(OH)2D3 catabolism by homogenates of liver and small intestinal mucosa was unchanged. In contrast to the effects of low-Ca diets, acute (7 h) pharmacological elevation of plasma 1,25(OH)2D3 to 1.5 ng/ml in normal rats did not influence the MCR. Thus chronically elevated 1,25(OH)2D3 levels are necessary to stimulate clearance. In conclusion, 1,25(OH)2D3 clearance in rats can be stimulated not only by chronic pharmacological doses of 1,25(OH)2D3 but also by the physiological stimulus of a low-Ca diet. Hence, plasma 1,25(OH)2D3 levels can be regulated by changes in both PR and MCR.

  5. Biological Evaluation of Double Point Modified Analogues of 1,25-Dihydroxyvitamin D2 as Potential Anti-Leukemic Agents

    PubMed Central

    Corcoran, Aoife; Nadkarni, Sharmin; Yasuda, Kaori; Sakaki, Toshiyuki; Brown, Geoffrey; Kutner, Andrzej; Marcinkowska, Ewa

    2016-01-01

    Structurally similar double-point modified analogues of 1,25-dihydroxyvitamin D2 (1,25D2) were screened in vitro for their pro-differentiating activity against the promyeloid cell line HL60. Their affinities towards human full length vitamin D receptor (VDR) and metabolic stability against human vitamin D 24-hydroxylase (CYP24A1) were also tested. The analogues (PRI-1730, PRI-1731, PRI-1732, PRI-1733 and PRI-1734) contained 5,6-trans modification of the A-ring and of the triene system, additional hydroxyl or unsaturation at C-22 in the side chain and reversed absolute configuration (24-epi) at C-24 of 1,25D2. As presented in this paper, introduction of selected structural modifications simultaneously in two distinct parts of the vitamin D molecule resulted in a divergent group of analogues. Analogues showed lower VDR affinity in comparison to that of the parent hormones, 1,25D2 and 1,25D3, and they caused effective HL60 cell differentiation only at high concentrations of 100 nM and above. Unexpectedly, introducing of a 5,6-trans modification combined with C-22 hydroxyl and 24-epi configuration switched off entirely the cell differentiation activity of the analogue (PRI-1734). However, this analogue remained a moderate substrate for CYP24A1, as it was metabolized at 22%, compared to 35% for 1,25D2. Other analogues from this series were either less (12% for PRI-1731 and PRI-1733) or more (52% for PRI-1732) resistant to the enzymatic deactivation. Although the inactive analogue PRI-1734 failed to show VDR antagonism, when tested in HL60 cells, its structure might be a good starting point for our design of a vitamin D antagonist. PMID:26840307

  6. Antiproliferative effects of 1alpha,25-dihydroxyvitamin D(3) and vitamin D analogs on tumor-derived endothelial cells.

    PubMed

    Bernardi, Ronald J; Johnson, Candace S; Modzelewski, Ruth A; Trump, Donald L

    2002-07-01

    Although there is abundant evidence that 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] inhibits the growth of several cancer cell types, inhibition of angiogenesis may also play a role in mediating the antitumor effects of 1,25-(OH)(2)D(3.) We examined the ability of 1,25-(OH)(2)D(3) to inhibit the growth of tumor-derived endothelial cells (TDECs) and normal endothelial cells and to modulate angiogenic signaling. 1,25-(OH)(2)D(3) inhibited the growth of TDECs from two tumor models at nanomolar concentrations, but was less potent against normal aortic or yolk sac endothelial cells. The vitamin D analogs Ro-25-6760, EB1089, and ILX23-7553 were also potent inhibitors of TDEC proliferation. Furthermore, the combination of 1,25-(OH)(2)D(3) and dexamethasone had greater activity than either agent alone. 1,25-(OH)(2)D(3) increased vitamin D receptor and p27(Kip1) protein levels in TDECs, whereas phospho-ERK1/2 and phospho-Akt levels were reduced. These changes were not observed in normal aortic endothelial cells. In squamous cell carcinoma and radiation-induced fibrosarcoma-1 cells, 1,25-(OH)(2)D(3) treatment caused a reduction in the angiogenic signaling molecule, angiopoietin-2. In conclusion, 1,25-(OH)(2)D(3) and its analogs directly inhibit TDEC proliferation at concentrations comparable to those required to inhibit tumor cells. Further, 1,25-(OH)(2)D(3) modulates cell cycle and survival signaling in TDECs and affects angiogenic signaling in cancer cells. Thus, our work supports the hypothesis that angiogenesis inhibition plays a role in the antitumor effects of 1,25-(OH)(2)D(3).

  7. 1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice

    NASA Technical Reports Server (NTRS)

    Bedalov, A.; Salvatori, R.; Dodig, M.; Kapural, B.; Pavlin, D.; Kream, B. E.; Clark, S. H.; Woody, C. O.; Rowe, D. W.; Lichtler, A. C.

    1998-01-01

    We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

  8. Effects of vitamin D analog, 22-oxa-1,25-dihydroxyvitamin D(3), on bone reconstruction by vascularized bone allograft.

    PubMed

    Merida, L; Shigetomi, M; Ihara, K; Tsubone, T; Ikeda, K; Yamaguchi, A; Sugiyama, T; Kawai, S

    2002-02-01

    We previously reported that vascularized bone allograft using immunosuppressants, such as cyclosporine A (CsA), is one approach for reconstruction of large bone defects in both experimental animals (Microsurgery 15:663; 1994) and clinically in humans (Lancet 347:970, 1996). Because immunosuppressive agents such as CsA induce significant side effects, including bone loss, other therapeutic agents supporting successful vascularized bone allografts have been sought after. We investigated the effects of 22-oxa-1,25-dihydroxyvitamin D(3) (OCT) on vascularized bone allograft, and compared its effects with CsA. Twelve-week-old DA rats with the major histocompatibility antigen (MHC) RT-1(a) were used as donors and age-matched Lewis rats with MHC RT-1(l) used as recipients. Allografted bones in rats treated with vehicle were rejected completely. Soft X-ray examination demonstrated that administration of OCT (0.5 microg/kg per day) for 12 weeks after bone graft induced bone union as effective as treatment for 12 weeks with CsA (10 mg/kg per day). Transplanted bones in OCT-treated rats showed higher bone mineral density than that in CsA-treated rats. Histologically, transplanted bones in OCT-treated rats at 12 weeks were nonvital, but these bones united with recipient vital bones. After cessation of 12 week treatment with OCT, new bone formation occurred around the grafted nonvital bones during a 9 month period. Transplanted bones in CsA-treated rats were vital and formed union with recipient bones, whereas cortical bones became thin when compared with nonvital bones in OCT-treated rats. Urinary deoxypyridinoline levels in rats treated with CsA were significantly higher than levels in rats treated with OCT, suggesting accelerated bone resorption in CsA-treated rats. These results suggest that OCT exerts an anabolic action on bone reconstruction by allogeneic bone transplantation.

  9. 1,25-dihydroxyvitamin D{sub 3} impairs NF-{kappa}B activation in human naive B cells

    SciTech Connect

    Geldmeyer-Hilt, Kerstin; Heine, Guido; Hartmann, Bjoern; Baumgrass, Ria; Radbruch, Andreas; Worm, Margitta

    2011-04-22

    Highlights: {yields} In naive B cells, VDR activation by calcitriol results in reduced NF-{kappa}B p105 and p50 protein expression. {yields} Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-{kappa}B p65. {yields} Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. {yields} Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1{alpha},25-dihydroxyvitamin D{sub 3} (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-{kappa}B p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-{kappa}B mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-{kappa}B activation by interference with NF-{kappa}B p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.

  10. Effects of Extracellular Calcium and 1,25 dihydroxyvitamin D3 on Sebaceous Gland Cells In vitro and In vivo.

    PubMed

    Zouboulis, Christos C; Seltmann, Holger; Abdel-Naser, M Badawy; Hossini, Amir M; Menon, Gopinathan K; Kubba, Raj

    2017-03-10

    Calcium and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) are promoters of epithelial cell functions; however their effects on sebaceous glands are unknown. In this study, morphology, ultrastructure, cell numbers, lipid synthesis and apoptosis of SZ95 sebocytes were assessed in vitro under different concentrations of extracellular calcium with or without 1,25(OH)2D3. Moreover, serum calcium and 1,25(OH)2D3 levels were assessed in acne and non-acne patients (controls). Under conditions of low extracellular calcium, lipogenesis and cell detachment were observed. Increasing extracellular calcium enhanced sebocyte numbers, induced epithelial morphology and reduced lipogenesis. Moreover, a reduction in extracellular calcium reduced E-cadherin and enhanced caspase 3/7 activity (apoptosis), whereas calcium chelation by EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) resulted in enhanced lipogenesis. 1,25(OH)2D3 decreased sebaceous lipogenesis, but also induced signs of autophagy. In the clinical study, patients and controls exhibited normal serum calcium levels. Younger acne patients presented lower 1,25(OH)2D3 levels than did older ones. In conclusion, extracellular calcium and 1,25(OH)2D3 regulate sebocyte morphology, increase cell numbers, decrease sebaceous lipogenesis and induce cell autophagy in vitro. The increased ionized calcium and the reduced 1,25(OH)2D3 levels detected in the serum of younger patients with acne may contribute respectively to increased sebaceous gland volume and enhanced lipogenesis.

  11. Suppression of epithelial ovarian cancer invasion into the omentum by 1α,25-dihydroxyvitamin D3 and its receptor

    PubMed Central

    Lungchukiet, Panida; Sun, Yuefeng; Kasiappan, Ravi; Quarni, Waise; Nicosia, Santo V.; Zhang, Xiaohong; Bai, Wenlong

    2015-01-01

    Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer death in women, mainly because it has spread to intraperitoneal tissues such as the omentum in the peritoneal cavity by the time of diagnosis. In the present study, we established in vitro assays, ex vivo omental organ culture system and syngeneic animal tumor models using wild type (WT) and vitamin D receptor (VDR) null mice to investigate the effects of 1α,25-dihydroxyvitamin D3 (1,25D3) and VDR on EOC invasion. Treatment of human EOC cells with 1,25D3 suppressed their migration and invasion in monolayer scratch and transwell assays and ability to colonize the omentum in the ex vivo system, supporting a role for epithelial VDR in interfering with EOC invasion. Furthermore, VDR knockdown in OVCAR3 cells increased their ability to colonize the omentum in the ex vivo system in the absence of 1,25D3, showing a potential ligand-independent suppression of EOC invasion by epithelial VDR. In syngeneic models, ID8 tumors exhibited an increased ability to colonize omenta of VDR null over that of WT mice; pre-treatment of WT, not VDR null, mice with EB1089 reduced ID8 colonization, revealing a role for stromal VDR in suppressing EOC invasion. These studies are the first to demonstrate a role for epithelial and stromal VDR in mediating the activity of 1,25D3 as well as a 1,25D3-independent action of the VDR in suppressing EOC invasion. The data suggest that VDR-based drug discovery may lead to the development of new intervention strategies to improve the survival of patients with EOC at advanced stages. This article is part of a Special Issue entitled “Vitamin D Workshop”. PMID:25448740

  12. 1,25-Dihydroxyvitamin D Modulates Antibacterial and Inflammatory Response in Human Cigarette Smoke-Exposed Macrophages.

    PubMed

    Heulens, Nele; Korf, Hannelie; Mathyssen, Carolien; Everaerts, Stephanie; De Smidt, Elien; Dooms, Christophe; Yserbyt, Jonas; Gysemans, Conny; Gayan-Ramirez, Ghislaine; Mathieu, Chantal; Janssens, Wim

    2016-01-01

    Cigarette smoking is associated with increased inflammation and defective antibacterial responses in the airways. Interestingly, vitamin D has been shown to suppress inflammation and to improve antibacterial defense. However, it is currently unknown whether vitamin D may modulate inflammation and antibacterial defects in human cigarette smoke (CS)-exposed airways. To explore these unresolved issues, alveolar macrophages obtained from non-smoking and smoking subjects as well as human cigarette smoke extract (CSE)-treated THP-1 macrophages were stimulated with 1,25-dihydroxyvitamin D (1,25(OH)2D) to address inflammatory and antibacterial responses. Although basal levels of inflammatory cytokines and chemokines did not differ between non-smoking and smoking subjects, 1,25(OH)2D did reduce levels of IL-6, TNF-α and MCP-1 in alveolar macrophages in response to LPS/IFN-γ, although not statistically significant for TNF-α and IL-6 in smokers. CSE did not significantly alter vitamin D metabolism (expression levels of CYP24A1 or CYP27B1) in THP-1 macrophages. Furthermore, stimulation with 1,25(OH)2D reduced mRNA expression levels and/or protein levels of IL-8, TNF-α and MCP-1 in CSE-treated THP-1 macrophages. 1,25(OH)2D did not improve defects in phagocytosis of E. coli bacteria or the oxidative burst response in CSE-treated THP-1 macrophages or alveolar macrophages from smokers. However, 1,25(OH)2D significantly enhanced mRNA expression and/or protein levels of the antimicrobial peptide cathelicidin in alveolar macrophages and THP-1 macrophages, independently of CS exposure. In conclusion, our results provide the first evidence that vitamin D could be a new strategy for attenuating airway inflammation and improving antibacterial defense in CS-exposed airways.

  13. 24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats

    PubMed Central

    Boyan, Barbara D.; Hyzy, Sharon L.; Pan, Qingfen; Scott, Kayla M.; Coutts, Richard D.; Healey, Robert; Schwartz, Zvi

    2016-01-01

    Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OH)D3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] accumulates in articular cartilage following injection of [3H]-25(OH)D3. Previously, we showed that 24R,25(OH)2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH)2D3 to prevent degenerative changes in articular cartilage in an OA-like environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1β with and without 24R,25(OH)2D3 or 1α,25(OH)2D3. 24R,25(OH)2D3 but not 1α,25(OH)2D3 blocked the effects of IL-1β in a dose-dependent manner, and its effect was partially mediated through the TGF-β1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)2D3 or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis. PMID:27575371

  14. Ethacrynic acid and 1 alpha,25-dihydroxyvitamin D3 cooperatively inhibit proliferation and induce differentiation of human myeloid leukemia cells.

    PubMed

    Makishima, M; Honma, Y

    1996-09-01

    The active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 (VD3), inhibits proliferation and induces differentiation of leukemia cells, but its clinical use is limited by the adverse effect of hypercalcemia. In this study we found that the loop diuretic ethacrynic acid, which is used to treat hypercalcemia, enhanced the differentiation of human leukemia cells induced by VD3. Ethacrynic acid alone inhibited the proliferation of human promyelocytic HL-60 cells while only slightly increasing differentiation markers such as nitroblue tetrazolium (NBT)-reducing and lysozyme activities. Ethacrynic acid effectively enhanced the growth-inhibiting action of VD3. In the presence of ethacrynic acid, VD3 increased the NBT-reducing and lysozyme activities and the CD11b expression of HL-60 cells more effectively than VD3 alone. Other loop diuretics, furosemide and bumetanide, also enhanced the differentiation of HL-60 cells induced by VD3, but to a lesser extent than ethacrynic acid. The differentiation of HL-60 cells induced by all-trans retinoic acid, dimethyl sulfoxide or phorbol-12-myristate 13-acetate was also enhanced by ethacrynic acid with increasing NBT-reducing and lysozyme activities and the expression of CD11b or CD14 surface antigen. Morphologically, ethacrynic acid enhanced the monocytic differentiation of HL-60 cells induced by VD3 and phorbol ester and the granulocytic differentiation by retinoic acid and dimethyl sulfoxide. Other human myelomonocytic leukemia ML-1, U937, P39/TSU and P31/FUJ cells were induced to differentiate by VD3 and this was also enhanced by ethacrynic acid. The long-term culture of HL-60 cells showed that ethacrynic acid plus VD3 induced the complete growth arrest of HL-60 cells. Therefore ethacrynic acid, which is used to treat hypercalcemia, enhanced the proliferation-inhibiting and differentiation-inducing activities of VD3 and the combination of ethacrynic acid and VD3 may be useful in therapy for myeloid leukemia.

  15. 1α,25-Dihydroxyvitamin D3 Inhibits Esophageal Squamous Cell Carcinoma Progression by Reducing IL6 Signaling.

    PubMed

    Chen, Ping-Tsung; Hsieh, Ching-Chuan; Wu, Chun-Te; Yen, Tzu-Chen; Lin, Paul-Yang; Chen, Wen-Cheng; Chen, Miao-Fen

    2015-06-01

    The aim of this study was to highlight the role of 1α,25-dihydroxyvitamin D3 (calcitriol) in esophageal squamous cell carcinoma (SCC). The human esophageal SCC cell lines CE81T and TE2 were selected for cellular and animal experiments to investigate the changes in tumor behavior after calcitriol supplementation and the underlying mechanisms. Moreover, we evaluated the relationship between calcitriol supplementation, myeloid-derived suppressor cell (MDSC) recruitment, IL6 levels, and tumor progression by a 4-nitroquinoline 1-oxide (4-NQO)-induced esophageal tumor animal model. In this study, we demonstrated that calcitriol supplementation inhibited aggressive tumor behavior both in vitro and in vivo. The underlying changes included increased cell death, a lower degree of epithelial-mesenchymal transition, and inhibited IL6 signaling. In the 4-NQO-induced esophageal tumor animal model, increased IL6 and MDSC recruitment were linked with invasive esophageal tumors. Supplementation with calcitriol attenuated the level of IL6, the induction of MDSCs, and the incidence of 4-NQO-induced invasive tumors. Moreover, the IL6-induced changes in C57 mice, including augmented MDSC recruitment, increased levels of ROS and p-Stat3 in MDSCs, and higher suppressive function of MDSCs in T-cell proliferation, which were abrogated by calcitriol supplementation. On the basis of our results, we concluded that calcitriol abrogated the IL6-induced aggressive tumor behavior and MDSC recruitment to inhibit esophageal tumor promotion. Therefore, we suggest that supplementation with vitamin D3 may be a promising strategy for the prevention and treatment of esophageal SCC.

  16. Role of carbonic anhydrase in bone resorption induced by 1,25 dihydroxyvitamin D3 in vitro

    NASA Technical Reports Server (NTRS)

    Hall, G. E.; Kenny, A. D.

    1985-01-01

    The calvaria of 5-to-6-day-old mice treated with 1 x 10 to the -8th M of 1,25(OH)2D3 in vitro for 48 hours are examined in order to study the function of carbonic anhydrase in bone resorption. Calcium concentrations in the culture were measured to assess bone resorption. It is observed that 1,25(OH)2D3 effectively stimulates bone resorption in vitro and the resorption is dose-dependent. The effects of azetazolamide on 1,25(OH)2D3-induced bone resorption are investigated. The data reveal that 1,25(OH)2D3-induced calcium release is associated with an increase in the carbonic anhydrase activity of bone, and bone alkaline phosphatase activity is decreased and acid phosphatase activity is increased in response to 1,25(OH)2D3. A two-fold mechanism for 1,25(OH)2D3-induced bone resorption is proposed; the first mechanism is an indirect activation of osteoclasts and the second involves an interaction between hormone and osteoclast precursors.

  17. Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) metabolism in vitamin D-deficient rats infused with 1,25-(OH)2D3

    SciTech Connect

    Yamato, H.; Matsumoto, T.; Fukumoto, S.; Ikeda, K.; Ishizuka, S.; Ogata, E.

    1989-01-01

    Previous studies revealed that administration of 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) to calcium (Ca)-deficient rats causes a dose-dependent reduction in markedly elevated serum 1,25-(OH)2D3 level. Although the results suggested that the metabolism of 1,25-(OH)2D3 was accelerated by 24,25-(OH)2D3, those experiments could not define whether the enhanced metabolism of 1,25-(OH)2D3 played a role in the reduction in the serum 1,25-(OH)2D3 level. In the present study, in order to address this issue more specifically, serum 1,25-(OH)2D3 was maintained solely by exogenous administration through miniosmotic pumps of 1,25-(OH)2D3 into vitamin D-deficient rats. Thus, by measuring the serum 1,25-(OH)2D3 concentration, the effect of 24,25-(OH)2D3 on the MCR of 1,25-(OH)2D3 could be examined. Administration of 24,25-(OH)2D3 caused a dose-dependent enhancement in the MCR of 1,25-(OH)2D3, and 1 microgram/100 g rat.day 24,25-(OH)2D3, which elevated serum 24,25-(OH)2D3 to 8.6 +/- 1.3 ng/ml, significantly increased MCR and suppressed serum levels of 1,25-(OH)2D3. The effect of 24,25-(OH)2D3 on 1,25-(OH)2D3 metabolism developed with a rapid time course, and the recovery of iv injected (1 beta-3H)1,25-(OH)2D3 in blood was significantly reduced within 1 h. In addition, there was an increase in radioactivity in the water-soluble fraction of serum as well as in urine, suggesting that 1,25-(OH)2D3 is rapidly degraded to a water-soluble metabolite(s). Furthermore, the reduction in serum 1,25-(OH)2D3 was associated with a reduction in both serum and urinary Ca levels. Because the conversion of (3H)24,25-(OH)2D3 to (3H)1,24,25-(OH)2D3 or other metabolites was minimal in these rats, 24,25-(OH)2D3 appears to act without being converted into other metabolites. These results demonstrate that 24,25-(OH)2D3 rapidly stimulates the metabolism of 1,25-(OH)2D3 and reduces its serum level.

  18. 24,25-dihydroxyvitamin D3 and Vitamin D Status of Community Dwelling Black and White Americans

    PubMed Central

    Berg, Anders H.; Powe, Camille E.; Evans, Michele K.; Wenger, Julia; Ortiz, Guillermo; Zonderman, Alan B.; Suntharalingam, Pirianthini; Lucchesi, Kathryn; Powe, Neil R.; Karumanchi, S. Ananth; Thadhani, Ravi I.

    2015-01-01

    BACKGROUND 24,25-dihydroxyvitamin D (24,25(OH)2D) is a metabolite of 25-hydroxyvitamin D (25D). Blacks frequently have low total 25D without manifestations of vitamin D deficiency, suggesting that total serum 25D may incorrectly reflect vitamin D status in different racial groups. The ratio of serum 24,25(OH)2D to 25D (Vitamin D Metabolite Ratio [VMR]) represents a new candidate biomarker for vitamin D status. METHODS We measured 24,25(OH)2D3 and 25D3 by mass spectrometry in a random community cohort of black (n=212) and white (n=164) Americans to evaluate VMR as a marker for vitamin D status. We measured parathyroid hormone concentrations by immunoassay to compare VMR and 25D3 against a physiological indicator of vitamin D deficiency. RESULTS Serum 24,25(OH)2D3 strongly correlated with 25D3 in both black and white subjects (r = 0.90, p<0.001 and r = 0.86, p<0.001 respectively). Blacks had lower mean 25D3 than whites (17.0±7.8 vs. 27.5±11.3 ng/mL (42.4±19.5 vs. 68.6±28.2 nmol/L), p<0.001) and lower mean 24,25(OH)2D3 (2.1±1.3 vs. 3.6±2.0 ng/mL (5.1±3.1 vs. 8.7±4.8 nmol/L)), p<0.001). In contrast to total 25D3 concentrations, mean VMR values were similar in blacks and whites (11.9±4.0 vs. 12.5±3.4, p=0.16, respectively) and were negatively correlated with parathyroid hormone concentrations in both races (rs= −0.26, p<0.001 and rs= −0.25, p<0.001, respectively). CONCLUSIONS Our results provide further evidence that measurement of total 25D for assessment of vitamin D status in patients of African descent deserves reevaluation, and suggests that alternative measures such as VMR should be considered. PMID:25922442

  19. 1,25-Dihydroxyvitamin D3 Does Not Affect MicroRNA Expression When Suppressing Human Th17 Differentiation

    PubMed Central

    Huang, Jian; Liang, Zibin; Kuang, Ying; Jia, Fujie; Yang, Yaqi; Kang, Miaomiao; Xie, Muke; Li, Feng

    2017-01-01

    Background Vitamin D is an import regulator of T helper 17 (Th17) differentiation, but our understanding of the underlying mechanisms remains limited. In the present study, we aimed to detect the expression levels of microRNAs (miRNAs) during human Th17 differentiation and evaluate the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the bioactive form of vitamin D, on Th17 differentiation and miRNA expression. Material/Methods We cultured human peripheral blood mononuclear cells (PBMC) in vitro and activated them with anti-CD3 and anti-CD28 antibodies in the presence of Th17-promoting cytokines interleukin (IL)-23, IL-1β, TGF-β1, and IL-6 for 72 hours. 1,25(OH)2D3 was added to the medium at a final concentration of 100 nM on day 0. The production of IL-17A in culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of miRNAs during Th17 differentiation were determined by quantitative polymerase chain reaction (qPCR). Results Six miRNAs were found to be dysregulated during human Th17 differentiation. Of these miRNAs, hsa-miR-155 was significantly up-regulated (median fold change: 3.61, P<0.05), whereas hsa-miR-20b, hsa-miR-21, hsa-miR-181a, hsa-miR-210, and hsa-miR-301a were significantly down-regulated (median fold change: 0.44, 0.37, 0.18, 0.15, and 0.26, respectively, P<0.05). 1,25(OH)2D3 treatment significantly decreased IL-17A production (median [interquartile range], 745.7 [473.5] pg/mL vs. 2535.4 [2153.3] pg/mL, P<0.05). However, expression of these miRNAs was not changed after 1,25(OH)2D3 treatment. Conclusions 1,25(OH)2D3 suppressed human Th17 differentiation without affecting miRNA expression. PMID:28133358

  20. Role of 1,25-Dihydroxyvitamin D3 on Intestinal Phosphate Absorption in Rats with a Normal Vitamin D Supply

    PubMed Central

    Rizzoli, R.; Fleisch, H.; Bonjour, J-P.

    1977-01-01

    In vitamin D-deficient rats, impaired intestinal phosphorus (P) absorption can be corrected by 1,25-dihydroxyvitamin D3[1,25-(OH)2D3]. In the present study, it was investigated whether changes in 1,25-(OH)2D3 production can influence intestinal P transport also in animals with a normal supply of vitamin D. The intestinal P absorption was evaluated in rats using both the in situ duodenal loop technique and the determination of the overall gastrointestinal absorption under three conditions known to influence the production of 1,25-(OH)2D3: (a) variation in dietary P, (b) thyroparathyroidectomy (TPTX) with or without administration of parathyroid hormone (PTH), and (c) treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). In all circumstances changes in duodenal absorption paralleled the changes in the overall fractional absorption. (a) Lowering dietary P stimulated P absorption. (b) TPTX decreased P absorption. This effect was corrected either by the administration of PTH or by the administration of 1,25-(OH)2D3. (c) EHDP, when given at a dose known to inhibit 1,25-(OH)2D3 formation, decreased the duodenal P absorption in both intact and TPTX animals. This effect was corrected by 1,25-(OH)2D3. In the TPTX-EHDP-treated animals, the administration of PTH did not rectify the low duodenal P absorption. These results support the thesis that, in rats with normal vitamin D supply, variations in the endogenous production of 1,25-(OH)2D3 change the rate of P absorption. However, these changes are in such magnitude that they are of relatively small importance when compared to the effect of variation in the dietary intake of P. These results also strongly suggest that the action of PTH on duodenal P transport is mediated by its effect on 1,25-(OH)2D3 production, inasmuch as the effect of the hormone is abolished after blocking the renal 1-hydroxylation with EHDP. PMID:893667

  1. Gene-by-Diet Interactions Affect Serum 1,25-Dihydroxyvitamin D Levels in Male BXD Recombinant Inbred Mice

    PubMed Central

    Replogle, Rebecca A.; Reyes-Fernandez, Perla; Wang, Libo; Zhang, Min; Clinkenbeard, Erica L.; White, Kenneth E.

    2016-01-01

    1,25-Dihydroxyvitamin D (1,25[OH]2D) regulates calcium (Ca), phosphate, and bone metabolism. Serum 1,25(OH)2D levels are reduced by low vitamin D status and high fibroblast growth factor 23 (FGF23) levels and increased by low Ca intake and high PTH levels. Natural genetic variation controls serum 25-hydroxyvitamin D (25[OH]D) levels, but it is unclear how it controls serum 1,25(OH)2D or the response of serum 1,25(OH)2D levels to dietary Ca restriction (RCR). Male mice from 11 inbred lines and from 51 BXD recombinant inbred lines were fed diets with either 0.5% (basal) or 0.25% Ca from 4 to 12 weeks of age (n = 8 per line per diet). Significant variation among the lines was found in basal serum 1,25(OH)2D and in the RCR as well as basal serum 25(OH)D and FGF23 levels. 1,25(OH)2D was not correlated to 25(OH)D but was negatively correlated to FGF23 (r = −0.5). Narrow sense heritability of 1,25(OH)2D was 0.67 on the 0.5% Ca diet, 0.66 on the 0.25% Ca diet, and 0.59 for the RCR, indicating a strong genetic control of serum 1,25(OH)2D. Genetic mapping revealed many loci controlling 1,25(OH)2D (seven loci) and the RCR (three loci) as well as 25(OH)D (four loci) and FGF23 (two loci); a locus on chromosome 18 controlled both 1,25(OH)2D and FGF23. Candidate genes underlying loci include the following: Ets1 (1,25[OH]2D), Elac1 (FGF23 and 1,25[OH]2D), Tbc1d15 (RCR), Plekha8 and Lyplal1 (25[OH]D), and Trim35 (FGF23). This report is the first to reveal that serum 1,25(OH)2D levels are controlled by multiple genetic factors and that some of these genetic loci interact with the dietary environment. PMID:26587785

  2. 1,25-Dihydroxyvitamin D3-glycoside of herbal origin exhibits delayed release pharmacokinetics when compared to its synthetic counterpart.

    PubMed

    Bachmann, Heinrich; Offord-Cavin, Elizabeth; Phothirath, Phoukham; Horcajada, Marie-Noelle; Romeis, Peter; Mathis, Georg A

    2013-07-01

    Vitamin D requires two metabolic steps to become biologically active. In a first step 25-hydroxyvitamin D3 is formed, which acts as storage form. After a tightly controlled step in kidney the active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is formed. Because kidney is the relevant metabolic organ for this conversion, 1,25(OH)2D3 needs to be supplemented in patients with kidney malfunction or kidney failure. Synthetic 1,25(OH)2D3 (calcitriol) has been available as a drug for decades. Due to its high potency and its kinetic profile (fast absorption and rapid elimination) its therapeutic windows has proven to be relatively narrow. A natural form of the active metabolite was identified in a few plants, such as Solanum glaucophyllum (SG) and suggested as alternative for animal and human health. An extract of a SG variety bred for high and uniform level of glycosylated 1,25(OH)2D3 was chemically characterized. Among the typical pharmaceutically inactive plant components (carbohydrates 54.3%, protein 24.9%, minerals 17.1% and water 4.1%) high levels of 1,25(OH)2D3 and a unique flavonoid content was found (1.11mg total quercetin/g extract) consisting exclusively of the quercetin glycosides hyperoside, isoquercetin, rutin and apinosylrutin. The molecular distribution of glycosyl moieties in 1,25(OH)2D3 extracted from SG as determined by gel permeation chromatography was found to be 1-10 hexose units per aglycone. 1,25(OH)2D3-1-β-glucopyranoside was identified in the SG extract, while a di- and triglycoside have been identified in SG by other groups. The pharmacokinetic properties of synthetic 1,25(OH)2D3 and glycosylated 1,25(OH)2D3 extracted from SG were compared in male rats. When compared to synthetic 1,25(OH)2D3, SG-derived 1,25(OH)2D3 exhibited delayed absorption and elimination characteristics, resulting in delayed Tmax (6-12h vs. 1h) and increased T½ (approximately 30h vs. 23h). This putative modified release pattern may be attributed to the glycosylation

  3. 1,25-Dihydroxyvitamin D3-mediated vesicular transport of calcium in intestine: time-course studies

    SciTech Connect

    Nemere, I.; Norman, A.W.

    1988-06-01

    Previous work has biochemically identified lysosomes containing calcium and calbindin-D28K (CaBP) in chick intestine that are sensitive to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) status. In the present work, lysosomal accumulation of 45Ca was optimal after 30 min of absorption from in situ ligated duodenal loops. The areas under the curves, defined as lysosomal fractions in Percoll gradients, were calculated, and values after 10, 20, 30, and 40 min of transport were (+D/-D ratio) 0.90, 1.62, 1.88, and 1.78, respectively. Lysosomal CaBP also increased in parallel with the time of absorption and was not due to nonspecific adsorption. When lysosomal 45Ca was determined 2.5, 5, 10, 15, and 43 h after administration of 1.3 nmol 1,25-(OH)2D3 or vehicle, the area ratios were 1.02, 1.47, 3.10, 1.88, and 1.29, respectively. Analyses of serum 45Ca in the same birds yielded a closely parallel time course with 1,25-(OH)2D3-dependent intestinal calcium absorption; values were 108 +/- 12% (+/- SE), 164 +/- 29%, 300 +/- 35%, 340 +/- 39%, and 169 +/- 8% of vitamin D-deficient control values at 2.5, 5, 10, 15, and 43 h, respectively. Immunoreactive CaBP in lysosomal fractions did not change significantly between 5-43 h after administration of seco-steroid. A similar series of experiments was conducted with microsomal membranes containing putative endocytic vesicles, which are believed to deliver calcium to the lysosomes. The brush border origin of the vesicles was supported by the internalization of anti-CaBP immunoglobulin G after 3 min of absorption. Accumulation of 45Ca by endocytic vesicles was subsequently found to be maximal after 20 min of absorption (+D/-D = 1.48), declining again at 30 min (+D/-D = 1.16), while CaBP levels in the same fractions remained unchanged between 0-30 min of absorption.

  4. Addition of chloride to a prepartal diet high in cations increases 1,25-dihydroxyvitamin D response to hypocalcemia preventing milk fever.

    PubMed

    Goff, J P; Horst, R L; Mueller, F J; Miller, J K; Kiess, G A; Dowlen, H H

    1991-11-01

    In this study, we present evidence that cows fed highly cationic diets are less responsive to parathyroid hormone than those fed a highly anionic diet. Forty-seven Jersey cows (55 mo of age) were fed an alfalfa haylage-based diet supplemented with either anions (Cl-) or cations (Na+). Cows fed the high cationic diet suffered significantly more cases of milk fever (6 out of 23) than those fed the high anionic diet (1 out of 24). Concentrations of Ca at parturition and the first 2 d of lactation were significantly higher in cows fed the anionic diet. Secretion of parathyroid hormone in response to developing hypocalcemia was similar in cows fed either diet. Plasma hydroxyproline concentration (an index of bone Ca resorption activity) was greater in cows fed the anionic diet, suggesting better utilization of bone Ca. Plasma 1,25-dihydroxyvitamin D concentration was correlated inversely with plasma Ca and related directly to plasma parathyroid hormone in both groups of cows. However, the magnitude of the response (the amount of 1,25-dihydroxyvitamin D produced per unit increase in parathyroid hormone) was reduced greatly in cows fed the high cation diet. Because parathyroid hormone regulates both bone Ca resorption and renal 1,25-dihydroxyvitamin D production, these data suggest that prepartal diets high in cations decrease the ability of bone and renal tissues to respond to parathyroid hormone. Addition of anions to prepartal diets can reduce the excess cation balance of diets, increasing tissue response to parathyroid hormone and enabling the cow to better adapt to the Ca demands of lactation.

  5. Inhibitory effects of 1alpha, 25dihydroxyvitamin D3 and Ajuga iva extract on oxidative stress, toxicity and hypo-fertility in diabetic rat testes.

    PubMed

    Hamden, K; Carreau, S; Jamoussi, K; Ayadi, F; Garmazi, F; Mezgenni, N; Elfeki, A

    2008-09-01

    The aim of the current study is to investigate the therapeutic and preventive effects of 1alpha, 25dihydroxyvitaminD3 (1,25 (OH)2 D3) and Afuga iva (AI) extract on diabetes toxicity in rats testes. Thus diabetic rats were treated with 1alpha, 25dihydroxyvitaminD3 or Ajuga iva extract as both therapeutic and preventive treatments on diabetes toxicity in rats testes. Our results showed that diabetes induced a decrease in testosterone and 17beta-estradiol levels in testes and plasma. Besides, a fall in testicular antioxidant capacity appeared by a decrease in both antioxidant (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities) and nonenzymatic antioxidant (copper (Cu), magnesium (Mg) and iron (Fe) levels). All theses changes enhanced testicular toxicity (increase in testicular aspartate amino transaminase (AST), alanine amino transaminase (ALT), lactate dehydrogenase (LDH) activities and the lipid peroxidation and triglyceride (TG) levels). In addition, a decrease in testicular total cholesterol (TCh) level was observed in diabetic rats testes. All the changes lead to a decrease in the total number and mobility of epididymal spermatozoa. The administration of 1alpha,25dihydroxyvitaminD3 and Ajuga iva extract three weeks before and after diabetes induction interfered and prevented diabetes toxicity in the reproductive system. 1,25 (OH)2 D3 and Ajuga iva extract blunted all changes observed in diabetic rats. To sum up, the data suggested that 1,25 (OH)2 D3 and Ajuga iva extract have a protective effect on alloxan-induced damage in reproductive system by enhancing the testosterone and 17beta-estradiol levels, consequently protecting from oxidative stress, cellular toxicity and maintaining the number and motility of spermatozoids.

  6. Evidence for altered osteoclastogenesis in splenocyte cultures from Cyp27b1 knockout mice.

    PubMed

    Reinke, Daniel C; Kogawa, Masakazu; Barratt, Kate R; Morris, Howard A; Anderson, Paul H; Atkins, Gerald J

    2016-11-01

    The association between increased serum 25-hydroxyvitamin D (25D) and reduced osteoclastic bone resorption is well known. Previously, we have demonstrated that mechanism by which this occurs, may include the conversion of 25D to 1,25-dihydroxyvitamin D (1,25D) by osteoclasts, catalysed by the CYP27B1 enzyme. Local 1,25D synthesis in osteoclasts was shown to regulate osteoclastogenesis and moderating resorptive activity. Thus, we hypothesised that osteoclasts differentiated from mice with global deletion of the Cyp27b1 gene (Cyp27b1 KO) would display enhanced resorptive capacity due to the lack of an ameliorating effect of 1,25D. Splenocytes isolated from Cyp27b1 KO mice or their wild-type (WT) littermates between 6 and 8 weeks of age were cultured under osteoclast-forming conditions for up to 14 days. Osteoclast formation was measured by staining for the osteoclast marker tartrate resistant acid phosphatase (TRAP). Bone resorption activity was measured by plating the cells on a bone-like substrate. In Cyp27b1 KO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p<0.05) when compared to wild-type (WT) levels. However, Cyp27b1 KO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts (p<0.03). In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in Cyp27b1 KO cultures, implying that these smaller osteoclasts survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of CYP27B1 expression, consistent with the notion that endogenous metabolism of 25D optimises osteoclastogenesis and ameliorates the resulting activity of mature osteoclasts.

  7. Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3.

    PubMed

    Braun, H J; Birkenhäger, J C; De Jonge, H R

    1984-07-11

    The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 X 10(-5) M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in Vmax of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50-70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.

  8. 1,25-Dihydroxyvitamin D3 Inhibits the Differentiation and Migration of TH17 Cells to Protect against Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Chang, Jae-Hoon; Cha, Hye-Ran; Lee, Dong-Sup; Seo, Kyoung Yul; Kweon, Mi-Na

    2010-01-01

    Background Vitamin D3, the most physiologically relevant form of vitamin D, is an essential organic compound that has been shown to have a crucial effect on the immune responses. Vitamin D3 ameliorates the onset of the experimental autoimmune encephalomyelitis (EAE); however, the direct effect of vitamin D3 on T cells is largely unknown. Methodology/Principal Findings In an in vitro system using cells from mice, the active form of vitamin D3 (1,25-dihydroxyvitamin D3) suppresses both interleukin (IL)-17-producing T cells (TH17) and regulatory T cells (Treg) differentiation via a vitamin D receptor signal. The ability of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to reduce the amount of IL-2 regulates the generation of Treg cells, but not TH17 cells. Under TH17-polarizing conditions, 1,25(OH)2D3 helps to increase the numbers of IL-10-producing T cells, but 1,25(OH)2D3's negative regulation of TH17 development is still defined in the IL-10−/− T cells. Although the STAT1 signal reciprocally affects the secretion of IL-10 and IL-17, 1,25(OH)2D3 inhibits IL-17 production in STAT1−/− T cells. Most interestingly, 1,25(OH)2D3 negatively regulates CCR6 expression which might be essential for TH17 cells to enter the central nervous system and initiate EAE. Conclusions/Significance Our present results in an experimental murine model suggest that 1,25(OH)2D3 can directly regulate T cell differentiation and could be applied in preventive and therapeutic strategies for TH17-mediated autoimmune diseases. PMID:20886077

  9. Simultaneous measurement of plasma vitamin D3 metabolites including 4β,25-dihydroxyvitamin D3 using liquid chromatography-tandem mass spectrometry

    PubMed Central

    Wang, Zhican; Senn, Tauri; Kalhorn, Tom; Zheng, Xi Emily; Zheng, Songmao; Davis, Connie L.; Hebert, Mary F.; Lin, Yvonne S.; Thummel, Kenneth E.

    2011-01-01

    Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we developed a specific LC-MS/MS method that permits the quantification of major circulating vitamin D3 metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid-liquid extraction and Diels-Alder derivatization procedure prior to LC-MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D3 peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D3 [4β,25(OH)2D3] and was found at concentrations comparable to 1α,25(OH)2D3. Quantification of 1α,25(OH)2D3 in plasma required complete chromatographic separation of 1α,25(OH)2D3 from 4β,25(OH)2D3. An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD3, 24R,25(OH)2D3, 1α,25(OH)2D3, and 4β,25(OH)2D3 in healthy individuals. The LC-MS/MS method developed and described here, could result in considerable improvement in the quantification of 1α,25(OH)2D3, as well as monitoring the newly identified circulating metabolite, 4β,25(OH)2D3. PMID:21784054

  10. The Effect of Analogues of 1α,25-Dihydroxyvitamin D₂ on the Regrowth and Gene Expression of Human Colon Cancer Cells Refractory to 5-Fluorouracil.

    PubMed

    Neska, Jacek; Swoboda, Paweł; Przybyszewska, Małgorzata; Kotlarz, Agnieszka; Bolla, Narasimha Rao; Miłoszewska, Joanna; Grygorowicz, Monika Anna; Kutner, Andrzej; Markowicz, Sergiusz

    2016-06-14

    This study aimed to evaluate the capacity of hypocalcemic analogues of 1α,25-dihydroxyvitamin D₂ (1,25D2) and 1α,25-dihydroxyvitamin D₃ (1,25D3) to inhibit regrowth and regulate the stemness-related gene expression in colon cancer cells undergoing renewal after exposure to 5-fluorouracil (5-FU). All of the tested analogues of 1,25D2 equally potently decreased the clonogenicity and the proliferative activity of HT-29 cells which survived the exposure to 5-FU, but differently regulated gene expression of these cells during their renewal. 1,25D2 and analogues (PRI-1907 and PRI-1917), as well as 1,25D3 and analogue PRI-2191, decreased the relative expression level of several stemness-related genes, such as NANOG, OCT3/4, PROM1, SOX2, ALDHA1, CXCR4, in HT-29/5-FU cells during their renewal, in comparison to untreated HT-29/5-FU cells. The other 1,25D2 analogues (PRI-1906 and PRI-1916) were not capable of downregulating the expression of these stemness-related genes as the analogues PRI-1907 and PRI-1917 did. All of the tested vitamin D analogues upregulated CDH1, the gene encoding E-cadherin associated with epithelial phenotype. Out of the series of analogues studied, side-chain branched analogues of 1,25D2 (PRI-1907, PRI-1917) and the analogue of 1,25D3 (PRI-2191) might be used to target cancer cells with stem-like phenotypes that survive conventional chemotherapy.

  11. Association of Arsenic and Metals with Concentrations of 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D among Adolescents in Torreón, Mexico

    PubMed Central

    Zamoiski, Rachel D.; Guallar, Eliseo; García-Vargas, Gonzalo G.; Rothenberg, Stephen J.; Resnick, Carol; Andrade, Marisela Rubio; Steuerwald, Amy J.; Parsons, Patrick J.; Weaver, Virginia M.; Navas-Acien, Ana

    2014-01-01

    Background: Limited data suggest that lead (Pb), cadmium (Cd), and uranium (U) may disrupt vitamin D metabolism and inhibit production of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active vitamin D metabolite, from 25-hydroxyvitamin D [25(OH)D] in the kidney. Objectives: We evaluated the association between blood lead (BPb) and urine arsenic (As), Cd, molybdenum (Mo), thallium (Tl), and U with markers of vitamin D metabolism [25(OH)D and 1,25(OH)2D]. Methods: We conducted a cross-sectional study of 512 adolescents in Torreón, a town in Mexico with a Pb smelter near residential areas. BPb was measured using atomic absorption spectrometry. Urine As, Cd, Mo, Tl, and U were measured using inductively coupled plasma mass spectrometry. Serum 25(OH)D and 1,25(OH)2D were measured using a chemiluminescent immunoassay and a radioimmunoassay, respectively. Multivariable linear models with vitamin D markers as the outcome were used to estimate associations of BPb and creatinine-corrected urine As and metal concentrations with serum vitamin D concentrations, controlling for age, sex, adiposity, smoking, socioeconomic status, and time outdoors. Results: Serum 25(OH)D was positively associated with urine Mo and Tl [1.5 (95% CI: 0.4, 2.6) and 1.2 (95% CI: 0.3, 2.1) ng/mL higher with a doubling of exposure, respectively]. Serum 1,25(OH)2D was positively associated with urine As and U [3.4 (95% CI: 0.9, 5.9) and 2.2 (95% CI: 0.7, 3.7) pg/mL higher, respectively], with little change in associations after additional adjustment for serum 25(OH)D. Pb and Cd were not associated with 25(OH)D or 1,25(OH)2D concentrations. Conclusions: Overall, our findings did not support a negative effect of As or metal exposures on serum 1,25(OH)2D concentrations. Additional research is needed to confirm positive associations between serum 1,25(OH)2D and urine U and As concentrations and to clarify potential underlying mechanisms. Citation: Zamoiski RD, Guallar E, García-Vargas GG, Rothenberg SJ

  12. 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 exert distinct effects on human skeletal muscle function and gene expression

    PubMed Central

    Hassan-Smith, Zaki K.; Jenkinson, Carl; Smith, David J.; Hernandez, Ivan; Morgan, Stuart A.; Crabtree, Nicola J.; Gittoes, Neil J.; Keevil, Brian G.; Stewart, Paul M.

    2017-01-01

    Age-associated decline in muscle function represents a significant public health burden. Vitamin D-deficiency is also prevalent in aging subjects, and has been linked to loss of muscle mass and strength (sarcopenia), but the precise role of specific vitamin D metabolites in determining muscle phenotype and function is still unclear. To address this we quantified serum concentrations of multiple vitamin D metabolites, and assessed the impact of these metabolites on body composition/muscle function parameters, and muscle biopsy gene expression in a retrospective study of a cohort of healthy volunteers. Active serum 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), but not inactive 25-hydroxyvitamin D3 (25OHD3), correlated positively with measures of lower limb strength including power (rho = 0.42, p = 0.02), velocity (Vmax, rho = 0.40, p = 0.02) and jump height (rho = 0.36, p = 0.04). Lean mass correlated positively with 1α,25(OH)2D3 (rho = 0.47, p = 0.02), in women. Serum 25OHD3 and inactive 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) had an inverse relationship with body fat (rho = -0.30, p = 0.02 and rho = -0.33, p = 0.01, respectively). Serum 25OHD3 and 24,25(OH)2D3 were also correlated with urinary steroid metabolites, suggesting a link with glucocorticoid metabolism. PCR array analysis of 92 muscle genes identified vitamin D receptor (VDR) mRNA in all muscle biopsies, with this expression being negatively correlated with serum 25OHD3, and Vmax, and positively correlated with fat mass. Of the other 91 muscle genes analysed by PCR array, 24 were positively correlated with 25OHD3, but only 4 were correlated with active 1α,25(OH)2D3. These data show that although 25OHD3 has potent actions on muscle gene expression, the circulating concentrations of this metabolite are more closely linked to body fat mass, suggesting that 25OHD3 can influence muscle function via indirect effects on adipose tissue. By contrast, serum 1α,25(OH)2D3 has limited effects on muscle gene expression

  13. Simultaneous Quantification of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Rats Shows Strong Correlations between Serum and Brain Tissue Levels.

    PubMed

    Xue, Ying; He, Xin; Li, Huan-De; Deng, Yang; Yan, Miao; Cai, Hua-Lin; Tang, Mi-Mi; Dang, Rui-Li; Jiang, Pei

    2015-01-01

    While vitamin D3 is recognized as a neuroactive steroid affecting both brain development and function, efficient analytical method in determining vitamin D3 metabolites in the brain tissue is still lacking, and the relationship of vitamin D3 status between serum and brain remains elusive. Therefore, we developed a novel analysis method by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to simultaneously quantify the concentrations of 25-hydroxyvitamin D3 (25(OH)D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in the serum and brain of rats fed with different dose of vitamin D3. We further investigated whether variations of serum vitamin D3 metabolites could affect vitamin D3 metabolite levels in the brain. Serum and brain tissue were analyzed by HPLC-MS/MS with electrospray ionization following derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). The method is highly sensitive, specific, and accurate to quantify 25(OH)D3 and 24,25(OH)2D3 in animal brain tissue. Vitamin D3 metabolites in brain tissue were significantly lower in rats fed with a vitamin D deficiency diet than in rats fed with high vitamin D3 diet. There was also a strong correlation of vitamin D3 metabolites in serum and brain. These results indicate that vitamin D3 status in serum affects bioavailability of vitamin D3 metabolites in the brain.

  14. Simultaneous Quantification of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Rats Shows Strong Correlations between Serum and Brain Tissue Levels

    PubMed Central

    Xue, Ying; He, Xin; Li, Huan-De; Deng, Yang; Yan, Miao; Cai, Hua-Lin; Tang, Mi-Mi; Dang, Rui-Li; Jiang, Pei

    2015-01-01

    While vitamin D3 is recognized as a neuroactive steroid affecting both brain development and function, efficient analytical method in determining vitamin D3 metabolites in the brain tissue is still lacking, and the relationship of vitamin D3 status between serum and brain remains elusive. Therefore, we developed a novel analysis method by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to simultaneously quantify the concentrations of 25-hydroxyvitamin D3 (25(OH)D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in the serum and brain of rats fed with different dose of vitamin D3. We further investigated whether variations of serum vitamin D3 metabolites could affect vitamin D3 metabolite levels in the brain. Serum and brain tissue were analyzed by HPLC-MS/MS with electrospray ionization following derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). The method is highly sensitive, specific, and accurate to quantify 25(OH)D3 and 24,25(OH)2D3 in animal brain tissue. Vitamin D3 metabolites in brain tissue were significantly lower in rats fed with a vitamin D deficiency diet than in rats fed with high vitamin D3 diet. There was also a strong correlation of vitamin D3 metabolites in serum and brain. These results indicate that vitamin D3 status in serum affects bioavailability of vitamin D3 metabolites in the brain. PMID:26713090

  15. 1,25-Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA, protein and activity level in a canine transitional carcinoma cell line.

    PubMed

    Middleton, R P; Nelson, R; Li, Q; Blanton, A; Labuda, J A; Vitt, J; Inpanbutr, N

    2015-12-01

    Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti-cancer effects of 1,25-dihydroxyvitamin D3 (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10(-7) M), and seocalcitol (10(-7) and 10(-9) M). MnSOD mRNA was decreased in response to calcitriol at 10(-7) M. Catalase was significantly increased in response to calcitriol (10(-7) and 10(-9) M), and seocalcitol (10(-9) M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10(-9) M). In addition, global gene expression analysis identified the involvement of mitogen-activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.

  16. Relationship between Structure and Conformational Change of the Vitamin D Receptor Ligand Binding Domain in 1α,25-Dihydroxyvitamin D3 Signaling.

    PubMed

    Wan, Lin-Yan; Zhang, Yan-Qiong; Chen, Meng-Di; Du, You-Qin; Liu, Chang-Bai; Wu, Jiang-Feng

    2015-11-18

    Vitamin D Receptor (VDR) belongs to the nuclear receptor (NR) superfamily. Whereas the structure of the ligand binding domain (LBD) of VDR has been determined in great detail, the role of its amino acid residues in stabilizing the structure and ligand triggering conformational change is still under debate. There are 13 α-helices and one β-sheet in the VDR LBD and they form a three-layer sandwich structure stabilized by 10 residues. Thirty-six amino acid residues line the ligand binding pocket (LBP) and six of these residues have hydrogen-bonds linking with the ligand. In 1α,25-dihydroxyvitamin D₃ signaling, H3 and H12 play an important role in the course of conformational change resulting in the provision of interfaces for dimerization, coactivator (CoA), corepressor (CoR), and hTAFII 28. In this paper we provide a detailed description of the amino acid residues stabilizing the structure and taking part in conformational change of VDR LBD according to functional domains.

  17. Effect of 1,25-dihydroxyvitamin D3 on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term imaging.

    PubMed

    Murata, Kaori; Takahashi, Ayumi; Morita, Takao; Nezu, Akihiro; Fukumoto, Satoshi; Saitoh, Masato; Tanimura, Akihiro

    2016-01-01

    Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca(2+) release from intracellular stores and subsequent Ca(2+) entry. The fluorescence intensity of these cells was increased more than 2-fold by stimulation with 10 μM ATP. We applied long-term imaging (for ~10 h) to monitor Ca(2+) responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca(2+) concentration in the presence of 100 nM 1,25-dihydroxyvitamin D3. Many of these Ca(2+) responses began at a specific location in the cytoplasm and spread throughout the entire cytoplasm. The combination of efficient gene delivery with an adenoviral vector and long-term imaging with a highly sensitive GECI enabled detection of intermittent Ca(2+) responses that occur only 3-10 times/h/100 cells. This method could be useful to study the effects of Ca(2+) responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.

  18. Duodenal active transport of calcium and phosphate in vitamin D-deficient rats: effects of nephrectomy, Cestrum diurnum, and 1alpha,25-dihydroxyvitamin D3.

    PubMed

    Walling, M W; Kimberg, D V; Wasserman, R H; Feinberg, R R

    1976-05-01

    Both the methanol:chloroform extractable material from the leaves of the Solanaceous plant, Cestrum diurnum (C.d.), and a 270 ng dose of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) increased the active absorption of calcium and phosphate across the proximal duodenum, studied in vitro, from sham-operated and nephrectomized (NPX) vitamin D-deficient rats. In these studies, conducted 24 h after surgery, the uremic state in the NPX animals markedly diminished the intestinal transport response to 1alpha,25-(OH)2D3 and also lowered baseline transport values across duodenum from the NPX vitamin D-deficient controls. Both C.d. and 1alpha, 25-(OH)2D3 elevated plasma Ca levels equally well in the sham-operated and NPX groups. The stimulation of intestinal Ca absorption in NPX animals indicates that, like the leaves of the South American plant, Solanum glaucophyllum, C.d. contains materials which can function in an analogous manner to compounds in the vitamin D group that have either a 1alpha hydroxyl group or its steric equivalent.

  19. CD8+ T cells are not necessary for 1α,25-dihydroxyvitamin D3 to suppress experimental autoimmune encephalomyelitis in mice

    PubMed Central

    Meehan, Terrence F.; DeLuca, Hector F.

    2002-01-01

    In addition to its role in calcium and phosphorous homeostasis, 1α,25-dihydroxyvitamin D3 [1,25-(OH)2D3] appears to be a modulator of the immune system. Administration of 1,25-(OH)2D3 prevents disease in several autoimmune animal models, including experimental autoimmune encephalomyelitis (EAE). The vitamin D receptor is believed to mediate this activity. Among cells of the immune system, CD8+ T cells have the highest levels of the vitamin D receptor. Because CD8+ T cells have been implicated as both suppressors and effectors of the inflammation associated with multiple sclerosis and EAE, we examined the question of whether the 1,25-(OH)2D3 suppression of EAE occurs through a CD8+ T cell-dependent mechanism. To test this hypothesis, mice that are homozygous knockouts for the α chain of the CD8 receptor and have been characterized as lacking functional CD8+ T cells (CD8+ −/−) were provided 1,25-(OH)2D3 in their diet before EAE induction. Although CD8+ −/− mice fed the same diet lacking 1,25-(OH)2D3 have a high incidence of EAE, EAE did not occur in CD8+ −/− mice fed the diet containing 1,25-(OH)2D3. We conclude that CD8+ T cells neither are needed nor do they play a role in the prevention of EAE by 1,25-(OH)2D3. PMID:11929984

  20. 1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium-regulating Components.

    PubMed

    Lee, Seong Min; Riley, Erin M; Meyer, Mark B; Benkusky, Nancy A; Plum, Lori A; DeLuca, Hector F; Pike, J Wesley

    2015-07-17

    1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney, and skeleton. Interestingly, although several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with vehicle or 1,25(OH)2D3 and evaluated 6 h later. RNA samples from the duodena were then subjected to RNA sequence analysis, and the data were analyzed bioinformatically. 1,25(OH)2D3 altered expression of large collections of genes in animals under either dietary condition. 45 genes were found common to both 1,25(OH)2D3-treated groups and were composed of genes previously linked to intestinal calcium uptake, including S100g, Trpv6, Atp2b1, and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. We then conducted a ChIP sequence analysis of binding sites for the vitamin D receptor (VDR) across the proximal intestine in vitamin D-sufficient normal mice treated with vehicle or 1,25(OH)2D3. The residual VDR cistrome was composed of 4617 sites, which was increased almost 4-fold following hormone treatment. Interestingly, the majority of the genes regulated by 1,25(OH)2D3 in each diet group as well as those found in common in both groups contained frequent VDR sites that likely regulated their expression. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.

  1. Residues of the human nuclear vitamin D receptor that form hydrogen bonding interactions with the three hydroxyl groups of 1alpha,25-dihydroxyvitamin D3.

    PubMed

    Reddy, Madhuri D; Stoynova, Ludmilla; Acevedo, Alejandra; Collins, Elaine D

    2007-03-01

    Most of the biological effects of 1,25-dihydroxyvitamin D(3) (hormone D) are mediated through the nuclear vitamin D receptor (VDR). Hormone binding induces conformational changes in VDR that enable the receptor to activate gene transcription. It is known that residues S237 and R274 form hydrogen bonds with the 1-hydroxyl group of hormone D, while residues Y143 and S278, and residues H305 and H397 form hydrogen bonds with the 3-hydroxyl and the 25-hydroxyl groups of the hormone. A series of VDR mutations were constructed (S237A, R274A, R274Q, Y143F, Y143A, S278A, H305A, and H397F; double mutants: S237A/R274A, Y143F/S278A, Y143A/S278A, and H305A/H397F). The relative binding affinities of the wild-type and variant VDRs were assessed. All of the mutants except H397F resulted in lower binding affinity compared to wild-type VDR. Binding to hormone was barely detectable in Y143F, H305A, and H305A/H397F mutants, and undetectable in mutants R274A, R274Q, Y143A, S237A/R274A, and Y143A/S278A, indicating the importance of these residues. Ability to activate gene transcription was also assessed. All of the VDR mutants, except the single mutant S278A, required higher doses of hormone D for half-maximal response. Defining the role of hormone D-VDR binding will lead to a better understanding of the vitamin D signal transduction pathway.

  2. Effects of Placental Ischemia Are Attenuated by 1,25-Dihydroxyvitamin D Treatment and Associated with Reduced Apoptosis and Increased Autophagy.

    PubMed

    Tian, Xiaoyu; Ma, Suling; Wang, Yaqi; Hou, Lianguo; Shi, Yun; Yao, Min; Wang, Xiaoning; Zhang, Huifeng; Jiang, Lingling

    2016-02-01

    We evaluated the effects of administration of 1,25-dihydroxyvitamin D (1,25(OH)2D) during pregnancy on relieving adverse outcomes of preeclampsia and the pathologic and biochemical changes in reduction in uteroplacental perfusion (RUPP) model of rats. On day 1, 7, and 14 of pregnancy, rats in pregnant RUPP plus 1,25(OH)2D (RUPP+VD) group (n = 15) received 120 ng/100 g body weight/week of 1,25(OH)2D by subcutaneous injection, while rats in normal pregnant (n = 12) and the RUPP group (n = 14) received 1,25(OH)2D vehicle (saline solution). On day 19 of pregnancy, after measure of blood pressure and cardiac function and urine collection, rats were euthanized, and fetal and maternal serum, placenta, and heart and kidney were collected. Fetal mortality, urinary protein, glucose, and parameters for kidney function in serum were measured. We evaluated vitamin D receptor expression and pathological and ultrastructural changes in rat heart, kidney, and placenta. Levels of oxidative stress, endoplasmic reticulum (ER) stress, apoptosis, and autophagy were measured in placenta. Compared to RUPP rats, 1,25(OH)2D decreased fetal mortality, mean blood pressure, 24-h urinary protein, urine microalbumin, and hyperglycemia in RUPP+VD rats. These were consistent with the improvements of structure impairment in heart, kidney, and placenta of RUPP rat by 1,25(OH)2D. In placenta of RUPP rat, the decrease in oxidative stress and ER stress by 1,25(OH)2D treatment was accompanied by autophagy activation and apoptosis attenuation. 1,25(OH)2D plays a beneficial effect on preeclampsia at the early gestation and might be used as a potential protective agent for preeclampsia. However, the RUPP model only recapitulated the hypoxic origin of preeclampsia; further randomized controlled trial is expected to be performed for validation and evaluation.

  3. Effect of lowered vitamin D binding protein levels on the biological activity and metabolism of 1,25-dihydroxyvitamin D/sub 3/

    SciTech Connect

    Kost, S.B.; Londowski, J.; Audran, M.; Kumar, R.

    1986-03-01

    The authors studied the effect of lowered vitamin D binding protein levels on the biological activity and metabolism of 1,25-dihydroxyvitamin D/sub 3/ (1,25(OH)/sub 2/D/sub 3/) in vivo. Estrogen administration to vitamin D-deficient rats resulted in decrease of plasma vitamin D binding protein concentrations by about 20%. The authors administered graded doses of 1,25(OH)/sub 2/D/sub 3/ (5 - 5000 pmol intravenously) to vitamin D-deficient rats given estrogen or vehicle, and studied the biological response in intestine and bone. Intestinal calcium transport, following the administration of 1,25(OH)/sub 2/D/sub 3/, was similar in the estrogen or vehicle-treated groups. Serum calcium concentrations were lower in the estrogen-treated rats when compared to rats given vehicle. Serum calcium in both groups, however, increased the same amount over the range of 1,25(OH)/sub 2/D/sub 3/ given. The uptake of (/sup 3/H) 1,25(OH)/sub 2/D/sub 3/ by the intestine and bone 8 hours after the administration of (/sup 3/H) 1,25(OH)/sub 2/D/sub 3/ was similar in estrogen- and vehicle-treated rats. The amount of (/sup 3/H) 1,25(OH)/sub 2/D/sub 3/ in plasma of estrogen-treated rats was the same as in vehicle-treated rats. The authors conclude that in estrogen-treated rats, lowered vitamin D binding protein levels do not alter the effect of 1,25(OH)/sub 2/D/sub 3/ on intestine or bone and do not alter the metabolism of 1,25(OH)/sub 2/D/sub 3/.

  4. Rapid changes in skeletal muscle calcium uptake induced in vitro by 1,25-dihydroxyvitamin D/sub 3/ are suppressed by calcium channel blockers

    SciTech Connect

    de Boland, A.R.; Boland, R.L.

    1987-05-01

    Previous investigations have shown that 1,25-dihydroxyvitamin D/sub 3/ (1,25-(OH)/sub 2/D/sub 3/) stimulates muscle Ca uptake through a nuclear mechanism. The possibility that 1,25-(OH)/sub 2/D/sub 3/ would induce rapid changes in muscle Ca fluxes independent of de novo protein synthesis was investigated in the present work. In vitro preparations of soleus muscles obtained from vitamin D-deficient chicks were used. A significant increase in /sup 45/Ca labeling of the tissue was already observed after 3-min treatment with 2.4 X 10(-10) M 1,25-(OH)/sub 2/D/sub 3/. This early stimulation in muscle Ca uptake became maximal at 10-15 min. Cycloheximide (50 microM) did not block the effect of the metabolite at 15 and 30 min. However, the antibiotic effectively blocked the increase in Ca uptake induced by 1,25-(OH)/sub 2/D/sub 3/ after 1-h treatment. The rapid 1,25-(OH)/sub 2/D/sub 3/-dependent stimulation of /sup 45/Ca labeling of soleus muscle was not associated to changes in lipid synthesis as assessed by measurements of /sup 3/H-glycerol incorporation into the tissue lipids. However, the calcium antagonists verapamil and nifedipine (50 microM) abolished the stimulation in Ca uptake produced by 1,25-(OH)/sub 2/D/sub 3/ in 5 min. These results suggest that 1,25-(OH)/sub 2/D/sub 3/ can act directly at the muscle membrane level affecting Ca fluxes through Ca channels.

  5. 1,25-dihydroxyvitamin D3 modulates effects of ionizing radiation (IR) on human keratinocytes: in vitro analysis of cell viability/proliferation, DNA-damage and -repair.

    PubMed

    Trémezaygues, Lea; Seifert, Markus; Vogt, Thomas; Tilgen, Wolfgang; Reichrath, Jörg

    2010-07-01

    We investigated the capacity of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to protect spontaneously immortalized human keratinocytes (HaCaT) and cutaneous squamous cell carcinoma cells (SCL-1) against the hazardous effects of ionizing radiation (IR). We pretreated HaCaT and SCL-1 cells in vitro with 1,25(OH)2D3 (10(-7) M) over 48 h and then irradiated them once with IR (1 Gy, 2 Gy, and 5 Gy). Using WST-1-assay and crystal violet (CV) assay, we compared viability/proliferation in 1,25(OH)2D3-pretreated cells with controls that were pretreated with the carrier substance ethanol alone. Additionally, we analyzed the effects of 1,25(OH)2D3 on the presence of IR-induced DNA-damage by immunocytochemical detection of gamma-H2AX-foci in HaCaT-keratinocytes. We demonstrate that 1,25(OH)2D3 (10(-7) M) inhibits proliferation of human keratinocytes and that IR (1-5 Gy) has no significant effect on proliferation and viability of HaCaT-keratinocytes and SCL-1 cells. Moreover, we show that IR modulates dose-dependently the number of gammaH2AX-foci in HaCaT-keratinocytes. Pretreatment of the cells with 1,25(OH)2D3 reduces the number of IR-induced gammaH2AX-foci after irradiation with 1 Gy and 2 Gy and increases it after irradiation with 5 Gy. To put it in a nutshell, our data support the hypothesis that 1,25(OH)2D3 modulates the effects of low-dose IR (1-5 Gy) on cultured human keratinocytes.

  6. 1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase.

    PubMed

    Wilmanski, Tomasz; Buhman, Kimberly; Donkin, Shawn S; Burgess, John R; Teegarden, Dorothy

    2017-02-01

    Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies, we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)₂D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)₂D (10 nM) for 5 and 7 days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)₂D treatment for 5 days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)₂D-mediated regulation and hypothesized that 1,25(OH)₂D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was down-regulated with 1,25(OH)₂D treatment at 2 (73%±6 relative to vehicle) and 5 (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at 5 days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)₂D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)₂D regulates lipid metabolism in malignant breast epithelial cells.

  7. Role of VDR in 1α,25-dihydroxyvitamin D3-dependent non-genomic activation of MAPKs, Src and Akt in skeletal muscle cells.

    PubMed

    Buitrago, Claudia; Pardo, Verónica Gonzalez; Boland, Ricardo

    2013-07-01

    1α,25-dihydroxyvitamin D3 [1,25D] is recognized as a steroid hormone that rapidly elicits intracellular signals in various tissues. In skeletal myoblasts, we have previously demonstrated that one of the 1,25D-induced non-genomic effects is the upstream stimulation of MAPKs through Src activation. In this work, the data obtained suggest that the classical receptor of vitamin D (VDR) participates in non-transcriptional actions of 1,25D. We significantly reduced VDR expression by infection of C2C12 murine myoblasts with lentiviral particles containing the pLKO.1 plasmid with information to express a shRNA against mouse VDR. In these cells (C2C12-shVDR), Western blot analyses show that 1,25D-induced p38 MAPK activation and Src tyr416 phosphorylation were abolished. In addition, 1,25D-dependent activity of ERK1/2 was diminished in cells lacking VDR but to a lesser extent (∼-60%). Phosphorylation of Akt by 1,25D, recently demonstrated in C2C12 cells, in the present work also appeared to be partially dependent on VDR expression (∼50% in C2C12-shVDR cells). Our results indicate that VDR is involved in 1,25D-induced rapid events related to survival/proliferation responses in skeletal muscle cells, providing relevant information on the mechanism of initiation of the non-genomic hormone signal. The participation of a VDR-independent non-genomic mechanism of action should also be taken into consideration. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

  8. Measurement of 25-hydroxyvitamin D2&3 and 1, 25-dihydroxyvitamin D2&3 by Tandem Mass Spectrometry: A Primate Multispecies Comparison

    PubMed Central

    Ziegler, Toni E.; Kapoor, Amita; Hedman, Curtis J.; Binkley, Neil; Kemnitz, Joseph W.

    2015-01-01

    Vitamin D metabolites are widely studied for their roles in bone health, immune functions and other potential physiologic roles in humans. However, the optimal blood levels of vitamin D metabolites are still unclear. Various methods for measuring vitamin D metabolites have been used and recently liquid chromatography tandem mass spectroscopy (LC-MS/MS) has been adopted as the gold standard for vitamin D metabolite measurement. Here we report the use of LC-MS/MS to measure 25-hydroxyvitamin D (25(OH)D2&3), and 1,25-dihydroxyvitamin D (1,25(OH)2D2&3), in three laboratory nonhuman primate species: common marmoset (Callithrix jacchus), rhesus macaque (Macaca mulatta), and cynomolgus macaque (Macaca fascicularis), and compare them to humans using the same technique. The nonhuman primates showed blood levels for 25(OH)D3 and 1,25(OH)2D3 significantly higher than human values with marmosets having the highest levels. Marmoset samples showed significantly more variability among individuals than those from macaques for both metabolites, but all three nonhuman primate species exhibited large variation within species for both 25(OH)D2&3 and 1,25(OH)2D2&3. Marmoset females had significantly lower values than the males for 25(OH)D3, while rhesus males showed a significant decrease in 25(OH)D3 with age. The most striking finding is the variation within species for vitamin D levels even in laboratory primates that have a controlled diet, UV exposure, and in some cases, genetic constraints. Similar variation in 25(OH)D responses to a fixed dose of oral vitamin D supplementation has been reported in humans. We suggest that these species can provide primate models for examining the factors influencing variation in the levels of vitamin D necessary for human and nonhuman primate health. PMID:25845705

  9. Development of a Sensitive LC/MS/MS Method for Vitamin D Metabolites: 1,25 Dihydroxyvitamin D2&3 Measurement Using a Novel Derivatization Agent

    PubMed Central

    Hedman, Curtis J.; Wiebe, Donald A.; Dey, Subhakar; Plath, Josh; Kemnitz, Joseph W.; Ziegler, Toni E.

    2014-01-01

    Active vitamin D metabolites 1,25-dihydroxyvitamin D2 [1,25-(OH)2-D2; derived from ergocalciferol] and D3 [1,25-(OH)2-D3; derived from cholecalciferol] are found in low levels in the circulation and require a very sensitive method for measurement. Radioimmunoassay (RIA) has been the method of choice, but it lacks the specificity needed to distinguish between 1,25-(OH)2-D2 and -D3whereas liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have the advantage of high specificity and sensitivity. Here, we compare a new derivative for ionizing 1,25-(OH)2-D to enhance the signal and provide the most sensitive assay for measuring vitamin D. We used the Amplifex diene method of derivatizing prior to LC/MS/MS and compared it to the standard RIA method and the 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) method of derivatizing prior to LC/MS/MS. In the evaluation of 20 human serum samples, all methods correlated strongly across the upper levels of the standard 1,25-(OH)2-D2 and - D3 ranges (Amplifex and RIA, pc = 0.97; Amplifex and PTAD, pc = 0.96) but less strongly on the lower levels of the standard range (Amplifex and RIA, pc = 0.81; Amplifex and PTAD, pc = 0.65) suggesting differences in the sensitivities between the assays. The Amplifex method was determined to be more sensitive than the PTAD method, as peak areas were significantly higher for the Amplifex method and provided for a 10 fold higher signal-to-noise ratio than PTAD. Therefore, the Amplifex LC/MS/MS method is the most sensitive and specific method available for measuring 1,25-(OH)2-D2 and -D3 while using the smallest sample volume. PMID:24576767

  10. 1α,25-dihydroxyvitamin D3 modulates the hair-inductive capacity of dermal papilla cells: therapeutic potential for hair regeneration.

    PubMed

    Aoi, Noriyuki; Inoue, Keita; Chikanishi, Toshihiro; Fujiki, Ryoji; Yamamoto, Hanako; Kato, Harunosuke; Eto, Hitomi; Doi, Kentaro; Itami, Satoshi; Kato, Shigeaki; Yoshimura, Kotaro

    2012-08-01

    Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D(3) (VD(3)) upregulates expression of transforming growth factor (TGF)-β2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD(3) actions on DPCs. VD(3) suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD(3) in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-β2, by VD(3) was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all-trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD(3)) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD(3) upregulation of Wnt10b, ALPL, and TGF-β2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD(3) significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD(3) may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies.

  11. Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3.

    PubMed Central

    Kristjansson, K; Rut, A R; Hewison, M; O'Riordan, J L; Hughes, M R

    1993-01-01

    We have identified and characterized two mutations in the hormone binding domain of the vitamin D receptor (VDR) in patients with hereditary vitamin D-resistant rickets. One patient was found to have a premature stop mutation (CAG to TAG) in the hinge region affecting amino acid 149 (Q149X) and the other demonstrated a missense mutation (CGC to CTC) resulting in the substitution of arginine 271 by leucine (R271L) in the steroid binding domain. Eukaryotic expression analyses in CV-1 cells showed the inability of both patients' VDR to induce transcription from the osteocalcin hormone gene response element at 10(-7) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Normal transcription levels could, however, be elicited by the missense mutated VDR (R271L) in the presence of 1,000-fold higher 1,25-(OH)2D3 concentrations than needed for the wild-type receptor. This shows that Arg 271 directly affects the affinity of the VDR for its ligand and its conversion to leucine decreases its affinity for 1,25(OH)2D3 by a factor of 1,000. Arg 271 is located immediately 3-prime to a 30 amino acid segment (VDR amino acids 241-270) that is conserved among members of the steroid/thyroid/retinoid hormone receptor superfamily. These results represent the first missense mutation identified in the hormone binding domain of VDR and further define the structure-function relationship of 1,25(OH)2D3 ligand binding to its nuclear receptor. Images PMID:8392085

  12. Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

    PubMed

    DeSmet, Marsha L; Fleet, James C

    2017-01-16

    High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH)2D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH)2D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH)2D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH)2D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention.

  13. Development of a sensitive LC/MS/MS method for vitamin D metabolites: 1,25 Dihydroxyvitamin D2&3 measurement using a novel derivatization agent.

    PubMed

    Hedman, Curtis J; Wiebe, Donald A; Dey, Subhakar; Plath, Josh; Kemnitz, Joseph W; Ziegler, Toni E

    2014-03-15

    Active vitamin D metabolites 1,25-dihydroxyvitamin D2 [1,25-(OH)2-D2; derived from ergocalciferol] and D3 [1,25-(OH)2-D3; derived from cholecalciferol] are found in low levels in the circulation and require a very sensitive method for measurement. Radioimmunoassay (RIA) has been the method of choice, but it lacks the specificity needed to distinguish between 1,25-(OH)2-D2 and -D3, whereas liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have the advantage of high specificity and sensitivity. Here, we compare a new derivative for ionizing 1,25-(OH)2-D to enhance the signal and provide the most sensitive assay for measuring vitamin D. We used the Amplifex diene method of derivatizing prior to LC/MS/MS and compared it to the standard RIA method and the 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) method of derivatizing prior to LC/MS/MS. In the evaluation of 20 human serum samples, all methods correlated strongly across the upper levels of the standard 1,25-(OH)2-D2 and -D3 ranges (Amplifex and RIA, pc=0.97; Amplifex and PTAD, pc=0.96) but less strongly on the lower levels of the standard range (Amplifex and RIA, pc=0.81; Amplifex and PTAD, pc=0.65) suggesting differences in the sensitivities between the assays. The Amplifex method was determined to be more sensitive than the PTAD method, as peak areas were significantly higher for the Amplifex method and provided for a 10 fold higher signal-to-noise ratio than PTAD. Therefore, the Amplifex LC/MS/MS method is the most sensitive and specific method available for measuring 1,25-(OH)2-D2 and -D3 while using the smallest sample volume.

  14. Keratinocyte G2/M growth arrest by 1,25-dihydroxyvitamin D3 is caused by Cdc2 phosphorylation through Wee1 and Myt1 regulation.

    PubMed

    Dai, Xiuju; Yamasaki, Kenshi; Yang, Lujun; Sayama, Koji; Shirakata, Yuji; Tokumara, Sho; Yahata, Yoko; Tohyama, Mikiko; Hashimoto, Koji

    2004-06-01

    1,25-dihydroxyvitamin D3 (1,25[OH]2VD3) has an antiproliferative effect on keratinocyte growth, and its derivatives are used for the treatment of psoriasis. It was reported previously that 1,25[OH]2VD3 induced cell cycle arrest not only at the G0/G1 phase but also at the G2/M phase. However, the mechanism of 1,25[OH]2VD3-induced G2/M phase arrest in keratinocytes has not been fully understood. The addition of 10(-8) to 10(-6) M 1,25[OH]2VD3 to cultured normal human keratinocytes enhanced the expression of Myt1 mRNA preceding Wee1 mRNA; 10(-6) M 1,25[OH]2VD3 unregulated Myt1 mRNA from 6 h to 24 h and Wee1 mRNA from 12 to 48 h. Interestingly, the levels of phosphorylated Cdc2 were increased between 6 h and 48 h after 1,25[OH]2VD3 treatment, although the expression levels of Cdc2 mRNA and its protein production were reduced. 1,25[OH]2VD3 also decreased the expression of cyclin B1, which forms a complex with Cdc2. These data indicated that the increase of Myt1 and Wee1 induced the phosphorylation of Cdc2 leading to G2/M arrest. In conclusion, the induction of Cdc2 phosphorylation due to the increase of Wee1 and Myt1 as well as the reduction of Cdc2 and cyclin B1 are involved in 1,25[OH]2VD3-induced G2/M arrest of keratinocytes.

  15. Regulation of gene expression by 1,25-dihydroxyvitamin D3 in bone cells: exploiting new approaches and defining new mechanisms

    PubMed Central

    Pike, J Wesley; Lee, Seong Min; Meyer, Mark B

    2014-01-01

    The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) serve both to orchestrate calcium and phosphorus homeostasis in higher vertebrates and to regulate a diverse set of cellular functions unrelated to control of mineral metabolism. With regard to bone, mesenchymal lineage cells, including both early and late osteoblasts as well as osteocytes represent classic targets of the vitamin D hormone. Accordingly, much of the early information regarding our current understanding of the mechanism of action of 1,25(OH)2D3, of which gene regulation is central, derives from a broad array of studies in these cell types. Indeed, a gene that provided both the earliest and perhaps the most extensive information regarding this and additional mechanisms was that of osteoblast-specific osteocalcin. Subsequent work has provided much additional detail as to how 1,25(OH)2D3, through the vitamin D receptor (VDR), mediates the modulation of many bone cell genes. In recent years, however, a series of technical advances involving the coupling of chromatin immunoprecipitation (ChIP) to unbiased methodologies that involve next-generation DNA sequencing techniques (ChIP-seq) have opened new avenues in the study of gene regulation. In this review, we summarize early work and then focus on more recent studies that have used ChIP-seq analysis and other approaches to provide insight into not only the regulation of specific genes such as the VDR, TNFSF11 (RANKL), LRP5, CBS and CYP24a1, but overarching genome-wide principles of gene regulation as well. The results of these studies highlight the value of these new approaches and the increased insight that can be gained. PMID:24466413

  16. 25-Hydroxyvitamin D3 induces osteogenic differentiation of human mesenchymal stem cells

    PubMed Central

    Lou, Yan-Ru; Toh, Tai Chong; Tee, Yee Han; Yu, Hanry

    2017-01-01

    25-Hydroxyvitamin D3 [25(OH)D3] has recently been found to be an active hormone. Its biological actions are demonstrated in various cell types. 25(OH)D3 deficiency results in failure in bone formation and skeletal deformation. Here, we investigated the effect of 25(OH)D3 on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We also studied the effect of 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], a metabolite of 25(OH)D3. One of the vitamin D responsive genes, 25(OH)D3-24-hydroxylase (cytochrome P450 family 24 subfamily A member 1) mRNA expression is up-regulated by 25(OH)D3 at 250–500 nM and by 1α,25-(OH)2D3 at 1–10 nM. 25(OH)D3 and 1α,25-(OH)2D3 at a time-dependent manner alter cell morphology towards osteoblast-associated characteristics. The osteogenic markers, alkaline phosphatase, secreted phosphoprotein 1 (osteopontin), and bone gamma-carboxyglutamate protein (osteocalcin) are increased by 25(OH)D3 and 1α,25-(OH)2D3 in a dose-dependent manner. Finally, mineralisation is significantly increased by 25(OH)D3 but not by 1α,25-(OH)2D3. Moreover, we found that hMSCs express very low level of 25(OH)D3-1α-hydroxylase (cytochrome P450 family 27 subfamily B member 1), and there is no detectable 1α,25-(OH)2D3 product. Taken together, our findings provide evidence that 25(OH)D3 at 250–500 nM can induce osteogenic differentiation and that 25(OH)D3 has great potential for cell-based bone tissue engineering. PMID:28211493

  17. Effects of 1α,25-Dihydroxyvitamin D3 on Intestinal Absorption and Disposition of Adefovir Dipivoxil and Its Metabolite, Adefovir, in Rats.

    PubMed

    Yoon, In-Soo; Son, Jun-Hyeng; Kim, Sang-Bum; Choi, Min-Koo; Maeng, Han-Joo

    2015-01-01

    The aim of this study was to investigate the effect of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), an active form of vitamin D, on the oral absorption and disposition of adefovir dipivoxil (P-glycoprotein (P-gp) substrate) and its major active metabolite, adefovir (multidrug resistance-associated protein 4 (Mrp4) substrate), in rats. The pharmacokinetics of intravenous adefovir and oral adefovir dipivoxil was evaluated in control and 1,25(OH)2D3-treated rats. The intestinal absorption of adefovir dipivoxil was investigated through an in situ closed loop study, and the tissue distribution of adefovir after oral administration of adefovir dipivoxil was evaluated in the two groups. There was no significant difference in pharmacokinetic parameters of intravenous adefovir between the two groups. Importantly, the total area under the plasma concentration-time curve from time zero to time infinity (AUC), peak plasma concentration (Cmax) and extent of absolute oral bioavailability (F) of adefovir after oral administration of adefovir dipivoxil were significantly higher in 1,25(OH)2D3-treated rats than in control rats. In the in situ closed loop study, there was no significant difference in the remaining fraction of adefovir dipivoxil in the duodenum, jejunum and ileum loops between the two groups. In the tissue distribution study after oral administration of adefovir dipivoxil, the tissue-to-plasma partition coefficients of adefovir in the liver, brain, kidney, and intestine were significantly lower in the 1,25(OH)2D3-treated rats than in control rats. The present study indicates that 1,25(OH)2D3 treatment can enhance the oral absorption of adefovir dipivoxil, likely via the induction of basolateral Mrp4 function in rat intestine. However, the impact of 1,25(OH)2D3 treatment on the pharmacokinetics of intravenous adefovir was limited. These results could lead to further studies in clinically significant P-gp and/or MRP4-mediated 1,25(OH)2D3-drug interactions.

  18. 1α,25 dihydroxyvitamin D3 enhances cellular defences against UV-induced oxidative and other forms of DNA damage in skin.

    PubMed

    Gordon-Thomson, Clare; Gupta, Ritu; Tongkao-on, Wannit; Ryan, Anthony; Halliday, Gary M; Mason, Rebecca S

    2012-12-01

    DNA damage induced by ultraviolet radiation is the key initiator for skin carcinogenesis since mutations may arise from the photoproducts and it also contributes to photoimmune suppression. The active vitamin D hormone, 1α,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) reduces thymine dimers, the major photoproduct found in human skin after UV exposure, and suppresses the accumulation of nitric oxide derivatives that lead to more toxic reactive nitrogen species (RNS). We examined whether other forms of DNA damage are reduced by 1,25(OH)(2)D(3), and hypothesized that photoprotection by 1,25(OH)(2)D(3) is, in part, due to the suppression of various forms of promutagenic DNA damage, including thymine dimers, through a reduction of genotoxic RNS. Different forms of UV-induced DNA damage were investigated in irradiated skin cells treated with or without 1,25(OH)(2)D(3), or inhibitors of metabolism and inducible nitric oxide synthase. Keratinocytes were also treated with nitric oxide donors in the absence of UV light. DNA damage was assessed by comet assay incorporating site specific DNA repair endonucleases, and by immunohistochemistry using antibodies to thymine dimers or 8-oxo-7,8-dihydro-2'-deoxyguanosine, and quantified by image analysis. Strand breaks in T4 endonuclease V, endonuclease IV and human 8-oxoguanine DNA glycosylase digests increased more than 2-fold in UV irradiated human keratinocytes, and were reduced by 1,25(OH)(2)D(3) treatment after UV exposure, and also by low temperature, sodium azide and an inhibitor of inducible nitric oxide synthase. Conversely, nitric oxide donors induced all three types of DNA damage in the absence of UV. We present data to show that 1,25(OH)(2)D(3) protects skin cells from at least three forms of UV-induced DNA damage, and provide further evidence to support the proposal that a reduction in RNS by 1,25(OH)(2)D(3) is a likely mechanism for its photoprotective effect against oxidative and nitrative DNA damage, as well as

  19. Human PXR-mediated induction of intestinal CYP3A4 attenuates 1α,25-dihydroxyvitamin D3 function in human colon adenocarcinoma LS180 cells

    PubMed Central

    Zheng, Xi Emily; Wang, Zhican; Liao, Michael Z.; Lin, Yvonne S.; Shuhart, Margaret C.; Schuetz, Erin G.; Thummel, Kenneth E.

    2012-01-01

    Oxidative catabolism of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is mediated by either CYP24A1 or CYP3A4. In this paper, we tested whether induction of CYP3A4 in the LS180 intestinal cell model enhances clearance of 1α,25(OH)2D3 and blunts its hormonal effect on expression of the apical membrane calcium transport protein, TRPV6. Treatment with the hPXR agonist rifampin significantly increased CYP3A4 mRNA content and catalytic activity, but had no effect on CYP24A1 or TRPV6 mRNA content. Pre-treating cells with rifampin for 48 hrs, prior to a 24 hr 1α,25(OH)2D3 treatment phase, was associated with a subsequent 48% increase in the elimination of 1α,25(OH)2D3 and a 35% reduction of peak TRPV6 mRNA. Introduction of the CYP3A4 inhibitor, 6′,7′-dihydroxybergamottin, an active inhibitor in grapefruit juice, reversed the effects of rifampin on 1α,25(OH)2D3 clearance and TRPV6 expression. Over-expression of hPXR in LS180 cells greatly enhanced the CYP3A4 responsiveness to rifampin pretreatment, and elicited a greater relative suppression of TRPV6 expression and an increase in 1α,25(OH)2D3 disappearance rate, compared to vector expressed cells, following hormone administration. Together, these results suggest that induction of CYP3A4 in the intestinal epithelium by hPXR agonists can result in a greater metabolic clearance of 1α,25(OH)2D3 and reduced effects of the hormone on the intestinal calcium absorption, which may contribute to an increased risk of drug-induced osteomalacia/osteoporosis in patients receiving chronic therapy with potent hPXR agonists. Moreover, ingestion of grapefruit juice in the at-risk patients could potentially prevent this adverse drug effect. PMID:22562045

  20. 1,25-Dihydroxyvitamin D induces the glutamate transporter SLC1A1 and alters glutamate handling in non-transformed mammary cells

    PubMed Central

    Beaudin, Sarah; Welsh, JoEllen

    2016-01-01

    Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3 is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters - SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1) - is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D treated cells. Taken together, these data identify 1,25D as a physiological

  1. Pakistanis living in Oslo have lower serum 1,25-dihydroxyvitamin D levels but higher serum ionized calcium levels compared with ethnic Norwegians. The Oslo Health Study

    PubMed Central

    Holvik, Kristin; Meyer, Haakon E; Søgaard, Anne Johanne; Haug, Egil; Falch, Jan A

    2007-01-01

    Background Persons of Pakistani origin living in Oslo have a much higher prevalence of vitamin D deficiency and secondary hyperparathyroidism but similar bone mineral density compared with ethnic Norwegians. Our objective was to investigate whether Pakistani immigrants living in Oslo have an altered vitamin D metabolism by means of compensatory higher serum levels of 1,25-dihydroxyvitamin D (s-1,25(OH)2D) compared with ethnic Norwegians; and whether serum levels of ionized calcium (s-Ca2+) differ between Pakistanis and Norwegians. Methods In a cross-sectional, population-based study venous serum samples were drawn from 94 Pakistani men and 67 Pakistani women aged 30–60 years, and 290 Norwegian men and 270 Norwegian women aged 45–60 years; in total 721 subjects. Results Pakistanis had lower s-1,25(OH)2D compared with Norwegians (p < 0.001). Age- and gender adjusted mean (95% CI) levels were 93 (86, 99) pmol/l in Pakistanis and 123 (120, 126) pmol/l in Norwegians, p < 0.001. The difference persisted after controlling for body mass index. There was a positive relation between serum 25-hydroxyvitamin D (s-25(OH)D) and s-1,25(OH)2D in both groups. S-Ca2+ was higher in Pakistanis; age-adjusted mean (95% CI) levels were 1.28 (1.27, 1.28) mmol/l in Pakistanis and 1.26 (1.26, 1.26) mmol/l in Norwegians, p < 0.001. In both groups, s-Ca2+ was inversely correlated to serum intact parathyroid hormone levels (s-iPTH). For any s-iPTH, s-Ca2+ was higher in Pakistanis, also when controlling for age. Conclusion Community-dwelling Pakistanis in Oslo with low vitamin D status and secondary hyperparathyroidism have lower s-1,25(OH)2D compared with ethnic Norwegians. However, the Pakistanis have higher s-Ca2+. The cause of the higher s-Ca2+ in Pakistanis in spite of their higher iPTH remains unclear. PMID:17945003

  2. Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1alpha,25-dihydroxyvitamin D3 synthesis in leptin-deficient mice.

    PubMed

    Tsuji, Kiyomi; Maeda, Toyonobu; Kawane, Tetsuya; Matsunuma, Ayako; Horiuchi, Noboru

    2010-08-01

    Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone-derived fibroblast growth factor 23 (FGF-23) as a mediator of the influence of leptin on renal 1alpha-hydroxylase mRNA expression in leptin-deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF-23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12-hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF-23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Administration of FGF-23 (5 microg i.p. at 12-hour intervals for 2 days) to ob/ob mice suppressed renal 1alpha-hydroxylase mRNA expression. The main site of FGF-23 mRNA expression was the bone, and leptin markedly increased the FGF-23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1alpha-hydroxylase and sodium-phosphate cotransporters (NaP(i)-IIa and NaP(i)-IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF-23 level and renal expression of 1alpha-hydroxylase mRNA were not influenced by administration of leptin to leptin receptor-deficient (db/db) mice. These results indicate that leptin directly stimulates FGF-23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH)(2)D(3) synthesis in these mice is at least partly due to elevated bone production of FGF-23.

  3. Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors.

    PubMed

    Chen, Jiaxuan; Dosier, Christopher R; Park, Jung Hwa; De, Subhendu; Guldberg, Robert E; Boyan, Barbara D; Schwartz, Zvi

    2016-01-01

    1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein-2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three-dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild-type (WT), Pdia3-silenced (Sh-Pdia3) and VDR-silenced (Sh-VDR) pre-osteoblastic MC3T3-E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε-caprolactone)-collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro-computed tomography (μCT) showed that the mineralized volume of untreated Sh-Pdia3 and Sh-VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh-VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3.

  4. Candidate Reference Measurement Procedure for the Determination of (24R),25-Dihydroxyvitamin D3 in Human Serum Using Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Tai, Susan S-C; Nelson, Michael A

    2015-08-04

    The two major forms of vitamin D, vitamin D3 and vitamin D2, are metabolized in the liver through hydroxylation to 25-hydroxyvitamin D species, and then further hydroxylated in the kidney to various dihydroxyvitamin D species. (24R),25-Dihydroxyvitamin D3 ((24R),25(OH)2D3) is a major catabolite of 25-hydroxyvitamin D metabolism and is an important vitamin D metabolite used as a catabolism marker and indicator of kidney disease. The National Institute of Standards and Technology has recently developed a reference measurement procedure for the determination of (24R),25(OH)2D3 in human serum using isotope-dilution LC-MS/MS. The (24R),25(OH)2D3 and added deuterated labeled internal standard (24R),25(OH)2D3-d6 were extracted from serum matrix using liquid-liquid extraction prior to LC-MS/MS analysis. Chromatographic separation was performed using a fused-core C18 column. Atmospheric pressure chemical ionization in the positive ion mode and multiple reaction monitoring were used for LC-MS/MS. The accuracy of the measurement of (24R),25(OH)2D3 was evaluated by recovery studies of measuring (24R),25(OH)2D3 in gravimetrically prepared spiked samples of human serum with known (24R),25(OH)2D3 levels. The recoveries of the added (24R),25(OH)2D3 averaged 99.0% (0.8% SD), and the extraction efficiencies averaged 95% (2% SD). Excellent repeatability was demonstrated with CVs of ∼1%. The limit of quantitation at a signal-to-noise ratio of ∼10 was 0.2 ng/g. Potential isomeric interferences from other endogenous species and from impurity components of the reference standard were investigated. LC baseline resolution of (24R),25(OH)2D3 from these isomers was achieved within 35 min. This method was used for value assignment of (24R),25(OH)2D3 in Standard Reference Materials of Vitamin D Metabolites in Human Serum, which can serve as an accuracy base for routine methods used in clinical laboratories.

  5. Protective effect of 1α,25-dihydroxyvitamin D3 on effector CD4+ T cell induced injury in human renal proximal tubular epithelial cells

    PubMed Central

    Chung, Byung Ha; Kim, Bo-Mi; Doh, Kyoung Chan; Cho, Mi-La

    2017-01-01

    Background The aim of this study was to investigate the protective effect of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] on effector CD4+ T cells or on inflammatory cytokine-induced injury in human renal proximal tubular epithelial cells (HRPTEpiC). Methods First, we investigated the effect of 1,25(OH)2D3 on CD4+ T cell proliferation. Second, we examined the effect of 1,25(OH)2D3 on inflammatory cytokine secretion or fibrosis in HRPTEpiC induced by inflammatory cytokines or activated CD4+ T cells using ELISA and real-time PCR. Lastly, we compared urine inflammatory-cytokine (IL-6, IL-8) or KIM-1 levels in kidney transplant recipients low serum 25-hydroxyvitamin D (25(OH)D) group (< 20 ng/mL) (n = 40) and normal 25(OH)D group (n = 50). Results Pre-incubation with 1,25(OH)2D3 significantly reduced the percentages of Th1 and Th17 cells compared to that of Th0 condition (P < 0.05 for each). In contrast, 1,25(OH)2D3 increased the proportion of Th2 and Treg cells in a dose-dependent manner (P < 0.05 for each). Treatment of HRPTEpiC with inflammatory cytokines (TNF-α, IL-17, and TGF-β) or effector CD4+ T cells resulted in increased production of IL-6, IL-8, or KIM-1 from HRPTEpiC in a dose-dependent manner. However, treatment with 1,25(OH)2D3 significantly reduced the level of these cytokines (P < 0.05 for all). Western blot analysis demonstrated that the mTOR/STAT3/ERK pathway was downregulated by 1,25(OH)2D3 in HRPTEpiC. Furthermore, the concentrations of urine IL-6/creatinine (P < 0.05) and Kim-1/creatinine (P < 0.05) were higher in the low 25(OH)D group than in the normal 25(OH)D group in kidney transplant recipients. Conclusion The results of this study suggests that vitamin D may have a significant role in the regulation of inflammation in allograft tissue in kidney transplant recipients. Trial registration All participants provided written informed consent in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of

  6. Abiraterone inhibits 1α,25-dihydroxyvitamin D3 metabolism by CYP3A4 in human liver and intestine in vitro.

    PubMed

    Deb, Subrata; Chin, Mei Yieng; Adomat, Hans; Guns, Emma S Tomlinson

    2014-10-01

    The chemopreventive and therapeutic effects of vitamin D3 are exerted through its dihydroxylated metabolite, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. Inactivation of 1α,25(OH)2D3 by cytochrome P450 3A4 (CYP3A4) may be an important determinant of its serum and tissue levels. Abiraterone, a steroidogenesis inhibitor used in late stage prostate cancer treatment, is a CYP17A1 inhibitor. The purpose of this study was to assess the potential of abiraterone to block hepatic and intestinal inactivation of biologically active vitamin D3in vitro and to evaluate if abiraterone can alter CYP3A4 marker substrate activities. Biotransformation reactions were initiated with NADPH regenerating solutions following initial preincubation of pooled human hepatic or intestinal microsomal protein or human recombinant CYP3A4 supersomes with 1α,25(OH)2D3, midazolam or triazolam for 10min at 37°C. Formation of hydroxylated metabolites of 1α,25(OH)2D3, midazolam or triazolam was analyzed by liquid chromatography-mass spectrometry method. Co-incubation of 1α,25(OH)2D3 with abiraterone at varying concentrations (0.2-100μM) led to up to ∼85% inhibition of formation of hydroxylated metabolites of 1α,25(OH)2D3 thus preventing inactivation of active vitamin D3. The IC50 values for individual metabolites of 1α,25(OH)2D3 ranged from 0.4 to 2.2μM in human liver microsomes or human intestinal microsomes. The mechanism of CYP3A4-mediated inhibition of 1α,25(OH)2D3 by abiraterone was competitive (apparent Ki 2.8-4.3μM). Similar inhibitory effects were also observed upon inclusion of abiraterone into midazolam or triazolam hydroxylation assays. In summary, our results suggest that abiraterone inhibits the CYP3A4-mediated inactivation of active vitamin D3 in human liver and intestine, potentially providing additional anti-cancer benefits to prostate cancer patients. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

  7. Suppressive effect of 1,25-dihydroxyvitamin D3 and its analogues EB 1089 and KH 1060 on T lymphocyte proliferation in active ulcerative colitis.

    PubMed

    Stio, M; Bonanomi, A G; d'Albasio, G; Treves, C

    2001-02-01

    This study examined the effect exerted by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and two vitamin D analogues, EB 1089 and KH 1060, on the proliferation of T lymphocytes obtained from ulcerative colitis (UC) patients and healthy controls. The proliferative response of T lymphocytes to phytohaemagglutinin treatment was first analyzed on days three, five, and seven of culture. Cell proliferation was significantly lower in UC patients than that observed in healthy controls. The highest proliferation value, in either controls or patients, was registered on day five of culture. On day seven, a decrease in proliferation occurred, less evident in patients with respect to controls, whereas on day three, controls and patients showed the same proliferation value. The response of T lymphocytes of either healthy controls or UC patients to 1,25(OH)2D3, EB 1089, or KH 1060 was then investigated, treating the cells for three, five, and seven days with 10 nM vitamin D derivatives. In the presence of these compounds, cell proliferation was significantly inhibited in both groups, but on day seven, the inhibition of lymphocyte proliferation was remarkable in controls, whereas in patients it was similar to that registered on day five. The highest inhibition values were always obtained in the presence of KH 1060, and the time dependence was continuous in controls, but in the presence of EB 1089 only in patients. T lymphocytes prepared from healthy controls and UC patients were then cultured for five days in the presence of vitamin D derivatives at three different concentrations (0.1, 1, and 10 nM). In the two groups, a dose-dependent inhibition was registered in the presence of 1,25(OH)2D3 or EB 1089, while the inhibition of proliferation exerted by KH 1060 was not dose-dependent. The results obtained suggest an option for the use of the two non-hypercalcemic vitamin D analogues in the therapy of UC patients, perhaps in association with other immunosuppressive drugs.

  8. Comparative effects of 1,25-dihydroxyvitamin D3 and EB 1089 on mouse renal and intestinal 25-hydroxyvitamin D3-24-hydroxylase.

    PubMed

    Roy, S; Martel, J; Tenenhouse, H S

    1995-12-01

    EB 1089 is a vitamin D analog that is less potent than 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in its calcemic action but more potent in its antiproliferative action. We characterized the interaction of 1,25(OH)2D3 and EB 1089 with renal 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway, and compared the effects of 1,25(OH)2D3 and EB 1089 on induction of 24-hydroxylase mRNA in mouse kidney and intestine. 1,25(OH)2D3 and EB 1089 were competitive inhibitors of 24-hydroxylase activity. However, the Ki for 1,25(OH)2D3 (5.2 +/- 2.5 nM) was significantly lower than that for EB 1089 (286 +/- 59 nM). In the kidney, the time course and extent of 24-hydroxylase mRNA induction, relative to 18S rRNA, was similar for 1,25(OH)2D3 and EB 1089 with a peak response at approximately equal to 6 h that was sustained for at least 16 h. In the intestine, however, induction of 24-hydroxylase mRNA, relative to 18S rRNA, was approximately 50% lower for EB 1089 than for 1,25(OH)2D3 at 3 h (p < 0.05) and 6 h (p < 0.05) while at 16 h 24-hydroxylase mRNA was no longer detectable. Moreover, while both 1,25(OH)2D3 and EB 10898 elicited a similar dose-dependent induction of 24-hydroxylase mRNA in the kidney (EC50 = 0.4 +/- 0.13 and 0.3 +/- 0.08 ng/g for EB 1089 and 1,25(OH)2D3, respectively), the EC50 for EB 1089 (6.6 +/- 1.7 ng/g) was significantly higher than that for 1,25(OH)2D3 (0.9 +/- 0.32 ng/g) in the intestine (p < 0.01). EB 1089 was also less effective than 1,25(OH)2D3 in the induction of intestinal but not renal calbindin-D9k mRNA. To determine the mechanism for tissue-specific differences in potency, we determined the binding affinity of 1,25(OH)2D3 and EB 1089 for the vitamin D receptor. In the kidney, Kd values for 1,25(OH)2D3 (0.40 +/- 0.95 nM) and EB 1089 (0.48 +/- 0.04 nM) were not different. However, in the intestine, the Kd for EB 1089 (1.43 +/- 0.19 nM) was significantly higher than that for 1,25(OH)2D3 (0.85 +/- 0.06 nM; p < 0

  9. Differential skeletal responses of hindlimb unloaded rats on a vitamin D-deficient diet to 1,25-dihydroxyvitamin D3 and its analog, seocalcitol (EB1089)

    NASA Technical Reports Server (NTRS)

    Narayanan, Ramesh; Allen, Matthew R.; Gaddy, Dana; Bloomfield, Susan A.; Smith, Carolyn L.; Weigel, Nancy L.

    2004-01-01

    Conditions of disuse in bed rest patients, as well as microgravity experienced by astronauts are accompanied by reduced mechanical loading, reduced calcium absorption, and lower serum levels of 1,25(OH)2D3 (1,25-D), the active metabolite of vitamin D, all contributing to bone loss. To determine whether 1,25-D or a less calcemic analog, Seocalcitol or EB1089 (1 alpha,25-dihydroxy-22,24-diene-24,26,27-trihomovitamin D3) can alleviate bone loss in a rat hindlimb unloading model of disuse osteopenia, mature male rats originally on a vitamin D replete diet containing 1.01% calcium were transferred to a vitamin D-deficient diet containing 0.48% calcium and then tail suspended and treated for 28 days with vehicle, 0.05 microg/kg 1,25-D, or 0.05 microg/kg EB1089. The vitamin D-deficient diet caused a substantial decrease in bone mineral density (-8%), which may be compounded by hindlimb unloading (-10%). Exogenous 1,25-D not only prevented the bone loss but also increased the bone mineral density to greater than the baseline level (+7%). EB1089 was less effective in preventing bone loss. Analysis of site and cell-specific effects of 1,25-D and EB1089 revealed that 1,25-D was more active than EB1089 in the intestine, the site of calcium absorption, and in inducing osteoclastogenesis and bone resorption whereas EB1089 was more effective in inducing osteoblast differentiation. These studies suggest that elevating circulating 1,25-D levels presumably increasing calcium absorption can counteract bone loss induced by disuse or microgravity with its associated reductions in circulating 1,25-D and decreased calcium absorption.

  10. A new point mutation in the deoxyribonuclic acid-binding domain of the vitamine D receptor in a kindred with hereditary 1,25-dihydroxyvitamin d-resistant rickets

    SciTech Connect

    Yagi, Hideki; Miyake, Hiroshi; Nagashima, Kanji; Kuroume, Takayoshi ); Ozone, K.; Pike, J.W. )

    1993-02-01

    Hereditary 1,25-dihydroxyvitamin D [1,25-(OH)[sub 2]D]-resistant rickets (HVDRR) is a rare disorder characterized by rickets, alopecia, hypocalcemia, secondary hyperparathyroidism, and normal or elevated serum 1,25-dihydroxyvitamin D levels. The authors describe a patient with typical clinical characteristics of HVDRR, except that elevated levels of serum phosphorus were present coincident with increased levels of serum intact PTH. The patient was treated with high dose calcium infusion after an ineffective treatment with 1[alpha]-hydroxyvitamin D[sub 3]; serum calcium and phosphorus as well as intact PTH and alkaline phosphatase levels were normalized. Evaluation of phytohemagglutinin-activated lymphocytes derived from this patient revealed that 1,25-(OH)[sub 2]D[sub 3] was unable to inhibit thymidine incooperation, a result that contrast with the capacity of 1,25-(OH)[sub 2]D[sub 3] to inhibit uptake into normal activated lymphocytes. 1,25-(OH)[sub 2]D[sub 3] did not induce human osteocalcin promoter activity after transfection of this DNA linked to a reporter gene into patient cells. Cointroduction of a human vitamin D receptor (VDR) cDNA expression vector with the reporter plasmid, however, restored the hormone response. Evaluation of extracts from the patient cells for VDR DNA binding revealed a defect in DNA binding. Analysis of genomic DNA from the patient's cells by PCR confirmed the presence of a point mutation in exon 2 of the VDR. This exon directs synthesis of a portion of the DNA-binding domain of the receptor. We conclude that the genetic basis for 1,25-(OH)[sub 2]D[sub 3] resistance in this kindred with VDR-positive HVDRR is due to a single base mutation in the VDR that leads to production of a receptor unable to interact appropriately with DNA. 20 refs., 3 figs., 1 tab.

  11. Effects of partial and total colectomy on mineral and acid-base homoeostasis in the rat: magnesium deficiency, hyperphosphaturia and osteopathy, in the presence of high serum 1,25-dihydroxyvitamin D but normal parathyroid hormone.

    PubMed

    Croner, R; Schwille, P O; Erben, R G; Gepp, H; Stahr, K; Rümenapf, G; Parth, R; Scheuerlein, H

    2000-06-01

    The effects of colectomy on acid-base status, extra-osseous and bone minerals, calciotropic hormones and bone morphology have not yet been studied. To rectify this, groups of normally fed male rats were subjected to distal (n=11), proximal (n=12) or total (n=12) colectomy. Sham-operated rats (n=12) served as controls. At 112 (+/-2) days after colectomy the following changes were noted: (1) weight gain was delayed; (2) faecal excretion of calcium and phosphorus was normal, whereas that of magnesium was increased; (3) intestinal calcium secretion and absorption of calcium and phosphorus were normal, but magnesium absorption was decreased; (4) urinary excretion of magnesium was also decreased, that of phosphorus was increased, and that of pyridinium and deoxypyridinium tended to be high; (5) the serum levels of ionized magnesium, total calcium, 25-hydroxyvitamin D and parathyroid hormone were normal, while that of 1,25-dihydroxyvitamin D was markedly elevated; and (6) bone magnesium and phosphorus content were decreased, but bone calcium was normal, and thus the bone calcium/phosphorus ratio was high. These abnormalities were associated with moderate metabolic acidosis, as reflected by high urinary ammonium, low citrate and low total CO(2), but normal blood gases. Significant structural abnormalities of bone were not detectable, but trabecular bone tended to show rarefication. Distal colectomy had the least effect, whereas proximal and total colectomies had a distinct effect, on these parameters. It is concluded that colectomy in the rat causes: (1) a syndrome of magnesium deficiency of intestinal origin, compensated metabolic acidosis, urinary phosphorus loss, and high circulating 1,25-dihydroxyvitamin D levels, with the degree depending on the extent of surgical resection; and (2) brittle bones, a feature characteristic of low bone magnesium and more generalized magnesium deficiency. The mechanisms leading to this syndrome are unknown, but altered tissue levels of

  12. The Effect of Bone Marrow Mesenchymal Stem Cells on Vitamin D3 Induced Monocytic Differentiation of U937 Cells

    PubMed Central

    Molaeipour, Zahra; Shamsasanjan, Karim; Movassaghpour, Ali Akbari; Akbarzadehlaleh, Parvin; Sabaghi, Fatemeh; Saleh, Mahshid

    2016-01-01

    Purpose: Mesenchymal stem cells (MSCs) are key components of the hematopoietic stem cells (HSCs) niche. They control the process of hematopoiesis by secreting regulatory cytokines, growth factors and expression of important cell adhesion molecules for cell-tocell interactions. In this research, we have investigated the effect of bone marrow derived MSCs on monocytic differentiation of U937 cells line. Methods: U937 cells were cultured in both direct co-culture with MSCs and MSCs conditioned medium (C.M) driven. This study used 1,25-dihydroxyvitamin D3(VitD3) as inductor of monocytic differentiation and U937 cells treated with VitD3 morphology was examined by Wright Giemsa staining. CD14 monocytic differentiation marker was measured by flow cytometry and monocytic gene expression was assessed by real time polymerase chain reaction (RT PCR). Results: The results of flow cytometric analysis showed that CD14 expression of U937 increased. The higher effect of MSCs co-culture on CD14 expression in U937 cells was observed, compared to the conditioned medium. Among ten monocytic related genes which were screened that was observed increase in 5 genes in which CXCR4 and CSF2RA showed significant increase. Conclusion: The results obtained show that MSCs have supportive effect on the monocytic differentiation of U937 cells. However, a distinct mechanism of that remains unclear. PMID:27123414

  13. Regulated expression of the MRP8 and MRP14 genes in human promyelocytic leukemic HL-60 cell treated with the differentiation-inducing agents mycophenolic acid and 1{alpha},25-Dihydroxyvitamin D{sub 3}

    SciTech Connect

    Warner-Bartnicki, A.L.; Murao, S.; Collart, F.R.; Huberman, E.

    1992-12-31

    The calcium-binding proteins MRP8 and MEP14 are present in mature monomyelocytic cells and are induced during differentiation. Previous studies have demonstrated that the proteins may mediate the growth arrest in differentiating HL-60 cells. We determined the levels of a protein complex (PC) containing MRP8 and MRP14 and investigated the mechanism by which the genes encoding these proteins are regulated in HL-60 cells treated with the differentiation-inducing agent mycophenorc acid (MPA)While the PC was barely detectable in untreated cells, MPA treatment resulted in elevated levels of the PC which were maximal at 3-4 d, and were found to directly parallel gains in the steady-state levels of MRP8 and MRP14 MRNA. Transcription studies with the use of nuclear run-on experiments revealed increased transcription initiation at the MRP8 and MRP14 promoters after MPA treatment. 1{alpha},25-Dihydroxyvitamin D{sub 3}, which induces HL-60 cell differentiation by another mechanism, was also found to increase transcription initiation at the MRP8 and MRP14 promoters. Our results suggest that this initiation is the major control of maturation agent-mediated increases in MRP8 and MRPl4 gene expression, and support a role for the PC in terminal differentiation of human monomyelocytic cells.

  14. 1,25-Dihydroxyvitamin D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated MLCK-P-MLC signaling pathway.

    PubMed

    Chen, Shanwen; Zhu, Jing; Chen, Guowei; Zuo, Shuai; Zhang, Junling; Chen, Ziyi; Wang, Xin; Li, Junxia; Liu, Yucun; Wang, Pengyuan

    2015-05-08

    Substantial studies have demonstrated the protective effect of 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) on intestinal barrier function, but the mechanisms are not fully illustrated. In this study, the effect of 1,25(OH)2D3 on TNF-α induced barrier dysfunction was further investigated in Caco-2 cell monolayers. The barrier function of Caco-2 monolayers was evaluated by measuring trans-epithelial electrical resistance (TEER) and FITC-Dextran 40,000 Da (FD-40) trans-membrane flux. ZO-1 and Occludin were chosen as markers of the localization of tight junction (TJ) proteins for immunofluorescence. The expression of MLCK and phosphorylation level of myosin light chain (MLC) were measured by immunoblotting. The activation of NF-kB p65 was analyzed by EMSA and immunofluorescence. The results suggest that 1,25(OH)2D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated activation of MLCK-P-MLC signaling pathway.

  15. QW-1624F2-2, a synthetic analogue of 1,25-dihydroxyvitamin D3, enhances the response to other deltanoids and suppresses the invasiveness of human metastatic breast tumor cells.

    PubMed

    Sundaram, Sujatha; Beckman, Matthew J; Bajwa, Amandeep; Wei, Jeffrey; Smith, Kathleen M; Posner, Gary H; Gewirtz, David A

    2006-11-01

    The enzyme 24-hydroxylase, also known as CYP24, metabolizes 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and is an established marker of vitamin D activity. Our studies evaluated the influence of a low-calcemic 1,25(OH)(2)D(3) analogue, QW-1624F2-2 (QW), on the regulation of CYP24 expression in MKL-4 cells, a metastatic mammary tumor cell model. 1,25(OH)(2)D(3) and its analogue, EB 1089, stimulated CYP24 induction at both protein and transcript levels. In contrast, QW failed to produce a sustained stimulation of CYP24, due, in large part, to a reduction in the stability of the CYP24 message. QW enhanced the capacity of 1,25(OH)(2)D(3) and EB 1089 to inhibit tumor cell proliferation by approximately 2-fold. QW also blocked the sustained induction of CYP24 expression by 1,25(OH)(2)D(3) and EB 1089, increased the potency of 1,25(OH)(2)D(3) and EB 1089, and inhibited breast tumor cell proliferation and invasion.

  16. Combination of lenalidomide with vitamin D3 induces apoptosis in mantle cell lymphoma via demethylation of BIK

    PubMed Central

    Brosseau, C; Dousset, C; Touzeau, C; Maïga, S; Moreau, P; Amiot, M; Le Gouill, S; Pellat-Deceunynck, C

    2014-01-01

    Mantle cell lymphoma (MCL) is a currently incurable B-cell malignancy. Lenalidomide (Len) has been demonstrated to be one of the most efficient new treatment options. Because Len and 1α,25-dihydroxyvitamin (VD3) synergize to kill breast cancer cells, we investigated whether VD3 could increase the ability of Len to induce MCL cell death. While MCL cells were weakly sensitive to Len (1 μM), the addition of VD3 at physiological dose (100 nM) strongly increased cell death, accompanied by slowdown in cell cycle progression in MCL cell lines (n=4 out of 6) and primary samples (n=5 out of 7). The Len/VD3 treatment markedly increased the expression of the BH3-only BCL2-interacting killer (Bik) without affecting the expression of other Bcl-2 molecules. Immunoprecipitation assays demonstrated that Bik was free from anti-apoptotic partners, Bcl-2 and Bcl-xL, in treated cells. Moreover, silencing of BIK prevented apoptosis induced by Len/VD3, confirming the direct involvement of Bik in cell death. Bik accumulation induced by Len/VD3 was related to an increase in BIK mRNA levels, which resulted from a demethylation of BIK CpG islands. The sensitivity of MCL cells to Len/VD3 was similar to the response to 5-azacytidine, which also induced demethylation of BIK CpG islands. These preclinical data provide the rationale to investigate the role of VD3 in vivo in the response to Len. PMID:25165875

  17. Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

    PubMed

    Nagasawa, M; Cooper, L F; Ogino, Y; Mendonca, D; Liang, R; Yang, S; Mendonca, G; Uoshima, K

    2016-03-01

    The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow-derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

  18. The proapoptotic protein Bim is up regulated by 1α,25-dihydroxyvitamin D3 and its receptor agonist in endothelial cells and transformed by viral GPCR associated to Kaposi sarcoma.

    PubMed

    Suares, Alejandra; Russo de Boland, Ana; Verstuyf, Annemieke; Boland, Ricardo; González-Pardo, Verónica

    2015-10-01

    We have previously shown that 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and its less calcemic analog TX 527 induce apoptosis via caspase-3 activation in endothelial cells (SVEC) and endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR). In this work, we studied whether intrinsic apoptotic pathway could be activated by changing the balance between anti and pro-apoptotic proteins. Time response qRT-PCR analysis demonstrated that the mRNA level of anti-apoptotic gene Bcl-2 decreased after 12h and increased after 48h treatment with 1α,25(OH)2D3 or TX 527 in SVEC and vGPCR cells, whereas its protein level remained unchanged through time. mRNA levels of pro-apoptotic gene Bax significantly increased only in SVEC after 24 and 48h treatment with 1α,25(OH)2D3 and TX 527 although its protein levels remained unchanged in both cell lines. Bim mRNA and protein levels increased in SVEC and vGPCR cells. Bim protein increase by 1α,25(OH)2D3 and TX 527 was abolished when the expression of vitamin D receptor (VDR) was suppressed. On the other hand, Bortezomib (0.25-1nM), an inhibitor of NF-κB pathway highly activated in vGPCR cells, increased Bim protein levels and induced caspase-3 cleavage. Altogether, these results indicate that 1α,25(OH)2D3 and TX 527 trigger apoptosis by Bim protein increase which turns into the activation of caspase-3 in SVEC and vGPCR cells. Moreover, this effect is mediated by VDR and involves NF-κB pathway inhibition in vGPCR.

  19. Expression of human kinase suppressor of Ras 2 (hKSR-2) gene in HL60 leukemia cells is directly upregulated by 1,25-dihydroxyvitamin D{sub 3} and is required for optimal cell differentiation

    SciTech Connect

    Wang Xuening; Wang, T.-T.; White, John H.; Studzinski, George P. . E-mail: studzins@umdnj.edu

    2007-08-15

    Induction of terminal differentiation of neoplastic cells offers potential for a novel approach to cancer therapy. One of the agents being investigated for this purpose in preclinical studies is 1,25-dihydroxyvitamin D{sub 3} (1,25D), which can convert myeloid leukemia cells into normal monocyte-like cells, but the molecular mechanisms underlying this process are not fully understood. Here, we report that 1,25D upregulates the expression of hKSR-2, a new member of a small family of proteins that exhibit evolutionarily conserved function of potentiating ras signaling. The upregulation of hKSR-2 is direct, as it occurs in the presence of cycloheximide, and occurs primarily at the transcriptional level, via activation of vitamin D receptor, which acts as a ligand-activated transcription factor. Two VDRE-type motifs identified in the hKSR-2 gene bind VDR-RXR alpha heterodimers present in nuclear extracts of 1,25D-treated HL60 cells, and chromatin immunoprecipitation assays show that these VDRE motifs bind VDR in 1,25D-dependent manner in intact cells, coincident with the recruitment of RNA polymerase II to these motifs. Treatment of the cells with siRNA to hKSR-2 reduced the proportion of the most highly differentiated cells in 1,25D-treated cultures. These results demonstrate that hKSR-2 is a direct target of 1,25D in HL60 cells, and is required for optimal monocytic differentiation.

  20. Deletion of the Intestinal Plasma Membrane Calcium Pump, Isoform 1, Atp2b1, in Mice is Associated with Decreased Bone Mineral Density and Impaired Responsiveness to 1, 25-Dihydroxyvitamin D3

    PubMed Central

    Ryan, Zachary C.; Craig, Theodore A.; Filoteo, Adelaida G.; Westendorf, Jennifer J.; Cartwright, Elizabeth J.; Neyses, Ludwig; Strehler, Emanuel E.; Kumar, Rajiv

    2016-01-01

    The physiological importance of the intestinal plasma membrane calcium pump, isoform 1, (Pmca1, Atp2b1), in calcium absorption and homeostasis has not been previously demonstrated in vivo. Since global germ-line deletion of the Pmca1 in mice is associated with embryonic lethality, we selectively deleted the Pmca1 in intestinal absorptive cells. Mice with loxP sites flanking exon 2 of the Pmca1 gene (Pmca1fl/fl) were crossed with mice expressing Cre recombinase in the intestine under control of the villin promoter to give mice in which the Pmca1 had been deleted in the intestine (Pmca1EKO mice). Pmca1EKO mice were born at a reduced frequency and were small at the time of birth when compared to wild-type (Wt) litter mates. At two months of age, Pmca1EKO mice fed a 0.81% calcium, 0.34% phosphorus, normal vitamin D diet had reduced whole body bone mineral density (P <0.037), and reduced femoral bone mineral density (P <0.015). There was a trend towards lower serum calcium and higher serum parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) concentrations in Pmca1EKO mice compared to Wt mice but the changes were not statistically significant. The urinary phosphorus/creatinine ratio was increased in Pmca1EKO mice (P <0.004). Following the administration of 200 ng of 1α,25(OH)2D3 intraperitoneally to Wt mice, active intestinal calcium transport increased ∼2-fold, whereas Pmca1EKO mice administered an equal amount of 1α,25(OH)2D3 failed to show an increase in active calcium transport. Deletion of the Pmca1 in the intestine is associated with reduced growth and bone mineralization, and a failure to up-regulate calcium absorption in response to 1α,25(OH)2D3. PMID:26392310

  1. 1α,25-dihydroxyvitamin D(3) mechanism of action: modulation of L-type calcium channels leading to calcium uptake and intermediate filament phosphorylation in cerebral cortex of young rats.

    PubMed

    Zanatta, Leila; Goulart, Paola Bez; Gonçalves, Renata; Pierozan, Paula; Winkelmann-Duarte, Elisa C; Woehl, Viviane Mara; Pessoa-Pureur, Regina; Silva, Fátima Regina Mena Barreto; Zamoner, Ariane

    2012-10-01

    The involvement of calcium-mediated signaling pathways in the mechanism of action of 1α,25-dihydroxyvitamin D(3) (1,25D) is currently demonstrated. In this study we found that 1,25D induces nongenomic effects mediated by membrane vitamin D receptor (VDRm) by modulating intermediate filament (IF) phosphorylation and calcium uptake through L-type voltage-dependent calcium channels (L-VDCC) in cerebral cortex of 10 day-old rats. Results showed that the mechanism of action of 1,25D involves intra- and extracellular calcium levels, as well as the modulation of chloride and potassium channels. The effects of L-VDCCs on membrane voltage occur over a broad potential range and could involve depolarizing or hyperpolarizing coupling modes, supporting a cross-talk among Ca(2+) uptake and potassium and chloride channels. Also, the Na(+)/K(+)-ATPase inactivation by ouabain mimicked the 1,25D action on (45)Ca(2+) uptake. The Na(+)/K(+)-ATPase inhibition observed herein might lead to intracellular Na(+) accumulation with subsequent L-VDCC opening and consequently increased (45)Ca(2+) (calcium, isotope of mass 45) uptake. Moreover, the 1,25D effect is dependent on the activation of the following protein kinases: cAMP-dependent protein kinase (PKA), Ca(2+)/calmodulin-dependent protein kinase (PKCaMII), phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase p38 (p38(MAPK)). The modulation of calcium entry into neural cells by the 1,25D we are highlighting, might take a role in the regulation of a plethora of intracellular processes. Considering that vitamin D deficiency can lead to brain illness, 1,25D may be a possible candidate to be used, at least as an adjuvant, in the pharmacological therapy of neuropathological conditions.

  2. Differential effect of 1{alpha},25-dihydroxyvitamin D{sub 3} on Hsp28 and PKC{beta} gene expression in the phorbol ester-resistant human myeloid HL-525 leukemic cells

    SciTech Connect

    Lee, Yong J.; Galoforo, S.S.; Berns, C.M.

    1997-08-01

    We investigated the effect of 1{alpha},25-dihydroxyvitamin D{sub 3} [1,25-(OH){sub 2}D{sub 3}] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC{beta}) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistance to phorbol ester-induced macrophage differentiation. Northern and Western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hps28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatment with 50-300 nM 1,25-(OH){sub 2}D{sub 3}, a marked reduction of hsp28 gene expression was not associated with heat shock transcription factor-heat shock element (HSF-HSE) binding activity. Our results suggest that the differential effect of 1,25-(OH){sub 2}D{sub 3} on hsp28 and PKC{beta} gene expression is due to the different sequence composition of the vitamin D response element in the in the promoter region as well as an accessory factor for each gene or that 1,25-(OH){sub 2}D{sub 3} increases PKC{beta} gene expression, which in turn negatively regulates the expression of the hsp28 gene, or vice versa.

  3. Comparative therapeutic effects of orally administered 1,25-dihydroxyvitamin D3 and 1alpha-hydroxyvitamin D3 on type-1 diabetes in non-obese diabetic mice fed a normal-calcaemic diet

    PubMed Central

    Driver, J P; Foreman, O; Mathieu, C; van Etten, E; Serreze, D V

    2008-01-01

    Frequent injections of the hormonal form of vitamin D3, 1,25 dihydroxyvitamin D3 (1,25D3) reportedly inhibits autoimmune type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by correcting some of the abnormalities in antigen-presenting cells which contribute the development of pathogenic T cell responses. This route of administration greatly elevates the levels of these compounds in the bloodstream for hours after treatment, which requires mice to be fed diets formulated to contain much reduced levels of Ca to avoid the toxic effects of hypercalcaemia. In the current work, we demonstrate that feeding 1,25D3 or its synthetic precursor, 1alpha(OH) vitamin D3 (1alphaD3), as part of a T1D supportive chow diet containing normal levels of Ca, is an effective means of reducing the incidence of disease in NOD mice, but the doses required for protection elicited hypercalcaemia. However, T1D protection elicited by D3 analogue feeding appears, at least partially, to have an immunological basis, as splenic T cells from treated mice had a decreased capacity to adoptively transfer disease. Protection is associated with an increased proportion of T cells with CD4+ forkhead box P3+ regulatory phenotype within the islet infiltrate of treated animals. The 1alphaD3 precursor is converted rapidly to the active 1,25D3 isoform in vivo. However, feeding the 1alphaD3 analogue elicited stronger T1D protection than the 1,25D3 compound, but also induced more severe hypercalcaemia. In future, the dietary supplementation of novel low-calcaemic D3 analogues may enable their continuous delivery at levels that inhibit T1D development in susceptible humans consuming normal levels of Ca. PMID:17983444

  4. Improved accuracy of an LC-MS/MS method measuring 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D metabolites in serum using unspiked controls and its application to determining cross-reactivity of a chemiluminescent microparticle immunoassay.

    PubMed

    Dowling, Kirsten G; Hull, George; Sundvall, Jouko; Lamberg-Allardt, Christel; Cashman, Kevin D

    2017-03-23

    Measurement of serum 25-hydroxyvitamin D [25(OH)D] is considered the best indicator of vitamin D status. Two minor vitamin D metabolites are common interferences encountered in 25(OH)D assays. The first is 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], which if not chromatographically resolved from 25-hydroxyvitamin D3 [25(OH)D3], can overestimate 25(OH)D concentrations. The second is 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], which can cross-react with the antibodies in 25(OH)D immunoassays. Our aim was to develop an LC-MS/MS method capable of detecting both 3-epi-25(OH)D3 and 24R,25(OH)2D3 in serum without the use of a derivatization agent. We report an isotope dilution LC-MS/MS method, with electrospray ionization in the positive mode, that can simultaneously detect 24R,25(OH)2D3, 25(OH)D3, 3-epi-25(OH)D3, and 25-hydroxyvitamin D2. The method employs a cost-effective liquid-liquid extraction using only 150μL of sera and a total run time of 10min. Method performance was assessed by using quality controls made from pooled sera as an alternative to sera spiked with analytes. Biobanked samples, originally analyzed by chemiluminescent microparticle immunoassay (CMIA), were re-analyzed with this method to determine the contribution of 24R,25(OH)2D3 cross-reactivity to 25(OH)D measurement bias. The CMIA over-estimation of 25(OH)D measurements relative to LC-MS/MS was found to depend on both 25(OH)D and 24R,25(OH)2D3 concentrations.

  5. Effects of 1 alpha,25-dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cells: role in cell differentiation, activation and phagocytosis.

    PubMed Central

    Spittler, A; Willheim, M; Leutmezer, F; Ohler, R; Krugluger, W; Reissner, C; Lucas, T; Brodowicz, T; Roth, E; Boltz-Nitulescu, G

    1997-01-01

    The effect of calcitriol/1 alpha,25-dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry. Both constitutive and interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and tumour necrosis factor-alpha (TNF-alpha)-induced human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ Ag expression on monocytes was significantly down-regulated by calcitriol, IL-10 and transforming growth factor-beta (TGF-beta). The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3-5 days. Modulation of HLA-DR by calcitriol and IFN-gamma at the protein level correlated with the amount of mRNA specific for the HLA-DR alpha-chain, as judged by Northern blot analysis. The basal as well as IL-4, IL-6, IFN-gamma, TNF-alpha and TGF-beta-driven levels of HLA-ABC Ag were significantly diminished by calcitriol. On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up-regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down-regulated by calcitriol. The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes. Both the basal level of CD71 and IL-4, IFN-gamma or TNF-alpha-driven expression was diminished on calcitriol-treated U937 cells. In addition, calcitriol suppressed the expression of CD71 Ag on monocytes. The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes. Images Figure 2 PMID:9135559

  6. The human analog of murine cystein rich protein 61 [correction of 16] is a 1alpha,25-dihydroxyvitamin D3 responsive immediate early gene in human fetal osteoblasts: regulation by cytokines, growth factors, and serum.

    PubMed

    Schütze, N; Lechner, A; Groll, C; Siggelkow, H; Hüfner, M; Köhrle, J; Jakob, F

    1998-04-01

    1Alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) is a potent mediator of differentiation and maintenance of specific functions of osteoblasts. To detect novel targets for 1,25-(OH)2D3 action, we applied differential display PCR to human fetal osteoblast-like cells and identified the human analog of murine cystein rich protein 61 (hCYR61) as a 1,25-(OH)2D3-responsive immediate early gene in differentiated fetal osteoblast-like cells. The murine gene CYR61 is important for cell-cell and cell-matrix interactions, and it belongs to an emerging gene family of cysteine-rich proteins. hCYR61 messenger RNA (mRNA) steady-state levels were stimulated 11-fold by 10 nM 1,25-(OH)2D3 by 1 h and declined to control levels by 4 h. This transient stimulation of hCYR61 mRNA was not inhibited by cycloheximide but was prevented by actinomycin D, indicating that the 1,25-(OH)2D3 effect involves transcriptional events and does not require de novo protein synthesis. hCYR61 mRNA stability was not influenced by 1,25(OH)2D3, whereas cycloheximide treatment stabilized hCYR61 mRNA. FCS, as well as growth factors and cytokines such as basic fibroblast growth factor, epidermal growth factor, tumor necrosis factor alpha, and interleukin-1, strongly elevated hCYR61 mRNA steady-state levels within 1 h. hCYR61 mRNA was expressed also in primary human osteoblasts and osteosarcoma cell lines. Using a commercial tissue blot, hCYR61 mRNA was only observed in skeletal muscle. The fast and transient response of hCYR 61 to 1,25-(OH)2D3, serum, growth factors, and cytokines suggests an important role of hCYR61 for osteoblast function and differentiation.

  7. Differential expression and regulation of vitamin D hydroxylases and inflammatory genes in prostate stroma and epithelium by 1,25-dihydroxyvitamin D in men with prostate cancer and an in vitro model.

    PubMed

    Giangreco, Angeline A; Dambal, Shweta; Wagner, Dennis; Van der Kwast, Theodorus; Vieth, Reinhold; Prins, Gail S; Nonn, Larisa

    2015-04-01

    Previous work on vitamin D in the prostate has focused on the prostatic epithelium, from which prostate cancer arises. Prostatic epithelial cells are surrounded by stroma, which has well-established regulatory control over epithelial proliferation, differentiation, and the inflammatory response. Here we examined the regulation of vitamin D-related genes and inflammatory genes by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D) in laser-capture microdissected prostate tissue from a vitamin D3 clinical trial and in an in vitro model that facilitates stromal-epithelial crosstalk. Analysis of the trial tissues showed that VDR was present in both cell types, whereas expression of the hydroxylases was the highest in the epithelium. Examination of gene expression by prostatic (1,25(OH)2D) concentrations showed that VDR was significantly lower in prostate tissues with the highest concentration of 1,25(OH)2D, and down-regulation of VDR by 1,25(OH) 2D was confirmed in the primary cell cultures. Analysis of inflammatory genes in the patient tissues revealed that IL-6 expression was the highest in the prostate stroma while PTGS2 (COX2) levels were lowest in the prostate cancer tissues from men in the highest tertile of prostatic 1,25(OH)2D. In vitro, TNF-α, IL-6 and IL-8 were suppressed by 1,25 (OH)2D in the primary epithelial cells, whereas TNF-α and PTGS2 were suppressed by 1,25(OH) 2D in the stromal cells. Importantly, the ability of 1,25(OH)2D to alter pro-inflammatory-induced changes in epithelial cell growth were dependent on the presence of the stromal cells. In summary, whereas both stromal and epithelial cells of the prostate express VDR and can presumably respond to 1,25(OH)2D, the prostatic epithelium appears to be the main producer of 1,25(OH)2D. Further, while the prostate epithelium was more responsive to the anti-inflammatory activity of 1,25 (OH)2D than stromal cells, stroma-epithelial crosstalk enhanced the phenotypic effects of 1,25(OH)2D and the inflammatory

  8. Differential response to 1α, 25-dihydroxyvitamin D3 (1α,25(OH)2D3) in non-small cell lung cancer cells with distinct oncogene mutations1

    PubMed Central

    Zhang, Qiuhong; Kanterewicz, Beatriz; Shoemaker, Suzanne; Hu, Qiang; Liu, Song; Atwood, Kristopher; Hershberger, Pamela

    2012-01-01

    We previously demonstrated that non-small cell lung cancer (NSCLC) cells and primary human lung tumors aberrantly express the vitamin D3-catabolizing enzyme, CYP24, and that CYP24 restricts transcriptional regulation and growth control by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in NSCLC cells. To ascertain the basis for CYP24 dysregulation, we assembled a panel of cell lines that represent distinct molecular classes of lung cancer: Cell lines were selected which harbored mutually exclusive mutations in either the K-ras or the Epidermal Growth Factor Receptor (EGFR) genes. We observed that K-ras mutant lines displayed a basal vitamin D receptor (VDR)lowCYP24high phenotype, whereas EGFR mutant lines had a VDRhighCYP24low phenotype. A mutation-associated difference in CYP24 expression was also observed in clinical specimens. Specifically, K-ras mutation was associated with a median 4.2-fold increase in CYP24 mRNA expression (p = 4.8 × 10−7) compared to EGFR mutation in a series of 147 primary lung adenocarcinoma cases. Because of their differential basal expression of VDR and CYP24, we hypothesized that NSCLC cells with an EGFR mutation would be more responsive to 1,25(OH)2D3 treatment than those with a K-ras mutation. To test this, we measured the ability of 1,25(OH)2D3 to increase reporter gene activity, induce transcription of endogenous target genes, and suppress colony formation. In each assay, the extent of 1,25(OH)2D3 response was greater in EGFR mutation-positive HCC827 and H1975 cells than in K-ras mutation-positive A549 and 128.88T cells. We subsequently examined the effect of combining 1,25(OH)2D3 with erlotinib, which is used clinically in the treatment of EGFR mutation-positive NSCLC. 1,25(OH)2D3/erlotinib combination resulted in significantly greater growth inhibition than either single agent in both the erlotinib-sensitive HCC827 cell line and the erlotinib-resistant H1975 cell line. These data are the first to suggest that EGFR mutations may

  9. 1,25-Dihydroxyvitamin-D3 prevents the development of diabetic cardiomyopathy in type 1 diabetic rats by enhancing autophagy via inhibiting the β-catenin/TCF4/GSK-3β/mTOR pathway.

    PubMed

    Wei, Huili; Qu, Hua; Wang, Hang; Ji, Baolan; Ding, Yao; Liu, Dan; Duan, Yang; Liang, Huimin; Peng, Chuan; Xiao, Xiaoqiu; Deng, Huacong

    2017-04-01

    Diabetic cardiomyopathy (DCM) can increase the risk of heart failure and death in diabetic patients. However, no effective approaches are available to prevent its progression and development. Studies have shown that vitamin D is greatly implicated in cardiac hypertrophy and fibrosis, and there is a high prevalence of vitamin D deficiency in diabetic patients. In this study, we investigated whether 1,25-Dihydroxyvitamin-D3 (1,25D3) can improve DCM through a vitamin D receptor (VDR)-dependent mechanism associated with autophagy and the β-catenin/T-cell factor/lymphoid enhancer factor (TCF4)/glycogen synthase kinase-3β (GSK-3β)/mammalian target of rapamycin (mTOR) pathway. In this study, streptozotocin (STZ)-induced type 1 diabetic rats were established and were treated with 1,25D3 and/or chloroquine and/or VDR gene silencing for 8 weeks before being sacrificed. Compared with untreated diabetic rats, 1,25D3 partly attenuated the myocardial hypertrophy and interstitial fibrosis, improved cardiac function and restored the impaired cardiac autophagy in diabetic rats, all of which were reversed by silencing the VDR gene in diabetic rats. In high-glucose cultured H9C2 cells, 1,25D3 increased autophagy in a dose-dependent manner. Besides, the β-catenin/TCF4/GSK-3β and mTOR signaling were activated both in diabetic rats and in high-glucose cultured H9C2 cells. Treatment with 1,25D3 inhibited the β-catenin/TCF4/GSK-3β and mTOR signaling in H9C2 cells, whereas co-treatment with lithium chloride (LiCl) reversed this situation and abolished the beneficial effect of 1,25D3 on autophagy. These data suggest that 1,25D3 may improve DCM in type 1 diabetic rats by modulating autophagy through the β-catenin/TCF4/GSK-3β and mTOR pathway. Vitamin D may exist as a new therapeutic target for the treatment of DCM.

  10. 1,25-dihydroxyvitamin D inhibits vitamin E succinate-induced apoptosis in C3H10T1/2 cells but not Harvey ras-transfected cells.

    PubMed

    Stedman, Lynn; Nickel, Kwangok P; Castillo, S Sianna; Andrade, Juan; Burgess, John R; Teegarden, Dorothy

    2003-01-01

    In this study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on regulation of apoptosis was compared in control C3H10T1/2 mouse fibroblast cells and those transfected with the Harvey ras oncogene. A known apoptotic stimulator, vitamin E succinate (VES), reduced cell number in a time- and dose-dependent manner in both cell types. In an assay for viable cells, there were significantly more C3H10T1/2 cells cotreated with VES and 1,25(OH)2D3 (-5.0 +/- 10.5% of vehicle-treated controls) compared to VES alone treated cells (-60.8 +/- 5.6%). In contrast, 1,25(OH)2D3 did not change the percentage of viable cells following treatment by VES in ras-transfected cells [-67.3 +/- 7.5%, VES alone compared to 57.3 +/-v 15.7% with VES and 1,25(OH)2D3 ]. Further studies confirmed that 1,25(OH)2D3 inhibited VES-mediated apoptosis (1.27 +/- 0.34-fold over vehicle control) compared to VES treatment alone (2.29 +/- 0.56-fold increase) in C3H10T1/2 cells, but not in ras-transfected cells [3.07 +/- 0.43-fold increase, VES treatment alone; 3.64 +/- 0.42-fold increase, VES and 1,25(OH)2D3]. Both C3H10T1/2 and ras-transfected cells treated with VES had increased concentrations of cellular VES with very little change in a-tocopherol, indicating that the cells took up VES intact. In addition, both cell lines contained similar levels of nuclear vitamin D receptor (VDR); however, the ras-transfected cells had reduced VDRE transcriptional activity. In conclusion, VES exerts its effect intact and 1,25(OH)2D3 preferentially protects C3H10T1/2 cells, whereas ras-transformed cells were not protected from VES-mediated apoptosis.

  11. 1,25-Dihydroxyvitamin D decreases HTRA1 promoter activity in the rhesus monkey--a plausible explanation for the influence of vitamin D on age-related macular degeneration?

    PubMed

    Pahl, Lisa; Schubert, Stephanie; Skawran, Britta; Sandbothe, Maria; Schmidtke, Jörg; Stuhrmann, Manfred

    2013-11-01

    Age-related macular degeneration is the major cause of blindness in the elderly worldwide and the risk is influenced by both environmental and genetic risk factors. One important disease-associated region in humans is located on 10q26 and includes the two candidate genes ARMS2 and HTRA1. However, determination of the causative gene has not yet been possible and examining the situation in the rhesus monkey may help understand the situation in humans. In a recent paper, we characterized the rhesus monkey 10q26-orthologue region on chromosome 9 in detail and identified the drusen-associated HTRA1 promoter SNP rs196357513 as a putative risk factor. In this study, we predicted 9 binding sites for the vitamin D-dependent transcription factor vitamin D receptor in the rhesus HTRA1 promoter, one of which is destroyed by the rs196357513-risk allele. As patients with vitamin D deficit are at increased risk for age-related macular degeneration, a luciferase assay in transiently transfected ARPE19-cells was performed to evaluate the influence of the SNP rs196357513 and of 1,25-dihydroxyvitamin D on the rhesus monkey HTRA1 promoter activity. This revealed that the luciferase activity of the promoter construct containing the rs196357513 wild type allele was significantly reduced after vitamin D stimulation. An in silico analysis and literature search imply that this regulation could also play a role in human HTRA1 expression. Moreover, HTRA1 promoter activity of the construct containing the rs196357513 risk allele appeared diminished in comparison to the construct with the wild type allele, albeit this difference was not significant. The lower promoter activity due to the rhesus monkey rs196357513 risk allele apparently contradicts the common hypothesis for the human HTRA1 promoter risk allele of SNP rs11200638, for which a higher promoter activity has been observed. Our data point to a yet unexpected effect of decreased HTRA1 expression on drusen pathogenesis. Thus not only a

  12. Ablation of the Stimulatory G Protein α-Subunit in Renal Proximal Tubules Leads to Parathyroid Hormone-Resistance With Increased Renal Cyp24a1 mRNA Abundance and Reduced Serum 1,25-Dihydroxyvitamin D.

    PubMed

    Zhu, Yan; He, Qing; Aydin, Cumhur; Rubera, Isabelle; Tauc, Michel; Chen, Min; Weinstein, Lee S; Marshansky, Vladimir; Jüppner, Harald; Bastepe, Murat

    2016-02-01

    PTH regulates serum calcium, phosphate, and 1,25-dihydroxyvitamin D (1,25(OH)2D) levels by acting on bone and kidney. In renal proximal tubules (PTs), PTH inhibits reabsorption of phosphate and stimulates the synthesis of 1,25(OH)2D. The PTH receptor couples to multiple G proteins. We here ablated the α-subunit of the stimulatory G protein (Gsα) in mouse PTs by using Cre recombinase driven by the promoter of type-2 sodium-glucose cotransporter (Gsα(Sglt2KO) mice). Gsα(Sglt2KO) mice were normophosphatemic but displayed, relative to controls, hypocalcemia (1.19 ±0.01 vs 1.23 ±0.01 mmol/L; P < .05), reduced serum 1,25(OH)2D (59.3 ±7.0 vs 102.5 ±12.2 pmol/L; P < .05), and elevated serum PTH (834 ±133 vs 438 ±59 pg/mL; P < .05). PTH-induced elevation in urinary cAMP excretion was blunted in Gsα(Sglt2KO) mice (2- vs 4-fold over baseline in controls; P < .05). Relative to baseline in controls, PTH-induced reduction in serum phosphate tended to be blunted in Gsα(Sglt2KO) mice (-0.39 ±0.33 vs -1.34 ±0.36 mg/dL; P = .07). Gsα(Sglt2KO) mice showed elevated renal vitamin D 24-hydroxylase and bone fibroblast growth factor-23 (FGF23) mRNA abundance (∼3.4- and ∼11-fold over controls, respectively; P < .05) and tended to have elevated serum FGF23 (829 ±76 vs 632 ±60 pg/mL in controls; P = .07). Heterozygous mice having constitutive ablation of the maternal Gsα allele (E1(m-/+)) (model of pseudohypoparathyroidism type-Ia), in which Gsα levels in PT are reduced, also exhibited elevated serum FGF23 (474 ±20 vs 374 ±27 pg/mL in controls; P < .05). Our findings indicate that Gsα is required in PTs for suppressing renal vitamin D 24-hydroxylase mRNA levels and for maintaining normal serum 1,25(OH)2D.

  13. Microgravity control of autophagy modulates osteoclastogenesis.

    PubMed

    Sambandam, Yuvaraj; Townsend, Molly T; Pierce, Jason J; Lipman, Cecilia M; Haque, Azizul; Bateman, Ted A; Reddy, Sakamuri V

    2014-04-01

    Evidence indicates that astronauts experience significant bone loss during space mission. Recently, we used the NASA developed rotary cell culture system (RCCS) to simulate microgravity (μXg) conditions and demonstrated increased osteoclastogenesis in mouse bone marrow cultures. Autophagy is a cellular recycling process of nutrients. Therefore, we hypothesize that μXg control of autophagy modulates osteoclastogenesis. Real-time PCR analysis of total RNA isolated from mouse bone marrow derived non-adherent cells subjected to modeled μXg showed a significant increase in autophagic marker Atg5, LC3 and Atg16L mRNA expression compared to ground based control (Xg) cultures. Western blot analysis of total cell lysates identified an 8.0-fold and 7.0-fold increase in the Atg5 and LC3-II expression, respectively. Confocal microscopy demonstrated an increased autophagosome formation in μXg subjected RAW 264.7 preosteoclast cells. RT(2) profiler PCR array screening for autophagy related genes identified that μXg upregulates intracellular signaling molecules associated with autophagy, autophagosome components and inflammatory cytokines/growth factors which coregulate autophagy in RAW 264.7 preosteoclast cells. Autophagy inhibitor, 3-methyladenine (3-MA) treatment of mouse bone marrow derived non-adherent mononuclear cells showed a significant decrease in μXg induced Atg5 and LC3 mRNA expression in the presence or absence of RANK ligand (RANKL) stimulation. Furthermore, RANKL treatment significantly increased (8-fold) p-CREB transcription factor levels under μXg as compared to Xg cultures and 3-MA inhibited RANKL increased p-CREB expression in these cells. Also, 3-MA suppresses μXg elevated osteoclast differentiation in mouse bone marrow cultures. Thus, our results suggest that μXg induced autophagy plays an important role in enhanced osteoclast differentiation and could be a potential therapeutic target to prevent bone loss in astronauts during space flight missions.

  14. 1,25-Dihydroxyvitamin D3 attenuates adenylyl cyclase activity in rat thyroid cells: reduction of thyrotropin receptor number and increase in guanine nucleotide-binding protein Gi-2 alpha.

    PubMed

    Berg, J P; Sandvik, J A; Ree, A H; Sørnes, G; Bjøro, T; Torjesen, P A; Gordeladze, J O; Haug, E

    1994-08-01

    1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the most potent of the naturally occurring vitamin D metabolites. In rat thyroid FRTL-5 cells, 1,25-(OH)2D3 attenuated the increase in TSH-stimulated adenylyl cyclase activity obtained by removing TSH from the culture medium. When cells were incubated with 1,25-(OH)2D3 (10 nmol/liter; 4 days), the binding capacity for specific [125I]TSH binding decreased from 20.1 +/- 1.8 to 8.8 +/- 1.6 fmol/10(6) cells (mean +/- SEM; n = 4; P < 0.01) compared to that in control cells. The Kd did not change (mean +/- SEM, 0.46 +/- 0.09 vs. 0.25 +/- 0.07 nmol/liter; n = 4; P = NS). Western blotting revealed no change in the membrane content of the adenylyl cyclase (AC) stimulatory guanine nucleotide-binding protein (G-protein) alpha-subunit (Gs alpha) during 1,25-(OH)2D3 treatment. Similarly, levels of the AC inhibitory G-protein Gi-3 alpha- and G-protein beta-subunits were not altered by 1,25-(OH)2D3. However, Western blotting with antibodies recognizing both Gi-1 alpha and Gi-2 alpha was augmented 4-fold, presumably representing an increase in Gi-2 alpha only, as Gi-1 alpha messenger RNA (mRNA) was not detected in FRTL-5 cells. 1,25-(OH)2D3 (10 nmol/liter; 4 days) reduced cholera toxin (10 nmol/liter)-stimulated AC activity to 85% of the control value (P < 0.05), whereas forskolin (100 mumol/liter)-stimulated direct activation of AC was inhibited by 39%. The TSH receptor mRNA level correlated to the beta-actin mRNA was 2-fold higher in control cells compared to that in 1,25-(OH)2D3-treated cells 12 h after TSH removal. Only minor alterations in the Gs alpha mRNA/beta-actin mRNA and Gi-3 alpha mRNA/beta-actin mRNA ratios were observed during 1,25-(OH)2D3 treatment, whereas Gi-2 alpha mRNA increased 3-fold compared to that in control cells. No change in the resting intracellular Ca2+ concentration could be detected after 4 days of 1,25-(OH)2D3 treatment. Our studies show that 1,25-(OH)2D3 attenuates AC activity by reducing the TSH receptor

  15. Cross-sectional and longitudinal associations between the active vitamin D metabolite (1,25 dihydroxyvitamin D) and haemoglobin levels in older Australian men: the Concord Health and Ageing in Men Project.

    PubMed

    Hirani, Vasant; Cumming, Robert G; Blyth, Fiona; Naganathan, Vasi; Le Couteur, David G; Waite, Louise M; Handelsman, David J; Seibel, Markus J

    2015-02-01

    Anaemia and low 25 hydroxyvitamin D (25D) and 1,25 dihydroxyvitamin D (1,25D) levels are common in older people and may adversely affect morbidity and mortality. While there is some evidence for an association between low serum 25D levels and anaemia, there are limited studies among community-dwelling older people. In addition, the relationship between anaemia and the active vitamin D metabolite, 1,25D, has not been investigated. The aim of this study was to examine the associations between serum 25D and 1,25D with anaemia in community-living men aged ≥70 years. Population-based, cross-sectional analysis of the baseline phase and longitudinal analysis of the Concord Health and Ageing in Men Project (CHAMP), a large epidemiological study conducted in Sydney among men aged 70 years and older, were performed; 1666 men were seen at baseline (2005-2007), 1314 men at a 2-year follow-up (2007-2009) and 917 at a 5-year follow-up (2012-2013). The main outcome measurement was haemoglobin levels as a continuous measure. Covariates included 25D and 1,25D, estimated glomerular filtration rate, demographic information, lifestyle measures, health conditions and medication information. The prevalence of anaemia (Hb < 13.0 g/dL, WHO definition) was 14.6 %. In cross-sectional analysis, serum 25D concentrations were positively associated with haemoglobin levels in unadjusted analysis (β value 0.004; 95 % confidence interval (CI) 0.0009, 0.007; p = 0.01), but the associations were no longer significant after multivariate adjustment. The association between 1,25D levels and haemoglobin levels was significant in unadjusted analysis (β value 0.003; 95 % CI 0.002, 0.004; p < 0.0001) and remained significant in adjusted analysis (β value 0.001; 95 % CI 0.004, 0.003; p = 0.01). Serum 1,25D (but not 25D) levels at baseline were significantly associated with changes in haemoglobin over 2 and 5 years in unadjusted (β value 0.002; 95 % CI 0.0009, 0.003; p < 0

  16. Crosstalk between the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and the vitamin D receptor (VDR) in human breast cancer cells: PPAR{gamma} binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} mediated transactivation

    SciTech Connect

    Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R.; Knethen, Andreas von; Choubey, Divaker; Mehta, Rajendra G.

    2012-11-15

    Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ER{alpha}) physically binds to peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and inhibits its transcriptional activity. The interaction between PPAR{gamma} and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPAR{gamma} and VDR signaling, and for the first time we show that PPAR{gamma} physically associates with VDR in human breast cancer cells. We found that overexpression of PPAR{gamma} decreased 1{alpha},25-dihydroxyvitamin D{sub 3} (1,25D{sub 3}) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPAR{gamma}'s hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPAR{gamma}'s AF2 domain attenuated its repressive action on 1,25D{sub 3} transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPAR{gamma} was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXR{alpha}). Overexpression of RXR{alpha} blocked PPAR{gamma}'s suppressive effect on 1,25D{sub 3} action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPAR{gamma} and VDR pathways. -- Highlights: PPAR{gamma}'s role on 1{alpha},25-dihydroxyvitamin D{sub 3} transcriptional activity is examined. Black-Right-Pointing-Pointer PPAR{gamma} physically binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} action. Black-Right-Pointing-Pointer PPAR{gamma}'s hinge and ligand binding domains are important for this inhibitory effect. Black-Right-Pointing-Pointer PPAR{gamma} competes with VDR for the availability of their binding partner, RXR{alpha}.

  17. The Modulatory Effects of Mesenchymal Stem Cells on Osteoclastogenesis

    PubMed Central

    Sharaf-Eldin, Wessam E.; Abu-Shahba, Nourhan; Mahmoud, Marwa; El-Badri, Nagwa

    2016-01-01

    The effect of mesenchymal stem cells (MSCs) on bone formation has been extensively demonstrated through several in vitro and in vivo studies. However, few studies addressed the effect of MSCs on osteoclastogenesis and bone resorption. Under physiological conditions, MSCs support osteoclastogenesis through producing the main osteoclastogenic cytokines, RANKL and M-CSF. However, during inflammation, MSCs suppress osteoclast formation and activity, partly via secretion of the key anti-osteoclastogenic factor, osteoprotegerin (OPG). In vitro, co-culture of MSCs with osteoclasts in the presence of high concentrations of osteoclast-inducing factors might reflect the in vivo inflammatory pathology and prompt MSCs to exert an osteoclastogenic suppressive effect. MSCs thus seem to have a dual effect, by stimulating or inhibiting osteoclastogenesis, depending on the inflammatory milieu. This effect of MSCs on osteoclast formation seems to mirror the effect of MSCs on other immune cells, and may be exploited for the therapeutic potential of MSCs in bone loss associated inflammatory diseases. PMID:26823668

  18. Inhibition of protein kinase CK2 reduces CYP24A1 expression and enhances 1,25-dihydroxyvitamin D3 anti-tumor activity in human prostate cancer cells

    PubMed Central

    Luo, Wei; Yu, Wei-Dong; Ma, Yingyu; Chernov, Mikhail; Trump, Donald L.; Johnson, Candace S.

    2013-01-01

    Vitamin D has broad range of physiological functions and anti-tumor effects. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme for degrading many forms of vitamin D including the most active form, 1,25D3. Inhibition of CYP24A1 enhances 1,25D3 anti-tumor activity. In order to isolate regulators of CYP24A1 expression in prostate cancer cells, we established a stable prostate cancer cell line PC3 with CYP24A1 promoter driving luciferase expression to screen a small molecular library for compounds that inhibit CYP24A1 promoter activity. From this screening, we identified, 4,5,6,7-tetrabromobenzimidazole (TBBz), a protein kinase CK2 selective inhibitor as a disruptor of CYP24A1 promoter activity. We show that TBBz inhibits CYP24A1 promoter activity induced by 1,25D3 in prostate cancer cells. In addition, TBBz downregulates endogenous CYP24A1 mRNA level in TBBz treated PC3 cells. Furthermore, siRNA-mediated CK2 knockdown reduces 1,25D3 induced CYP24A1 mRNA expression in PC3 cells. These results suggest that CK2 contributes to 1,25D3 mediated target gene expression. Lastly, inhibition of CK2 by TBBz or CK2 siRNA significantly enhanced 1,25D3 mediated anti-proliferative effect in vitro and in vivo in a xenograft model. In summary, our findings reveal that protein kinase CK2 is involved in the regulation of CYP24A1 expression by 1,25D3 and CK2 inhibitor enhances 1,25D3 mediated anti-tumor effect. PMID:23358686

  19. SHP2 regulates osteoclastogenesis by promoting preosteoclast fusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genes that regulate osteoclast development and function under physiological and disease conditions remain incompletely understood. Shp2, a ubiquitously expressed cytoplasmic protein tyrosine phosphatase, was implicated in regulating M-CSF and RANKL-evoked signaling, its role in osteoclastogenesis an...

  20. The molecular mechanism of osteoclastogenesis in rheumatoid arthritis

    PubMed Central

    Udagawa, Nobuyuki; Kotake, Shigeru; Kamatani, Naoyuki; Takahashi, Naoyuki; Suda, Tatsuo

    2002-01-01

    Bone-resorbing osteoclasts are formed from hemopoietic cells of the monocyte–macrophage lineage under the control of bone-forming osteoblasts. We have cloned an osteoblast-derived factor essential for osteoclastogenesis, the receptor activator of NF-κB ligand (RANKL). Synovial fibroblasts and activated T lymphocytes from patients with rheumatoid arthritis also express RANKL, which appears to trigger bone destruction in rheumatoid arthritis as well. Recent studies have shown that T lymphocytes produce cytokines other than RANKL such as IL-17, granulocyte–macrophage colony-stimulating factor and IFN-γ, which have powerful regulatory effects on osteoclastogenesis. The possible roles of RANKL and other cytokines produced by T lymphocytes in bone destruction are described. PMID:12223101

  1. RBP-J-Regulated miR-182 Promotes TNF-α-Induced Osteoclastogenesis.

    PubMed

    Miller, Christine H; Smith, Sinead M; Elguindy, Mahmoud; Zhang, Tuo; Xiang, Jenny Z; Hu, Xiaoyu; Ivashkiv, Lionel B; Zhao, Baohong

    2016-06-15

    Increased osteoclastogenesis is responsible for osteolysis, which is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α-induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating the expression of microRNAs (miRNAs) important for this process. Using high-throughput sequencing of miRNAs, we obtained the first, to our knowledge, genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow-derived macrophages/osteoclast precursors during inflammatory osteoclastogenesis. Furthermore, we identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J-deficient cells. Complementary loss- and gain-of-function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and B lymphocyte-induced maturation protein-1. Moreover, we identified that direct miR-182 targets, Foxo3 and Maml1, play important inhibitory roles in TNF-α-mediated osteoclastogenesis. Thus, RBP-J-regulated miR-182 promotes TNF-α-induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α-induced osteoclastogenesis. Our results provide a novel miRNA-mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J-miR-182-Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone

  2. Role of actin filaments in fusopod formation and osteoclastogenesis.

    PubMed

    Wang, Yongqiang; Brooks, Patricia Joyce; Jang, Janet Jinyoung; Silver, Alexandra Shade; Arora, Pamma D; McCulloch, Christopher A; Glogauer, Michael

    2015-07-01

    Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width >2 μm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell-cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.

  3. Vitamin E suppresses ex vivo osteoclastogenesis in ovariectomized rats.

    PubMed

    Johnson, Sarah A; Feresin, Rafaela G; Soung, Do Y; Elam, Marcus L; Arjmandi, Bahram H

    2016-03-01

    Postmenopausal osteoporosis may be caused, in part, by oxidative stress and inflammation. Vitamin E is a strong antioxidant which has been shown to have anti-inflammatory and bone-protective effects. The objective of this study was to investigate the effects of various doses of supplemental vitamin E on osteoclastogenesis in ovariectomized rats. Sixty 12-month-old female Sprague-Dawley rats were sham-operated (Sham) or ovariectomized (Ovx; 4 groups) and fed a diet containing basal levels of vitamin E (75 mg D-α tocopherol acetate per kg diet) for 220 days. Rats in three of the Ovx groups were given supplemental doses of vitamin E (300, 525, and 750 mg D-α tocopherol acetate per kg diet) for the last 100 days. Femoral bone marrow cells were isolated, cultured, and osteoclasts were counted and normalized to 1000 total bone marrow cells. Blood monocyte and lymphocyte counts were also determined. Osteoclast number was significantly higher in the Ovx control group and was suppressed by all three doses of vitamin E, although more effectively in the Ovx group that received 300 mg per kg diet vitamin E. Additionally, vitamin E suppressed the Ovx-induced increase in monocyte and lymphocyte production. The results of this study suggest that vitamin E supplementation suppresses osteoclastogenesis, possibly by inhibiting monocyte and lymphocyte production.

  4. γ-Glutamyltranspeptidase is an endogenous activator of Toll-like receptor 4-mediated osteoclastogenesis

    PubMed Central

    Moriwaki, Sawako; Into, Takeshi; Suzuki, Keiko; Miyauchi, Mutsumi; Takata, Takashi; Shibayama, Keigo; Niida, Shumpei

    2016-01-01

    Chronic inflammation-associated bone destruction, which is observed in rheumatoid arthritis (RA) and periodontitis, is mediated by excessive osteoclastogenesis. We showed previously that γ-glutamyltranspeptidase (GGT), an enzyme involved in glutathione metabolism, acts as an endogenous activator of such pathological osteoclastogenesis, independent of its enzymatic activity. GGT accumulation is clinically observed in the joints of RA patients, and, in animals, the administration of recombinant GGT to the gingival sulcus as an in vivo periodontitis model induces an increase in the number of osteoclasts. However, the underlying mechanisms of this process remain unclear. Here, we report that Toll-like receptor 4 (TLR4) recognizes GGT to activate inflammation-associated osteoclastogenesis. Unlike lipopolysaccharide, GGT is sensitive to proteinase K treatment and insensitive to polymyxin B treatment. TLR4 deficiency abrogates GGT-induced osteoclastogenesis and activation of NF-κB and MAPK signaling in precursor cells. Additionally, GGT does not induce osteoclastogenesis in cells lacking the signaling adaptor MyD88. The administration of GGT to the gingival sulcus induces increased osteoclastogenesis in wild-type mice, but does not induce it in TLR4-deficient mice. Our findings elucidate a novel mechanism of inflammation-associated osteoclastogenesis, which involves TLR4 recognition of GGT and subsequent activation of MyD88-dependent signaling. PMID:27775020

  5. AG490 inhibits NFATc1 expression and STAT3 activation during RANKL induced osteoclastogenesis

    SciTech Connect

    Li, Chang-hong; Zhao, Jin-xia; Sun, Lin; Yao, Zhong-qiang; Deng, Xiao-li; Liu, Rui; Liu, Xiang-yuan

    2013-06-14

    Highlights: •AG490 inhibits RANKL-induced osteoclastogenesis in RAW264.7 cells. •AG490 affects cell proliferation and cell cycle distribution. •AG490 reduces NFATc1 expression during RANKL-induced osteoclastogenesis. •AG490 disrupts the activation of RANKL-mediated JAK2/STAT3 signaling pathway. •STAT3 depletion partly mimics the effect of AG490 on RANKL-induced osteoclastogenesis. -- Abstract: Commonly, JAK/STAT relays cytokine signals for cell activation and proliferation, and recent studies have shown that the elevated expression of JAK/STAT is associated with the immune rejection of allografts and the inflammatory processes of autoimmune disease. However, the role which JAK2/STAT3 signaling plays in the receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis is unknown. In this study, we investigated the effects of AG490, specific JAK2 inhibitor, on osteoclast differentiation in vitro. AG490 significantly inhibited osteoclastogenesis in murine osteoclast precursor cell line RAW264.7 induced by RANKL. AG490 suppressed cell proliferation and delayed the G1 to S cell cycle transition. Furthermore, AG490 also suppressed the expression of nuclear factor of activated T cells (NFAT) c1 but not c-Fos in RAW264.7. Subsequently, we investigated various intracellular signaling components associated with osteoclastogenesis. AG490 had no effects on RANKL-induced activation of Akt, ERK1/2. Interestingly, AG490 partly inhibited RANKL-induced phosphorylation of Ser{sup 727} in STAT3. Additionally, down-regulation of STAT3 using siRNA resulted in suppression of TRAP, RANK and NFATc1 expression. In conclusion, we demonstrated that AG490 inhibited RANKL-induced osteoclastogenesis by suppressing NFATc1 production and cell proliferation via the STAT3 pathway. These results suggest that inhibition of JAK2 may be useful for the treatment of bone diseases characterized by excessive osteoclastogenesis.

  6. TNF-induced osteoclastogenesis and inflammatory bone resorption are inhibited by transcription factor RBP-J

    PubMed Central

    Zhao, Baohong; Grimes, Shannon N.; Hu, Xiaoyu

    2012-01-01

    Tumor necrosis factor (TNF) plays a key role in the pathogenesis of inflammatory bone resorption and associated morbidity in diseases such as rheumatoid arthritis and periodontitis. Mechanisms that regulate the direct osteoclastogenic properties of TNF to limit pathological bone resorption in inflammatory settings are mostly unknown. Here, we show that the transcription factor recombinant recognition sequence binding protein at the Jκ site (RBP-J) strongly suppresses TNF-induced osteoclastogenesis and inflammatory bone resorption, but has minimal effects on physiological bone remodeling. Myeloid-specific deletion of RBP-J converted TNF into a potent osteoclastogenic factor that could function independently of receptor activator of NF-κB (RANK) signaling. In the absence of RBP-J, TNF effectively induced osteoclastogenesis and bone resorption in RANK-deficient mice. Activation of RBP-J selectively in osteoclast precursors suppressed inflammatory osteoclastogenesis and arthritic bone resorption. Mechanistically, RBP-J suppressed induction of the master regulator of osteoclastogenesis (nuclear factor of activated T cells, cytoplasmic 1) by attenuating c-Fos activation and suppressing induction of B lymphocyte–induced maturation protein-1, thereby preventing the down-regulation of transcriptional repressors such as IRF-8 that block osteoclast differentiation. Thus, RBP-J regulates the balance between activating and repressive signals that regulate osteoclastogenesis. These findings identify RBP-J as a key upstream negative regulator of osteoclastogenesis that restrains excessive bone resorption in inflammatory settings. PMID:22249448

  7. Anti-inflammatory effects of 1,25-dihydroxyvitamin D3 in monocytes cultured in serum from patients with type 2 diabetes mellitus and diabetic nephropathy with uremia via Toll-like receptor 4 and nuclear factor-κB p65.

    PubMed

    Yang, Mengxue; Xu, Jie; Yu, Jie; Yang, Bo; Gan, Hua; Li, Sicheng; Li, Xianwen

    2015-12-01

    Type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) seriously affect the quality of human life. 1,25-dihydroxyvitamin D3 (VD3) is known to exert anti‑inflammatory and immunomodulatory effects. The present study investigated the effects of VD3 on Toll‑like receptor 4 (TLR4) and nuclear factor (NF)‑κB p65 expression in monocytes from patients with T2DM and investigated the underlying mechanisms. Serum from subjects of the control, T2DM and DN with uremia groups was isolated, and THP‑1 monocytes were cultured in these sera with or without VD3. After treatment with lipopolysaccharide (LPS) and interleukin (IL)‑15, monocytes and culture supernatants were collected. The expression of TLR4, TLR9 and IL‑15 mRNA was detected using reverse‑transcription polymerase chain reaction analysis. Furthermore, the protein expression of TLR4, NF‑κB p65 and inhibitor of NF‑κB (IκB) was determined using western blot analysis. The levels of IL‑6 and monocyte chemotactic protein in culture supernatants were detected using ELISAs. The impact of LPS, IL‑15 and VD3 on the TLR/NF‑κB signaling pathway in the T2DM and DN uremia groups was also investigated. In the T2DM and DN uremia groups, LPS and IL‑15 downregulated the expression of IκB and upregulated levels of proteins, including TLR4, NF‑κB p65, IL‑6 and monocyte chemoattractant protein 1, as well as TLR4 and IL‑15 mRNA. There was no significant difference in TLR9 mRNA protein expression among the three groups. VD3 partially blocked the above effects; However, pre‑treatment with VD3 had no significant effect on TLR4 mRNA expression, protein expression of NF-κB p65 and IκB, or the levels of associated inflammatory cytokines. In conclusion, the present study indicated that anti‑inflammatory effects of VD3 in inflammatory immune responses in T2DM and DN uremia are associated with the TLR4/NF-κB p65 signaling pathway.

  8. Pasteurella multocida Toxin Triggers RANKL-Independent Osteoclastogenesis.

    PubMed

    Chakraborty, Sushmita; Kloos, Bianca; Harre, Ulrike; Schett, Georg; Kubatzky, Katharina F

    2017-01-01

    Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL

  9. Pasteurella multocida Toxin Triggers RANKL-Independent Osteoclastogenesis

    PubMed Central

    Chakraborty, Sushmita; Kloos, Bianca; Harre, Ulrike; Schett, Georg; Kubatzky, Katharina F.

    2017-01-01

    Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL

  10. MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis

    SciTech Connect

    Guo, Li-Juan; Liao, Lan; Yang, Li; Li, Yu; Jiang, Tie-Jian

    2014-02-15

    MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease. - Highlights: • MiR-125a was significantly down-regulated in osteoclastogenesis of CD14+ PBMCs. • MiR-125a inhibited osteoclast differentiation by targeting TRAF6. • NFATc1 inhibited miR-125a transciption by binding to the promoter of miR-125a. • TRAF6/NFATc1 and miR-125a form a regulatory feedback loop in osteoclastogenesis.

  11. Enterococcus faecalis promotes osteoclastogenesis and semaphorin 4D expression.

    PubMed

    Wang, Shuai; Deng, Zuhui; Seneviratne, Chaminda J; Cheung, Gary S P; Jin, Lijian; Zhao, Baohong; Zhang, Chengfei

    2015-10-01

    Enterococcus faecalis is considered a major bacterial pathogen implicated in endodontic infections and contributes considerably to periapical periodontitis. This study aimed to investigate the potential mechanisms by which E. faecalis accounts for the bone destruction in periapical periodontitis in vitro. Osteoclast precursor RAW264.7 cells were treated with E. faecalis ATCC 29212 and a wild strain of E. faecalis derived clinically from an infected root canal. The results showed that, to some extent, E. faecalis induced the RAW264.7 cells to form tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast-like cells. This pathogen markedly stimulated RAW264.7 cells to express semaphorin 4D (Sema4D), which inhibits bone formation. Once RAW264.7 cells were primed by low-dose receptor activator of nuclear factor-kappa B ligand (RANKL), E. faecalis could significantly increase the production of TRAP-positive multinucleated cells and up-regulate the expression of osteoclast-specific markers, including NFATc1, TRAP and cathepsin K. Both p38 and ERK1/2 MAPK signaling pathways were activated by E. faecalis in RANKL-primed RAW264.7 cells, and meanwhile the expression of Sema4D was highly increased. In conclusion, E. faecalis may greatly contribute to the bone resorption in periapical periodontitis by promoting RANKL-dependent osteoclastogenesis and expression of Sema4D through activation of p38 and ERK1/2 MAPK signaling pathways.

  12. Analysis of Osteoclastogenesis/Osteoblastogenesis on Nanotopographical Titania Surfaces.

    PubMed

    Silverwood, Robert K; Fairhurst, Paul G; Sjöström, Terje; Welsh, Findlay; Sun, Yuxin; Li, Gang; Yu, Bin; Young, Peter S; Su, Bo; Meek, Robert M D; Dalby, Matthew J; Tsimbouri, Penelope M

    2016-04-20

    A focus of orthopedic research is to improve osteointegration and outcomes of joint replacement. Material surface topography has been shown to alter cell adhesion, proliferation, and growth. The use of nanotopographical features to promote cell adhesion and bone formation is hoped to improve osteointegration and clinical outcomes. Use of block-copolymer self-assembled nanopatterns allows nanopillars to form via templated anodization with control over height and order, which has been shown to be of cellular importance. This project assesses the outcome of a human bone marrow-derived co-culture of adherent osteoprogenitors and osteoclast progenitors on polished titania and titania patterned with 15 nm nanopillars, fabricated by a block-copolymer templated anodization technique. Substrate implantation in rabbit femurs is performed to confirm the in vivo bone/implant integration. Quantitative and qualitative results demonstrate increased osteogenesis on the nanopillar substrate with scanning electron microscopy, histochemical staining, and real-time quantitative reverse-transcription polymerase chain reaction analysis performed. Osteoblast/osteoclast co-culture analysis shows an increase in osteoblastogenesis-related gene expression and reduction in osteoclastogenesis. Supporting this in vitro finding, in vivo implantation of substrates in rabbit femora indicates increased implant/bone contact by ≈20%. These favorable osteogenic characteristics demonstrate the potential of 15 nm titania nanopillars fabricated by the block-copolymer templated anodization technique.

  13. Hemin activation of innate cellular response blocks human immunodeficiency virus type-1-induced osteoclastogenesis

    SciTech Connect

    Takeda, Kazuyo; Adhikari, Rewati; Yamada, Kenneth M.; Dhawan, Subhash

    2015-08-14

    The normal skeletal developmental and homeostatic process termed osteoclastogenesis is exacerbated in numerous pathological conditions and causes excess bone loss. In cancer and HIV-1-infected patients, this disruption of homeostasis results in osteopenia and eventual osteoporesis. Counteracting the factors responsible for these metabolic disorders remains a challenge for preventing or minimizing this co-morbidity associated with these diseases. In this report, we demonstrate that a hemin-induced host protection mechanism not only suppresses HIV-1 associated osteoclastogenesis, but it also exhibits anti-osteoclastogenic activity for non-infected cells. Since the mode of action of hemin is both physiological and pharmacological through induction of heme oxygenase-1 (HO-1), an endogenous host protective response to an FDA-licensed therapeutic used to treat another disease, our study suggests an approach to developing novel, safe and effective therapeutic strategies for treating bone disorders, because hemin administration in humans has previously met required FDA safety standards. - Highlights: • HIV-1 infection induced osteoclastogenesis in primary human macrophages. • Heme oxygenase-1 (HO-1) induction inhibited HIV-1-induced osteoclastogenesis in macrophages. • HO-1 induction suppressed RANKL-enhanced osteoclastogenesis in HIV-1-infected macrophages. • This inverse relationship between HO-1 and HIV-1 pathogenesis may define a novel host defense response against HIV-1 infection.

  14. RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis

    SciTech Connect

    Li, Changhong; Zhao, Jinxia; Sun, Lin; Yao, Zhongqiang; Liu, Rui; Huang, Jiansheng; Liu, Xiangyuan

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer CXCR6 is down-regulated during RANKL-induced osteoclastogenesis in RAW264.7 cells. Black-Right-Pointing-Pointer CXCR6 reduction was nearly reversed by inhibition of JAK2/STAT3 signaling pathway. Black-Right-Pointing-Pointer CXCL16 alone does not positively regulate osteoclastogenesis. -- Abstract: The receptor activator of nuclear factor-{kappa}B ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which may be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis.

  15. Estrogens antagonize RUNX2-mediated osteoblast-driven osteoclastogenesis through regulating RANKL membrane association.

    PubMed

    Martin, Anthony; Xiong, Jian; Koromila, Theodora; Ji, Jie S; Chang, Stephanie; Song, Yae S; Miller, Jonathan L; Han, Chun-Ya; Kostenuik, Paul; Krum, Susan A; Chimge, Nyam-Osor; Gabet, Yankel; Frenkel, Baruch

    2015-06-01

    In addition to its thoroughly investigated role in bone formation, the osteoblast master transcription factor RUNX2 also promotes osteoclastogenesis and bone resorption. Here we demonstrate that 17β-estradiol (E2), strongly inhibits RUNX2-mediated osteoblast-driven osteoclastogenesis in co-cultures. Towards deciphering the underlying mechanism, we induced premature expression of RUNX2 in primary murine pre-osteoblasts, which resulted in robust differentiation of co-cultured splenocytes into mature osteoclasts. This was attributable to RUNX2-mediated increase in RANKL secretion, determined by ELISA, as well as to RUNX2-mediated increase in RANKL association with the osteoblast membrane, demonstrated using confocal fluorescence microscopy. The increased association with the osteoblast membrane was recapitulated by transiently expressed GFP-RANKL. E2 abolished the RUNX2-mediated increase in membrane-associated RANKL and GFP-RANKL, as well as the concomitant osteoclastogenesis. RUNX2-mediated RANKL cellular redistribution was attributable in part to a decrease in Opg expression, but E2 did not influence Opg expression either in the presence or absence of RUNX2. Diminution of RUNX2-mediated osteoclastogenesis by E2 occurred regardless of whether the pre-osteoclasts were derived from wild type or estrogen receptor alpha (ERα)-knockout mice, suggesting that activated ERα inhibited osteoblast-driven osteoclastogenesis by acting in osteoblasts, possibly targeting RUNX2. Indeed, microarray analysis demonstrated global attenuation of the RUNX2 response by E2, including abrogation of Pstpip2 expression, which likely plays a critical role in membrane trafficking. Finally, the selective ER modulators (SERMs) tamoxifen and raloxifene mimicked E2 in abrogating the stimulatory effect of osteoblastic RUNX2 on osteoclast differentiation in the co-culture assay. Thus, E2 antagonizes RUNX2-mediated RANKL trafficking and subsequent osteoclastogenesis. Targeting RUNX2 and

  16. Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

    SciTech Connect

    Nakamura, Haruhiko; Nakashima, Tomoki; Hayashi, Mikihito; Izawa, Naohiro; Yasui, Tetsuro; Aburatani, Hiroyuki; Tanaka, Sakae; Takayanagi, Hiroshi

    2014-12-12

    Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion.

  17. CD147 deficiency blocks IL-8 secretion and inhibits lung cancer-induced osteoclastogenesis

    SciTech Connect

    Wang, Hongkai; Zhuo, Yunyun; Hu, Xu; Shen, Weiwei; Zhang, Ying; Chu, Tongwei

    2015-03-06

    Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and poor prognosis; however, the molecular basis of this process is still unknown. This study investigated the role of extracellular matrix metalloproteinase inducer (also known as cluster of differentiation (CD)147) in osteoclastogenesis resulting from bone metastasis, based on the enrichment of this glycoprotein on the surface of many malignant bone tumors. RNA interference was used to silence CD147 expression in A549 human lung cancer cells. Compared with conditioned medium (CM) from control cells (A549-CM), CM from CD147-deficient cells (A549-si-CM) suppressed receptor activator of nuclear factor κB ligand-stimulated osteoclastogenesis in RAW 264.7 cells and bone marrow-derived macrophages. The mRNA levels of osteoclast-specific genes such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K were also reduced in the presence of A549-si-CM. CD147 knockdown in A549 cells decreased interleukin (IL)-8mRNA and protein expression. IL-8 is present in large amounts in A549-CM and mimicked its inductive effect on osteoclastogenesis; this was reversed by depletion of IL-8 from the medium. Taken together, these results indicate that CD147 promotes lung cancer-induced osteoclastogenesis by modulating IL-8 secretion, and suggest that CD147 is a potential therapeutic target for cancer-associated bone resorption in lung cancer patients. - Highlights: • Bone loss frequently results from lung cancer metastasis. • Cluster of differentiation (CD)147 was depleted in A549 lung adenocarcinoma cells. • RAW 264.7 cell osteoclastogenesis was blocked by medium from CD147-deficient cells. • Interleukin (IL)-8 level was reduced in the conditioned medium. • Osteoclastogenesis induced by lung tumor cells requires CD147-mediated IL-8 release.

  18. Excavatolide B Attenuates Rheumatoid Arthritis through the Inhibition of Osteoclastogenesis

    PubMed Central

    Lin, Yen-You; Jean, Yen-Hsuan; Lee, Hsin-Pai; Lin, Sung-Chun; Pan, Chieh-Yu; Chen, Wu-Fu; Wu, Shu-Fen; Su, Jui-Hsin; Tsui, Kuan-Hao; Sheu, Jyh-Horng; Sung, Ping-Jyun; Wen, Zhi-Hong

    2017-01-01

    Osteoclasts are multinucleated giant cells of macrophage/monocyte lineage, and cell differentiation with the upregulation of osteoclast-related proteins is believed to play a major role in the destruction of the joints in the course of rheumatoid arthritis (RA). Pro-inflammatory cytokines, such as interleukin-17A (IL-17A) and macrophage colony-stimulating factor (M-CSF), can be overexpressed in RA and lead to osteoclastogenesis. In a previous study, we found that cultured-type soft coral-derived excavatolide B (Exc-B) exhibited anti-inflammatory properties. In the present study, we thus aimed to evaluate the anti-arthritic activity of Exc-B in in vitro and in vivo models. The results demonstrated that Exc-B inhibits LPS-induced multinucleated cell and actin ring formation, as well as TRAP, MMP-9, and cathepsin K expression. Additionally, Exc-B significantly attenuated the characteristics of RA in adjuvant (AIA) and type II collagen-induced arthritis (CIA) in rats. Moreover, Exc-B improved histopathological features, and reduced the number of TRAP-positive multinucleated cells in the in vivo AIA and CIA models. Immunohistochemical analysis showed that Exc-B attenuated the protein expression of cathepsin K, MMP-2, MMP-9, CD11b, and NFATc1 in ankle tissues of AIA and CIA rats. Level of interleukin-17A and macrophage colony-stimulating factor were also decreased by Exc-B. These findings strongly suggest that Exc-B could be of potential use as a therapeutic agent by inhibiting osteoclast differentiation in arthritis. Moreover, this study also illustrates the use of the anti-inflammatory marine compound, Exc-B, as a potential therapeutic strategy for RA. PMID:28067799

  19. Transforming growth factor-{beta} enables NFATc1 expression during osteoclastogenesis

    SciTech Connect

    Fox, S.W. Evans, K.E.; Lovibond, A.C.

    2008-02-01

    Osteoclastogenesis is dependent on distinct stimuli that prime and activate osteoclast differentiation. One cytokine needed to prime monocytes for osteoclastogenesis is TGF-{beta}, which enables and augments RANKL and TNF-{alpha}-induced osteoclast differentiation. However, the precise time-period during which this occurs and the molecular mechanism mediating this action are unknown. We report here TGF-{beta} prime monocytes for osteoclast formation within 24 h by regulating expression of NFATc1, a key osteoclastic transcription factor. TGF-{beta} directly induces cytoplasmic NFATc1 expression within 24 h, but is unable to stimulate NFATc1 nuclear translocation. Furthermore, RANKL-induced NFATc1 expression is dependent on the presence of TGF-{beta} during the early stages of osteoclastogenesis. Similarly, TNF-{alpha} activates osteoclastogenesis by stimulating translocation of TGF-{beta}-induced NFATc1. In light of these findings, it is apparent that osteoclast formation is dependent on coordinated interactions between TGF-{beta} and RANKL/TNF-{alpha} that regulate the expression and intracellular distribution of NFATc1 during early stages of osteoclast differentiation.

  20. CD147 deficiency blocks IL-8 secretion and inhibits lung cancer-induced osteoclastogenesis.

    PubMed

    Wang, Hongkai; Zhuo, Yunyun; Hu, Xu; Shen, Weiwei; Zhang, Ying; Chu, Tongwei

    2015-03-06

    Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and poor prognosis; however, the molecular basis of this process is still unknown. This study investigated the role of extracellular matrix metalloproteinase inducer (also known as cluster of differentiation (CD)147) in osteoclastogenesis resulting from bone metastasis, based on the enrichment of this glycoprotein on the surface of many malignant bone tumors. RNA interference was used to silence CD147 expression in A549 human lung cancer cells. Compared with conditioned medium (CM) from control cells (A549-CM), CM from CD147-deficient cells (A549-si-CM) suppressed receptor activator of nuclear factor κB ligand-stimulated osteoclastogenesis in RAW 264.7 cells and bone marrow-derived macrophages. The mRNA levels of osteoclast-specific genes such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K were also reduced in the presence of A549-si-CM. CD147 knockdown in A549 cells decreased interleukin (IL)-8mRNA and protein expression. IL-8 is present in large amounts in A549-CM and mimicked its inductive effect on osteoclastogenesis; this was reversed by depletion of IL-8 from the medium. Taken together, these results indicate that CD147 promotes lung cancer-induced osteoclastogenesis by modulating IL-8 secretion, and suggest that CD147 is a potential therapeutic target for cancer-associated bone resorption in lung cancer patients.

  1. Smad4 is required to inhibit osteoclastogenesis and maintain bone mass

    PubMed Central

    Morita, Mayu; Yoshida, Shigeyuki; Iwasaki, Ryotaro; Yasui, Tetsuro; Sato, Yuiko; Kobayashi, Tami; Watanabe, Ryuichi; Oike, Takatsugu; Miyamoto, Kana; Takami, Masamichi; Ozato, Keiko; Deng, Chu-Xia; Aburatani, Hiroyuki; Tanaka, Sakae; Yoshimura, Akihiko; Toyama, Yoshiaki; Matsumoto, Morio; Nakamura, Masaya; Kawana, Hiromasa; Nakagawa, Taneaki; Miyamoto, Takeshi

    2016-01-01

    Bone homeostasis is maintained as a delicate balance between bone-resorption and bone-formation, which are coupled to maintain appropriate bone mass. A critical question is how bone-resorption is terminated to allow bone-formation to occur. Here, we show that TGFβs inhibit osteoclastogenesis and maintain bone-mass through Smad4 activity in osteoclasts. We found that latent-TGFβ1 was activated by osteoclasts to inhibit osteoclastogenesis. Osteoclast-specific Smad4 conditional knockout mice (Smad4-cKO) exhibited significantly reduced bone-mass and elevated osteoclast formation relative to controls. TGFβ1-activation induced expression of Irf8 and Bcl6, both of which encode factors inhibiting osteoclastogenesis, by blocking their negative regulator, Prdm1, in osteoclasts in a Smad4-dependent manner. Reduced bone-mass and accelerated osteoclastogenesis seen in Smad4-cKO were abrogated by Prdm1 deletion. Administration of latent-TGFβ1-Fc to wild-type mice antagonized LPS-induced bone destruction in a model of activated osteoclast-mediated bone destruction. Thus, latent-TGFβ1-Fc could serve as a promising new therapeutic agent in bone diseases marked by excessive resorption. PMID:27731422

  2. Myeloid thrombomodulin lectin-like domain inhibits osteoclastogenesis and inflammatory bone loss.

    PubMed

    Cheng, Tsung-Lin; Lai, Chao-Han; Shieh, Shyh-Jou; Jou, Yin-Bo; Yeh, Jwu-Lai; Yang, Ai-Lun; Wang, Yan-Hsiung; Wang, Chau-Zen; Chen, Chung-Hwan; Shi, Guey-Yueh; Ho, Mei-Ling; Wu, Hua-Lin

    2016-06-17

    Osteoclastogenesis is an essential process during bone metabolism which can also be promoted by inflammatory signals. Thrombomodulin (TM), a transmembrane glycoprotein, exerts anti-inflammatory activities such as neutralization of proinflammatory high-mobility group box 1 (HMGB1) through TM lectin-like domain. This study aimed to identify the role of myeloid TM (i.e., endogenous TM expression on the myeloid lineage) in osteoclastogenesis and inflammatory bone loss. Using human peripheral blood mononuclear cells and mouse bone marrow-derived macrophages, we observed that the protein levels of TM were dramatically reduced as these cells differentiated into osteoclasts. In addition, osteoclastogenesis and extracellular HMGB1 accumulation were enhanced in primary cultured monocytes from myeloid-specific TM-deficient mice (LysMcre/TM(flox/flox)) and from TM lectin-like domain deleted mice (TM(LeD/LeD)) compared with their respective controls. Micro-computerized tomography scans showed that ovariectomy-induced bone loss was more pronounced in TM(LeD/LeD) mice compared with controls. Finally, the inhibiting effects of recombinant TM lectin-like domain (rTMD1) on bone resorption in vitro, and bone loss in both the ovariectomized model and collagen antibody-induced arthritis model has been detected. These findings suggested that the myeloid TM lectin-like domain may inhibit osteoclastogenesis by reducing HMGB1 signaling, and rTMD1 may hold therapeutic potential for inflammatory bone loss.

  3. Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

    PubMed Central

    2012-01-01

    Introduction Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis. Methods Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb). Results Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast, blockade of a Notch ligand Jagged1 enhanced osteoclastogenesis and immobilized Jagged1-Fc suppressed it. Enhancement of osteoclastogenesis by agonistic anti-Notch2 mAb suggested that Dll1 promoted osteoclastogenesis via Notch2, while suppression by agonistic anti-Notch1 mAb suggested that Jagged1 suppressed osteoclastogenesis via Notch1. Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed osteoclastogenesis, implying that Notch2/Dll1-mediated enhancement was dominant. Actually, blockade of Dll1 ameliorated arthritis induced by K/BxN serum transfer, reduced the number of osteoclasts in the affected joints and suppressed ovariectomy-induced bone loss. Conclusions The differential regulation of osteoclastogenesis by Notch2/Dll1 and Notch1/Jagged1 axes may be a novel target for amelioration of bone erosion in RA patients. PMID:22390640

  4. Mechanical loading prevents the stimulating effect of IL-1{beta} on osteocyte-modulated osteoclastogenesis

    SciTech Connect

    Kulkarni, Rishikesh N.; Bakker, Astrid D.; Everts, Vincent; Klein-Nulend, Jenneke

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Osteocyte incubation with IL-1{beta} stimulated osteocyte-modulated osteoclastogenesis. Black-Right-Pointing-Pointer Conditioned medium from IL-1{beta}-treated osteocytes increased osteoclastogenesis. Black-Right-Pointing-Pointer IL-1{beta} upregulated RANKL and downregulated OPG gene expression by osteocytes. Black-Right-Pointing-Pointer CYR61 is upregulated in mechanically stimulated osteocytes. Black-Right-Pointing-Pointer Mechanical loading of osteocytes may abolish IL-1{beta}-induced osteoclastogenesis. -- Abstract: Inflammatory diseases such as rheumatoid arthritis are often accompanied by higher plasma and synovial fluid levels of interleukin-1{beta} (IL-1{beta}), and by increased bone resorption. Since osteocytes are known to regulate bone resorption in response to changes in mechanical stimuli, we investigated whether IL-1{beta} affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. MLO-Y4 osteocytes were pre-incubated with IL-1{beta} (0.1-1 ng/ml) for 24 h. Cells were either or not subjected to mechanical loading by 1 h pulsating fluid flow (PFF; 0.7 {+-} 0.3 Pa, 5 Hz) in the presence of IL-1{beta} (0.1-1 ng/ml). Conditioned medium was collected after 1 h PFF or static cultures. Subsequently mouse bone marrow cells were seeded on top of the IL-1{beta}-treated osteocytes to determine osteoclastogenesis. Conditioned medium from mechanically loaded or static IL-1{beta}-treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Gene expression of cysteine-rich protein 61 (CYR61/CCN1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) by osteocytes was determined immediately after PFF. Incubation of osteocytes with IL-1{beta}, as well as conditioned medium from static IL-1{beta}-treated osteocytes increased the formation of osteoclasts. However, conditioned medium from mechanically loaded IL

  5. STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis

    PubMed Central

    Lee, Jongwon; Seong, Semun; Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Jeong, Byung-chul; Nam, Kwang-Il; Kim, Kyung Keun; Hennighausen, Lothar; Kim, Nacksung

    2016-01-01

    Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways. PMID:27485735

  6. Hemin activation of innate cellular response blocks human immunodeficiency virus type-1-induced osteoclastogenesis

    PubMed Central

    Takeda, Kazuyo; Adhikari, Rewati; Yamada, Kenneth M.; Dhawan, Subhash

    2017-01-01

    The normal skeletal developmental and homeostatic process termed osteoclastogenesis is exacerbated in numerous pathological conditions and causes excess bone loss. In cancer and HIV-1-infected patients, this disruption of homeostasis results in osteopenia and eventual osteoporesis. Counteracting the factors responsible for these metabolic disorders remains a challenge for preventing or minimizing this comorbidity associated with these diseases. In this report, we demonstrate that a hemin-induced host protection mechanism not only suppresses HIV-1 associated osteoclastogenesis, but it also exhibits anti-osteoclastogenic activity for non-infected cells. Since the mode of action of hemin is both physiological and pharmacological through induction of heme oxygenase-1 (HO-1), an endogenous host protective response to an FDA-licensed therapeutic used to treat another disease, our study suggests an approach to developing novel, safe and effective therapeutic strategies for treating bone disorders, because hemin administration in humans has previously met required FDA safety standards. PMID:25998388

  7. Hemin activation of innate cellular response blocks human immunodeficiency virus type-1-induced osteoclastogenesis.

    PubMed

    Takeda, Kazuyo; Adhikari, Rewati; Yamada, Kenneth M; Dhawan, Subhash

    2015-08-14

    The normal skeletal developmental and homeostatic process termed osteoclastogenesis is exacerbated in numerous pathological conditions and causes excess bone loss. In cancer and HIV-1-infected patients, this disruption of homeostasis results in osteopenia and eventual osteoporesis. Counteracting the factors responsible for these metabolic disorders remains a challenge for preventing or minimizing this co-morbidity associated with these diseases. In this report, we demonstrate that a hemin-induced host protection mechanism not only suppresses HIV-1 associated osteoclastogenesis, but it also exhibits anti-osteoclastogenic activity for non-infected cells. Since the mode of action of hemin is both physiological and pharmacological through induction of heme oxygenase-1 (HO-1), an endogenous host protective response to an FDA-licensed therapeutic used to treat another disease, our study suggests an approach to developing novel, safe and effective therapeutic strategies for treating bone disorders, because hemin administration in humans has previously met required FDA safety standards.

  8. TGF-β in dentin matrix extract induces osteoclastogenesis in vitro.

    PubMed

    Sriarj, Wannakorn; Aoki, Kazuhiro; Ohya, Keiichi; Takahashi, Mariko; Takagi, Yuzo; Shimokawa, Hitoyata

    2015-01-01

    Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in α-MEM containing macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA levels of osteoclast-related genes, assayed by real-time RT-PCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factor-β1 (TGF-β1), SB431542, a TGF-β receptor inhibitor, and ELISA were used to determine the role of TGF-β1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-β1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-β1 involvement. The addition of exogenous TGF-β1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-β1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-β1.

  9. DDR2 (discoidin domain receptor 2) suppresses osteoclastogenesis and is a potential therapeutic target in osteoporosis.

    PubMed

    Zhang, Yan; Su, Jin; Wu, Shufang; Teng, Yue; Yin, Zhanhai; Guo, Yan; Li, Jing; Li, Kun; Yao, Libo; Li, Xu

    2015-03-24

    Osteoporosis is a disease characterized by abnormal skeletal fragility due to an imbalance in bone homeostasis. Finding therapeutic targets that promote bone formation is valuable for treating osteoporosis. Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that promotes the differentiation of bone-depositing cells, osteoblasts, and bone formation. We investigated the role of DDR2 in osteoclastogenesis, the formation of cells responsible for bone resorption. We found that, although differentiated osteoclasts had catalytically active DDR2, its abundance was decreased. Overexpression of DDR2 inhibited the expression of osteoclastic markers, osteoclast maturation, and osteoclast-mediated bone resorption in a culture model of bone matrix. Conversely, knocking down Ddr2 through RNA interference accelerated osteoclast differentiation and bone resorption in mice. Further, we identified the co-receptor Neuropilin-1 (Nrp1) as a DDR2-interacting protein. DDR2 facilitated the binding of Nrp1 and the co-receptor PlexinA1 by forming a DDR2-Nrp1-PlexinA1 complex, which blocked PlexinA1-mediated stimulation of osteoclastogenesis. DDR2 prevented PlexinA1 from interacting with the receptor TREM2 and the adaptor DAP12. Moreover, knocking down Nrp1 rescued the expression of osteoclastic markers observed in DDR2-overexpressing cells, whereas ectopic expression of Nrp1 in DDR2-silenced cells inhibited the induction of osteoclastogenesis. Nrp1 enhanced the function of DDR2 in promoting osteoblast differentiation and bone formation in mice. In an ovariectomized mouse model of osteoporosis, adenovirus-mediated delivery of DDR2 to the femur bone alleviated osteopenic phenotypes. Our results revealed that DDR2 functions as an inhibitor of osteoclastogenesis, as well as a promoter of osteoblastogenesis, suggesting that enhancing DDR2 may be therapeutic for patients with osteoporosis.

  10. Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors.

    PubMed

    Haywood, Joel; Qi, Jianxun; Chen, Chun-Chi; Lu, Guangwen; Liu, Yingxia; Yan, Jinghua; Shi, Yi; Gao, George F

    2016-01-26

    Human osteoclast-associated receptor (OSCAR) is an immunoglobulin (Ig)-like collagen receptor that is up-regulated on osteoclasts during osteoclastogenesis and is expressed in a range of myeloid cells. As a member of the leukocyte receptor complex family of proteins, OSCAR shares a high degree of sequence and structural homology with other collagen receptors of this family, including glycoprotein VI, leukocyte-associated Ig-like receptor-1, and leukocyte Ig-like receptor B4, but recognizes a unique collagen sequence. Here, we present the crystal structures of OSCAR in its free form and in complex with a triple-helical collagen-like peptide (CLP). These structures reveal that the CLP peptide binds only one of the two Ig-like domains, the membrane-proximal domain (domain 2) of OSCAR, with the middle and trailing chain burying a total of 661 Å(2) of solvent-accessible collagen surface. This binding mode is facilitated by the unusual topography of the OSCAR protein, which displays an obtuse interdomain angle and a rotation of domain 2 relative to the membrane-distal domain 1. Moreover, the binding of the CLP to OSCAR appears to be mediated largely by tyrosine residues and conformational changes at a shallow Phe pocket. Furthermore, we investigated CLP peptides as inhibitors of osteoclastogenesis and found that a peptide length of 40 amino acids is required to ensure adequate inhibition of osteoclastogenesis in vitro. These findings provide valuable structural insights into the mode of collagen recognition by OSCAR and into the use of synthetic peptide matrikines for osteoclastogenesis inhibition.

  11. Cysteine cathepsin activity suppresses osteoclastogenesis of myeloid-derived suppressor cells in breast cancer.

    PubMed

    Edgington-Mitchell, Laura E; Rautela, Jai; Duivenvoorden, Hendrika M; Jayatilleke, Krishnath M; van der Linden, Wouter A; Verdoes, Martijn; Bogyo, Matthew; Parker, Belinda S

    2015-09-29

    Cysteine cathepsin proteases contribute to many normal cellular functions, and their aberrant activity within various cell types can contribute to many diseases, including breast cancer. It is now well accepted that cathepsin proteases have numerous cell-specific functions within the tumor microenvironment that function to promote tumor growth and invasion, such that they may be valid targets for anti-metastatic therapeutic approaches. Using activity-based probes, we have examined the activity and expression of cysteine cathepsins in a mouse model of breast cancer metastasis to bone. In mice bearing highly metastatic tumors, we detected abundant cysteine cathepsin expression and activity in myeloid-derived suppressor cells (MDSCs). These immature immune cells have known metastasis-promoting roles, including immunosuppression and osteoclastogenesis, and we assessed the contribution of cysteine cathepsins to these functions. Blocking cysteine cathepsin activity with multiple small-molecule inhibitors resulted in enhanced differentiation of multinucleated osteoclasts. This highlights a potential role for cysteine cathepsin activity in suppressing the fusion of osteoclast precursor cells. In support of this hypothesis, we found that expression and activity of key cysteine cathepsins were downregulated during MDSC-osteoclast differentiation. Another cysteine protease, legumain, also inhibits osteoclastogenesis, in part through modulation of cathepsin L activity. Together, these data suggest that cysteine protease inhibition is associated with enhanced osteoclastogenesis, a process that has been implicated in bone metastasis.

  12. Bu-Shen-Ning-Xin decoction suppresses osteoclastogenesis via increasing dehydroepiandrosterone to prevent postmenopausal osteoporosis.

    PubMed

    Gui, Yuyan; Qiu, Xuemin; Xu, Yingping; Li, Dajin; Wang, Ling

    2015-06-01

    Bu-Shen-Ning-Xin decoction (BSNXD), a traditional Chinese medicine, has been used to prevent and treat age-related diseases such as postmenopausal osteoporosis (PMO) for decades. This study sought to investigate the underlying mechanisms of BSNXD in terms of receptor activation of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro because of the critical roles of bone resorption in the development and progression of osteoporosis. In mice, serum levels of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), and 17-β-estradiol (E2) were evaluated with an enzyme immunoassay kit after ovariectomy. Levels of DHEA and DHEAS increased significantly following administration of BSNXD while the level of E2 did not. In addition, tartrate-resistance acid phosphatase staining showed that DHEA profoundly inhibited RANKL-induced osteoclastogenesis in vitro in a dose-dependent manner via estrogen receptor α (ERα) but not via estrogen receptor β or androgen receptors. Cytotoxicity was not detected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These data suggest that BSNXD prevents PMO by increasing DHEA via the ERαpathway to suppress osteoclastogenesis.

  13. Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis

    PubMed Central

    Muschter, Dominique; Schäfer, Nicole; Stangl, Hubert; Straub, Rainer H.; Grässel, Susanne

    2015-01-01

    Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors. PMID:26431344

  14. Artesunate Inhibits RANKL-induced Osteoclastogenesis and Bone Resorption In Vitro and Prevents LPS-induced Bone Loss In Vivo.

    PubMed

    Wei, Cheng-Ming; Liu, Qian; Song, Fang-Ming; Lin, Xi-Xi; Su, Yi-Ji; Xu, Jiake; Huang, Lin; Zong, Shao-Hui; Zhao, Jin-Min

    2017-03-15

    Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side-effects caused by the currently available drugs, a continuous search for novel bone-protective therapies is essential. Artesunate (Art), the water-soluble derivative of artemisinin has been investigated owing to its anti-malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, the mRNA expression of osteoclastic-specific genes, and resorption pit formation in a dose-dependent manner in primary bone marrow-derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL-induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF-κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)-induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL-induced osteoclastogenesis by suppressing the NF-κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases. This article is protected by copyright. All rights reserved.

  15. Intravenous Immunoglobulin (IVIG) Attenuates TNF-induced Pathologic Bone Resorption and Suppresses Osteoclastogenesis by Inducing A20 Expression

    PubMed Central

    Mun, Sehwan; Bae, Seyeon; Murata, Koichi; Ivashkiv, Lionel B.; Park-Min, Kyung-Hyun

    2016-01-01

    Investigations on the therapeutic effects of intravenous immunoglobulin (IVIG) have focused on the suppression of autoantibody- and immune complex-mediated inflammatory pathogenesis. Inflammatory diseases such as rheumatoid arthritis are often accompanied by excessive bone erosion but the effect of IVIG on osteoclasts, bone-resorbing cells, has not been studied. Here, we investigate whether IVIG directly regulates osteoclast differentiation and has therapeutic potential for suppressing osteoclast-mediated pathologic bone resorption. IVIG or cross-linking of Fcγ receptors with plate-bound IgG suppressed receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and expression of osteoclast-related genes such as integrin β3 and cathepsin K in a dose-dependent manner. Mechanistically, IVIG or plate-bound IgG suppressed osteoclastogenesis by downregulating RANKL-induced expression of NFATC1, the master regulator of osteoclastogenesis. IVIG suppressed NFATC1 expression by attenuating RANKL-induced NF-κB signaling, explained in part by induction of the inflammatory signaling inhibitor A20. IVIG administration attenuated in vivo osteoclastogenesis and suppressed bone resorption in the tumor necrosis factor (TNF)-induced calvarial osteolysis model. Our findings show that, in addition to suppressing inflammation, IVIG directly inhibits osteoclastogenesis through a mechanism involving suppression of RANK signaling. Direct suppression of osteoclast differentiation may provide beneficial effects on preserving bone mass when IVIG is used to treat rheumatic disorders. PMID:26189496

  16. SOD2 and Sirt3 Control Osteoclastogenesis by Regulating Mitochondrial ROS.

    PubMed

    Kim, Haemin; Lee, Yong Deok; Kim, Hyung Joon; Lee, Zang Hee; Kim, Hong-Hee

    2017-02-01

    Reactive oxygen species (ROS) are an indispensable element of cellular signal transduction in various cell types, including bone cells. In particular, osteoclasts (OCs), cells specialized for bone resorption, utilize ROS as second messengers during receptor activator of NF-κB ligand (RANKL)-induced differentiation and activation. In addition, because of the high energy demands of bone-resorbing activity, OCs contain large amounts of mitochondria, the source of the majority of total ROS. In this study, we focused on the regulation of ROS generated from mitochondria during osteoclastogenesis. We observed that the level of mitochondrial superoxide dismutase 2 (SOD2), an enzyme responsible for reducing superoxide radicals in mitochondria, was increased by RANKL. siRNA-mediated knockdown (KD) of SOD2 increased ROS levels and enhanced OC differentiation. Conversely, overexpression of SOD2 reduced osteoclastogenesis by decreasing ROS levels. Moreover, we found that NAD-dependent deacetylase sirtuin 3 (Sirt3), an activator of SOD2 in mitochondria, was induced by RANKL. Sirt3-targeted siRNA decreased SOD2 activity by reducing deacetylation of lysine 68 of SOD2, leading to increased osteoclastogenesis. Furthermore, in vivo KD of SOD2 or Sirt3 in ICR mouse calvariae decreased bone volume and increased OC surface, supporting the results of in vitro experiments. Taken together, our findings demonstrate for the first time to our knowledge that the regulation of mitochondrial ROS by SOD2 and Sirt3 plays an important role in fine-tuning the OC differentiation program. © 2016 American Society for Bone and Mineral Research.

  17. Mechanisms of enhanced osteoclastogenesis in girls and young women with Turner's Syndrome.

    PubMed

    Faienza, Maria Felicia; Brunetti, Giacomina; Ventura, Annamaria; Piacente, Laura; Messina, Maria Francesca; De Luca, Filippo; Ciccarelli, Maria; Oranger, Angela; Mori, Giorgio; Natale, Maria Pia; Gigante, Margherita; Ranieri, Elena; Gesualdo, Loreto; Colucci, Silvia; Cavallo, Luciano; Grano, Maria

    2015-12-01

    Subjects with hypergonadotropic hypogonadism due to Turner's syndrome show low cortical mineral density, osteoporosis and risk of fractures. It is not clear if this bone fragility derives from chromosomal abnormalities or is the result of inadequate bone formation due to estrogen deficiency. The aim of this study was to investigate the cellular mechanisms underlying bone fragility in subjects with Turner's syndrome before induction of puberty and after hormonal replacement therapy (HRT). For this purpose, we have evaluated the osteoclastogenic potential of non-fractioned and T-cell depleted cultures of peripheral blood mononuclear cells (PBMCs) belonging to girls with Turner's syndrome who had not been treated with HRT yet, girls and young women who were on HRT and age-matched controls. Untreated subjects showed high FSH serum levels, whereas the other subjects displayed normal FSH serum levels. T-cell immunophenotype was analyzed through flow cytometry. Biochemical and DXA analyses were performed. Spontaneous osteoclastogenesis in non-fractioned and T-cell depleted cultures of PBMC belonging to girls with high FSH levels was more evident than in cultures of subjects with normal FSH levels. In the former, osteoclastogenesis was sustained by monocytes expressing high levels of c-fms, TNF-α and RANK, and T-cells producing high RANKL and TNF-α; in the latter it was supported by T-cells expressing high RANKL levels. CD4(+)CD25(high) T-cells were reduced in all subjects, whereas CD3(+)/CD16(+)/CD56(+) NKT-cells were increased in those with high FSH levels. High RANKL and CTX levels were detected in the sera. Bone impairment was already detectable by DXA in subjects aged under 10, although it became more evident with aging. In conclusion, our results demonstrated that bone fragility in subjects with Turner's syndrome is associated to enhanced osteoclastogenesis. This process seems to be due to high FSH serum levels before HRT, whereas it is caused by high RANKL during

  18. RBP-J imposes a requirement for ITAM-mediated costimulation of osteoclastogenesis.

    PubMed

    Li, Susan; Miller, Christine H; Giannopoulou, Eugenia; Hu, Xiaoyu; Ivashkiv, Lionel B; Zhao, Baohong

    2014-11-01

    Osteoclastogenesis requires activation of RANK signaling as well as costimulatory signals from immunoreceptor tyrosine-based activation motif-containing (ITAM-containing) receptors/adaptors, predominantly tyrosine kinase-binding proteins DAP12 and FcRγ, in osteoclast precursors. It is not well understood how costimulatory signals are regulated and integrated with RANK signaling. Here, we found that osteopetrotic bone phenotypes in mice lacking DAP12 or DAP12 and FcRγ are mediated by the transcription factor RBP-J, as deletion of Rbpj in these mice substantially rescued the defects of bone remodeling. Using a TNF-α-induced model of inflammatory bone resorption, we determined that RBP-J deficiency enables TNF-α to induce osteoclast formation and bone resorption in DAP12-deficient animals. Thus, RBP-J imposes a requirement for ITAM-mediated costimulation of RANKL or TNF-α-induced osteoclastogenesis. Mechanistically, RBP-J suppressed induction of key osteoclastogenic factors NFATc1, BLIMP1, and c-FOS by inhibiting ITAM-mediated expression and function of PLCγ2 and activation of downstream calcium-CaMKK/PYK2 signaling. Moreover, RBP-J suppressed Plcg2 expression and downstream calcium oscillations indirectly by a TGF-β/PLCγ2/calcium axis. Together, our findings indicate that RBP-J suppresses ITAM-mediated costimulation, thereby limiting crosstalk between ITAM and RANK/TNFR signaling and allowing fine tuning of osteoclastogenesis during bone homeostasis and under inflammatory conditions. Furthermore, these data suggest that environmental cues that regulate RBP-J expression/function potentially modulate the requirement for costimulatory signaling for osteoclast differentiation and bone remodeling.

  19. Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process

    SciTech Connect

    Hong, Huixian; Shi, Zhenqi; Qiao, Ping; Li, Hui; McCoy, Erin M.; Mao, Ping; Xu, Hui; Feng, Xu; Wang, Shunqing

    2013-11-01

    Highlights: •IL-3 treatment of bone marrow cells generates a population of hematopoietic cells. •IL-3-dependent hematopoietic cells are capable of differentiating into osteoclasts. •Osteoclasts derived from IL-3-dependent hematopoietic cells are functional. •IL-3 promotes the development of osteoclast progenitors. •IL-3 inhibits the osteoclastogenic process. -- Abstract: Interleukin (IL)-3, a multilineage hematopoietic growth factor, is implicated in the regulation of osteoclastogenesis. However, the role of IL-3 in osteoclastogenesis remains controversial; whereas early studies showed that IL-3 stimulates osteoclastogenesis, recent investigations demonstrated that IL-3 inhibits osteoclast formation. The objective of this work is to further address the role of IL-3 in osteoclastogenesis. We found that IL-3 treatment of bone marrow cells generated a population of cells capable of differentiating into osteoclasts in tissue culture dishes in response to the stimulation of the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). The IL-3-dependent hematopoietic cells were able to further proliferate and differentiate in response to M-CSF stimulation and the resulting cells were also capable of forming osteoclasts with M-CSF and RANKL treatment. Interestingly, IL-3 inhibits M-CSF-/RANKL-induced differentiation of the IL-3-dependent hematopoietic cells into osteoclasts. The flow cytometry analysis indicates that while IL-3 treatment of bone marrow cells slightly affected the percentage of osteoclast precursors in the surviving populations, it considerably increased the percentage of osteoclast precursors in the populations after subsequent M-CSF treatment. Moreover, osteoclasts derived from IL-3-dependent hematopoietic cells were fully functional. Thus, we conclude that IL-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the

  20. Desferrioxamine reduces ultrahigh-molecular-weight polyethylene-induced osteolysis by restraining inflammatory osteoclastogenesis via heme oxygenase-1

    PubMed Central

    Kang, Hui; Yan, Yufei; Jia, Peng; Yang, Kai; Guo, Changjun; Chen, Hao; Qi, Jin; Qian, Niandong; Xu, Xing; Wang, Fei; Li, Changwei; Guo, Lei; Deng, Lianfu

    2016-01-01

    As wear particles-induced osteolysis still remains the leading cause of early implant loosening in endoprosthetic surgery, and promotion of osteoclastogenesis by wear particles has been confirmed to be responsible for osteolysis. Therapeutic agents targeting osteoclasts formation are considered for the treatment of wear particles-induced osteolysis. In the present study, we demonstrated for the first time that desferrioxamine (DFO), a powerful iron chelator, could significantly alleviate osteolysis in an ultrahigh-molecular-weight polyethylene (UHMWPE) particles-induced mice calvaria osteolysis model. Furthermore, DFO attenuated calvaria osteolysis by restraining enhanced inflammatory osteoclastogenesis induced by UHMWPE particles. Consistent with the in vivo results, we found DFO was also able to inhibit osteoclastogenesis in a dose-dependent manner in vitro, as evidenced by reduction of osteoclasts formation and suppression of osteoclast specific genes expression. In addition, DFO dampened osteoclasts differentiation and formation at early stage but not at late stage. Mechanistically, the reduction of osteoclastogenesis by DFO was due to increased heme oxygenase-1 (HO-1) expression, as decreased osteoclasts formation induced by DFO was significantly restored after HO-1 was silenced by siRNA, while HO-1 agonist COPP treatment enhanced DFO-induced osteoclastogenesis inhibition. In addition, blocking of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway promoted DFO-induced HO-1 expression, implicating that p38 signaling pathway was involved in DFO-mediated HO-1 expression. Taken together, our results suggested that DFO inhibited UHMWPE particles-induced osteolysis by restraining inflammatory osteoclastogenesis through upregulation of HO-1 via p38MAPK pathway. Thus, DFO might be used as an innovative and safe therapeutic alternative for treating wear particles-induced aseptic loosening. PMID:27787522

  1. Neuronal TRPV1 activation regulates alveolar bone resorption by suppressing osteoclastogenesis via CGRP

    PubMed Central

    Takahashi, Naoki; Matsuda, Yumi; Sato, Keisuke; de Jong, Petrus R.; Bertin, Samuel; Tabeta, Koichi; Yamazaki, Kazuhisa

    2016-01-01

    The transient receptor potential vanilloid 1 (TRPV1) channel is abundantly expressed in peripheral sensory neurons where it acts as an important polymodal cellular sensor for heat, acidic pH, capsaicin, and other noxious stimuli. The oral cavity is densely innervated by afferent sensory neurons and is a highly specialized organ that protects against infections as well as physical, chemical, and thermal stresses in its capacity as the first part of the digestive system. While the function of TRPV1 in sensory neurons has been intensively studied in other organs, its physiological role in periodontal tissues is unclear. In this study we found that Trpv1−/− mice developed severe bone loss in an experimental model of periodontitis. Chemical ablation of TRPV1-expressing sensory neurons recapitulated the phenotype of Trpv1−/− mice, suggesting a functional link between neuronal TRPV1 signaling and periodontal bone loss. TRPV1 activation in gingival nerves induced production of the neuropeptide, calcitonin gene-related peptide (CGRP), and CGRP treatment inhibited osteoclastogenesis in vitro. Oral administration of the TRPV1 agonist, capsaicin, suppressed ligature-induced bone loss in mice with fewer tartrate-resistant acid phosphatase (TRAP)-positive cells in alveolar bone. These results suggest that neuronal TRPV1 signaling in periodontal tissue is crucial for the regulation of osteoclastogenesis via the neuropeptide CGRP. PMID:27388773

  2. Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures

    PubMed Central

    Movérare-Skrtic, Sofia; Henning, Petra; Liu, Xianwen; Nagano, Kenichi; Saito, Hiroaki; Börjesson, Anna E; Sjögren, Klara; Windahl, Sara H; Farman, Helen; Kindlund, Bert; Engdahl, Cecilia; Koskela, Antti; Zhang, Fu-Ping; Eriksson, Emma E; Zaman, Farasat; Hammarstedt, Ann; Isaksson, Hanna; Bally, Marta; Kassem, Ali; Lindholm, Catharina; Sandberg, Olof; Aspenberg, Per; Sävendahl, Lars; Feng, Jian Q; Tuckermann, Jan; Tuukkanen, Juha; Poutanen, Matti; Baron, Roland; Lerner, Ulf H; Gori, Francesca; Ohlsson, Claes

    2015-01-01

    The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need. PMID:25306233

  3. Hepatocyte growth factor can substitute for M-CSF to support osteoclastogenesis

    SciTech Connect

    Adamopoulos, Iannis E. . E-mail: iadamopoulos@path.wustl.edu; Xia Zhidao; Lau, Y.S.; Athanasou, Nicholas A.

    2006-11-17

    Osteopetrotic mice lacking functional macrophage-colony stimulating factor (M-CSF) recover with ageing, suggesting that alternative osteoclastogenesis pathways exist. Hepatocyte growth factor (HGF) and M-CSF signal through tyrosine kinase receptors and phosphorylate common transducers and effectors such as Src, Grb2, and PI3-Kinase. HGF is known to play a role in osteoclast formation, and in this study we have determined whether HGF could replace M-CSF to support human osteoclastogenesis. We found that the HGF receptor, c-Met, is expressed by the CD14{sup +} monocyte fraction of human peripheral blood mononuclear cells (PBMC). HGF was able to support monocyte-osteoclast differentiation in the presence of receptor activator for nuclear factor {kappa}B ligand as evidenced by the formation of numerous multinucleated tartrate-resistant acid phosphatase and vitronectin receptor positive cells which formed F-actin rings and were capable of lacunar resorption. The addition of a neutralising antibody to M-CSF did not inhibit osteoclast differentiation. HGF is a well-established survival factor and viability assays and live/dead staining showed that it promoted the survival and proliferation of monocytes and osteoclasts in a manner similar to M-CSF. Our findings indicate that HGF can substitute for M-CSF to support human osteoclast formation.

  4. Akermanite bioceramics promote osteogenesis, angiogenesis and suppress osteoclastogenesis for osteoporotic bone regeneration.

    PubMed

    Xia, Lunguo; Yin, Zhilan; Mao, Lixia; Wang, Xiuhui; Liu, Jiaqiang; Jiang, Xinquan; Zhang, Zhiyuan; Lin, Kaili; Chang, Jiang; Fang, Bing

    2016-02-25

    It is a big challenge for bone healing under osteoporotic pathological condition with impaired angiogenesis, osteogenesis and remodeling. In the present study, the effect of Ca, Mg, Si containing akermanite bioceramics (Ca2MgSi2O7) extract on cell proliferation, osteogenic differentiation and angiogenic factor expression of BMSCs derived from ovariectomized rats (BMSCs-OVX) as well as the expression of osteoclastogenic factors was evaluated. The results showed that akermanite could enhance cell proliferation, ALP activity, expression of Runx2, BMP-2, BSP, OPN, OCN, OPG and angiogenic factors including VEGF and ANG-1. Meanwhile, akermanite could repress expression of osteoclastogenic factors including RANKL and TNF-α. Moreover, akermanite could activate ERK, P38, AKT and STAT3 signaling pathways, while crosstalk among these signaling pathways was evident. More importantly, the effect of akermanite extract on RANKL-induced osteoclastogenesis was evaluated by TRAP staining and real-time PCR assay. The results showed that akermanite could suppress osteoclast formation and expression of TRAP, cathepsin K and NFATc1. The in vivo experiments revealed that akermanite bioceramics dramatically stimulated osteogenesis and angiogenesis in an OVX rat critical-sized calvarial defect model. All these results suggest that akermanite bioceramics with the effects of Mg and Si ions on osteogenesis, angiogenesis and osteoclastogenesis are promising biomaterials for osteoporotic bone regeneration.

  5. SH3BP2 is an activator of NFAT activity and osteoclastogenesis

    SciTech Connect

    Lietman, Steven A. Yin Lihong; Levine, Michael A.

    2008-07-11

    Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Osteoclasts differentiate after binding of sRANKL to RANK induces a number of downstream signaling effects, including activation of the calcineurin/NFAT (nuclear factor of activated T cells) pathway. Here, we have investigated the functional significance of SH3BP2 protein on osteoclastogenesis in the presence of sRANKL. Our results indicate that SH3BP2 both increases nuclear NFATc1 in sRANKL treated RAW 264.7 preosteoclast cells and enhances expression of tartrate resistant acid phosphatase (TRAP), a specific marker of osteoclast differentiation. Moreover, overexpression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLC{gamma}1 and 2, thus providing a mechanistic pathway for the rapid translocation of NFATc1 into the nucleus and increased osteoclastogenesis in cherubism.

  6. Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

    SciTech Connect

    Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa . E-mail: baekjh@snu.ac.kr

    2006-08-18

    Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.

  7. Osteoactivin inhibition of osteoclastogenesis is mediated through CD44-ERK signaling

    PubMed Central

    Sondag, Gregory R; Mbimba, Thomas S; Moussa, Fouad M; Novak, Kimberly; Yu, Bing; Jaber, Fatima A; Abdelmagid, Samir M; Geldenhuys, Werner J; Safadi, Fayez F

    2016-01-01

    Osteoactivin is a heavily glycosylated protein shown to have a role in bone remodeling. Previous studies from our lab have shown that mutation in Osteoactivin enhances osteoclast differentiation but inhibits their function. To date, a classical receptor and a signaling pathway for Osteoactivin-mediated osteoclast inhibition has not yet been characterized. In this study, we examined the role of Osteoactivin treatment on osteoclastogenesis using bone marrow-derived osteoclast progenitor cells and identify a signaling pathway relating to Osteoactivin function. We reveal that recombinant Osteoactivin treatment inhibited osteoclast differentiation in a dose-dependent manner shown by qPCR, TRAP staining, activity and count. Using several approaches, we show that Osteoactivin binds CD44 in osteoclasts. Furthermore, recombinant Osteoactivin treatment inhibited ERK phosphorylation in a CD44-dependent manner. Finally, we examined the role of Osteoactivin on receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteolysis in vivo. Our data indicate that recombinant Osteoactivin inhibits RANKL-induced osteolysis in vivo and this effect is CD44-dependent. Overall, our data indicate that Osteoactivin is a negative regulator of osteoclastogenesis in vitro and in vivo and that this process is regulated through CD44 and ERK activation. PMID:27585719

  8. RANK Expression and Osteoclastogenesis in Human Monocytes in Peripheral Blood from Rheumatoid Arthritis Patients

    PubMed Central

    Kobashigawa, Tsuyoshi

    2016-01-01

    Rheumatoid arthritis (RA) appears as inflammation of synovial tissue and joint destruction. Receptor activator of NF-κB (RANK) is a member of the TNF receptor superfamily and a receptor for the RANK ligand (RANKL). In this study, we examined the expression of RANKhigh and CCR6 on CD14+ monocytes from patients with RA and healthy volunteers. Peripheral blood samples were obtained from both the RA patients and the healthy volunteers. Osteoclastogenesis from monocytes was induced by RANKL and M-CSF in vitro. To study the expression of RANKhigh and CCR6 on CD14+ monocytes, two-color flow cytometry was performed. Levels of expression of RANK on monocytes were significantly correlated with the level of osteoclastogenesis in the healthy volunteers. The expression of RANKhigh on CD14+ monocyte in RA patients without treatment was elevated and that in those receiving treatment was decreased. In addition, the high-level expression of RANK on CD14+ monocytes was correlated with the high-level expression of CCR6 in healthy volunteers. Monocytes expressing both RANK and CCR6 differentiate into osteoclasts. The expression of CD14+RANKhigh in untreated RA patients was elevated. RANK and CCR6 expressed on monocytes may be novel targets for the regulation of bone resorption in RA and osteoporosis. PMID:27822475

  9. Akermanite bioceramics promote osteogenesis, angiogenesis and suppress osteoclastogenesis for osteoporotic bone regeneration

    PubMed Central

    Xia, Lunguo; Yin, Zhilan; Mao, Lixia; Wang, Xiuhui; Liu, Jiaqiang; Jiang, Xinquan; Zhang, Zhiyuan; Lin, Kaili; Chang, Jiang; Fang, Bing

    2016-01-01

    It is a big challenge for bone healing under osteoporotic pathological condition with impaired angiogenesis, osteogenesis and remodeling. In the present study, the effect of Ca, Mg, Si containing akermanite bioceramics (Ca2MgSi2O7) extract on cell proliferation, osteogenic differentiation and angiogenic factor expression of BMSCs derived from ovariectomized rats (BMSCs-OVX) as well as the expression of osteoclastogenic factors was evaluated. The results showed that akermanite could enhance cell proliferation, ALP activity, expression of Runx2, BMP-2, BSP, OPN, OCN, OPG and angiogenic factors including VEGF and ANG-1. Meanwhile, akermanite could repress expression of osteoclastogenic factors including RANKL and TNF-α. Moreover, akermanite could activate ERK, P38, AKT and STAT3 signaling pathways, while crosstalk among these signaling pathways was evident. More importantly, the effect of akermanite extract on RANKL-induced osteoclastogenesis was evaluated by TRAP staining and real-time PCR assay. The results showed that akermanite could suppress osteoclast formation and expression of TRAP, cathepsin K and NFATc1. The in vivo experiments revealed that akermanite bioceramics dramatically stimulated osteogenesis and angiogenesis in an OVX rat critical-sized calvarial defect model. All these results suggest that akermanite bioceramics with the effects of Mg and Si ions on osteogenesis, angiogenesis and osteoclastogenesis are promising biomaterials for osteoporotic bone regeneration. PMID:26911441

  10. CD200:CD200R Interactions Regulate Osteoblastogenesis and Osteoclastogenesis in Space

    NASA Astrophysics Data System (ADS)

    Kos, Olha; Lee, Lydia; Gorezynski, Reginald M.

    2008-06-01

    We report data from studies on a recent FOTON mission, using an eOSTEO cell culture system developed by Systems Technologies Canada Inc., showing that in space overexpression of CD200 (using cell cultures derived from transgenic mice expressing CD200 under control of a doxycycline-inducible promoter) is associated with an attenuation in the suppression of mRNA markers of osteoblastogeneis (including BSP, OPG) with concomitant loss of the preferential increased osteoclastogenesis which is otherwise seen in the absence of CD200. In separate cultures we also explored the additional effect of altered inflammatory cytokines on the perturbation of expression of these bone-related genes, using cells from cytokine-receptor knockout mice. Our data suggest that while exogenous inflammatory cytokines (TNFα+IL1β) increased mRNAs typical for osteoclastogenesis under ground conditions, they appeared to produce no further modification of mRNA expression in flight. We suggest that altered mRNA expression in flight is not primarily driven by altered expression of inflammatory cytokines.

  11. Neuronal TRPV1 activation regulates alveolar bone resorption by suppressing osteoclastogenesis via CGRP.

    PubMed

    Takahashi, Naoki; Matsuda, Yumi; Sato, Keisuke; de Jong, Petrus R; Bertin, Samuel; Tabeta, Koichi; Yamazaki, Kazuhisa

    2016-07-08

    The transient receptor potential vanilloid 1 (TRPV1) channel is abundantly expressed in peripheral sensory neurons where it acts as an important polymodal cellular sensor for heat, acidic pH, capsaicin, and other noxious stimuli. The oral cavity is densely innervated by afferent sensory neurons and is a highly specialized organ that protects against infections as well as physical, chemical, and thermal stresses in its capacity as the first part of the digestive system. While the function of TRPV1 in sensory neurons has been intensively studied in other organs, its physiological role in periodontal tissues is unclear. In this study we found that Trpv1(-/-) mice developed severe bone loss in an experimental model of periodontitis. Chemical ablation of TRPV1-expressing sensory neurons recapitulated the phenotype of Trpv1(-/-) mice, suggesting a functional link between neuronal TRPV1 signaling and periodontal bone loss. TRPV1 activation in gingival nerves induced production of the neuropeptide, calcitonin gene-related peptide (CGRP), and CGRP treatment inhibited osteoclastogenesis in vitro. Oral administration of the TRPV1 agonist, capsaicin, suppressed ligature-induced bone loss in mice with fewer tartrate-resistant acid phosphatase (TRAP)-positive cells in alveolar bone. These results suggest that neuronal TRPV1 signaling in periodontal tissue is crucial for the regulation of osteoclastogenesis via the neuropeptide CGRP.

  12. Regulators of G protein signaling 12 promotes osteoclastogenesis in bone remodeling and pathological bone loss

    PubMed Central

    Yuan, X; Cao, J; Liu, T; Li, Y-P; Scannapieco, F; He, X; Oursler, M J; Zhang, X; Vacher, J; Li, C; Olson, D; Yang, S

    2015-01-01

    Regulators of G protein signaling (Rgs) have pivotal roles in controlling various cellular processes, such as cell differentiation. How Rgs proteins regulate osteoclast (OC) differentiation, function and bone homeostasis is poorly understood. It was previously demonstrated that Rgs12, the largest protein in the Rgs family, is predominantly expressed in OCs and regulates OC differentiation in vitro. To further understand the role and mechanism of Rgs12 in OC differentiation and bone diseases in vivo, we created OC-targeted Rgs12 knockout mice by using inducible Mx1-Cre and CD11b-Cre. Deletion of Rgs12 in hematopoietic cells or specifically in OC precursors resulted in increased bone mass with decreased OC numbers. Loss of Rgs12 impaired OC differentiation and function with impaired Ca2+ oscillations and reduced nuclear factor of activated T cells (NFAT) 2 expression. The introduction of wild-type osteoblasts did not rescue the defective osteoclastogenesis. Ectopic expression of NFAT2 rescued defective OC differentiation in CD11b;Rgs12fl/fl cells and promoted normal OC differentiation. Moreover, deletion of Rgs12 significantly inhibited pathological osteoclastogenesis and bone destruction in Rgs12-deficient mice that were subjected to ovariectomy and lipodysaccharide for bone loss. Thus our findings demonstrate that Rgs12 is an important regulator in OC differentiation and function and identify Rgs12 as a potential therapeutic target for osteoporosis and inflammation-induced bone loss. PMID:25909889

  13. In vitro investigation of the roles of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1 in murine osteoclastogenesis.

    PubMed

    Jules, Joel; Feng, Xu

    2014-01-01

    Whereas the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-кB ligand (RANKL) are essential and sufficient for osteoclastogenesis, a number of other cytokines including two proinflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1), can exert profound effects on the osteoclastogenic process. However, the precise mode of action of TNF-α and IL-1 in osteoclastogenesis remains controversial. While some groups demonstrated that these two cytokines can promote murine osteoclastogenesis in vitro in the presence of M-CSF only, we and others showed that TNF-α-/IL-1-mediated osteoclastogenesis requires permissive levels of RANKL. This chapter describes the method that we have used to investigate the effects of TNF-α and IL-1 on osteoclast formation in in vitro osteoclastogenesis assays using primary murine bone marrow macrophages (BMMs). Detailed experimental conditions are provided and critical points are discussed to help the reader use the method to independently evaluate the roles of TNF-α and IL-1 in osteoclastogenesis in vitro. Moreover, this method can be used to further elucidate the signaling mechanisms by which these two cytokines act in concert with RANKL or with each other to modulate osteoclastogenesis.

  14. Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathway

    PubMed Central

    Wu, Mengrui; Chen, Wei; Lu, Yun; Zhu, Guochun; Hao, Liang; Li, Yi-Ping

    2017-01-01

    Many positive signalling pathways of osteoclastogenesis have been characterized, but negative signalling pathways are less well studied. Here we show by microarray and RNAi that guanine nucleotide-binding protein subunit α13 (Gα13) is a negative regulator of osteoclastogenesis. Osteoclast-lineage-specific Gna13 conditional knockout mice have a severe osteoporosis phenotype. Gna13-deficiency triggers a drastic increase in both osteoclast number and activity (hyper-activation), mechanistically through decreased RhoA activity and enhanced Akt/GSK3β/NFATc1 signalling. Consistently, Akt inhibition or RhoA activation rescues hyper-activation of Gna13-deficient osteoclasts, and RhoA inhibition mimics the osteoclast hyperactivation resulting from Gna13-deficiency. Notably, Gα13 gain-of-function inhibits Akt activation and osteoclastogenesis, and protects mice from pathological bone loss in disease models. Collectively, we reveal that Gα13 is a master endogenous negative switch for osteoclastogenesis through regulation of the RhoA/Akt/GSK3β/NFATc1 signalling pathway, and that manipulating Gα13 activity might be a therapeutic strategy for bone diseases. PMID:28102206

  15. Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops.

    PubMed

    Li, Jing; Zeng, Li; Xie, Juan; Yue, Zhiying; Deng, Huayun; Ma, Xueyun; Zheng, Chunbing; Wu, Xiushan; Luo, Jian; Liu, Mingyao

    2015-12-01

    Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

  16. Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops

    PubMed Central

    Li, Jing; Zeng, Li; Xie, Juan; Yue, Zhiying; Deng, Huayun; Ma, Xueyun; Zheng, Chunbing; Wu, Xiushan; Luo, Jian; Liu, Mingyao

    2015-01-01

    Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca2+/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis. PMID:26620037

  17. Determination of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 in human serum using liquid chromatography with tandem mass spectrometry.

    PubMed

    Fang, Huiling; Yu, Songlin; Cheng, Qian; Cheng, Xinqi; Han, Jianhua; Qin, Xuzhen; Xia, Liangyu; Jiang, Xiaomei; Qiu, Ling

    2016-08-01

    Vitamin D plays important roles in skeletal metabolism and many other diseases, including chronic renal failure, hypoparathyroidism, sarcoidosis and rickets. 1α,25-dihydroxy vitamin D (1α,25(OH)2D), the active form of vitamin D, exhibits an extremely low serum concentration, which makes its quantification very challenging. High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is considered to be the "gold standard" for the determination of these chemicals, which are found in low concentrations in the serum, but conventionally, it needs tedious sample pretreatment procedures, such as solid phase extraction and derivatization. Herein, we describe a simple and rapid HPLC-MS/MS method for the simultaneous quantification of 1α,25-dihydroxy vitamin D3 (1α,25(OH)2D3) and 1α,25-dihydroxy vitamin D2 (1α,25(OH)2D2). The analytes were extracted from the matrix by liquid-liquid extraction, centrifuged to dryness and reconstituted with 75% methanol. Lithium acetate was employed to improve the ionization efficiency of 1α,25(OH)2D. The assay was sensitive with a low limit of quantitation of 10.0pg/mL for both 1α,25(OH)2D3 and 1α,25(OH)2D2 using a 0.5mL sample aliquot. Linearity was obtained over the range of 10.0pg/mL to 500pg/mL. Both the inter-assay and intra-assay precisions were <15%, and the analytical recoveries were within 100±5%. The performance evaluation of this assay demonstrated that it was a practical, sensitive and specific method for measuring the serum 1α,25(OH)2D3 and 1α,25(OH)2D2 concentrations.

  18. Nucleus pulposus cells derived IGF-1 and MCP-1 enhance osteoclastogenesis and vertebrae disruption in lumbar disc herniation

    PubMed Central

    Zhu, Zhongjiao; Huang, Peng; Chong, Yanxue; George, Suraj K; Wen, Bingtao; Han, Na; Liu, Zhiqiang; Kang, Lixin; Lin, Nie

    2014-01-01

    Study design: Chronic strained lumbar disc herniation (LDH) cases were classified into bulging LDH, herniated LDH and prolapse LDH types according to imaging examination, and vertebrae disruptions were evaluated. Cytokines derived from the nucleus pulposus cells were detected, and their effects on osteoclastogenesis, as well as the mechanisms involved, were studied via an in vitro osteoclast differentiation system. Objective: To clarify the mechanisms of lumbar vertebrae resorption induced by lumbar herniation. Summary and background data: Chronic strained lumbar disc herniation induced vertebrae erosion exacerbates quality of patients’ life and clinical outcome. Although nucleus pulposus cells derived cytokines were reported to play an important role in this pathogenesis, the fundamental mechanisms underlying this process are still unclear. Methods: Chronic strained lumbar disc herniation patients were diagnosed with CT scan and T2-weighted magnetic resonance imaging. RNA was extracted from 192 surgical specimens of the herniated lumbar disc and 29 surgical excisions of the lumbar disc from spinal injury patients. The expressions of osteoclastogenesis related cytokines and chemokines were examined using real time PCR. Monocytes were induced into osteoclast with M-CSF and RANKL in vitro, while the IGF-1 and MCP-1 were added into the differentiation procedure in order to evaluate the effects and explore the molecular mechanisms. Results: Vertebrae erosion had a positive relationship with lumbar disc herniation severity types. In all of the osteoclastogenesis related cytokines, the IGF-1 and MCP-1 were the most highly expressed in the nucleus pulposus cells. IGF-1 enhances activation of NF-kB signaling directly, but MCP-1 upregulated the expression of RANK, so that enhanced cellular sensitivity to RANKL resulted in increasing osteoclastogenesis and activity. Conclusion: Lumbar herniation induced overexpression of IGF-1 and MCP-1 in nucleus pulposus cells aggravated

  19. Bone Resorption Is Regulated by Circadian Clock in Osteoblasts.

    PubMed

    Takarada, Takeshi; Xu, Cheng; Ochi, Hiroki; Nakazato, Ryota; Yamada, Daisuke; Nakamura, Saki; Kodama, Ayumi; Shimba, Shigeki; Mieda, Michihiro; Fukasawa, Kazuya; Ozaki, Kakeru; Iezaki, Takashi; Fujikawa, Koichi; Yoneda, Yukio; Numano, Rika; Hida, Akiko; Tei, Hajime; Takeda, Shu; Hinoi, Eiichi

    2016-12-07

    We have previously shown that endochondral ossification is finely regulated by the Clock system expressed in chondrocytes during postnatal skeletogenesis. Here we show a sophisticated modulation of bone resorption and bone mass by the Clock system through its expression in bone-forming osteoblasts. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Period1 (Per1) were expressed with oscillatory rhythmicity in the bone in vivo, and circadian rhythm was also observed in cultured osteoblasts of Per1::luciferase transgenic mice. Global deletion of murine Bmal1, a core component of the Clock system, led to a low bone mass, associated with increased bone resorption. This phenotype was recapitulated by the deletion of Bmal1 in osteoblasts alone. Co-culture experiments revealed that Bmal1-deficient osteoblasts have a higher ability to support osteoclastogenesis. Moreover, 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]-induced receptor activator of nuclear factor κB ligand (Rankl) expression was more strongly enhanced in both Bmal1-deficient bone and cultured osteoblasts, whereas overexpression of Bmal1/Clock conversely inhibited it in osteoblasts. These results suggest that bone resorption and bone mass are regulated at a sophisticated level by osteoblastic Clock system through a mechanism relevant to the modulation of 1,25(OH)2 D3 -induced Rankl expression in osteoblasts. © 2016 American Society for Bone and Mineral Research.

  20. Potentiation of osteoclastogenesis by adipogenic conversion of bone marrow-derived mesenchymal stem cells.

    PubMed

    Mori, Keisuke; Suzuki, Keiji; Hozumi, Akira; Goto, Hisataka; Tomita, Masato; Koseki, Hironobu; Yamashita, Shunichi; Osaki, Makoto

    2014-01-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) are the indispensable component of the bone marrow, being the common precursors for adipocytes and osteoblasts. We show here that adipogenic differentiation resulted in increase in the production of adipocyte markers, such as adiponectin,fatty-acid binding proteins (FABP4), peroxisome proliferator-activated receptor γ (PPARγ), as well as the receptor activator of nuclear-κB ligand (RANKL). Co-culture of osteoclast precursors (OCPs) with BMSCs-derived adipocytes significantly enhanced osteoclast differentiation with low-dose RANKL, whose levels alone could not promote osteoclastogenesis. These results demonstrate for the first time that adipogenic differentiation of BMSCs plays a pivotal role in maintaining bone homeostasis.

  1. GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation

    PubMed Central

    Liu, Weiqing; Zhou, Liyan; Zhou, Chenchen; Zhang, Shiwen; Jing, Junjun; Xie, Liang; Sun, Ningyuan; Duan, Xiaobo; Jing, Wei; Liang, Xing; Zhao, Hu; Ye, Ling; Chen, Qianming; Yuan, Quan

    2016-01-01

    Osteoporosis is an age-related disease that affects millions of people. Growth differentiation factor 11 (GDF11) is a secreted member of the transforming growth factor beta (TGF-β) superfamily. Deletion of Gdf11 has been shown to result in a skeletal anterior–posterior patterning disorder. Here we show a role for GDF11 in bone remodelling. GDF11 treatment leads to bone loss in both young and aged mice. GDF11 inhibits osteoblast differentiation and also stimulates RANKL-induced osteoclastogenesis through Smad2/3 and c-Fos-dependent induction of Nfatc1. Injection of GDF11 impairs bone regeneration in mice and blocking GDF11 function prevents oestrogen-deficiency-induced bone loss and ameliorates age-related osteoporosis. Our data demonstrate that GDF11 is a previously unrecognized regulator of bone remodelling and suggest that GDF11 is a potential target for treatment of osteoporosis. PMID:27653144

  2. Reactive oxygen species and oxidative stress in osteoclastogenesis, skeletal aging and bone diseases.

    PubMed

    Callaway, Danielle A; Jiang, Jean X

    2015-07-01

    Osteoclasts are cells derived from bone marrow macrophages and are important in regulating bone resorption during bone homeostasis. Understanding what drives osteoclast differentiation and activity is important when studying diseases characterized by heightened bone resorption relative to formation, such as osteoporosis. In the last decade, studies have indicated that reactive oxygen species (ROS), including superoxide and hydrogen peroxide, are crucial components that regulate the differentiation process of osteoclasts. However, there are still many unanswered questions that remain. This review will examine the mechanisms by which ROS can be produced in osteoclasts as well as how it may affect osteoclast differentiation and activity through its actions on osteoclastogenesis signaling pathways. In addition, the contribution of ROS to the aging-associated disease of osteoporosis will be addressed and how targeting ROS may lead to the development of novel therapeutic treatment options.

  3. Imatinib mesylate inhibits osteoclastogenesis and joint destruction in rats with collagen-induced arthritis (CIA).

    PubMed

    Ando, Wataru; Hashimoto, Jun; Nampei, Akihide; Tsuboi, Hideki; Tateishi, Kosuke; Ono, Takeshi; Nakamura, Norimasa; Ochi, Takahiro; Yoshikawa, Hideki

    2006-01-01

    Macrophage colony-stimulating factor (M-CSF) is a key factor for osteoclastogenesis at the bone-pannus interface in patients with rheumatoid arthritis as well as a receptor activator of NF-kappaB ligand (RANKL). Imatinib mesylate inhibits the phosphorylation of c-fms, a receptor for M-CSF. The present study investigates the effect of imatinib mesylate on joint destruction in rats with collagen-induced arthritis (CIA) and on osteoclastogenesis in vitro. Imatinib mesylate (50 or 150 mg/kg), dexamethasone, or vehicle was administered daily to CIA rats for 4 weeks from the onset of arthritis. Hind-paw swelling and body weight were measured weekly. At weeks 2 and 4, the metatarsophalangeal (MTP) joints and the ankle and subtalar joints were radiographically and histologically assessed. The effect of imatinib mesylate on osteoclast formation from rat bone marrow cells with M-CSF and soluble RANKL (sRANKL) in vitro was also examined. Radiographic assessment showed that 150 mg/kg imatinib mesylate suppressed the destruction of the MTP and the ankle and subtalar joints at week 2, and MTP joint destruction at week 4 in CIA rats, although hind-paw swelling was not suppressed. The number of TRAP-positive cells at the bone-pannus interface was significantly reduced in the group administered with 150 mg/kg imatinib mesylate compared with that given vehicle at week 4. Imatinib mesylate dose-dependently inhibited the proliferation of M-CSF-dependent osteoclast precursor cells in vitro as well as osteoclast formation induced by M-CSF and sRANKL. These findings suggest that imatinib mesylate could prevent joint destruction in patients with rheumatoid arthritis.

  4. Herbacetin inhibits RANKL-mediated osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo.

    PubMed

    Li, Liang; Sapkota, Mahesh; Kim, Se-woong; Soh, Yunjo

    2016-04-15

    Herbacetin is an active flavonol (a type of flavonoid) that has various biologic effects such as antioxidant, antitumor, and anti-inflammatory activities. However, one of its novel effects remains to be investigated, that is, the induction of osteoclastogenesis by the receptor activator of nuclear factor-κB ligand (RANKL). In this study, we examined the effects and mechanisms of action of herbacetin on osteoclastogenesis in RANKL-treated bone marrow-derived macrophages (BMMs) and murine macrophage RAW264.7 cells in vitro and on lipopolysaccharide (LPS)-induced bone destruction in vivo. Herbacetin significantly inhibited RANKL-induced osteoclast formation and differentiation in BMMs and RAW264.7 cells in a dose-dependent manner. Moreover, the suppressive effect of herbacetin resulted in a decrease in osteoclast-related genes, including RANK, tartrate-resistant acid phosphatase, cathepsin K, and matrix metalloproteinase-2 and -9 (MMP-9). Consistent with mRNA results, we confirmed that herbacetin treatment downregulated protein expression of MMP-9 and cathepsin K. Herbacetin also decreased induction of the osteoclastogenic transcription factor c-Fos and nuclear factor of activated T cells c1 (NFATc1) and blocked RANKL-mediated activation of Jun N-terminal kinase (JNK) and nuclear factor-κB. Herbacetin clearly inhibited the bone resorption activity of osteoclasts on plates coated with fluorescein-labeled calcium phosphate. More importantly, the application of herbacetin significantly reduced LPS-induced inflammatory bone loss in mice in vivo. Taken together, our results indicate that herbacetin has potential for use as a therapeutic agent in disorders associated with bone loss.

  5. uPA Attenuated LPS-induced Inflammatory Osteoclastogenesis through the Plasmin/PAR-1/Ca2+/CaMKK/AMPK Axis

    PubMed Central

    Kanno, Yosuke; Ishisaki, Akira; Kawashita, Eri; Kuretake, Hiromi; Ikeda, Kanako; Matsuo, Osamu

    2016-01-01

    Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis-caused bone destruction, results from an increase of bone-resorbing osteoclasts (OCs) induced by inflammation. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated that the effect of urokinase-type plasminogen activator (uPA) on inflammatory osteoclastogenesis induced by lipopolysaccharide (LPS), which is a potent stimulator of bone resorption in inflammatory diseases. We found that the uPA deficiency promoted inflammatory osteoclastogenesis and bone loss induced by LPS. We also showed that LPS induced the expression of uPA, and the uPA treatment attenuated the LPS-induced inflammatory osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells. Additionally, we showed that the uPA-attenuated inflammatory osteoclastgenesis is associated with the activation of plasmin/protease-activated receptor (PAR)-1 axis by uPA. Moreover, we examined the mechanism underlying the effect of uPA on inflammatory osteoclastogenesis, and found that uPA/plasmin/PAR-1 activated the adenosine monophosphate-activated protein kinase (AMPK) pathway through Ca2+/calmodulin dependent protein kinase kinase (CaMKK) activation, and attenuated inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 cells. These data suggest that uPA attenuated inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK axis. Our findings may provide a novel therapeutic approach to bone loss caused by inflammatory diseases. PMID:26722218

  6. Xanthohumol modulates the expression of osteoclast-specific genes during osteoclastogenesis in RAW264.7 cells.

    PubMed

    Suh, Kwang Sik; Rhee, Sang Youl; Kim, Young Seol; Lee, Young Soon; Choi, Eun Mi

    2013-12-01

    RANKL has been shown to play a critical role in osteoclast formation and bone resorption. Thus, agents that suppress RANKL signaling have a potential to suppress bone loss. In this study, we examined the ability of xanthohumol, a structurally simple prenylated chalcone, to suppress RANKL signaling during osteoclastogenesis in RAW264.7 cells. Xanthohumol markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclasts formation, and resorption-pit formation. In experiments to elucidate its mechanism of action, xanthohumol was found to suppress RANKL-induced expression of TRAF6, GAB2, ERK, c-Src, PI3K, and Akt genes. Moreover, RANKL-induced expressions of c-Fos and NFATc1, which are crucial transcription factors for osteoclastogenesis, were reduced by treatment with xanthohumol. Xanthohumol also inhibited RANKL-induced expression of bone-resorption related osteoclast-specific genes (carbonic anhydrase II, TCIRG, CLCN7, OSTM1, cathepsin K, and MMP-9). These data demonstrate that xanthohumol inhibits osteoclastogenesis by modulating RANKL signaling and may be useful for the prevention of bone-destructive diseases such as osteoporosis, arthritis and periodontitis.

  7. Acro-osteolysis is associated with enhanced osteoclastogenesis and higher blood VEGF levels in systemic sclerosis

    PubMed Central

    Park, Jin Kyun; Fava, Andrea; Carrino, John; Del Grande, Filippo; Rosen, Antony; Boin, Francesco

    2015-01-01

    Objective Bone resorption of distal phalanges or acro-osteolysis (AO) can develop in systemic sclerosis (SSc) causing pain and functional limitation. We investigated whether AO may be associated with abnormal osteoclastogenesis in SSc patients and whether hypoxia may be involved in this process. Methods Peripheral blood mononuclear cells (PBMCs) obtained from 26 SSc patients (11 with and 15 without AO) and 14 healthy controls (HC) were cultured in the presence of receptor activator of NF-κB-ligand (RANKL) and macrophage colony-stimulating factor for 9 days. Tartrate resistant acid phosphatase (TRAP)+ multinucleated giant cells (MNGs) containing 3 or more nuclei were counted as osteoclasts (OCs). Plasma levels and effects of vascular endothelial growth factor (VEGF) on OC formation were evaluated. Results SSc patients with AO formed significantly more OCs after 9 days than did subjects without AO (142.4±67.0 vs. 27.2±17.6 MNGs/well, p<0.001) or HC (18.7±27.0 MNGs/well, p <0.001). No significant difference in OC formation was noted between the patients without AO and HC. Plasma levels of VEGF were higher in SSc patients with AO compared to those without (142.4± 69.6 pg/mL vs. 88.1±38.2 pg/mL, p<0.001) or HC (54.2±24.6 pg/mL, p=0.018). Priming with VEGF-A for 24 hours significantly increased OC generation by 5.3±1.9 fold (p=0.03). The radiographic extent of AO was associated with increased OC formation (Spearman rho=0.741, p=0.01). Conclusion Increased OC formation and higher VEGF levels may contribute to AO in SSc patients.Further studies are needed to elucidate whether targeting osteoclastogenesis may provide a specific therapeutic option for SSc-AO. PMID:26361270

  8. The Hemoglobin Receptor Protein of Porphyromonas gingivalis Inhibits Receptor Activator NF-κB Ligand-Induced Osteoclastogenesis from Bone Marrow Macrophages

    PubMed Central

    Fujimura, Yuji; Hotokezaka, Hitoshi; Ohara, Naoya; Naito, Mariko; Sakai, Eiko; Yoshimura, Mamiko; Narita, Yuka; Kitaura, Hideki; Yoshida, Noriaki; Nakayama, Koji

    2006-01-01

    Extracellular proteinaceous factors of Porphyromonas gingivalis, a periodontal pathogen, that influence receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis from bone marrow macrophages were investigated. The culture supernatant of P. gingivalis had the ability to inhibit RANKL-induced in vitro osteoclastogenesis. A major protein of the culture supernatant, hemoglobin receptor protein (HbR), suppressed RANKL-induced osteoclastogenesis in a dose-dependent fashion. HbR markedly inhibited RANKL-induced osteoclastogenesis when present in the culture for the first 24 h after addition of RANKL, whereas no significant inhibition was observed when HbR was added after 24 h or later, implying that HbR might interfere with only the initial stage of RANKL-mediated differentiation. HbR tightly bound to bone marrow macrophages and had the ability to induce phosphorylation of ERK, p38, NF-κB, and Akt. RANKL-induced phosphorylation of ERK, p38, and NF-κB was not suppressed by HbR, but that of Akt was markedly suppressed. HbR inhibited RANKL-mediated induction of c-Fos and NFATc1. HbR could induce beta interferon (IFN-β) from bone marrow macrophages, but the induction level of IFN-β might not be sufficient to suppress RANKL-mediated osteoclastogenesis, implying presence of an IFN-β-independent pathway in HbR-mediated inhibition of osteoclastogenesis. Since rapid and extensive destruction of the alveolar bone causes tooth loss, resulting in loss of the gingival crevice that is an anatomical niche for periodontal pathogens such as P. gingivalis, the suppressive effect of HbR on osteoclastogenesis may help the microorganism exist long in the niche. PMID:16622189

  9. β-catenin activity in late hypertrophic chondrocytes locally orchestrates osteoblastogenesis and osteoclastogenesis

    PubMed Central

    Houben, Astrid; Weissenböck, Martina; Graf, Julian; Teufel, Stefan; von der Mark, Klaus

    2016-01-01

    Trabecular bone formation is the last step in endochondral ossification. This remodeling process of cartilage into bone involves blood vessel invasion and removal of hypertrophic chondrocytes (HTCs) by chondroclasts and osteoclasts. Periosteal- and chondrocyte-derived osteoprogenitors utilize the leftover mineralized HTC matrix as a scaffold for primary spongiosa formation. Here, we show genetically that β-catenin (encoded by Ctnnb1), a key component of the canonical Wnt pathway, orchestrates this remodeling process at multiple levels. Conditional inactivation or stabilization of β-catenin in HTCs by a Col10a1-Cre line locally modulated osteoclastogenesis by altering the Rankl:Opg ratio in HTCs. Lack of β-catenin resulted in a severe decrease of trabecular bone in the embryonic long bones. Gain of β-catenin activity interfered with removal of late HTCs and bone marrow formation, leading to a continuous mineralized hypertrophic core in the embryo and resulting in an osteopetrotic-like phenotype in adult mice. Furthermore, β-catenin activity in late HTCs is required for chondrocyte-derived osteoblastogenesis at the chondro-osseous junction. The latter contributes to the severe trabecular bone phenotype in mutants lacking β-catenin activity in HTCs. PMID:27621061

  10. Carvacrol Inhibits Osteoclastogenesis and Negatively Regulates the Survival of Mature Osteoclasts.

    PubMed

    Deepak, Vishwa; Kasonga, Abe; Kruger, Marlena Cathorina; Coetzee, Magdalena

    2016-07-01

    Bone is a dynamic tissue that undergoes continuous remodeling coupled with the action of osteoblasts and osteoclasts. Osteoclast activity is elevated during osteoporosis and periodontitis resulting in excessive loss of trabecular and alveolar bone. Osteoclasts are formed in an inflammatory response to cytokine production receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL) and bacterial challenge lipopolysaccharide (LPS). Carvacrol, a monoterpenic phenol present in Origanum vulgare and Thymus vulgaris, is a natural compound with known medicinal properties. We investigated the effects of carvacrol on osteoclast formation induced by RANKL and LPS. Carvacrol suppressed RANKL-induced formation of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells in RAW264.7 macrophages and human CD14(+) monocytes. Furthermore, carvacrol inhibited LPS-induced osteoclast formation in RAW264.7 macrophages. Investigation of the underlying molecular mechanisms revealed that carvacrol downregulated RANKL-induced NF-κB activation in a dose-dependent manner. Furthermore, the suppression of NF-κB activation correlated with inhibition of inhibitor of kappaB (IκB) kinase (IKK) activation and attenuation of inhibitor of NF-κB (IκBa) degradation. Carvacrol potentiated apoptosis in mature osteoclasts by caspase-3 activation and DNA fragmentation. Moreover, carvacrol did not affect the viability of proliferating MC3T3-E1 osteoblast-like cells. Collectively, these results demonstrate that carvacrol mitigates osteoclastogenesis by impairing the NF-κB pathway and induction of apoptosis in mature osteoclasts.

  11. Bacterial Infections and Osteoclastogenesis Regulators in Men and Women with Cholesteatoma.

    PubMed

    Likus, Wirginia; Siemianowicz, Krzysztof; Markowski, Jarosław; Wiaderkiewicz, Jan; Kostrząb-Zdebel, Anna; Jura-Szołtys, Edyta; Dziubdziela, Włodzimierz; Wiaderkiewicz, Ryszard; Łos, Marek J

    2016-06-01

    One of the most distinct features of middle ear cholesteatoma is bone destruction. Aetiology of cholesteatoma is thought to be multifactorial. Endotoxins produced by bacteria are thought to initiate the inflammation process in the middle ear leading to cholesteatoma. There are physiological differences in bone metabolism between men and women. The aim of our study was the immunohistochemical evaluation of the contents of two key components of the OPG/RANK/RANKL triad-RANKL and OPG in cholesteatoma, to analyse if there are any differences between the sexes and to evaluate the bacteria species isolated from cholesteatoma just before surgical treatment and to evaluate their plausible influence on the expression of OPG and RANKL in cholesteatoma. Twenty-one adult patients with acquired cholesteatoma who underwent surgery were analysed. There were no statistically significant differences in the expression of both regulators of osteoclastogenesis between the sexes. In 38.1 % patients cholesteatoma was not infected, whereas in 61.9 % patients various bacterial infections or mycosis were found. The most frequently isolated species was Pseudomonas aeruginosa (14.29 % infections) followed by Staphylococcus aureus (9.52 % infections). There were no statistically significant differences in expression of both OPG and RANKL between uninfected and infected cholesteatomas.

  12. Suppression of NF-κB Activation By Gentian Violet Promotes Osteoblastogenesis and Suppresses Osteoclastogenesis

    PubMed Central

    Yamaguchi, M.; Vikulina, T.; Arbiser, J.L.; Weitzmann, M.N.

    2015-01-01

    Skeletal mass is regulated by the coordinated action of bone forming osteoblasts and bone resorbing osteoclasts. Accelerated rates of bone resorption relative to bone formation lead to net bone loss and the development of osteoporosis, a devastating disease that predisposes the skeleton to fractures. Bone fractures are associated with significant morbidity and in the case of hip fractures, high mortality. Gentian violet (GV), a cationic triphenylmethane dye, has long been used as an antifungal and antibacterial agent and is presently under investigation as a potential chemotherapeutic and antiangiogenic agent. However, effects on bone cells have not been previously reported and the mechanisms of action of GV, are poorly understood. In this study we show that GV suppresses receptor activator of NF-κB ligand (RANKL)-induced differentiation of RAW264.7 osteoclast precursors into mature osteoclasts, but paradoxically stimulates the differentiation of MC3T3 cells into mineralizing osteoblasts. These actions stem from the capacity of GV to suppress activation of the nuclear factor kappa B (NF-κB) signal transduction pathway that is required for osteoclastogenesis, but inhibitory to osteoblast differentiation and activity. Our data reveal that GV is an inhibitor of NF-κB activation and may hold promise for modulation of bone turnover to promote a balance between bone formation and bone resorption, favorable to gain of bone mass. PMID:25056540

  13. Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene.

    PubMed

    Guha, Manti; Srinivasan, Satish; Koenigstein, Alexander; Zaidi, Mone; Avadhani, Narayan G

    2016-01-01

    Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its end point. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca(2) ](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages, this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Ctsk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Ctsk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Ctsk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss.

  14. DC-STAMP, the key fusion-mediating molecule in osteoclastogenesis.

    PubMed

    Zhang, Chengcheng; Dou, C E; Xu, Jianzhong; Dong, Shiwu

    2014-10-01

    As a member of the mononuclear phagocyte system, osteoclasts (OC) absorb the bone matrix and participate in bone modeling by keeping a balance with osteoblasts (OB) and stromal cells. Mature OC derive from the fusion of mononuclear osteoclasts (mOC) and the fusion is considered as the indispensable process for the osteoclastogenesis and absorbing activity of OC. DC-STAMP (dendritic cell-specific transmembrane protein) has been validated playing a key role in the fusion of mOC. DC-STAMP is mainly expressed in OC, macrophages and dendritic cells (DC). While DC-STAMP was discovered in DC, more attentions have been paid to DC-STAMP in OC in this decade. This review will mainly focus on the function of DC-STAMP in OC. Studies on DC-STAMP in DC may also provide new sight for the study of DC-STAMP in OC. Since the function of DC-STAMP is still poorly understood and few studies have been implemented for illustration, many issues are still unknown and need to be revealed. We will also discuss these questions in this review.

  15. Identification of Nedd9 as a TGF-β-Smad2/3 Target Gene Involved in RANKL-Induced Osteoclastogenesis by Comprehensive Analysis

    PubMed Central

    Yasui, Tetsuro; Hirose, Jun; Izawa, Naohiro; Matsumoto, Takumi; Imai, Yuuki; Seo, Sachiko; Kurokawa, Mineo; Tsutsumi, Shuichi; Kadono, Yuho; Morimoto, Chikao; Aburatani, Hiroyuki; Miyamoto, Takeshi; Tanaka, Sakae

    2016-01-01

    TGF-ß is a multifunctional cytokine that is involved in cell proliferation, differentiation and function. We previously reported an essential role of the TGF-ß -Smad2/3 pathways in RANKL-induced osteoclastogenesis. Using chromatin immunoprecipitation followed by sequencing, we comprehensively identified Smad2/3 target genes in bone marrow macrophages. These genes were enriched in the gene population upregulated by TGF-ß and downregulated by RANKL. Recent studies have revealed that histone modifications, such as trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), critically regulate key developmental steps. We identified Nedd9 as a Smad2/3 target gene whose histone modification pattern was converted from H3K4me3(+)/H3K4me27(+) to H3K4me3(+)/H3K4me27(-) by TGF-ß. Nedd9 expression was increased by TGF-ß and suppressed by RANKL. Overexpression of Nedd9 partially rescued an inhibitory effect of a TGF-ß inhibitor, while gene silencing of Nedd9 suppressed RANKL-induced osteoclastogenesis. RANKL-induced osteoclastogenesis were reduced and stimulatory effects of TGF-ß on RANKL-induced osteoclastogenesis were partially abrogated in cells from Nedd9-deficient mice although knockout mice did not show abnormal skeletal phenotypes. These results suggest that Nedd9 is a Smad2/3 target gene implicated in RANKL-induced osteoclastogenesis. PMID:27336669

  16. Trapidil, a platelet-derived growth factor antagonist, inhibits osteoclastogenesis by down-regulating NFATc1 and suppresses bone loss in mice.

    PubMed

    Kim, Sun-Don; Kim, Ha-Neui; Lee, Jong-Ho; Jin, Won Jong; Hwang, Soon Jung; Kim, Hong-Hee; Ha, Hyunil; Lee, Zang Hee

    2013-09-15

    Trapidil, a platelet-derived growth factor antagonist, was originally developed as a vasodilator and anti-platelet agent and has been used to treat patients with ischemic coronary heart, liver, and kidney disease. In this study, we investigated the effects of trapidil on osteoclastogenesis and elucidated the possible mechanism of action of trapidil. Trapidil strongly inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblasts without affecting receptor activator of NF-κB ligand (RANKL) or osteoprotegerin expression in osteoblasts. In addition, trapidil suppressed RANKL-induced osteoclast formation from osteoclast precursors. Trapidil reduced RANKL-induced expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis, without affecting the expression of c-Fos that functions as a key upstream activator of NFATc1 during osteoclastogenesis. Ectopic expression of a constitutively active form of NFATc1 reversed the anti-osteoclastogenic effect of trapidil, indicating that NFATc1 is a critical target of the anti-osteoclastogenic action of trapidil. RANKL-induced calcium oscillation and Pim-1 expression, which are required for NFATc1 induction and osteoclastogenesis, were abrogated by trapidil. Consistent with the in vitro results, trapidil had a potent inhibitory effect on osteoclast formation and bone resorption induced by interleukin-1 in an animal model. Taken together, our data demonstrate that trapidil abrogates RANKL-induced calcium oscillation and Pim-1 expression required for NFATc1 induction, thereby inhibiting osteoclastogenesis.

  17. Curcumin analog UBS109 prevents bone marrow osteoblastogenesis and osteoclastogenesis disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro.

    PubMed

    Yamaguchi, Masayoshi; Zhu, Shijun; Weitzmann, M Neale; Snyder, James P; Shoji, Mamoru

    2015-03-01

    UBS109 is a curcumin analog that possesses antitumor properties has been shown to stimulate osteoblastogenesis and suppress osteoclastogenesis in vitro. This study was undertaken to determine whether UBS109 might alleviate the inhibitory activity of breast cancer cells on osteoblastic mineralization and stimulatory effects on osteoclastogenesis. Mouse bone marrow cells were cocultured with breast cancer MDA-MB-231 bone metastatic cells in vitro. UBS109 stimulated osteoblastic mineralization and suppressed adipogenesis and osteoclastogenesis in bone marrow culture. Coculture with MDA-MB-231 cells suppressed osteoblastic mineralization and enhanced osteoclastogenesis in bone marrow culture. Effects that were reserved by UBS109 (50-200 nM). Mineralization in preosteoblastic MC3T3-E1 cells was suppressed by coculture with MDA-MB-231 cells, while MDA-MB-231 cells did not have effects on osteoclastogenesis of RAW267.4 cells in vitro. UBS109 (500 nM) revealed toxic effects on MDA-MB-231 bone metastatic cells. This study demonstrates that UBS109, which is an antitumor agent, reveals restorative effects on bone marrow cell differentiation disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro. This in vitro model may be a useful tool to evaluate the mechanism of breast cancer cell bone metastasis.

  18. 1,25 Dihydroxyvitamin D3 Inhibits TGFβ1-Mediated Primary Human Cardiac Myofibroblast Activation

    PubMed Central

    Meredith, Anna; Boroomand, Seti; Carthy, Jon; Luo, Zongshu; McManus, Bruce

    2015-01-01

    Aims Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av), and investigated the relationship between circulating vitamin D (25(OH)D3) and cardiac fibrosis in human myocardial samples. Methods and Results Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OH)D3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGF)β1 to induce activation, in the presence or absence of active vitamin D (1,25(OH)2D3). Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation. Conclusions Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis. PMID:26061181

  19. Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription*

    PubMed Central

    Hidalgo, Alejandro A.; Deeb, Kristin K.; Pike, J. Wesley; Johnson, Candace S.; Trump, Donald L.

    2011-01-01

    Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. PMID:21868377

  20. Multidirectional effects of Sr-, Mg-, and Si-containing bioceramic coatings with high bonding strength on inflammation, osteoclastogenesis, and osteogenesis.

    PubMed

    Wu, Chengtie; Chen, Zetao; Yi, Deliang; Chang, Jiang; Xiao, Yin

    2014-03-26

    Ideal coating materials for implants should be able to induce excellent osseointegration, which requires several important parameters, such as good bonding strength, limited inflammatory reaction, and balanced osteoclastogenesis and osteogenesis, to gain well-functioning coated implants with long-term life span after implantation. Bioactive elements, like Sr, Mg, and Si, have been found to play important roles in regulating the biological responses. It is of great interest to combine bioactive elements for developing bioactive coatings on Ti-6Al-4 V orthopedic implants to elicit multidirectional effects on the osseointegration. In this study, Sr-, Mg-, and Si-containing bioactive Sr2MgSi2O7 (SMS) ceramic coatings on Ti-6Al-4 V were successfully prepared by the plasma-spray coating method. The prepared SMS coatings have significantly higher bonding strength (∼37 MPa) than conventional pure hydroxyapatite (HA) coatings (mostly in the range of 15-25 MPa). It was also found that the prepared SMS coatings switch the macrophage phenotype into M2 extreme, inhibiting the inflammatory reaction via the inhibition of Wnt5A/Ca(2+) and Toll-like receptor (TLR) pathways of macrophages. In addition, the osteoclastic activities were also inhibited by SMS coatings. The expression of osteoclastogenesis-related genes (RANKL and MCSF) in bone-marrow-derived mesenchymal cells (BMSCs) with the involvement of macrophages was decreased, whereas OPG expression was enhanced on SMS coatings compared to HA coatings, indicating that SMS coatings also downregulated the osteoclastogenesis. However, the osteogenic differentiation of BMSCs with the involvement of macrophages was comparable between SMS and HA coatings. Therefore, the prepared SMS coatings showed multidirectional effects, such as improving bonding strength, reducing inflammatory reaction, and downregulating osteoclastic activities, but maintaining a comparable osteogenesis, as compared with HA coatings. The combination of

  1. Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

    SciTech Connect

    Choi, Bongkun; Kang, Soon-Suk; Kang, Sang-Wook; Min, Bon-Hong; Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min; Song, Youngsup; Yoon, Seung-Yong; Chang, Eun-Ju

    2014-07-18

    Highlights: • We describe the expression and secretion of clusterin in osteoclasts. • Endogenous clusterin deficiency does not affect osteoclast formation. • Exogenous treatment with secretory clusterin decreases osteoclast differentiation. • Secretory clusterin attenuates osteoclast precursor cell proliferation by inhibiting M-CSF-mediated ERK activation. - Abstract: Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU{sup −/−} mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

  2. Comparison of the alendronate and irradiation with a light-emitting diode (LED) on murine osteoclastogenesis.

    PubMed

    Sohn, Hong Moon; Ko, Youngjong; Park, Mineon; Kim, Bora; Park, Jung Eun; Kim, Donghwi; Moon, Young Lae; Lim, Wonbong

    2017-01-01

    Photomodulation therapy (PBMT) using light-emitting diode (LED) has been proposed as an alternative to conventional osteoporosis therapies. Our aim was to determine the effect of irradiation with a light-emitting diode on receptor activator of NF-κB ligand (RANKL)-mediated differentiation of mouse bone marrow macrophages into osteoclasts and compare it to alendronate treatment. The cells were irradiated with LED at 635±10 nm, 9-cm spot size, 5 mW/cm(2), and 18 J for 60 min/day in a CO2 incubator. The differentiation of irradiated and untreated RANKL-stimulated bone marrow macrophages into osteoclasts was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and by molecular methods. These included assessing messenger RNA (mRNA) expression of osteoclastic markers such as TRAP, c-Fos, Atp6v0d2, DC-STAMP, NFATc1, cathepsin K, MMP9 and OSCAR; phosphorylation of various MAPKs, including extracellular signal-regulated kinase ERK1/2, P38, and JNK; NF-κB translocation; and resorption pit formation. Results were compared to those obtained with sodium alendronate. Production of reactive oxygen species was measured by a 2',7'-dihydrodichlorofluorescein diacetate assay. LED irradiation and alendronate inhibited mRNA expression of osteoclast-related genes, such as TRAP, c-Fos, and NFATc1, and reduced the osteoclast activity of RANKL-stimulated bone marrow macrophages. LED irradiation, but not alendronate, also inhibited the production of reactive oxygen species (ROS); phosphorylation of ERK, P38, and IκB; and NF-κB translocation. These findings suggest that LED irradiation downregulates osteoclastogenesis by ROS production; this effect could lead to reduced bone loss and may offer a new therapeutic tool for managing osteoporosis.

  3. β-Caryophyllene promotes osteoblastic mineralization, and suppresses osteoclastogenesis and adipogenesis in mouse bone marrow cultures in vitro

    PubMed Central

    Yamaguchi, Masayoshi; Levy, Robert M.

    2016-01-01

    Osteoporosis is induced by the reduction in bone mass through decreased osteoblastic osteogenesis and increased osteoclastic bone resorption, and it is associated with obesity and diabetes. Osteoblasts and adipocytes are derived from bone marrow mesenchymal stem cells. The prevention of osteoporosis is an important public health concern in aging populations. β-caryophyllene, a component of various essential oils, is a selective agonist of the cannabinoid receptor type 2 and exerts cannabimimetic anti-inflammatory effects in animals. The present study aimed to identify the effect of β-caryophyllene on adipogenesis, osteoblastic mineralization and osteoclastogenesis in mouse bone marrow cell cultures in vitro. Bone marrow cells obtained from mouse femoral tissues were cultured in the presence of β-caryophyllene (0.1–100 µM) in vitro. The results revealed that β-caryophyllene stimulated osteoblastic mineralization, and suppressed adipogenesis and osteoclastogenesis. Thus, β-caryophyllene may be used as a therapeutic agent for the prevention and treatment of osteoporosis. PMID:28105093

  4. Polygonatum sibiricum polysaccharide inhibits osteoporosis by promoting osteoblast formation and blocking osteoclastogenesis through Wnt/β-catenin signalling pathway

    PubMed Central

    Du, Li; Nong, Meng-Ni; Zhao, Jin-Min; Peng, Xiao-Ming; Zong, Shao-Hui; Zeng, Gao-Feng

    2016-01-01

    Bone homeostasis is maintained by a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoporosis occurs when osteoclast activity surpasses osteoblast activity. Our previous studies showed the plant-derived natural polysaccharide (Polygonatum sibiricum polysaccharide or PSP) had significant anti-ovariectomy (OVX)-induced osteoporosis effects in vivo, but the mechanisms of PSP’s anti-osteoporosis effect remains unclear. In this study, we assessed PSP’s effect on the generation of osteoblast and osteoclast in vitro. This study showed that PSP promoted the osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) without affecting BMPs signaling pathway. This effect was due to the increased nuclear accumulation of β-catenin, resulting in a higher expression of osteoblast-related genes. Furthermore, the study showed PSP could inhibit the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and exert prophylatic protection against LPS-induced osteolysis in vivo. This effect was also related to the increased nuclear accumulation of β-catenin, resulting in the decreased expression of osteoclast-related genes. In conclusion, our results showed that PSP effectively promoted the osteogenic differentiation of mouse BMSCs and suppressed osteoclastogenesis; therefore, it could be used to treat osteoporosis. PMID:27554324

  5. LIS1 Regulates Osteoclastogenesis through Modulation of M-SCF and RANKL Signaling Pathways and CDC42

    PubMed Central

    Ye, Shiqiao; Fujiwara, Toshifumi; Zhou, Jian; Varughese, Kottayil I; Zhao, Haibo

    2016-01-01

    We have previously reported that depletion of LIS1, a key regulator of microtubules and cytoplasmic dynein motor complex, in osteoclast precursor cells by shRNAs attenuates osteoclastogenesis in vitro. However, the underlying mechanisms remain unclear. In this study, we show that conditional deletion of LIS1 in osteoclast progenitors in mice led to increased bone mass and decreased osteoclast number on trabecular bone. In vitro mechanistic studies revealed that loss of LIS1 had little effects on cell cycle progression but accelerated apoptosis of osteoclast precursor cells. Furthermore, deletion of LIS1 prevented prolonged activation of ERK by M-CSF and aberrantly enhanced prolonged JNK activation stimulated by RANKL. Finally, lack of LIS1 abrogated M-CSF and RANKL induced CDC42 activation and retroviral transduction of a constitutively active form of CDC42 partially rescued osteoclastogenesis in LIS1-deficient macrophages. Therefore, these data identify a key role of LIS1 in regulation of cell survival of osteoclast progenitors by modulating M-CSF and RANKL induced signaling pathways and CDC42 activation. PMID:27994513

  6. Osteoclastogenesis in peripheral blood mononuclear cell cultures of periprosthetic osteolysis patients and the phenotype of T cells localized in periprosthetic tissues.

    PubMed

    Roato, Ilaria; Caldo, Davide; D'Amico, Lucia; D'Amelio, Patrizia; Godio, Laura; Patanè, Salvatore; Astore, Franco; Grappiolo, Guido; Boggio, Maurizio; Scagnelli, Roberto; Molfetta, Luigi; Ferracini, Riccardo

    2010-10-01

    Arthroplasty is a very successful medical procedure. Failures depend on aseptic loosening caused by periprosthetic osteolysis, where T cells have a contradictory role. We analyzed osteoclastogenesis in peripheral blood mononuclear cell (PBMC) cultures of periprosthetic osteolysis patients and the phenotype of T cells localized in periprosthetic tissues. We enrolled 45 subjects with periprosthetic osteolysis (15), stable prosthesis (15) and healthy controls (15). We performed PBMC cultures to study osteoclastogenesis. Osteoclasts and T cell phenotype were examined by immunohistochemistry, immunofluorescence and flow citometry. Periprosthetic osteolysis patients showed spontaneous osteoclastogenesis, which was inhibited by RANK-Fc and T cell depletion. In periprosthetic osteolysis patients' PBMC cultures, CD4 and CD8 T cells increased and CD8 T cells did not express CD25. In periprosthetic tissues T cells were close to osteoclasts, suggesting their interaction. Local CD8 T cells showed a regulatory phenotype, expressing CD25 and FoxP3, while CD4 T cells did not express activation markers. Our data suggest that, in an early stage of periprosthetic osteolysis, T cells may promote osteoclastogenesis, whereas subsequently osteoclasts activate FoxP3/CD8 T cells, which inhibit CD4 effector T cells. This mechanism may explain the previous finding of non-active T cells in periprosthetic tissues.

  7. Thiocolchicoside suppresses osteoclastogenesis induced by RANKL and cancer cells through inhibition of inflammatory pathways: a new use for an old drug

    PubMed Central

    Reuter, Simone; Gupta, Subash C; Phromnoi, Kanokkarn; Aggarwal, Bharat B

    2012-01-01

    BACKGROUND AND PURPOSE Most patients with cancer die not because of the tumour in the primary site, but because it has spread to other sites. Common tumours, such as breast, multiple myeloma, and prostate tumours, frequently metastasize to the bone. To search for an inhibitor of cancer-induced bone loss, we investigated the effect of thiocolchicoside, a semi-synthetic colchicoside derived from the plant Gloriosa superba and clinically used as a muscle relaxant, on osteoclastogenesis induced by receptor activator of NF-κB ligand (RANKL) and tumour cells. EXPERIMENTAL APPROACH We used RAW 264.7 (murine macrophage) cells, a well-established system for osteoclastogenesis, and evaluated the effect of thiocolchicoside on RANKL-induced NF-κB signalling and osteoclastogenesis as well as on osteoclastogenesis induced by tumour cells. KEY RESULTS Thiocolchicoside suppressed osteoclastogenesis induced by RANKL, and by breast cancer and multiple myeloma cells. Inhibition of the NF-κB pathway was responsible for this effect since the colchicoside inhibited RANKL-induced NF-κB activation, activation of IκB kinase (IKK) and suppressed inhibitor of NF-κBα (IκBα) phosphorylation and degradation, an inhibitor of NF-κB. Furthermore, an inhibitor of the IκBα kinase γ or NF-κB essential modulator, the regulatory component of the IKK complex, demonstrated that the NF-κB signalling pathway is mandatory for osteoclastogenesis induced by RANKL. CONCLUSIONS AND IMPLICATIONS Together, these data suggest that thiocolchicoside significantly suppressed osteoclastogenesis induced by RANKL and tumour cells via the NF-κB signalling pathway. Thus, thiocolchicoside, a drug that has been used for almost half a century to treat muscle pain, may also be considered as a new treatment for bone loss. LINKED ARTICLE This article is commented on by Micheau et al., pp. 2124–2126 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01792.x PMID:21955206

  8. Macrophage-elicited osteoclastogenesis in response to bacterial stimulation requires Toll-like receptor 2-dependent tumor necrosis factor-alpha production.

    PubMed

    Ukai, Takashi; Yumoto, Hiromichi; Gibson, Frank C; Genco, Caroline Attardo

    2008-02-01

    The receptor activator of NF-kappaB ligand (RANKL) and the proinflammatory cytokines are believed to play important roles in osteoclastogenesis. We recently reported that the innate immune recognition receptor, Toll-like receptor 2 (TLR2), is crucial for inflammatory bone loss in response to infection by Porphyromonas gingivalis, the primary organism associated with chronic inflammatory periodontal disease. However, the contribution of macrophage-expressed TLRs to osteoclastogenesis has not been defined. In this study, we defined a requirement for TLR2 in tumor necrosis factor-alpha (TNF-alpha)-elicited osteoclastogenesis in response to exposure to P. gingivalis. Culture supernatant (CS) fluids from P. gingivalis-stimulated macrophages induced bone marrow macrophage-derived osteoclastogenesis. This activity was dependent on TNF-alpha and occurred independently of RANKL, interleukin-1beta (IL-1beta), and IL-6. CS fluids from P. gingivalis-stimulated TLR2(-/-) macrophages failed to express TNF-alpha, and these fluids induced significantly less osteoclast formation compared with that of the wild-type or the TLR4(-/-) macrophages. In addition, P. gingivalis exposure induced up-regulation of TLR2 expression on the cell surface of macrophages, which was demonstrated to functionally react to reexposure to P. gingivalis, as measured by a further increase in TNF-alpha production. These results demonstrate that macrophage-dependent TLR2 signaling is crucial for TNF-alpha-dependent/RANKL-independent osteoclastogenesis in response to P. gingivalis infection. Furthermore, the ability of P. gingivalis to induce the cell surface expression of TLR2 may contribute to the chronic inflammatory state induced by this pathogen.

  9. Inter-relationship of vitamins A, D and K in incidence of renal calcification in A/J mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix gamma-carboxyglutamic acid protein (MGP), a vitamin K-dependent protein, is involved in regulation of tissue calcification. In mice, 9-cis retinoic acid (9-cis RA) mitigates 1alpha, 25 dihydroxyvitamin D3 [1,25(OH)2D3]-induced renal calcification. It is not known if the mechanism(s) underlyin...

  10. Toll-Like Receptor 2 Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL

    PubMed Central

    Kassem, Ali; Lindholm, Catharina; Lerner, Ulf H

    2016-01-01

    Severe Staphylococcus aureus (S. aureus) infections pose an immense threat to population health and constitute a great burden for the health care worldwide. Inter alia, S. aureus septic arthritis is a disease with high mortality and morbidity caused by destruction of the infected joints and systemic bone loss, osteoporosis. Toll-Like receptors (TLRs) are innate immune cell receptors recognizing a variety of microbial molecules and structures. S. aureus recognition via TLR2 initiates a signaling cascade resulting in production of various cytokines, but the mechanisms by which S. aureus causes rapid and excessive bone loss are still unclear. We, therefore, investigated how S. aureus regulates periosteal/endosteal osteoclast formation and bone resorption. S. aureus stimulation of neonatal mouse parietal bone induced ex vivo bone resorption and osteoclastic gene expression. This effect was associated with increased mRNA and protein expression of receptor activator of NF-kB ligand (RANKL) without significant change in osteoprotegerin (OPG) expression. Bone resorption induced by S. aureus was abolished by OPG. S. aureus increased the expression of osteoclastogenic cytokines and prostaglandins in the parietal bones but the stimulatory effect of S. aureus on bone resorption and Tnfsf11 mRNA expression was independent of these cytokines and prostaglandins. Stimulation of isolated periosteal osteoblasts with S. aureus also resulted in increased expression of Tnfsf11 mRNA, an effect lost in osteoblasts from Tlr2 knockout mice. S. aureus stimulated osteoclastogenesis in isolated periosteal cells without affecting RANKL-stimulated resorption. In contrast, S. aureus inhibited RANKL-induced osteoclast formation in bone marrow macrophages. These data show that S. aureus enhances bone resorption and periosteal osteoclast formation by increasing osteoblast RANKL production through TLR2. Our study indicates the importance of using different in vitro approaches for studies of how S

  11. Geraniin suppresses RANKL-induced osteoclastogenesis in vitro and ameliorates wear particle-induced osteolysis in mouse model

    SciTech Connect

    Xiao, Fei; Zhai, Zanjing; Jiang, Chuan; Liu, Xuqiang; Li, Haowei; Qu, Xinhua; Ouyang, Zhengxiao; Fan, Qiming; Tang, Tingting; Qin, An; Gu, Dongyun

    2015-01-01

    Wear particle-induced osteolysis and subsequent aseptic loosening remains the most common complication that limits the longevity of prostheses. Wear particle-induced osteoclastogenesis is known to be responsible for extensive bone erosion that leads to prosthesis failure. Thus, inhibition of osteoclastic bone resorption may serve as a therapeutic strategy for the treatment of wear particle induced osteolysis. In this study, we demonstrated for the first time that geraniin, an active natural compound derived from Geranium thunbergii, ameliorated particle-induced osteolysis in a Ti particle-induced mouse calvaria model in vivo. We also investigated the mechanism by which geraniin exerts inhibitory effects on osteoclasts. Geraniin inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, evidenced by reduced osteoclast formation and suppressed osteoclast specific gene expression. Specially, geraniin inhibited actin ring formation and bone resorption in vitro. Further molecular investigation demonstrated geraniin impaired osteoclast differentiation via the inhibition of the RANKL-induced NF-κB and ERK signaling pathways, as well as suppressed the expression of key osteoclast transcriptional factors NFATc1 and c-Fos. Collectively, our data suggested that geraniin exerts inhibitory effects on osteoclast differentiation in vitro and suppresses Ti particle-induced osteolysis in vivo. Geraniin is therefore a potential natural compound for the treatment of wear particle induced osteolysis in prostheses failure. - Highlights: • Geraniin suppresses osteoclasts formation and function in vitro. • Geraniin impairs RANKL-induced nuclear factor-κB and ERK signaling pathway. • Geraniin suppresses osteolysis in vivo. • Geraniin may be used for treating osteoclast related diseases.

  12. CCN Family 2/Connective Tissue Growth Factor (CCN2/CTGF) Promotes Osteoclastogenesis via Induction of and Interaction with Dendritic Cell–Specific Transmembrane Protein (DC-STAMP)

    PubMed Central

    Nishida, Takashi; Emura, Kenji; Kubota, Satoshi; Lyons, Karen M; Takigawa, Masaharu

    2013-01-01

    CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF-κB ligand (GST-RANKL), and a combination of recombinant CCN2 (rCCN2) and GST-RANKL significantly enhanced tartrate-resistant acid phosphatase (TRACP)–positive multinucleated cell formation compared with GST-RANKL alone. Therefore, we suspected the involvement of CCN2 in cell-cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real-time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid-phase binding assay of CCN2 and dendritic cell–specific transmembrane protein (DC-STAMP), which is involved in cell-cell fusion. The results showed that CCN2 induced and interacted with DC-STAMP. Furthermore, GST-RANKL–induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC-STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC-STAMP. PMID:20721934

  13. Thymoquinone prevents RANKL-induced osteoclastogenesis activation and osteolysis in an in vivo model of inflammation by suppressing NF-KB and MAPK Signalling.

    PubMed

    Thummuri, Dinesh; Jeengar, Manish Kumar; Shrivastava, Shweta; Nemani, Harishankar; Ramavat, Ravindar Naik; Chaudhari, Pradip; Naidu, V G M

    2015-09-01

    Osteoclasts are multinuclear giant cells responsible for bone resorption in inflammatory bone diseases such as osteoporosis, rheumatoid arthritis and periodontitis. Because of deleterious side effects with currently available drugs the search continues for novel effective and safe therapies. Thymoquinone (TQ), the major bioactive component of Nigella sativa has been investigated for its anti-inflammatory, antioxidant and anticancer activities. However, its effects in osteoclastogenesis have not been reported. In the present study we show for the first time that TQ inhibits nuclear factor-KB ligand (RANKL) induced osteoclastogenesis in RAW 264.7 and primary bone marrow derived macrophages (BMMs) cells. RANKL induced osteoclastogenesis is associated with increased expression of multiple transcription factors via activation of NF-KB, MAPKs signalling and reactive oxygen species (ROS). Mechanistically TQ blocked the RANKL induced NF-KB activation by attenuating the phosphorylation of IkB kinase (IKKα/β). Interestingly, in RAW 264.7 cells TQ inhibited the RANKL induced phosphorylation of MAPKs and mRNA expression of osteoclastic specific genes such as TRAP, DC-STAMP, NFATc1 and c-Fos. In addition, TQ also decreased the RANKL stimulated ROS generation in macropahges (RAW 264.7) and H2O2 induced ROS generation in osteoblasts (MC-3T3-E1). Consistent with in vitro results, TQ inhibited lipopolysaccharide (LPS) induced bone resorption by suppressing the osteoclastogenesis. Indeed, micro-CT analysis showed that bone mineral density (BMD) and bone architecture parameters were positively modulated by TQ. Taken together our data demonstrate that TQ has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-KB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

  14. CXCR4(+) CD45(-) Cells are Niche Forming for Osteoclastogenesis via the SDF-1, CXCL7, and CX3CL1 Signaling Pathways in Bone Marrow.

    PubMed

    Goto, Yoh; Aoyama, Mineyoshi; Sekiya, Takeo; Kakita, Hiroki; Waguri-Nagaya, Yuko; Miyazawa, Ken; Asai, Kiyofumi; Goto, Shigemi

    2016-06-24

    Bone homeostasis comprises the balance between bone-forming osteoblasts and bone-resorbing osteoclasts (OCs), with an acceleration of osteoclastic bone resorption leading to osteoporosis. OCs can be generated from bone marrow cells (BMCs) under the tightly regulated local bone environment. However, it remained difficult to identify the critical cells responsible for providing an osteoclastogenesis niche. In this study, we used a fluorescence-activated cell sorting technique to determine the cell populations important for forming an appropriate microenvironment for osteoclastogenesis and to verify the associated interactions between osteoclast precursor cells and non-OCs. We isolated and removed a small cell population specific for osteoclastogenesis (CXCR4(+) CD45(-) ) from mouse BMCs and cultured the remaining cells with receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage-colony stimulating factor. The resulting cultures showed significantly less large osteoclast formation. Quantitative RT-PCR analysis revealed that these CXCR4(+) CD45(-) cells expressed low levels of RANK and RANKL, but high levels of critical chemokines including stromal cell derived factor 1 (SDF-1), chemokine (C-X-C motif) ligand 7 (CXCL7), and chemokine (C-X3-C motif) ligand 1 (CX3CL1). Furthermore, an SDF-1-specific antibody strongly suppressed OC formation in RAW264.7 cells and antibodies against SDF-1, CXCL7, and CX3CL1 suppressed OC formation in BMCs. These results suggest that isolated CXCR4(+) CD45(-) cells support an appropriate microenvironment for osteoclastogenesis with a direct effect on the cells expressing SDF-1, CXCL7, and CX3CL1 receptors. The regulation of CXCR4(+) CD45(-) cell function might therefore inform therapeutic strategies for diseases involving loss of bone homeostasis. Stem Cells 2016.

  15. Wedelolactone enhances osteoblastogenesis by regulating Wnt/β-catenin signaling pathway but suppresses osteoclastogenesis by NF-κB/c-fos/NFATc1 pathway

    PubMed Central

    Liu, Yan-Qiu; Hong, Zhi-Lai; Zhan, Li-Bin; Chu, Hui-Ying; Zhang, Xiao-Zhe; Li, Guo-Hui

    2016-01-01

    Bone homeostasis is maintained by formation and destruction of bone, which are two processes tightly coupled and controlled. Targeting both stimulation on bone formation and suppression on bone resorption becomes a promising strategy for treating osteoporosis. In this study, we examined the effect of wedelolactone, a natural product from Ecliptae herba, on osteoblastogenesis as well as osteoclastogenesis. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone stimulated osteoblast differentiation and bone mineralization. At the molecular level, wedelolactone directly inhibited GSK3β activity and enhanced the phosphorylation of GSK3β, thereafter stimulated the nuclear translocation of β-catenin and runx2. The expression of osteoblastogenesis-related marker gene including osteorix, osteocalcin and runx2 increased. At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. Further, wedelolactone blocked NF-kB/p65 phosphorylation and abrogated the NFATc1 nuclear translocation. As a result, osteoclastogenesis-related marker gene expression decreased, including c-src, c-fos, and cathepsin K. In ovariectomized mice, administration of wedelolactone prevented ovariectomy-induced bone loss by enhancing osteoblast activity and inhibiting osteoclast activity. Together, these data demonstrated that wedelolactone facilitated osteoblastogenesis through Wnt/GSK3β/β-catenin signaling pathway and suppressed RANKL-induced osteoclastogenesis through NF-κB/c-fos/NFATc1 pathway. These results suggested that wedelolacone could be a novel dual functional therapeutic agent for osteoporosis. PMID:27558652

  16. Morphoproteomics provides support for TGF-β pathway signaling in the osteoclastogenesis and immune dysregulation of osteolytic Langerhans cell histiocytosis

    PubMed Central

    Alexandrescu, Sanda; Tatevian, Nina; Czerniak, Bogdan A; Covinsky, Michael H; Burns, Nadja K; Brown, Robert E

    2012-01-01

    Langerhans cell histiocytosis (LCH) has a challenging and still unclear pathogenesis. A body of literature points to impaired maturation of the lesional dendritic cells, and to immune dysregulation in the form of increased FoxP3 cells. Various cytokine abnormalities such as expression of transforming growth factor (TGF)-β have been reported, as well as abnormalities in lipid content in LCH cells. Morphoproteomic techniques were applied to identify the signal transduction pathways that could influence histogenesis and immune regulation in osteolytic LCH. Five pediatric cases of osteolytic LCH were examined, using antibodies against CD1a, S100, CD68, CD8, FoxP3, phosphorylated (p)-STAT3 (Tyr705), protein kinase C (PKC)-α, phospholipase (PL)D1, fatty acid synthase (FASN), and zinc finger protein, Gli2. Positive and negative controls were performed. A FoxP3(+)/CD8(+) cell ratio was calculated by counting the FoxP3+ and CD8+ cells in 10 high power fields for each case. There is induction of sonic hedgehog (SHH) mediators consistent with TGF-β signaling pathway through Smad3-dependent activation of Gli2, findings supported by the plasmalemmal and cytoplasmic expression of PKC-α and PLD1, and nuclear expression of Gli2, in lesional cells. The FoxP3+/CD8+ cell ratio is increased, ranging from 1.7-7.94. There is moderate cytoplasmic expression of FASN in most of the Langerhans cells, a finding that supports previously published phospholipid abnormalities in LCH and is consistent with PKC-α/PLD1/TGF-β signaling. With our study, we strongly suggest that the TGF-β cell signaling pathway is a major player in the pathogenesis of LCH, leading to non-canonical induction of nuclear Gli2 expression, thereby contributing to osteoclastogenesis in LCH histiocytes. It could also cause a state of immune frustration in LCH, by inducing the transformation of CD4(+)CD25(-) cells into CD4(+)/FoxP3(+) cells. This coincides with the clinical evidence of a response to thalidomide in

  17. t10c12-CLA maintains higher bone mineral density during aging by modulating osteoclastogenesis and bone marrow adiposity.

    PubMed

    Rahman, Md M; Halade, Ganesh V; Williams, Paul J; Fernandes, Gabriel

    2011-09-01

    Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Earlier, we showed that CLA (equal mixture of c9t11-CLA and t10c12-CLA) could protect age-associated bone loss by modulating inflammatory markers and osteoclastogenesis. Since, c9t11-CLA and t10c12-CLA isomers differentially regulate functional parameters and gene expression in different cell types, we examined the efficacy of individual CLA isomers against age-associated bone loss using 12 months old C57BL/6 female mice fed for 6 months with 10% corn oil (CO), 9.5% CO + 0.5% c9t11-CLA, 9.5% CO + 0.5% t10c12-CLA or 9.5% CO + 0.25% c9t11-CLA + 0.25% t10c12-CLA. Mice fed a t10c12-CLA diet maintained a significantly higher bone mineral density (BMD) in femoral, tibial and lumbar regions than those fed CO and c9t11-CLA diets as measured by dual-energy-X-ray absorptiometry (DXA). The increased BMD was accompanied by a decreased production of osteoclastogenic factors, that is, RANKL, TRAP5b, TNF-alpha and IL-6 in serum. Moreover, a significant reduction of high fat diet-induced bone marrow adiposity was observed in t10c12-CLA fed mice as compared to that of CO and c9t11-CLA fed mice, as measured by Oil-Red-O staining of bone marrow sections. In addition, a significant reduction of osteoclast differentiation and bone resorbing pit formation was observed in t10c12-CLA treated RAW 264.7 cell culture stimulated with RANKL as compared to that of c9t11-CLA and linoleic acid treated cultures. In conclusion, these findings suggest that t10c12-CLA is the most potent CLA isomer and it exerts its anti-osteoporotic effect by modulating osteoclastogenesis and bone marrow adiposity.

  18. Clomipramine causes osteoporosis by promoting osteoclastogenesis via E3 ligase Itch, which is prevented by Zoledronic acid

    PubMed Central

    Li, Xing; Sun, Wen; Li, Jinbo; Wang, Mengmeng; Zhang, Hengwei; Pei, Lingpeng; Boyce, Brendan F.; Wang, Zhiyu; Xing, Lianping

    2017-01-01

    Patients taking antidepressants, including Clomipramine (CLP), have an increased risk of osteoporotic fracture. However, the effects of CLP on bone metabolism are unknown. Here, we demonstrate that WT mice treated with CLP for 2 weeks had significantly reduced trabecular bone volume and cortical bone thickness, associated with increased osteoclast (OC) numbers, but had no change in osteoblast numbers or bone formation rate. Bone marrow cells from CLP-treated mice had normal OC precursor frequency, but formed significantly more OCs when they were cultured with RANKL and M-CSF. CLP promoted OC formation and bone resorption and expression of OC-associated genes. CLP-induced bone loss was prevented by Zoledronic acid. At the molecular level, CLP inhibited the activity of the ubiquitin E3 ligase Itch. CLP did not promote OC formation from bone marrow cells of Itch−/− mice in vitro nor induce bone loss in Itch−/− mice. Our findings indicate that CLP causes bone loss by enhancing Itch-mediated osteoclastogenesis, which was prevented by Zoledronic acid. Thus, anti-resorptive therapy could be used to prevent bone loss in patients taking antidepressants, such as CLP. PMID:28145497

  19. CRM1 inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular mechanisms and therapeutic implications

    PubMed Central

    Tai, Y-T; Landesman, Y; Acharya, C; Calle, Y; Zhong, MY; Cea, M; Tannenbaum, D; Cagnetta, A; Reagan, M; Munshi, AA; Senapedis, W; Saint-Martin, J-R; Kashyap, T; Shacham, S; Kauffman, M; Gu, Y; Wu, L; Ghobrial, I; Zhan, F; Kung, AL; Schey, SA; Richardson, P; Munshi, NC; Anderson, KC

    2013-01-01

    The key nuclear export protein CRM1/XPO1 may represent a promising novel therapeutic target in human multiple myeloma (MM). Here we showed that chromosome region maintenance 1 (CRM1) is highly expressed in patients with MM, plasma cell leukemia cells and increased in patient cells resistant to bortezomib treatment. CRM1 expression also correlates with increased lytic bone and shorter survival. Importantly, CRM1 knockdown inhibits MM cell viability. Novel, oral, irreversible selective inhibitors of nuclear export (SINEs) targeting CRM1 (KPT-185, KPT-330) induce cytotoxicity against MM cells (ED50<200 nM), alone and cocultured with bone marrow stromal cells (BMSCs) or osteoclasts (OC). SINEs trigger nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins followed by growth arrest and apoptosis in MM cells. They further block c-myc, Mcl-1, and nuclear factor κB (NF-κB) activity. SINEs induce proteasome-dependent CRM1 protein degradation; concurrently, they upregulate CRM1, p53-targeted, apoptosis-related, anti-inflammatory and stress-related gene transcripts in MM cells. In SCID mice with diffuse human MM bone lesions, SINEs show strong anti-MM activity, inhibit MM-induced bone lysis and prolong survival. Moreover, SINEs directly impair osteoclastogenesis and bone resorption via blockade of RANKL-induced NF-κB and NFATc1, with minimal impact on osteoblasts and BMSCs. These results support clinical development of SINE CRM1 antagonists to improve patient outcome in MM. PMID:23588715

  20. Potential Effects of Phytoestrogen Genistein in Modulating Acute Methotrexate Chemotherapy-Induced Osteoclastogenesis and Bone Damage in Rats

    PubMed Central

    King, Tristan J.; Shandala, Tetyana; Lee, Alice M.; Foster, Bruce K.; Chen, Ke-Ming; Howe, Peter R.; Xian, Cory J.

    2015-01-01

    Chemotherapy-induced bone damage is a frequent side effect which causes diminished bone mineral density and fracture in childhood cancer sufferers and survivors. The intensified use of anti-metabolite methotrexate (MTX) and other cytotoxic drugs has led to the need for a mechanistic understanding of chemotherapy-induced bone loss and for the development of protective treatments. Using a young rat MTX-induced bone loss model, we investigated potential bone protective effects of phytoestrogen genistein. Oral gavages of genistein (20 mg/kg) were administered daily, for seven days before, five days during, and three days after five once-daily injections (sc) of MTX (0.75 mg/kg). MTX treatment reduced body weight gain and tibial metaphyseal trabecular bone volume (p < 0.001), increased osteoclast density on the trabecular bone surface (p < 0.05), and increased the bone marrow adipocyte number in lower metaphyseal bone (p < 0.001). Genistein supplementation preserved body weight gain (p < 0.05) and inhibited ex vivo osteoclast formation of bone marrow cells from MTX-treated rats (p < 0.001). However, MTX-induced changes in bone volume, trabecular architecture, metaphyseal mRNA expression of pro-osteoclastogenic cytokines, and marrow adiposity were not significantly affected by the co-administration of genistein. This study suggests that genistein may suppress MTX-induced osteoclastogenesis; however, further studies are required to examine its potential in protecting against MTX chemotherapy-induced bone damage. PMID:26258775

  1. MicroRNA-106b inhibits osteoclastogenesis and osteolysis by targeting RANKL in giant cell tumor of bone

    PubMed Central

    Wang, Ting; Yin, Huabin; Wang, Jing; Li, Zhenxi; Wei, Haifeng; Liu, Zhi'an; Wu, Zhipeng; Yan, Wangjun; Liu, Tielong; Song, Dianwen; Yang, Xinghai; Huang, Quan; Zhou, Wang; Xiao, Jianru

    2015-01-01

    Giant cell tumor (GCT) of bone consists of three major cell types: giant cells, monocytic cells, and stromal cells. From microarray analysis, we found that miR-106b was down-regulated in GCT clinical samples and further determined by fluorescence in situ hybridization. In addition, the expression of novel potential target of miR-106b, RANKL, was elevated in GCT along with previously determined targets in other tumors such as IL-8, MMP2 and TWIST. In a RANKL 3′UTR luciferase reporter assays, agomiR-106b repressed the luciferase activity and the effect was eliminated when the targeting site in the reporter was mutated, suggesting a direct regulation of miR-106b on RANKL mRNA. Moreover, overexpression of miR-106b in GCTSCs through TALEN-mediated site-specific knockin clearly inhibited osteoclastogenesis and osteolysis. By grafting the GCT onto the chick CAM, we confirmed the inhibitory effect of miR-106b on RANKL expression and giant cell formation. Furthermore, in an OVX mouse model, silencing of miR-106b increased RANKL protein expression and promoted bone resorption, while up-regulation of miR-106b inhibited bone resorption. These results suggest that miR-106b is a novel suppressor of osteolysis by targeting RANKL and some other cytokines, which indicates that miR-106b may be a potential therapeutic target for the treatment of GCT. PMID:26053181

  2. NF-kappaB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor.

    PubMed

    Kubota, Tetsuo; Hoshino, Machiko; Aoki, Kazuhiro; Ohya, Keiichi; Komano, Yukiko; Nanki, Toshihiro; Miyasaka, Nobuyuki; Umezawa, Kazuo

    2007-01-01

    Inhibition of NF-kappaB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-kappaB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-kappaB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-kappaB inhibitors to rheumatoid arthritis

  3. Lentivirus-mediated TNF-α gene silencing and overexpression of osteoprotegerin inhibit titanium particle-induced inflammatory response and osteoclastogenesis in vitro.

    PubMed

    Peng, Li; Wang, Hongzhi; Song, Keguan; Wang, Hai; Liu, Ping

    2016-01-01

    Macrophages and osteoclasts release proinflammatory factors and promote osteoclastogenesis following the phagocytosis of wear particles. During this pathological process, receptor of nuclear factor κB ligand (RANKL) and tumor necrosis factor (TNF)-α are critical factors contributing to resorption and the inflammatory response. The present study aimed to construct recombination lentivirus vectors carrying TNF-α small interfering (si)RNA and osteoprotegerin (OPG) cDNA, and to examine the effects of Lenti‑siTNFα‑OPG on the wear particle‑induced inflammatory response and osteoclastogenesis in a titanium (Ti) particle‑induced‑inflammatory response cell model. Lenti‑siTNFα‑OPG vectors were constructed and transnfected into RAW264.7 and MC3T3‑E1 cells, respectively, prior to particle stimulation. The protein levels of TNF‑α, OPG and RANKL were evaluated using western blot analysis and enzyme‑linked immunosorbent assays, and the mRNA expression levels of the inflammatory factors, TNF‑α, interleukin (IL)‑1β and IL‑6, as well as OPG and RANKL, were measured using reverse transcription‑quantitative polymerase chain reaction analysis. The activity of alkaline phosphatase (ALP) was examined using an ALP kit. In the presence of the Lenti‑siTNFα‑OPG vector, the mRNA expression levels of the inflammatory factors and RANKL were downregulated, as were the protein levels of TNF‑α. The mRNA expression and protein levels of OPG were upregulated, and ALP activity was increased. These findings suggested that Lenti‑siTNFα‑OPG transfection inhibited the wear particle‑induced inflammatory response and osteoclastogenesis, which warrants further investigation for the prevention and/or treatment of wear particle-induced osteolysis.

  4. 25-hydroxycholesterol promotes RANKL-induced osteoclastogenesis through coordinating NFATc1 and Sp1 complex in the transcription of miR-139-5p.

    PubMed

    Zhang, Lishan; Lv, Yinping; Xian, Guozhe; Lin, Yanliang

    2017-04-15

    25-hydroxycholesterol (25-HC) is implicated in many processes, including lipid metabolism and the immune response. However, the role of 25-HC in RANKL-induced osteoclastogenesis remains largely unknown. Our results showed that 25-HC inhibited miR-139-5p expression in mouse bone marrow macrophages (BMMs) cultured in receptor activator of NF-κB ligand (RANKL) and monocyte macrophage colony-stimulating factor (M-CSF). Further investigation suggested that 25-HC promoted the expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and Sp1, especially in the presence of RANKL and M-CSF. Meanwhile, 25-HC induced nuclear translocation of NFATc1, resulting in the interaction between NFATc1 and Sp1 that was confirmed by co-immunoprecipitation. Chromatin immunoprecipitation assay indicated that Sp1 could bind to miR-139-5p promoter, but NFATc1 had no binding capacity. Although forming NFATc1/Sp1 complex increased its binding to miR-139-5p promoter, the complex inhibited the transcriptional activity of Sp1. Inhibition of NFATc1 increase the expression of miR-139-5p, which might be due to the release of free Sp1 that could bind to the promoter of miR-139-5p. Enforced expression of miR-139-5p impaired osteoclastogenesis induced by co-treatment with 25-HC and RANKL. These results suggested that 25-HC induced the interaction between NFATc1 and Sp1, reducing the level of free Sp1 to inhibit miR-139-5p expression and promote osteoclastogenesis.

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

    PubMed

    Zhou, Feng; Shen, Yi; Liu, Bo; Chen, Xia; Wan, Lu; Peng, Dan

    2017-03-18

    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.

  6. Class A Scavenger Receptor Exacerbates Osteoclastogenesis by an Interleukin-6-Mediated Mechanism through ERK and JNK Signaling Pathways

    PubMed Central

    Guo, Shuyu; Ni, Yuanyuan; Ben, Jingjing; Xia, Yang; Zhou, Tingting; Wang, Dongyue; Ni, Jieli; Bai, Hui; Wang, Lin; Ma, Junqing; Chen, Qi

    2016-01-01

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, which are important for bone health. Class A scavenger receptor (SR-A) is a multifunctional molecule that functions during differentiation of monocyte into macrophages and osteoclasts. To further characterize the role of SR-A in osteoclasts, we used the murine tooth movement model (TM) and the murine anterior cruciate ligament transection model of osteoarthritis (ACLT OA). In these two models the bones involved are of different origin and have different properties. Bone resorption was decreased in SR-A-/- mice compared to SR-A+/+ mice. Further evaluation showed that the number of multinucleated osteoclasts in SR-A-/- mice, compared to SR-A+/+ mice, was significantly decreased both in vivo and in vitro. The levels of interleukin-6 (IL-6) produced by osteoclasts were reduced in SR-A-/- mice compared to SR-A+/+ mice. In the in vitro marrow-derived osteoclast formation assay and in both mouse models, osteoclastogenesis was restored to normal in SR-A-/- mice by administration of recombinant murine IL-6. Moreover, neutralization of IL-6 reduced the number of osteoclasts formed in SR-A+/+ mice of TM model. Both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38, signaling pathways were downregulated in receptor activator of nuclear factor-κB ligand (RANKL)-stimulated SR-A-/- osteoclasts. Importantly, when treated with either ERK or JNK inhibitor, the numbers of osteoclasts generated from RANKL-induced bone marrow derived-macrophages of SR-A+/+ mice, and their IL-6 production, were significantly decreased. This suggests that SR-A activates the ERK and JNK signaling pathways, and promotes production of IL-6 by osteoclasts to further stimulate osteoclast formation. PMID:27766031

  7. A novel HSP90 inhibitor delays castrate resistant prostate cancer without altering serum PSA levels and inhibits osteoclastogenesis

    PubMed Central

    Lamoureux, Francois; Thomas, Christian; Yin, Min-Jean; Kuruma, Hidetoshi; Fazli, Ladan; Gleave, Martin E; Zoubeidi, Amina

    2015-01-01

    Purpose Prostate cancer responds initially to anti-androgen therapies, however, progression to castration resistant disease frequently occurs. Therefore there is an urgent need for novel therapeutic agents that can prevent the emergence of castration resistant prostate cancer (CRPC). Hsp90 is a molecular chaperone involved in the stability of many client proteins including Akt and androgen receptor (AR). 17-AAG have been reported to inhibit tumor growth in various cancers, however induces tumor progression in the bone microenvironment. Methods Cell growth, apoptosis, and AR transactivation were examined by crystal violet assay, flow cytometry and luciferase assays respectively. The consequence of HSP90 therapy in vivo was evaluated in LNCaP xenograft model. The consequence of PF-04928473 therapy on bone metastasis was studied using an osteoclastogenesis in vitro assay. Results PF-04928473 inhibits cell growth in a panel of prostate cancer cells, induces cell cycle arrest at sub-G1 and leads to apoptosis and increased caspase-3 activity. These biologic events were accompanied by decreased activation of Akt and Erk as well as decreased expression of Her2, and decreased AR expression and activation in vitro. In contrast to 17-AAG, PF-04928473 abrogates RANKL-induced osteoclast differentiation by affecting NF-kB activation and Src phosphorylation. Finally, PF-04929113 inhibited tumor growth and prolonged survival compared to controls. Surprisingly, PF-04929113 did not reduce serum PSA levels in vivo in parallel these decreases in tumor volume. Conclusion These data identify significant anti-cancer activity of PF-04929113 in CRPC but suggest that serum PSA may not prove useful as pharmaco-dynamic tool for this drug. PMID:21349995

  8. Conjugated linoleic acid prevents ovariectomy-induced bone loss in mice by modulating both osteoclastogenesis and osteoblastogenesis

    PubMed Central

    Rahman, Md Mizanur; Fernandes, Gabriel; Williams, Paul

    2014-01-01

    Postmenopausal osteoporosis due to estrogen deficiency is associated with severe morbidity and mortality. Beneficial effects of conjugated linoleic acid (CLA) on bone mineral density (BMD) have been reported in mice, rats and humans, but the effect of long term CLA supplementation against ovariectomy-induced bone loss in mice and the mechanisms underlying this effect have not been studied yet. Eight weeks old ovariectomized (Ovx) and sham operated C57BL/6 mice were fed either a diet containing 0.5% safflower oil (SFO) or 0.5% CLA for 24 weeks to examine BMD, bone turn over markers and osteotropic factors. Bone marrow (BM) cells were cultured to determine the effect on inflammation, osteoclastogenesis, and osteoblastogenesis. SFO/Ovx mice had significantly lower femoral, tibial and lumbar BMD compared to SFO/Sham mice; whereas, no difference was found between CLA/OVX and CLA/Sham mice. CLA inhibited bone resorption markers whereas enhanced bone formation markers in Ovx mice as compared to SFO fed mice. RT-PCR and FACS analyses of splenocytes revealed that CLA inhibited pro-osteoclastogenic RANKL and stimulated decoy receptor of RANKL, OPG expression. CLA also inhibited pro-inflammatory cytokine and enhanced anti-inflammatory cytokine production of LPS-stimulated splenocytes and bone marrow cells. Furthermore, CLA inhibited osteoclast differentiation in BM and stimulated osteoblast differentiation in BM stromal cells as confirmed by TRAP and Alizarin Red staining, respectively. In conclusion, CLA may prevent postmenopausal bone loss not only by inhibiting excessive bone resorption due to estrogen deficiency but also by stimulating new bone formation. CLA might be a potential alternative therapy against osteoporotic bone loss. PMID:24338525

  9. Design, synthesis and biological properties of seco-d-ring modified 1α,25-dihydroxyvitamin D3 analogues.

    PubMed

    Szybinski, Marcin; Sektas, Katarzyna; Sicinski, Rafal R; Plum, Lori A; Frelek, Jadwiga; DeLuca, Hector F

    2017-03-08

    As a continuation of our efforts directed to the structure-activity relationship studies of vitamin D compounds, we present in this paper the synthesis of new analogues of 1α,25-(OH)2D3 characterized by numerous structural modifications, especially a cleaved D ring. Total synthesis of the CD fragment required for the construction of the target vitamins was based on the Stork approach. The structure of the key intermediate - bicyclic hydroxy lactone - was established by crystallographic and electronic circular dichroism (ECD) spectral analysis. Following the attachment of the hydroxyalkyl side chain, the formed D-seco Grundmann ketone was subjected to Wittig-Horner coupling with the corresponding A-ring phosphine oxides providing two desired D-seco analogues of 19-nor-1α,25-(OH)2D3, one without a substituent at C-2 and the other possessing a 2-exomethylene group. Both compounds were biologically tested and the latter was found to be more active in in vitro tests. Despite so many structural changes introduced in its structure, the biological activity of the 2-methylene analogue approached that of the natural hormone. The synthesized D-seco vitamins, however, proved to be inactive on bone and intestine in vivo.

  10. Effect of estrogen/gestagen and 24R,25-dihydroxyvitamin D3 therapy on bone formation in postmenopausal women

    SciTech Connect

    Thomsen, K.; Riis, B.; Christiansen, C.

    1986-12-01

    The effect of two different estrogen/gestagen regimens and 24R,25-(OH)2-cholecalciferol on bone formation was studied in a randomized trial with 144 healthy postmenopausal women. Urinary excretion (UE) of /sup 99m/technetium-diphosphonate and serum alkaline phosphatase (AP) was determined before and then once a year for 2 years of treatment. Both estimates of bone formation showed highly significant decreases (p less than .001) to normal premenopausal levels in women receiving unopposed 17 beta-estradiol or in a sequential combination with progestagen, whereas unchanged high values were found in the groups receiving 24R,25-(OH)2D3 and placebo. The data show that bone turnover increases in early postmenopausal women concomitantly with the loss of bone mass, and that hormonal substitutional therapy normalizes the total skeletal turnover as well as preventing bone loss.

  11. The vitamin D receptor and its ligand 1alpha,25-dihydroxyvitamin D3 in Atlantic salmon (Salmo salar).

    PubMed

    Lock, E J; Ornsrud, R; Aksnes, L; Spanings, F A T; Waagbø, R; Flik, G

    2007-06-01

    Seaward migration of Salmo salar is preceded by preparatory physiological adaptations (parr-smolt transformation) to allow for a switch from freshwater (FW) to seawater (SW), which also means a switch in ambient calcium from hypocalcic (<1 mM Ca(2+)) to the plasma (~1.25 mM Ca(2+)) and to strongly hypercalcic (8-12 mM Ca(2+)). Uptake, storage (skeleton, scales) and excretion of calcium need careful regulation. In fish, the vitamin D endocrine system plays a rather enigmatic role in calcium physiology. Here, we give direct evidence for calcitriol involvement in SW migration. We report the full sequence of the nuclear vitamin D receptor (sVDR0) and two alternatively spliced variants resulting from intron retention (sVDR1 and sVDR2). In FW parr, SW adapting smolts, and in SW adults, plasma concentrations of 25(OH)D(3) and 24,25(OH)(2)D(3) did not change significantly. Plasma calcitriol concentrations were lowest in FW parr, doubled during smoltification and remained elevated in SW adults. Increased calcitriol coincided with a twofold decrease in sVDR mRNA levels in gill, intestine, and kidney of FW smolts and SW adults, when compared with parr. Clearly, there was a negative feedback and dynamic response of the vitamin D endocrine system during parr-smolt transformation. The onset of these dynamic changes in FW parr warrants a further search for the endocrines that initiate these changes. We speculate that the vitamin D system plays a crucial role in calcium and phosphorus handling in Atlantic salmon.

  12. High-dose 1,25-dihydroxyvitamin D supplementation elongates the lifespan of Huntington's disease transgenic mice.

    PubMed

    Molnár, Máté Fort; Török, Rita; Szalárdy, Levente; Sümegi, Evelin; Vécsei, László; Klivényi, Péter

    2016-01-01

    Huntington's disease is an autosomal dominant progressive neurodegenerative disease, which results in a decreased quality of life and an early death. A high prevalence of vitamin D deficiency was first described in a 2013 study in patients with manifest Huntington's disease, where serum vitamin D level was found to be associated with motor capabilities of the patients. Our objective was to investigate the effect of a high-dose vitamin D3 supplementation on a transgenic mouse model of Huntington's disease. Our study was performed on N171-82Q Huntington's disease transgenic mice in age- and gender-matched groups. We collected data on the motor state and survival of the mice. The results demonstrate that though vitamin D3 had no effect on the motor performance of transgenic mice, but significantly increased the lifespan of transgenic animals (Kaplan-Meier survival curves: vehicle-supplemented group: 73 (67-94) days vs. vitamin D3-supplemented group: 101 (74-109) days, p=0.048 Mantel-Cox log rank test). Further investigations are needed to determine whether a neuroprotective or a general corroborative effect of vitamin D leads to the measured effect. Our findings support the potential influence of vitamin D deficiency on the disease course and propose that vitamin D may be an effective supplementary treatment to beneficially influence clinical features of Huntington's disease.

  13. 25-Hydroxyvitamin D Can Interfere With a Common Assay for 1,25-Dihydroxyvitamin D in Vitamin D Intoxication

    PubMed Central

    Hawkes, Colin P.; Schnellbacher, Sarah; Singh, Ravinder J.

    2015-01-01

    Context: Vitamin D intoxication is characterized by elevated serum 25-hydroxyvitamin D (25(OH)D) and suppressed serum 1,25-dihydroxvitamin D (1,25(OH)2D). We evaluated two adolescents with hypercalcemia due to vitamin D intoxication; both had elevated serum 1,25(OH)2D by Diasorin RIA, but normal serum 1,25(OH)2D concentrations by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Objective: This study aimed to determine the effect of 25(OH)D2 and 25(OH)D3 on 1,25(OH)2D concentration determined using RIA and LC-MS/MS. Methods: Pools of normal serum and an artificial serum matrix were prepared and aliquots were spiked with >99% pure 25(OH)D2 or 25(OH)D3 (50–700 ng/mL). Samples were maintained at 4°C or heated to 56°C, and the concentrations of vitamin D metabolites were measured by LC-MS/MS and Diasorin RIA. Results: Median 1,25(OH)2D increased by 114% with RIA and 21% with LC-MS/MS with addition of 100 ng/mL 25(OH)D3, and 349% (RIA) and 117% (LC-MS/MS) with 700 ng/mL of 25(OH)D3. Each 1-ng/mL increase in 25(OH)D3 increased 1,25(OH)2D by 0.231 pg/mL (RIA) and 0.121 pg/mL (LC-MS/MS). Spiking with 25(OH)D2 led to similar changes. Heat inactivation of serum, and using an artificial serum matrix, were associated with similar effects of 25(OH)D on 1,25(OH)2D assays. Conclusions: Vitamin D intoxication with high serum levels of 25(OH)D2 or 25(OH)D3 can be associated with elevated levels of 1,25(OH)2D due to interference in a commonly used RIA. A similar but attenuated effect also occurs when 1,25(OH)2D is measured using LC-MS/MS but does not seem to be clinically significant. The basis for this effect on the LC-MS/MS assay is presently uncertain. PMID:26120794

  14. Transcriptional effects of 1,25 dihydroxyvitamin D3 physiological and supra-physiological concentrations in breast cancer organotypic culture

    PubMed Central

    2013-01-01

    Background Vitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)2D3 (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)2D3 in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)2D3 at concentrations that can be attained in vivo. Methods Tumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)2D3 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR≤0.1) or RT-qPCR (p≤0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)2D3 0.5nM, using RT-qPCR, western blot or immunocytochemistry. Results 1,25(OH)2D3 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)2D3 near physiological concentration. Genes up-modulated by both 1,25(OH)2D3 concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)2D3 was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)2D3 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)2D3 0.5nM was detected. Conclusions In breast cancer specimens a short period of 1,25(OH)2D3 exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)2D3 effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment. PMID:23497279

  15. 1, 25-dihydroxyvitamin D3, a potential role player in the development of thyroid disorders in schizophrenics

    PubMed Central

    Malik, Arif; Saleem, Shamaila; Basit Ashraf, Muhammad Abdul; Qazi, Mahmood Husain

    2016-01-01

    Objective: The present study was designed to assess the role of vitamin-D, in the development of autoimmune thyroid dysfunction in newly diagnosed schizophrenics. Methods: For the present study 100 patients and 100 controls were screened out and studied for their thyroid antibodies, GSH, homocysteine, NOS and vitamin D levels by appropriate protocols to assess the underlying mechanism involved in the schizophrenics susceptible to autoimmune thyroid diseases. Results: The results of the present study depicted that in schizophrenics, levels of cytokines like IL-6 (7.98±0.67 pg/ml), TNF-α, (40.76±6.98 pg/ml), homocysteine (16.98±1.09 µmol/L), Tg-Ab (30.93±3.87 IU/L), TPO-Ab (10.33±1.78 IU/L) and TSHr-Ab (3.76±0.055 IU/L) increased whereas, those of Vit-D (12.76±0.99 pmol/L), NOS (5.99±0.87 IU/L), GSH (4.48±.965 µg/dl) and NO (16.87±3.98 ng/ml) were decreased in the patients as compared to healthy control subjects. Conclusion: Vitamin-D in schizophrenia is involved in augmentation of hyperhomocysteinemia, inflammation, oxidative stress and thyroid antibodies, thereby playing a significant role not only in induction of schizophrenic symptoms but may also result in autoimmune thyroid diseases. Thus, earlier detection and rectification of its levels are helpful to limit the miseries of schizophrenia. PMID:28083028

  16. Toll-like receptor signaling increases production of 1,25-dihydroxyvitamin D3 in bovine macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Activation of macrophages can occur through Toll-like receptor (TLR) recognition of pathogen associated molecular patterns (PAMP). Recently, it has been discovered that TLR signaling can increase 1alpha-hydroxylase (Cyp27B1) expression in human and mouse macrophages. The enzymatic activity of 1alp...

  17. Periodontal disease-associated compensatory expression of osteoprotegerin is lost in type 1 diabetes mellitus and correlates with alveolar bone destruction by regulating osteoclastogenesis.

    PubMed

    Silva, Juliete Aparecida F; Lopes Ferrucci, Danilo; Peroni, Luis Antônio; de Paula Ishi, Eduardo; Rossa-Junior, Carlos; Carvalho, Hernandes F; Stach-Machado, Dagmar Ruth

    2012-01-01

    Alveolar bone resorption results from the inflammatory response to periodontal pathogens. Systemic diseases that affect the host response, such as type 1 diabetes mellitus (DM1), can potentiate the severity of periodontal disease (PD) and accelerate bone resorption. However, the biological mechanisms by which DM1 modulates PD are not fully understood. The aim of this study was to determine the influence of DM1 on alveolar bone resorption and to evaluate the role of receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin (OPG) in osteoclastogenesis in rats. PD was induced by means of ligature in nondiabetic and in streptozotocyn-induced DM1 rats. Morphological and morphometric analyses, stereology and osteoclast counting were performed. RANKL and OPG mRNA levels, protein content, and location were determined. PD caused alveolar bone resorption, increased the number of osteoclasts in the alveolar bone crest and also promoted changes in RANKL/OPG mRNA expression. DM1 alone showed alveolar bone destruction and an increased number of osteoclasts at the periapical and furcal regions. DM1 exacerbated these characteristics, with a greater impact on bone structure, resulting in a low OPG content and a higher RANKL/OPG ratio, which correlated with prominent osteoclastogenesis. This work demonstrates that the effects of PD and DM1 enhance bone destruction, confirms the importance of the RANKL signaling pathway in bone destruction in DM1 in animal models and suggests the existence of alternative mechanisms potentiating bone degradation in PD.

  18. Ajulemic acid, a nonpsychoactive cannabinoid acid, suppresses osteoclastogenesis in mononuclear precursor cells and induces apoptosis in mature osteoclast-like cells.

    PubMed

    George, Kerri L; Saltman, Laura H; Stein, Gary S; Lian, Jane B; Zurier, Robert B

    2008-03-01

    Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, prevents joint tissue injury in rats with adjuvant induced arthritis. Because activation of osteoclasts is central to the pathogenesis of bone erosion in patients with rheumatoid arthritis (RA), we investigated the influence of AjA on osteoclast differentiation and survival. Osteoclast cultures were established by stimulation of RAW264.7 cells and primary mouse bone marrow cultures with receptor activator of NF-kappaB ligand (RANKL). Simultaneous addition of AjA (15 and 30 microM) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose dependent manner, as determined by quantification of multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells. AjA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AjA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V and propidium iodide staining, and caspase activity. The anti-osteoclastogenic effects of AjA did not require the continuous presence of AjA in the cell cultures. Based on these findings, we propose that AjA or other nonpsychoactive synthetic analogs of Cannabis constituents may be useful therapy for diseases such as RA and osteoporosis in which bone resorption is a central feature.

  19. Diosgenin inhibits osteoclastogenesis, invasion, and proliferation through the downregulation of Akt, I kappa B kinase activation and NF-kappa B-regulated gene expression.

    PubMed

    Shishodia, S; Aggarwal, B B

    2006-03-09

    Diosgenin, a steroidal saponin present in fenugreek (Trigonella foenum graecum) and other plants, has been shown to suppress inflammation, inhibit proliferation, and induce apoptosis in a variety of tumor cells, but through a mechanism that is poorly understood. In the present study, we report that diosgenin inhibits receptor-activated nuclear factor-kappaB ligand-induced osteoclastogenesis, suppresses tumor necrosis factor (TNF)-induced invasion, and blocks the proliferation of tumor cells, all activities known to be regulated by NF-kappaB. Diosgenin suppressed TNF-induced NF-kappaB activation as determined by DNA binding, activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation through inhibition of Akt activation. NF-kappaB-dependent reporter gene expression was also abrogated by diosgenin. TNF-induced expression of NF-kappaB-regulated gene products involved in cell proliferation (cyclin D1, COX-2, c-myc), antiapoptosis (IAP1, Bcl-2, Bcl-X(L), Bfl-1/A1, TRAF1 and cFLIP), and invasion (MMP-9) were also downregulated by the saponin. Diosgenin also potentiated the apoptosis induced by TNF and chemotherapeutic agents. Overall, our results suggest that diosgenin suppresses proliferation, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression and enhances apoptosis induced by cytokines and chemotherapeutic agents.

  20. The correlation of Salvia miltiorrhiza extract-induced regulation of osteoclastogenesis with the amount of components tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone.

    PubMed

    Kim, Hae-Kyung; Woo, Eun-Rhan; Lee, Hae-Won; Park, Hyung-Rae; Kim, Hyun-Nam; Jung, Yeon-Kwan; Choi, Je-Yong; Chae, Soo-Wan; Kim, Hyung-Ryong; Chae, Han-Jung

    2008-01-01

    Tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone are compounds that have been isolated from the root of Salvia miltiorrhiza (SM), which is also known as "Danshen." The SM extract has been used successfully in China for treating postmenopausal syndrome. Furthermore, it was previously reported that SM had inhibitory effect on osteoporosis in ovariectomized rats. Another study reported that the four components, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone, prevented osteoclast function in an in vitro system. However, there are no reports of a correlation between SM and its components on osteoporosis and osteoclast function. This study was undertaken to examine the effect of SM on osteoclastogenesis and osteoblast differentiation, which are two important markers of the bone physiology. Through a rapid, sensitive and specific isocratic liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of four diterpenoids, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone in SM, the authors tried to correlate the amount of tanshinone compounds in SM into the antiosteoclast activity. The SM fraction (methanol and ethanol isolated) with a low concentration of tanshinone IIA (1 mug/mL) had no effect on the alkaline phosphotase activity (osteoblast differentiation), but completely inhibited osteoclastogenesis. Although the tanshinone compound itself showed similar effects, the concentrations of commercially available tanshinone (diterpenoids, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone) needed for antiosteoclast activity was almost 1000 times more than that of tanshinone in SM fraction. This suggests that there are other unknown compounds in the SM extract that have a synergistic effect with tanshinone. These results also suggest that tanshinone can be a good marker compound to explain the antiosteoporotic function of SM.

  1. Muscadine grape skin extract can antagonize Snail-cathepsin L-mediated invasion, migration and osteoclastogenesis in prostate and breast cancer cells

    PubMed Central

    Burton, Liza J.; Smith, Basil A.; Smith, Bethany N.; Loyd, Quentin; Nagappan, Peri; McKeithen, Danielle; Wilder, Catera L.; Platt, Manu O.; Hudson, Tamaro

    2015-01-01

    To develop new and effective chemopreventive agents against bone metastasis, we assessed the effects of muscadine grape skin extract (MSKE), whose main bioactive component is anthocyanin, on bone turnover, using prostate and breast cancer cell models overexpressing Snail transcription factor. MSKE has been shown previously to promote apoptosis in prostate cancer cells without affecting normal prostate epithelial cells. Snail is overexpressed in prostate and breast cancer, and is associated with increased invasion, migration and bone turnover/osteoclastogenesis. Cathepsin L (CatL) is a cysteine cathepsin protease that is overexpressed in cancer and involved in bone turnover. Snail overexpression in prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells led to increased CatL expression/activity and phosphorylated STAT-3 (pSTAT-3), compared to Neo vector controls, while the reverse was observed in C4-2 (the aggressive subline of LNCaP) cells with Snail knockdown. Moreover, CatL expression was higher in prostate and breast tumor tissue compared to normal tissue. MSKE decreased Snail and pSTAT3 expression, and abrogated Snail-mediated CatL activity, migration and invasion. Additionally, Snail overexpression promoted osteoclastogenesis, which was significantly inhibited by the MSKE as effectively as Z-FY-CHO, a CatL-specific inhibitor, or osteoprotegerin, a receptor activator of nuclear factor kappa B ligand (RANKL) antagonist. Overall, these novel findings suggest that Snail regulation of CatL may occur via STAT-3 signaling and can be antagonized by MSKE, leading to decreased cell invasion, migration and bone turnover. Therefore, inhibition using a natural product such as MSKE could potentially be a promising bioactive compound for bone metastatic cancer. PMID:26069256

  2. The natural flavonoid galangin inhibits osteoclastic bone destruction and osteoclastogenesis by suppressing NF-κB in collagen-induced arthritis and bone marrow-derived macrophages.

    PubMed

    Huh, Jeong-Eun; Jung, In-Tae; Choi, Junyoung; Baek, Yong-Hyeon; Lee, Jae-Dong; Park, Dong-Suk; Choi, Do-Young

    2013-01-05

    We investigated the effect of galangin, a natural flavonoid, on osteoclastic bone destruction in collagen-induced arthritis and examined the molecular mechanisms by which galangin affects osteoclastogenesis in bone marrow derived macrophages. In mice with collagen-induced arthritis, administration of galangin significantly reduced the arthritis clinical score, edema and severity of disease without toxicity. Interestingly, galangin treatment during a later stage of collagen-induced arthritis, using mice with a higher clinical arthritis score, still significantly slowed the progression of the disease. Extensive cartilage and bone erosive changes as well as synovial inflammation, synovial hyperplasia and pannus formation were dramatically inhibited in arthritic mice treated with galangin. Furthermore, galangin-treated arthritic mice showed a significant reduction in the concentrations of IL-1β, TNF-α and IL-17. We found that galangin inhibited osteoclastogenic factors and osteoclast formation in bone marrow-derived macrophages and osteoblast co-cultured cells, and increased osteoprotegerin (OPG) levels in osteoblasts. Galangin and NF-κB siRNA suppressed RANKL-induced phosphorylation of the c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Also, the JNK inhibitor SP600125 and p38 inhibitor SB203580 reduced RANKL-induced expressions of phospho-c-Jun, c-fos and NFATc1 genes during osteoclast development. In addition, galangin suppressed RANKL-induced phosphorylation of NF-κB, phospho-IκBα, inflammatory cytokines and osteoclast formation in bone marrow-derived macrophages. Our data suggest that galangin prevented osteoclastic bone destruction and osteoclastogenesis in osteoclast precursors as well as in collagen-induced arthritis mice without toxicity via attenuation of RANKL-induced activation of JNK, p38 and NF-κB pathways.

  3. The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-{kappa}B and MAPK pathways

    SciTech Connect

    Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit; Pudhom, Khanitha; Palaga, Tanapat

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer A gedunin type limonoid from seeds of mangroves, 7-oxo-7-deacetoxygedunin, exhibits strong anti-osteoclastogenic activity. Black-Right-Pointing-Pointer Treatment with this limonoid results in significant decrease in expression of NFATc1 and osteoclast-related genes. Black-Right-Pointing-Pointer The mode of action of this limonoid is by inhibiting activation of the NF-{kappa}B and MAPK pathways which are activated by RANKL. -- Abstract: Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-{kappa}B (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 {mu}M. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-{kappa}B p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.

  4. Methylation status of a single CpG locus 3 bases upstream of TATA-box of receptor activator of nuclear factor-kappaB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis.

    PubMed

    Kitazawa, Riko; Kitazawa, Sohei

    2007-01-01

    Receptor activator of nuclear factor-kappaB ligand (RANKL) expression is tissue specific and limited to certain subsets of T-lymphocytes and stromal/osteoblastic cells. Even among osteoblasts, RANKL is expressed on about 20% of osteoblasts of the normal mouse. To clarify the mechanism of population-specific RANKL expression, we analyzed the effect of CpG methylation on its transcription, mRNA and protein expression as well as on osteoclastogenesis. Subpopulations of ST2 cells were used: P9, which expresses RANKL and supports osteoclastogenesis, and P16, which does not. By sodium bisulfite mapping, the rate of CpG methylation of the -65/+350 region, especially of CpG locus no. 1 three bases upstream of the TATA-box, was higher in P16 than in P9 ST2 cells. ChIP and gel shift assay showed that methylated CpG locus no. 1 was a target of MeCP2 binding that, in turn, blocked the binding of the TATA-box binding protein to the TATA-box. In vitro methylation by SssI of the promoter construct reduced its transcriptional activity at the steady state and its response to 1alpha,25(OH)2 vitamin D3. Conversely, treatment with DNA methylase inhibitor, 5-aza-2'-deoxycytidine, significantly restored RANKL expression and osteoclastogenesis in P16 cells. Except for primary cultured osteoblasts, CpG locus no. 1 was frequently methylated in various normal mouse tissues. We propose that the methylation status of the CpG locus three bases upstream of the TATA-box modulates the control of cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. The heterogeneity of stromal/ osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter.

  5. Dickkopf-1 negatively regulates the expression of osteoprotegerin, a key osteoclastogenesis inhibitor, by sequestering Lrp6 in primary and metastatic lytic bone lesions.

    PubMed

    Wang, Jian-Hang; Zhang, Yuanjin; Li, Hong-Yan; Liu, Yun-Yan; Sun, Tao

    2016-06-01

    Recently, an inverse role for Wnt signaling in the development of osteoclasts in the bone was demonstrated. In the present study, we examined whether there is a commonality in the mechanism of bone resorption and lysis that occur in a diverse set of bone metastatic lesions, as well as in primary bone lesions. Compared with control bone tissue and bone biopsies from patients with nonmetastatic primary tumors (i.e., breast carcinoma, lung adenocarcinoma, and prostate carcinoma), patients with bone metastatic lesions from the three aforementioned primary tumors, as well as osteolytic lesions obtained from the bone biopsies of patients with multiple myeloma, demonstrated an upregulated expression of the glycoprotein Dickkopf-1 at both the mRNA and protein levels. Additionally, by coimmunoprecipitation, Dickkopf-1 pulled-down low-density lipoprotein receptor-related protein 6 (Lrp6), which is a key downstream effector of the Wnt signaling pathway. The expression of Lrp6 was unaltered in the osteometastatic lesions. This negative regulation was associated with a lowered expression of osteoprotegerin in the osteometastatic lesions, an observation that was previously reported to promote osteoclastogenesis. These findings provide a common mechanism for the inverse relationship between the Wnt signaling pathway and the development of primary or metastatic bone lesions. Pharmacological modulation of the Wnt signaling pathway might benefit the clinical management of primary and metastatic bone lesions.

  6. Harpagoside Inhibits RANKL-Induced Osteoclastogenesis via Syk-Btk-PLCγ2-Ca(2+) Signaling Pathway and Prevents Inflammation-Mediated Bone Loss.

    PubMed

    Kim, Ju-Young; Park, Sun-Hyang; Baek, Jong Min; Erkhembaatar, Munkhsoyol; Kim, Min Seuk; Yoon, Kwon-Ha; Oh, Jaemin; Lee, Myeung Su

    2015-09-25

    Harpagoside (HAR) is a natural compound isolated from Harpagophytum procumbens (devil's claw) that is reported to have anti-inflammatory effects; however, these effects have not been investigated in the context of bone development. The current study describes for the first time that HAR inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro and suppresses inflammation-induced bone loss in a mouse model. HAR also inhibited the formation of osteoclasts from mouse bone marrow macrophages (BMMs) in a dose-dependent manner as well as the activity of mature osteoclasts, including filamentous actin (F-actin) ring formation and bone matrix breakdown. This involved a HAR-induced decrease in extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) phosphorylation, leading to the inhibition of Syk-Btk-PLCγ2-Ca(2+) in RANKL-dependent early signaling, as well as the activation of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which resulted in the down-regulation of various target genes. Consistent with these in vitro results, HAR blocked lipopolysaccharide (LPS)-induced bone loss in an inflammatory osteoporosis model. However, HAR did not prevent ovariectomy-mediated bone erosion in a postmenopausal osteoporosis model. These results suggest that HAR is a valuable agent against inflammation-related bone disorders but not osteoporosis induced by hormonal abnormalities.

  7. Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α

    PubMed Central

    Wang, Ling; Qiu, Xue-Min; Gui, Yu-Yan; Xu, Ying-Ping; Gober, Hans-Jürgen; Li, Da-Jin

    2015-01-01

    Introduction Bu-Shen-Ning-Xin decoction (BSNXD) is a traditional Chinese medicinal composition that has been used as a remedy for postmenopausal osteoporosis, but the mechanisms affecting bone metabolism are not fully understood. Purpose We investigated the molecular mechanism and signaling pathway underlying the effect of BSNXD on osteoclastogenesis. Materials and methods A postmenopausal osteoporosis animal model generated by ovariectomy was administered BSNXD and drug-derived serum was prepared. An enzyme immunoassay was conducted to measure the 17-β-estradiol (E2) concentration in the drug-derived serum. Bone marrow-derived monocyte/macrophage precursor cells were treated with drug-derived serum, and tartrate-resistance acid phosphatase staining was conducted to observe osteoclastogenesis. A bone resorption assay was performed to analyze the effect on osteoclastic resorptive function. Real-time PCR, flow cytometry, Western blotting, transfection, and luciferase assays were conducted to explore the related mechanism. Results E2 was not elevated in BSNXD-derived serum. BSNXD-derived serum suppressed receptor activation of nuclear factor κB ligand (RANKL)-activated osteoclastogenesis in a dose-dependent manner; this effect could be reversed by estrogen receptor α antagonist methyl-piperidino-pyrazole. The serum suppressed RANKL-induced NF-κB transcription and inhibited the accumulation of nuclear factor of activated T-cells, cytoplasmic 1 in osteoclast precursor cells; the inhibitory effect was abolished by methyl-piperidino-pyrazole but not the estrogen receptor β antagonist or androgen receptor antagonist. Conclusion These results collectively suggest that administration of BSNXD presents inhibitory effects on osteoclast differentiation by abrogating the RANKL-induced nuclear factor of activated T-cells, cytoplasmic 1 and NF-κB signaling pathways downstream of estrogen receptor α, thereby contributing to the inhibitory effect on bone resorption. PMID

  8. Combination of IL-6 and sIL-6R differentially regulate varying levels of RANKL-induced osteoclastogenesis through NF-κB, ERK and JNK signaling pathways

    PubMed Central

    Feng, Wei; Liu, Hongrui; Luo, Tingting; Liu, Di; Du, Juan; Sun, Jing; Wang, Wei; Han, Xiuchun; Yang, Kaiyun; Guo, Jie; Amizuka, Norio; Li, Minqi

    2017-01-01

    Interleukin (IL)-6 is known to indirectly enhance osteoclast formation by promoting receptor activator of nuclear factor kappa-B ligand (RANKL) production by osteoblastic/stromal cells. However, little is known about the direct effect of IL-6 on osteoclastogenesis. Here, we determined the direct effects of IL-6 and its soluble receptor (sIL-6R) on RANKL-induced osteoclast formation by osteoclast precursors in vitro. We found IL-6/sIL-6R significantly promoted and suppressed osteoclast differentiation induced by low- (10 ng/ml) and high-level (50 ng/ml) RANKL, respectively. Using a bone resorption pit formation assay, expression of osteoclastic marker genes and transcription factors confirmed differential regulation of RANKL-induced osteoclastogenesis by IL-6/sIL-6R. Intracellular signaling transduction analysis revealed IL-6/sIL-6R specifically upregulated and downregulated the phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), ERK (extracellular signal–regulated kinase) and JNK (c-Jun N-terminal kinase) induced by low- and high level RANKL, respectively. Taken together, our findings demonstrate that IL-6/sIL-6R differentially regulate RANKL-induced osteoclast differentiation and activity through modulation of NF-κB, ERK and JNK signaling pathways. Thus, IL-6 likely plays a dual role in osteoclastogenesis either as a pro-resorption factor or as a protector of bone, depending on the level of RANKL within the local microenvironment. PMID:28128332

  9. KMUP-1 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss: Roles of MAPKs, Akt, NF-κB and Calcium/Calcineurin/NFATc1 Pathways

    PubMed Central

    Lin, I-Ling; Ho, Mei-Ling; Hsu, Pei-Chuan; Chen, Li-Wen; Chen, Ing-Jun; Yeh, Jwu-Lai

    2013-01-01

    Background KMUP-1 is a xanthine derivative with inhibitory activities on the phosphodiesterase (PDE) 3,4 and 5 isoenzymes to suppress the degradation of cyclic AMP and cyclic GMP. However, the effects of KMUP-1 on osteoclast differentiation are still unclear. In this study, we investigated whether KMUP-1 inhibits osteoclastogenesis induced by RANKL in RAW 264.7 cells and bone loss induced by ovariectomy in mice, and the underlying mechanisms. Principal Findings In vitro, KMUP-1 inhibited RANKL-induced TRAP activity, the formation of multinucleated osteoclasts and resorption-pit formation. It also inhibited key mediators of osteoclastogenesis including IL-1β, IL-6, TNF-α and HMGB1. In addition, KMUP-1 inhibited RANKL-induced activation of signaling molecules (Akt, MAPKs, calcium and NF-κB), mRNA expression of osteoclastogensis-associated genes (TRAP, MMP-9, Fra-1, and cathepsin K) and transcription factors (c-Fos and NFATc1). Furthermore, most inhibitory effects of KMUP-1 on RANKL-mediated signal activations were reversed by a protein kinase A inhibitor (H89) and a protein kinase G inhibitor (KT5823). In vivo, KMUP-1 prevented loss of bone mineral content, preserved serum alkaline phosphate and reduced serum osteocalcin in ovariectomized mice. Conclusions KMUP-1 inhibits RANKL-induced osteoclastogenesis in vitro and protects against ovariectomy-induced bone loss in vivo. These effects are mediated, at least in part, by cAMP and cGMP pathways. Therefore, KMUP-1 may have a role in pharmacologic therapy of osteoporosis. PMID:23936022

  10. Enhanced but hypofunctional osteoclastogenesis in an autosomal dominant osteopetrosis type II case carrying a c.1856C>T mutation in CLCN7

    PubMed Central

    Chen, Xiang; Zhang, Kun; Hock, Janet; Wang, Chunyu; Yu, Xijie

    2016-01-01

    Type II autosomal dominant osteopetrosis (ADO2), which is the most common form of osteopetrosis, is caused by heterozygous mutations in the chloride channel 7 (CLCN7) gene. The osteopetrosis of ADO2 has been attributed to hypofunctional osteoclasts. The mechanism underlying the abnormality in osteoclast function remains largely unknown. This study was designed to investigate gene mutations and osteoclast function in a case that was clinically diagnosed as ADO2. Genomic DNA was extracted from blood samples of this patient, and the 25 exons of CLCN7 were amplified. Peripheral blood from the ADO2 subject and a healthy age- and sex-matched control was used to evaluate osteoclastogenesis, osteoclast morphology, and bone resorption. Analysis of DNA from the patient showed a germline heterozygous missense mutation, c.1856C>T (p.P619L), in exon 20 of CLCN7. A similar homozygous mutation at this site was previously reported in a patient with autosomal recessive osteopetrosis. When cultured, the peripheral blood mononuclear cells (PBMCs) from the ADO2 patient spontaneously differentiated into mature osteoclasts in vitro. The ADO2 patient’s PBMCs formed enhanced, but heterogeneous, osteoclasts in both the presence and absence of macrophage-colony stimulating factor, and nuclear factor-ĸB ligand. Bone resorption was reduced in the ADO2 patient’s osteoclasts, which exhibited aberrant morphology and abnormal distribution of integrin avβ3. Gene analysis found increased c-fos expression and reduced RhoA and integrin beta 3 expression in ADO2 cells. In conclusion, our data suggest that enhanced, heterogeneous osteoclast induction may be an intrinsic characteristic of ADO2. PMID:27990310

  11. Inhibition of titanium-particle-induced inflammatory osteolysis after local administration of dopamine and suppression of osteoclastogenesis via D2-like receptor signaling pathway.

    PubMed

    Yang, Huilin; Xu, Yaozeng; Zhu, Mo; Gu, Ye; Zhang, Wen; Shao, Hongguo; Wang, Yijun; Ping, Zichuan; Hu, Xuanyang; Wang, Liangliang; Geng, Dechun

    2016-02-01

    Chronic inflammation and extensive osteoclast formation play critical roles in wear-debris-induced peri-implant osteolysis. We investigated the potential impact of dopamine on titanium-particle-induced inflammatory osteolysis in vivo and in vitro. Twenty-eight C57BL/6J mice were randomly assigned to four groups: sham control (PBS treatment), titanium (titanium/PBS treatment), low- (titanium/2 μg kg(-1) day(-1) dopamine) and high-dopamine (titanium/10 μg kg(-1) day(-1) dopamine). After 2 weeks, mouse calvariae were collected for micro-computed tomography (micro-CT) and histomorphometry analysis. Bone-marrow-derived macrophages (BMMs) were isolated to assess osteoclast differentiation. Dopamine significantly reduced titanium-particle-induced osteolysis compared with the titanium group as confirmed by micro-CT and histomorphometric data. Osteoclast numbers were 34.9% and 59.7% (both p < 0.01) lower in the low- and high-dopamine-treatment groups, respectively, than in the titanium group. Additionally, low RANKL, tumor necrosis factor-α, interleukin-1β and interleukin-6 immunochemistry staining were noted in dopamine-treatment groups. Dopamine markedly inhibited osteoclast formation, osteoclastogenesis-related gene expression and pro-inflammatory cytokine expression in BMMs in a dose-dependent manner. Moreover, the resorption area was decreased with 10(-9) M and 10(-8) M dopamine to 40.0% and 14.5% (both p < 0.01), respectively. Furthermore, the inhibitory effect of dopamine was reversed by the D2-like-receptor antagonist haloperidol but not by the D1-like-receptor antagonist SCH23390. These results suggest that dopamine therapy could be developed into an effective and safe method for osteolysis-related disease caused by chronic inflammation and excessive osteoclast formation.

  12. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion

    PubMed Central

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán

    2015-01-01

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage. PMID:26459511

  13. Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3.

    PubMed

    Kerner, S A; Scott, R A; Pike, J W

    1989-06-01

    Osteoblast-specific expression of the bone protein osteocalcin is controlled at the transcriptional level by the steroid hormone 1 alpha,25-dihydroxyvitamin D3. As this protein may represent a marker for bone activity in human disease, we examined the regulation of its expression at the molecular level by evaluating human osteocalcin gene promoter function. We describe regions within the promoter that contribute to basal expression of the gene in osteoblast-like cells in culture. Further, we define a 21-base-pair DNA element with the sequence 5'-GTGACTCACCGGGTGAACGGG-3', which acts in cis to mediate 1 alpha,25-dihydroxyvitamin D3 inducibility of the osteocalcin gene. This response element bears sequence similarity with other short DNA segments, particularly those for estrogen and thyroid hormone, which act together with their respective trans-acting receptors to modulate gene transcription.

  14. SH3BP2 cherubism mutation potentiates TNF-α-induced osteoclastogenesis via NFATc1 and TNF-α-mediated inflammatory bone loss.

    PubMed

    Mukai, Tomoyuki; Ishida, Shu; Ishikawa, Remi; Yoshitaka, Teruhito; Kittaka, Mizuho; Gallant, Richard; Lin, Yi-Ling; Rottapel, Robert; Brotto, Marco; Reichenberger, Ernst J; Ueki, Yasuyoshi

    2014-12-01

    Cherubism (OMIM# 118400) is a genetic disorder with excessive jawbone resorption caused by mutations in SH3 domain binding protein 2 (SH3BP2), a signaling adaptor protein. Studies on the mouse model for cherubism carrying a P416R knock-in (KI) mutation have revealed that mutant SH3BP2 enhances tumor necrosis factor (TNF)-α production and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in myeloid cells. TNF-α is expressed in human cherubism lesions, which contain a large number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, and TNF-α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2(KI/KI) ). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF-α-mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF-α-mediated osteoclast formation and bone loss. Here, we show that bone marrow-derived M-CSF-dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2(KI/+) ) mice are highly responsive to TNF-α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves spleen tyrosine kinase (SYK) and phospholipase Cγ2 (PLCγ2) phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF-α injection model as well as in a human TNF-α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP-positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF-α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF-α-induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for

  15. Compound Heterozygous Mutations in the Vitamin D Receptor in a Patient With Hereditary 1,25-Dihydroxyvitamin D–Resistant Rickets With Alopecia

    PubMed Central

    Zhou, Yulin; Wang, Jining; Malloy, Peter J; Dolezel, Zdenek; Feldman, David

    2009-01-01

    Hereditary vitamin D–resistant rickets (HVDRR) is a rare recessive genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young girl with clinical features of HVDRR including rickets, hypophosphatemia, and elevated serum 1,25(OH)2D. The girl also had total alopecia. Two mutations were found in the VDR gene: a nonsense mutation (R30X) in the DNA-binding domain and a unique 3-bp in-frame deletion in exon 6 that deleted the codon for lysine at amino acid 246 (ΔK246). The child and her mother were both heterozygous for the 3-bp deletion, whereas the child and her father were both heterozygous for the R30X mutation. Fibroblasts from the patient were unresponsive to 1,25(OH)2D3 as shown by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)2D3 responsiveness. [3H]1,25(OH)2D3 binding and immunoblot analysis showed that the patient's cells expressed the VDRΔK246 mutant protein; however, the amount of VDRΔK246 mutant protein was significantly reduced compared with wildtype controls. In transactivation assays, the recreated VDRΔK246 mutant was unresponsive to 1,25(OH)2D3. The ΔK246 mutation abolished heterodimerization of the mutant VDR with RXRα and binding to the coactivators DRIP205 and SRC-1. However, the ΔK246 mutation did not affect the interaction of the mutant VDR with the corepressor Hairless (HR). In summary, we describe a patient with compound heterozygous mutations in the VDR that results in HVDRR with alopecia. The R30X mutation truncates the VDR, whereas the ΔK246 mutation prevents heterodimerization with RXR and disrupts coactivator interactions. PMID:19049339

  16. Pulmonary administration of 1,25-dihydroxyvitamin D3 to the lungs induces alveolar regeneration in a mouse model of chronic obstructive pulmonary disease.

    PubMed

    Horiguchi, Michiko; Hirokawa, Mai; Abe, Kaori; Kumagai, Harumi; Yamashita, Chikamasa

    2016-07-10

    Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disease with several causes, including smoking, and no curative therapeutic agent is available, particularly for destructive alveolar lesions. In this study, we investigated the differentiation-inducing effect on undifferentiated lung cells (Calu-6) and the alveolar regenerative effect of the active vitamin 1,25-dihydroxy vitamin D3 (VD3) with the ultimate goal of developing a novel curative drug for COPD. First, the differentiation-inducing effect of VD3 on Calu-6 cells was evaluated. Treatment with VD3 increased the proportions of type I alveolar epithelial (AT-I) and type II alveolar epithelial (AT-II) cells constituting alveoli in a concentration- and treatment time-dependent manner, demonstrating the potent differentiation-inducing activity of VD3 on Calu-6 cells. We thus administered VD3 topically to the mice lung using a previously developed intrapulmonary administration via self-inhalation method. To evaluate the alveolus-repairing effect of VD3, we administered VD3 intrapulmonarily to elastase-induced COPD model mice and computed the mean distance between the alveolar walls as an index of the extent of alveolar injury. Results showed significant decreases in the alveolar wall distance in groups of mice that received 0.01, 0.1, and 1μg/kg of intrapulmonary VD3, revealing excellent alveolus-regenerating effect of VD3. Furthermore, we evaluated the effect of VD3 on improving respiratory function using a respiratory function analyzer. Lung elasticity and respiratory competence [forced expiratory volume (FEV) 1 s %] are reduced in COPD, reflecting advanced emphysematous changes. In elastase-induced COPD model mice, although lung elasticity and respiratory competence were reduced, VD3 administered intrapulmonarily twice weekly for 2weeks recovered tissue elastance and forced expiratory volume in 0.05s to the forced vital capacity, which are indicators of lung elasticity and respiratory competence, respectively, to levels comparable to those in normal mice. These results revealed the potent activity of VD3 in inducing differentiation of the Calu-6 cells and the effect of topical administration of VD3 to the lungs to induce lung regeneration at histological and functional levels, demonstrating the potential of VD3 as a curative agent for alveolar destruction in COPD.

  17. Decreased Conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 Following Cholecalciferol Therapy in Patients with CKD

    PubMed Central

    Zhang, Shiqin; Friedman, Peter A.; Nolin, Thomas D.

    2014-01-01

    Background and objectives Elevated concentrations of fibroblast growth factor 23 (FGF23) are postulated to promote 25-hydroxyvitamin D (25[OH]D) insufficiency in CKD by stimulating 24-hydroxylation of this metabolite, leading to its subsequent degradation; however, prospective human studies testing this relationship are lacking. Design, setting, participants, & measurements An open-label prospective study was conducted from October 2010 through July 2012 to compare the effect of 8 weeks of oral cholecalciferol therapy (50,000 IU twice weekly) on the production of 24,25(OH)2D3 in vitamin D–insufficient patients with CKD (n=15) and controls with normal kidney function (n=15). Vitamin D metabolites were comprehensively profiled at baseline and after treatment, along with FGF23 and other mineral metabolism parameters. Results Vitamin D3 and 25(OH)D3 concentrations increased equivalently in the CKD and control groups following cholecalciferol treatment (median D3 change, 8.6 ng/ml [interquartile range, 3.9–25.6 ng/ml] for controls versus 12.6 ng/ml [6.9–41.2 ng/ml] for CKD [P=0.15]; 25(OH)D3 change, 39.2 ng/ml [30.9–47.2 ng/ml] for controls versus 39.9 ng/ml [31.5–44.1 ng/ml] for CKD [P=0.58]). Likewise, the absolute increase in 1α,25(OH)2D3 was similar between CKD participants and controls (change, 111.2 pg/ml [64.3–141.6 pg/ml] for controls versus 101.1 pg/ml [74.2–123.1 pg/ml] for CKD; P=0.38). Baseline and post-treatment 24,25(OH)2D3 concentrations were lower in the CKD group; moreover, the absolute increase in 24,25(OH)2D3 after therapy was markedly smaller in patients with CKD (change, 2.8 ng/ml [2.3–3.5 ng/ml] for controls versus 1.2 ng/ml [0.6–1.9 ng/ml] for patients with CKD; P<0.001). Furthermore, higher baseline FGF23 concentrations were associated with smaller increments in 24,25(OH)2D3 for individuals with CKD; this association was negated after adjustment for eGFR by multivariate analysis. Conclusions Patients with CKD exhibit an altered ability to increase serum 24,25(OH)2D3 after cholecalciferol therapy, suggesting decreased 24-hydroxylase activity in CKD. The observed relationship between baseline FGF23 and increments in 24,25(OH)2D3 further refutes the idea that FGF23 directly contributes to 25(OH)D insufficiency in CKD through stimulation of 24-hydroxylase activity. PMID:25183657

  18. Adverse effects of a high-glucose diet on body weight and plasma calcium and 1,25-dihydroxyvitamin D3 levels in calcium-deficient growing rats.

    PubMed

    Clark, S A; Boass, A; Toverud, S U

    1989-03-01

    We tested the hypothesis that dietary calcium would lead to greater impairment of body weight gain and calcium homeostasis if rats are fed a diet with a high glucose content compared with our standard diet in which the carbohydrate is supplied by whole wheat flour. Groups of female rats at 21 days of age were given either of two equivalent calcium-deficient diets with carbohydrate supplied either by glucose (LCaG) or by wheat flour (LCaW). Control rats were fed the wheat-flour diet containing 0.4% calcium. Since previous studies indicated divergent effects of glucose-based and flour-based diets on body weight in vitamin D-deficient rats, we designed a parallel study with vitamin D-deprived rats. Compared with rats fed the LCaW diet, the rats fed the LCaG diet had inferior body weight gain and more severe hypocalcemia (1-2 mg/ml lower) over a 40-day period, and no significant elevation of the plasma 1,25(OH)2D3 level at 61 days of age. Rats fed the LCaW diet maintained a 3-fold elevation of plasma 1,25(OH)2D3 relative to the level of control rats fed the 0.4% calcium diet. The dry weight and percent ash of tibias were similarly reduced in the two calcium-restricted groups compared to the control group. Among the vitamin D-deprived rats, those fed the glucose diet had poorer weight gain than those fed the wheat flour diet. However, both groups had similarly depressed serum calcium level, tibia ash content and 1,25(OH)2D3 level. Thus, a glucose diet combined with calcium restriction or vitamin D deprivation appears to accentuate the impairment of body weight gain and, when combined with calcium restriction, it also accentuates the impairment of calcium homeostasis and interferes with the adaptive increase in plasma 1,25(OH)2D3.

  19. 1,25-Dihydroxyvitamin D3 and fetal lung maturation: immunogold detection of VDR expression in pneumocytes type II cells and effect on fructose 1,6 bisphosphatase.

    PubMed

    Nguyen, M; Trubert, C L; Rizk-Rabin, M; Rehan, V K; Besançon, F; Cayre, Y E; Garabédian, M

    2004-05-01

    Lung maturation before birth includes type II pneumocyte differentiation with progressive disappearance of glycogen content and onset of surfactant synthesis. We have shown previously that 1,25-(OH)2D3 increases surfactant synthesis and secretion by type II cells and decreases their glycogen content in fetal rat lung explants. Recently, the gene coding fructose 1,6 bisphosphatase (F1,6BP), a regulatory enzyme of gluconeogenesis, has been identified in type II cells and its promoter bears a Vitamin D response element. Present results show:The coexistence of type II cells at different stages of maturation. in rat fetal lung on day 21 of gestation (electron microscopy), and the association between maturation of type II cells and disappearance of their glycogen content. The immunogold labeling of all type II cells when using the 9A7g VDR-antibody, with significantly more abundant gold particles in cells exhibiting an intermediate glycogen content. The expression of F1,6BP mRNA in a human type II cell line (NCI-H441) and the increase of this expression after 18h incubation with 1,25-(OH)2D3 (10(-8)M). These results bring further evidence for a physiological role of 1,25-(OH)2D3 during type II pneumocyte maturation. Activation of F1,6BP may participate to the 1,25-(OH)2D3 action on surfactant synthesis via the gluconeogenesis pathway.

  20. Transplantation of bone marrow-derived mesenchymal stem cells rescues partially rachitic phenotypes induced by 1,25-Dihydroxyvitamin D deficiency in mice

    PubMed Central

    Zhang, Zengli; Yin, Shaomeng; Xue, Xian; Ji, Ji; Tong, Jian; Goltzman, David; Miao, Dengshun

    2016-01-01

    To determine whether the transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) can improve the 1,25(OH)2D deficiency-induced rachitic phenotype, 2×106 BM-MSCs from wild-type mice or vehicle were transplanted by tail vein injection into mice deficient in 1,25(OH)2D due to targeted deletion of 1α(OH)ase (1α(OH)ase-/-). Our results show that 1α(OH)ase mRNA was expressed in the BM-MSCs derived from wild-type mice, and was detected in long bone, kidney and intestine from BM-MSC-transplanted 1α(OH)ase-/- recipients. Serum calcium, 1,25(OH)2D3 levels and body weight were significantly increased in BM-MSC-transplanted 1α(OH)ase-/- recipients compared to vehicle-treated 1α(OH)ase-/- mice. Skeletal mineralization improved in 1α(OH)ase-/- recipients as demonstrated by BMD measurement, micro-CT analysis and von Kossa staining of undecalcified sections. Expression levels of type I collagen, osteocalcin, bone sialoprotein and vitronectin and the size of calcified nodules were decreased in BM-MSC cultures from 1α(OH)ase-/- mice compared with those from wild-type mice, however, these parameters were increased in those from BM-MSCs-transplanted 1α(OH)ase-/- recipients compared with those from vehicle-treated 1α(OH)ase-/- mice. This study indicates that donor BM-MSCs cells can relocate to multiple tissues where they synthesize 1α(OH)ase and produce 1,25(OH)2D that contributes to the improvement of serum calcium and skeletal mineralization. Results from this study suggest that BM-MSC transplantation may provide a therapeutic approach to treatment of pseudovitamin D-deficiency rickets. PMID:27830022

  1. Eldecalcitol (ED-71), an analog of 1α,25-dihydroxyvitamin D3 as a potential anti-cancer agent for oral squamous cell carcinomas.

    PubMed

    Shintani, T; Rosli, S N Z; Takatsu, F; Choon, Y F; Hayashido, Y; Toratani, S; Usui, E; Okamoto, T

    2016-11-01

    We have previously reported that 1,25(OH)2D3 inhibits NF-κB activity and thus inhibits growth of OSCC cells in serum-free culture and down-regulates HBp17/FGFBP-1 expression, which is important for cancer cell growth and angiogenesis. Here, we have investigated the effects of ED-71, an analog of vitamin D3 (VD) on OSCC cell lines in serum-free culture. It is known that ED-71 has a stronger inhibitory effect on bone resorption compared to VD and other VD analogs. To the best of our knowledge, there was no report examining the potential of ED-71 as an anti-cancer agent for OSCC. We found that ED-71 is able to inhibit the growth of cancer cell lines at a concentration of hundred times lower than calcitriol. As Cyp24A1 was reportedly induced in cancer cells, we measured the expression of CYP24A1 in OSCC cell lines (NA and UE), A431 epidermoid carcinoma and normal fibroblast cell (gfi) in serum-free culture. As a result, CYP24A1 mRNA and the protein expression in the OSCC cells treated with ED-71 increased in a dose-dependent manner. However, in vivo experiment, in which the A431 cells were implanted in mice, tumor formation was reduced by the ED-71 treatment with no significant difference between Cyp24A1 expression in the tumors of ED-71-treated and control group, as analyzed by western blotting and immunohistochemistry. These results suggest that ED-71 is a potential anti-cancer agent for OSCC.

  2. Effects of 1 alpha,25- and 24R,25-dihydroxyvitamin D3 on aluminum-induced rickets in growing uremic rats.

    PubMed

    Vukicević, S; Krempien, B; Stavljenić, A

    1987-12-01

    Rats were subjected to a two-stage subtotal nephrectomy or sham operation, and treated with aluminum (Al) or both aluminum and vitamin D3 metabolites for 5 weeks with a cumulative dose of 13.6 mg aluminum. Animals were injected with 3H-thymidine and 3H-proline. The following analyses were performed: quantitative histology of tibial metaphyses and cytomorphometric electron microscopy of osteoclasts, quantitative (ICP-spectroscopy) and qualitative determination (histochemical staining) of aluminum within organs, and serum biochemistry (Ca, P, Mg, vitamin D3 metabolites, alkaline phosphatase, urea). The following new facts of the aluminum-related bone disease became evident: (a) Application of aluminum to growing uremic rats induced rickets, whose major epiphyseal growth plate changes were 1 alpha,25(OH)2D3-dependent. Addition of 1 alpha,25(OH)2D3 prevented the formation of rachitic metaphysis, but failed to prevent osteoid accumulation on epiphyseal and metaphyseal trabecular surfaces. Moreover, calcitriol produced hyperosteoidosis and osteosclerosis in the same rats. Aluminum did not alter the function of osteoblasts, while osteoclasts seemed inactivated. (b) The development of rickets was associated with suppressed serum levels of 1,25(OH)2D3, reduced phosphorus level and the high content of aluminum in the bone, kidney, and liver. The addition of 24R,25(OH)2D3 markedly exaggerated the reduction of serum levels of calcitriol. We suggested that aluminum induces rickets in growing uremic rats, which consists of two components: vitamin D refractory osteomalacia and 1 alpha,25(OH)2D3-dependent epiphyseal growth plate changes.

  3. 1,25-Dihydroxyvitamin D3 enhances the expression of transforming growth factor beta 1 and its latent form binding protein in cultured breast carcinoma cells.

    PubMed

    Koli, K; Keski-Oja, J

    1995-04-01

    Transforming growth factor beta s (TGF-beta s) are a family of polypeptide growth factors that regulate cellular growth, phenotype, and differentiation. TGF-beta s are synthesized as latent high molecular weight complexes that include the NH2-terminal remnant of the TGF-beta precursor (latency-associated protein) and, frequently, latent TGF-beta binding protein. After activation, TGF-beta s act as local mediators of hormonal responses in target tissues. TGF-beta functions as a negative growth regulator for both breast cancer cells and normal mammary epithelial cells. Vitamin D3 is growth inhibitory for the estrogen receptor-negative breast cancer cell line BT-20 and regulates TGF-beta expression in cultured keratinocytes. We studied here the effects of vitamin D3 and its analogues on TGF-beta expression and activity in BT-20 cells. It was found that vitamin D3 enhanced both TGF-beta 1 mRNA and secretion of the protein in a time- and dose-dependent manner. Analyses of the vitamin D3 responses in the presence of cycloheximide or actinomycin D indicated that the TGF-beta 1 mRNA induction was dependent on both protein and RNA synthesis. The amounts of latent TGF-beta binding protein were also increased in the conditioned medium but not in the pericellular matrix of vitamin D3-treated cultures. The amounts of active TGF-beta were enhanced in vitamin D3-treated cultures as well, suggesting autocrine or paracrine functions for the secreted growth factor. Some analogues of vitamin D3 (EB 1089, MC 903, and KH 1060) that are known to be potent inhibitors of breast cancer cell growth both in vitro and in vivo had similar or more pronounced inducing effects on TGF-beta 1 mRNA levels. The present results indicate that vitamin D3 and its analogues are potent inducers of both active and latent forms of TGF-beta 1 in BT-20 breast carcinoma cells and provide evidence for coordinated regulation of latent TGF-beta binding protein and TGF-beta 1.

  4. WITHDRAWN: VDR involvement in 1a,25-dihydroxyvitamin D3-action on cellular cycle in C2C12 skeletal muscle cells.

    PubMed

    Irazoqui, Ana P; Boland, Ricardo L; Buitrago, Claudia G

    2013-11-01

    This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

  5. Active vitamin D (1,25-dihydroxyvitamin D3) increases host susceptibility to Citrobacter rodentium by suppressing mucosal Th17 responses

    PubMed Central

    Ryz, Natasha R.; Patterson, Scott J.; Zhang, Yiqun; Ma, Caixia; Huang, Tina; Bhinder, Ganive; Wu, Xiujuan; Chan, Justin; Glesby, Alexa; Sham, Ho Pan; Dutz, Jan P.; Levings, Megan K.; Jacobson, Kevan

    2012-01-01

    Vitamin D deficiency affects more that 1 billion people worldwide and is associated with an increased risk of developing a number of inflammatory/autoimmune diseases, including inflammatory bowel disease (IBD). At present, the basis for the impact of vitamin D on IBD and mucosal immune responses is unclear; however, IBD is known to reflect exaggerated immune responses to luminal bacteria, and vitamin D has been shown to play a role in regulating bacteria-host interactions. Therefore, to test the effect of active vitamin D on host responses to enteric bacteria, we gave 1,25(OH)2D3 to mice infected with the bacterial pathogen Citrobacter rodentium, an extracellular microbe that causes acute colitis characterized by a strong Th1/Th17 immune response. 1,25(OH)2D3 treatment of infected mice led to increased pathogen burdens and exaggerated tissue pathology. In association with their increased susceptibility, 1,25(OH)2D3-treated mice showed substantially reduced numbers of Th17 T cells within their infected colons, whereas only modest differences were noted in Th1 and Treg numbers. In accordance with the impaired Th17 responses, 1,25(OH)2D3-treated mice showed defects in their production of the antimicrobial peptide REG3γ. Taken together, these studies show that 1,25(OH)2D3 suppresses Th17 T-cell responses in vivo and impairs mucosal host defense against an enteric bacterial pathogen. PMID:23019194

  6. Roles for the heliodynamic hormones, all trans retinoic acid and 1 alpha, 25-dihydroxyvitamin D3, in control of the hematopoietic cell cycle.

    PubMed

    Blazsek, I; Comisso, M; Farabos, C; Misset, J L

    1991-01-01

    It is now well established that the production of primary hematopoietic cells is controlled at different levels of the biological organization. Bone marrow (BM) stromal cells, the extracellular matrix (ECM), polypeptide hematopoietic growth factors (HGF) as well as endogenous cell-division cycle (CDC) related factors play a dominant role in this control. Recent information suggest that the 2 lipophilic hormones, transRA and 1 alpha,25D3, depending on and/or perhaps mediating solar energy, play a role in the maintenance of BM homeostasis. Here we show that both transRA and 1 alpha,25D3: a) modulate the growth and/or stimulate the adipocytic differentiation of fibroblastic stromal cells (F-CFU); b) inhibit the synthesis and extracellular processing but stimulate the solubilization of matrix collagen; c) modulate the clonal growth of myeloid progenitor cells (GM-CFU) in synergy with HGFs; and d) inhibit the production of lactic acid in standard, normal long-term BM cultures (LTBMC). Comparative analysis of normal, preleukemic and leukemic BM cells in LTBMC indicated a positive correlation between the induction of terminal differentiation and reduced lactate production elicited by transRA or 1 alpha,25D3. These results raise a hypothesis according to which the terminal differentiation induced by the helicodynamic hormones is dependent on the mitochondrial aerobic ATP-generating system whose impairment may be a critical step during the process of leukemic transformation.

  7. 1,25-Dihydroxyvitamin D3 Suppresses TLR8 Expression and TLR8-Mediated Inflammatory Responses in Monocytes In Vitro and Experimental Autoimmune Encephalomyelitis In Vivo

    DTIC Science & Technology

    2013-03-14

    International Agency for Research on Cancer , Lyon, France, 3Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma...vitamin D, was able to suppress inflammatory cytokine production in the inflamed EAE spinal cords and effectively ameliorate EAE [6], [7], [8], [9...was recorded daily and presented in the Figure S1A. Immunostaining of the spinal cord sections for microglia/ macrophage maker F4/80 revealed a severe

  8. Modulation of mouse RANKL gene expression by Runx2 and vitamin D3.

    PubMed

    Kitazawa, Riko; Mori, Kiyoshi; Yamaguchi, Akira; Kondo, Takeshi; Kitazawa, Sohei

    2008-12-01

    The expression of receptor activator of nuclear factor-kappaB ligand (RANKL) is regulated by bone-seeking hormones such as PTH and 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Runx2, a master gene for osteoblastic differentiation, also modulates osteoclastogenesis by regulating the RANKL gene. To elucidate the mechanism whereby runx2 and 1,25(OH)2D3 regulate RANKL expression, we studied the function of runx2 on the chromatin structure and on the proximal binding sites using osteoblastic cell lines derived from normal (ST2) and runx2-deficient mice (RD-C6). Although the expression of RANKL in the steady-state was higher in RD-C6 than in ST2, 1,25(OH)2D3-treatment of the cells increased it 20-fold in ST2 but only 1.8-fold in RD-C6. Transient transfection studies with proximal RANKL 2kb promoter, runx2 knock-down in ST2, and forced expression of runx2 in RD-C6 all confirmed that runx2 set the steady-state expression of the RANKL gene at a low level, but exerted a positive effect on enhanced transcriptional activity in response to 1,25(OH)2D3. Also, assessment of the acetylation status of the area spanning 40 kb upstream of the basic promoter in ST2 and RD-C6 by ChIP assay revealed that whereas H3 and H4 histone acetylation was detected even in the steady-state in RD-C6, it was detected only with 1,25(OH)2D3 in ST2. In the steady-state, runx2 may suppress RANKL gene by condensing the chromatin structure; however, it exerts a positive effect on 1,25(OH)2D3-induced RANKL transcription when the proximal runx2 sites are accessible. Thus, RANKL expression in stromal/osteoblastic cells is keenly regulated by 1,25(OH)2D3 which transactivates the gene at two different levels

  9. Resveratrol-mediated SIRT-1 interactions with p300 modulate receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB signaling and inhibit osteoclastogenesis in bone-derived cells.

    PubMed

    Shakibaei, Mehdi; Buhrmann, Constanze; Mobasheri, Ali

    2011-04-01

    Resveratrol is a polyphenolic phytoestrogen that has been shown to exhibit potent anti-oxidant, anti-inflammatory, and anti-catabolic properties. Increased osteoclastic and decreased osteoblastic activities result in bone resorption and loss of bone mass. These changes have been implicated in pathological processes in rheumatoid arthritis and osteoporosis. Receptor activator of NF-κB ligand (RANKL), a member of the TNF superfamily, is a major mediator of bone loss. In this study, we investigated the effects of resveratrol on RANKL during bone morphogenesis in high density bone cultures in vitro. Untreated bone-derived cell cultures produced well organized bone-like structures with a bone-specific matrix. Treatment with RANKL induced formation of tartrate-resistant acid phosphatase-positive multinucleated cells that exhibited morphological features of osteoclasts. RANKL induced NF-κB activation, whereas pretreatment with resveratrol completely inhibited this activation and suppressed the activation of IκBα kinase and IκBα phosphorylation and degradation. RANKL up-regulated p300 (a histone acetyltransferase) expression, which, in turn, promoted acetylation of NF-κB. Resveratrol inhibited RANKL-induced acetylation and nuclear translocation of NF-κB in a time- and concentration-dependent manner. In addition, activation of Sirt-1 (a histone deacetylase) by resveratrol induced Sirt-1-p300 association in bone-derived and preosteoblastic cells, leading to deacetylation of RANKL-induced NF-κB, inhibition of NF-κB transcriptional activation, and osteoclastogenesis. Co-treatment with resveratrol activated the bone transcription factors Cbfa-1 and Sirt-1 and induced the formation of Sirt-1-Cbfa-1 complexes. Overall, these results demonstrate that resveratrol-activated Sirt-1 plays pivotal roles in regulating the balance between the osteoclastic versus osteoblastic activity result in bone formation in vitro thereby highlighting its therapeutic potential for treating

  10. Responsiveness to phytoestrogens in primary human osteoblasts is modulated differentially by a "less-calcemic" analog of 1,25 dihydroxyvitamin D(3): JK 1624F(2)-2 (JKF).

    PubMed

    Somjen, Dalia; Katzburg, Sara; Kohen, Fortune; Gayer, Batya; Sharon, Orly; Hendel, David; Posner, Gary H; Kaye, Alvin M

    2006-02-01

    We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and raloxifene (Ral), and male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. We also found that pre-treatment with the less-calcemic analog of Vitamin D, JK 1624 F(2)-2 (JKF), upregulated the response of CK to E(2) and Ral. In this study, we analyzed the response of human bone cells from pre- and post-menopausal females and males, to phytoestrogens. Bone cells derived from pre-menopausal women showed greater stimulation of CK than cells from post-menopausal women, after treatment with E(2) (30 nM), daidzein (D, 3000 nM), genistein (G, 3000 nM) and Ral (3000 nM); whereas the responses to biochainin A (BA 3000 nM), quecertin (Qu 3000 nM) or the carboxy derivative of G (cG 300 nM) were not age-dependent. Male-derived cells did not respond to phytoestrogens. When cells derived from female bones at both age groups were pre-treated with JKF, by daily addition of 1nM, for 3 days, there was an upregulation of the response to E(2), Ral, G and D but not to BA or Qu. Nuclear binding of (3)[H] E(2) was characteristic of the different phytoestrogens, with increase of the specific binding after pre-treatment with JKF. In contrast, the membranal binding of E(2)-Ov-Eu, which was specific for the estrogenic compounds except Ral, was inhibited by pre-treatment with JKF except for ICI 161480 (ICI). Male bone cells did not bind E(2)-Ov-Eu but bound T-BSA-Eu; this binding was abolished by pre-treatment with JKF. Pre-treatment with JKF increased mRNA for ERalpha and decreased mRNA for ERbeta in bone cells from both age groups of females and from males, all of which expressed both ERs, with a ratio of ERalpha to ERbeta of 121:1 in pre- and 78:1 in post-menopausal and 105:1 in male bone cells. This study raises the possibility of combined Vitamin D analog and phytoestrogen for hormonal replacement therapy to prevent post-menopausal osteoporosis, which is a subject of ongoing research in animal models.

  11. Effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and its analogues (EB1089 and analog V) on canine adenocarcinoma (CAC-8) in nude mice.

    PubMed

    Kunakornsawat, Sunee; Rosol, Thomas John; Capen, Charles Chabert; Reddy, Gudimetla Satyanarayana; Binderup, Lise; Inpanbutr, Nongnuch

    2002-05-01

    The aim of this study is to determine the effects of 1,25(OH)2D3 and its analogues on tumor growth and body weight, changes in plasma ionized calcium, parathyroid hormone-related protein (PTHrP) production, bone resorption, and the distribution of the 1,25(OH)2D3 receptor (VDR) on tumors in nude mice-bearing the canine adenocarcinoma (CAC-8). Thirty-seven nude mice were implanted subcutaneously with CAC-8. Two weeks after implantation, the mice were divided into 5 groups and injected intraperitoneally 3 times/week for 4 weeks with 5 different substrates. Group I (nontumor-bearing mice) were injected with vehicle. Groups II through V were CAC-8-bearing mice injected with the following: Grp. II, vehicle; Grp. III, analog V; Grp. IV, 1,25(OH)2D3; and Grp. V, EB1089. Our results showed that mice body weight (% change) of CAC-8-bearing mice was significantly lower than those of nontumor-bearing mice (p<0.05). CAC-8-bearing mice treated with analog V maintained their body weight better than CAC-8-bearing mice treated with either vehicle, 1,25(OH)2D3, or EB1089. A reduction of tumor growth was observed in CAC-8-bearing mice treated with 1,25(OH)2D3 and its analogues; however, the reduction was not statistically significant compared to the vehicle-treated CAC-8-bearing mice. All CAC-8-bearing mice increased osteoclastic bone resorption and hypercalcemia. Immunohistochemical staining of CAC-8 with VDR antibody demonstrated a positive reaction in nuclei of tumor cells. In conclusion, CAC-8-bearing mice treated with analog V were more active and maintained their body weight better than other CAC-8-bearing groups. Analog V-treated mice also showed no toxic side effects of hypercalcemia despite an increase in plasmaionized calcium comparable to nontumor-bearing mice. Tumor volumes of CAC-8-bearing mice treated with 1,25(OH)2D3 and its analogues were smaller than vehicle-treated CAC-8-bearing mice. This finding suggested an inhibitory effect on tumor cell growth.

  12. 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1alpha,25(OH)2D3 in hypertrophic cells.

    PubMed

    Boyan, B D; Hurst-Kennedy, J; Denison, T A; Schwartz, Z

    2010-07-01

    Previously we showed that costochondral growth plate resting zone (RC) chondrocytes response primarily to 24R,25(OH)2D3 whereas prehypertrophic and hypertrophic (GC) cells respond to 1alpha,25(OH)2D3. 24R,25(OH)2D3 increases RC cell proliferation and inhibits activity of matrix processing enzymes, suggesting it stabilizes cells in the reserve zone, possibly by inhibiting the matrix degradation characteristic of apoptotic hypertrophic GC cells. To test this, apoptosis was induced in rat RC cells by treatment with exogenous inorganic phosphate (Pi). 24R,25(OH)2D3 blocked apoptotic effects in a dose-dependent manner. Similarly, apoptosis was induced in ATDC5 cell cultures and 24R,25(OH)2D3 blocked this effect. Further studies indicated that 24R,25(OH)2D3 acts via at least two independent pathways. 24R,25(OH)2D3 increases LPA receptor-1 (LPA R1) expression and production of lysophosphatidic acid (LPA), and subsequent LPA R1/3-dependent signaling, thereby decreasing p53 abundance. LPA also increases the Bcl-2/Bax ratio. In addition, 24R,25(OH)2D3 acts by increasing PKC activity. 24R,25(OH)2D3 stimulates 1-hydroxylase activity, resulting in increased levels of 1,25(OH)2D3, and it increases levels of phospholipase A2 activating protein, which is required for rapid 1alpha,25(OH)2D3-dependent activation of PKC in GC cells. These results suggest that 24R,25(OH)2D3 modulates growth plate development by controlling the rate and extent of RC chondrocyte transition to a GC chondrocyte phenotype.

  13. Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation.

    PubMed

    Knippenberg, Marlene; Helder, Marco N; Doulabi, Behrouz Zandieh; Semeins, Cornelis M; Wuisman, Paul I J M; Klein-Nulend, Jenneke

    2005-01-01

    To engineer bone tissue, mechanosensitive cells are needed that are able to perform bone cell-specific functions, such as (re)modeling of bone tissue. In vivo, local bone mass and architecture are affected by mechanical loading, which is thought to provoke a cellular response via loading-induced flow of interstitial fluid. Adipose tissue is an easily accessible source of mesenchymal stem cells for bone tissue engineering, and is available in abundant amounts compared with bone marrow. We studied whether adipose tissue-derived mesenchymal stem cells (AT-MSCs) are responsive to mechanical loading by pulsating fluid flow (PFF) on osteogenic stimulation in vitro. We found that ATMSCs show a bone cell-like response to fluid shear stress as a result of PFF after the stimulation of osteogenic differentiation by 1,25-dihydroxyvitamin D3. PFF increased nitric oxide production, as well as upregulated cyclooxygenase-2, but not cyclooxygenase-1, gene expression in osteogenically stimulated AT-MSCs. These data suggest that AT-MSCs acquire bone cell-like responsiveness to pulsating fluid shear stress on 1,25-dihydroxyvitamin D3-induced osteogenic differentiation. ATMSCs might be able to perform bone cell-specific functions during bone (re)modeling in vivo and, therefore, provide a promising new tool for bone tissue engineering.

  14. Vitamin MK-7 enhances vitamin D3-induced osteogenesis in hMSCs: modulation of key effectors in mineralization and vascularization.

    PubMed

    Gigante, A; Brugè, F; Cecconi, S; Manzotti, S; Littarru, G P; Tiano, L

    2015-06-01

    The osteoblast is the bone-forming cell and is derived from mesenchymal stem cells (MSCs). Osteo-inductive substances could represent a useful therapeutic approach during the fracture repair process. The aim of this work was to evaluate the effects of vitamin MK-7, alone or in association with vitamin D3, in differentiating human MSCs (hMSCs) in vitro along the osteoblastic lineage. In particular, primary endpoints of the study include gene and protein markers of osteoblast differentiation. Considering genes involved in bone formation and mineralization, our data show that vitamin MK-7 enhances vitamin D3 gene induction of osteocalcin (OC). Among genes related to cell growth and differentiation, a specific effect of vitamin MK-7 was observed for growth differentiation factor-10 (GDF10) and insulin-like growth factor 1 (IGF1), the latter being also involved in the induction of vascular endothelial growth factors (VEGFA). Accordingly, vitamin co-supplementation greatly affected VEGFA and its receptor fms-related tyrosine kinase 1 (FLT1), a key factor in both angiogenic and osteogenic processes. These results stress the relevance of MK-7 and D3 co-supplementation in the bone-healing process as able to modulate the expression of genes involved in both mineralization and angiogenesis. Moreover, at the protein level co-association of vitamins might provide an optimal balance between induction and carboxylation of osteocalcin, essential for its functionality in the extracellular matrix (ECM). Our results may provide hints for therapeutic application of hMSCs in bone disease, clarifying mechanisms involved in stem cell-mediated bone development, and they also highlight the relevance of co-supplementation strategies, since single supplementations might result in a suboptimal effect.

  15. The Effect of 1α,25(OH)2D3 on Osteogenic Differentiation of Stem Cells from Dental Pulp of Exfoliated Deciduous Teeth

    PubMed Central

    Mojarad, Farzad; Amiri, Iraj; Rafatjou, Rezvan; Janeshin, Atousa; Farhadian, Maryam

    2016-01-01

    Statement of the Problem: Stem cells from human exfoliated deciduous teeth (SHEDs) are a population of highly proliferative cells, being capable of differentiating into osteogenic, odontogenic, adipocytes, and neural cells. Vitamin D3 metabolites such as 1α, 25-dihydroxyvitamin D3 are key factors in the regulation of bone metabolism. Purpose: The aim of this study was to investigate the effect of 1α, 25-dihydroxyvitamin D3 on osteogenic differentiation (alkaline phosphatase activity and alizarin red staining) of stem cells of exfoliated deciduous teeth. Materials and Method: Dental pulp was removed from freshly extracted primary teeth and immersed in a digestive solution. Then, the dental pulp cells were immersed in α-MEM (minimum essential medium) to which 10% fetal bovine serum was added. After the third passage, the cells were isolated from the culture plate and were used for osteogenic differentiation. As a control group, the cells were cultured in osteogenic cell culture medium. As the case group, the cells were cultured in osteogenic culture medium supplemented with 100 nM 1α,25 (OH)2D3. The alkaline phosphatase (ALP) activity and alizarin red staining were analyzed to evaluate the osteogenic differentiation at day 21. The results were analyzed by using t-test. Results: Compared with the control group, significant increase was observed in ALP activity of SHEDs after being treated with 1α,25(OH)2D3 (p= 0.002). Alizarin red staining demonstrated that the cells exposed to 1α,25(OH)2D3 induced higher mineralized nodules (p< 0.001). Conclusion: Osteoblast differentiation in SHEDs was stimulated by 1α,25(OH) 2D3. It can be concluded that 1α,25(OH)2D3 can improve osteoblastic differentiation. PMID:27942551

  16. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  17. Vitamin D Promotes Odontogenic Differentiation of Human Dental Pulp Cells via ERK Activation

    PubMed Central

    Woo, Su-Mi; Lim, Hae-Soon; Jeong, Kyung-Yi; Kim, Seon-Mi; Kim, Won-Jae; Jung, Ji-Yeon

    2015-01-01

    The active metabolite of vitamin D such as 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) is a well-known key regulatory factor in bone metabolism. However, little is known about the potential of vitamin D as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro. The purpose of this study was to evaluate the effect of vitamin D3 metabolite, 1α,25(OH)2D3, on odontoblastic differentiation in HDPCs. HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured with 1α,25(OH)2D3 in the absence of differentiation-inducing factors. Treatment of HDPCs with 1α,25(OH)2D3 at a concentration of 10 nM or 100 nM significantly upregulated the expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein1 (DMP1), the odontogenesis-related genes. Also, 1α,25(OH)2D3 enhanced the alkaline phosphatase (ALP) activity and mineralization in HDPCs. In addition, 1α,25(OH)2D3 induced activation of extracellular signal-regulated kinases (ERKs), whereas the ERK inhibitor U0126 ameliorated the upregulation of DSPP and DMP1 and reduced the mineralization enhanced by 1α,25(OH)2D3. These results demonstrated that 1α,25(OH)2D3 promoted odontoblastic differentiation of HDPCs via modulating ERK activation. PMID:26062551

  18. Mechanisms involved in vitamin D mediated intestinal calcium absorption and in non-classical actions of vitamin D.

    PubMed

    Christakos, Sylvia; Dhawan, Puneet; Ajibade, Dare; Benn, Bryan S; Feng, Jingjing; Joshi, Sneha S

    2010-07-01

    Recent studies in our laboratory using calbindin-D9k null mutant mice as well as mice lacking the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inducible epithelial calcium channel TRPV6 provide evidence for calbindin-D9k and TRPV6 independent regulation of active intestinal calcium absorption. These findings suggest that in the knock out (KO) mice there is compensation by another calcium channel or protein and that other novel factors are involved in 1,25(OH)2D3 mediated active intestinal calcium absorption. In addition, 1,25(OH)2D3 mediated paracellular transport of calcium may have contributed to the normalization of serum calcium in the null mutant mice. 1,25(OH)2D3 downregulates cadherin-17 and upregulates claudin-2 and claudin-12 in the intestine, suggesting that 1,25(OH)2D3, by regulating these epithelial cell junction proteins, can route calcium through the paracellular path. With regard to non-classical actions, 1,25(OH)2D3 has been reported to inhibit the proliferation of a number of malignant cells and to regulate adaptive as well as innate immunity. This article will review new developments related to the function and regulation of vitamin D target proteins in classical and non-classical vitamin D target tissues that have provided novel insight into mechanisms of vitamin D action.

  19. RhoA-ROCK and p38MAPK-MSK1 mediate vitamin D effects on gene expression, phenotype, and Wnt pathway in colon cancer cells.

    PubMed

    Ordóñez-Morán, Paloma; Larriba, María Jesús; Pálmer, Héctor G; Valero, Ruth A; Barbáchano, Antonio; Duñach, Mireia; de Herreros, Antonio García; Villalobos, Carlos; Berciano, María Teresa; Lafarga, Miguel; Muñoz, Alberto

    2008-11-17

    The active vitamin D metabolite 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits proliferation and promotes differentiation of colon cancer cells through the activation of vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Additionally, 1,25(OH)(2)D(3) has several nongenomic effects of uncertain relevance. We show that 1,25(OH)(2)D(3) induces a transcription-independent Ca(2+) influx and activation of RhoA-Rho-associated coiled kinase (ROCK). This requires VDR and is followed by activation of the p38 mitogen-activated protein kinase (p38MAPK) and mitogen- and stress-activated kinase 1 (MSK1). As shown by the use of chemical inhibitors, dominant-negative mutants and small interfering RNA, RhoA-ROCK, and p38MAPK-MSK1 activation is necessary for the induction of CDH1/E-cadherin, CYP24, and other genes and of an adhesive phenotype by 1,25(OH)(2)D(3). RhoA-ROCK and MSK1 are also required for the inhibition of Wnt-beta-catenin pathway and cell proliferation. Thus, the action of 1,25(OH)(2)D(3) on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA-ROCK and p38MAPK-MSK1.

  20. Vitamin D-induced ectodomain shedding of TNF receptor 1 as a nongenomic action: D3 vs D2 derivatives.

    PubMed

    Yang, Won Seok; Yu, Hoon; Kim, Jin Ju; Lee, Mee Jeong; Park, Su-Kil

    2016-01-01

    As a nongenomic action, 1,25-dihydroxyvitamin D3 (1,25D3) induces L-type Ca(2+) channel-mediated extracellular Ca(2+) influx in human aortic smooth muscle cells (HASMCs), which activates a disintegrin and metalloprotease 10 (ADAM10) to cleave and shed the ectodomain of tumor necrosis factor receptor 1 (TNFR1). In this study, we examined the potencies of other vitamin D3 and D2 analogs to stimulate the ectodomain shedding of TNFR1 in HASMCs. 25-Hydroxyvitamin D3 (25D3), a precursor of 1,25D3, and elocalcitol, an analog of 1,25D3, caused ectodomain shedding of TNFR1 within 30 min, whereas 1,25-dihydroxyvitamin D2 (1,25D2) and paricalcitol, a derivative of 1,25D2, did not. Both 25D3 and elocalcitol rapidly induced extracellular Ca(2+) influx and markedly increased intracellular Ca(2+), while 1,25D2 and paricalcitol caused only small increases in intracellular Ca(2+). 25D3- and elocalcitol-induced TNFR1 ectodomain sheddings were abolished by verapamil and in Ca(2+)-free media. Both 25D3 and elocalcitol caused the translocation of ADAM10 to the cell surface, which was inhibited by verapamil, while 1,25D2 and paricalcitol did not cause ADAM10 translocation. When ADAM10 was depleted by ADAM10-siRNA, 25D3 and elocalcitol could not induce ectodomain shedding of TNFR1. The plasma membrane receptor, endoplasmic reticulum stress protein 57 (ERp57), but not the classic vitamin D receptor, mediated the nongenomic action of vitamin D to induce ectodomain shedding of TNFR1. In summary, like 1,25D3, 25D3 and elocalcitol caused ADAM10-mediated ectodomain shedding of TNFR1, whereas 1,25D2 and paricalcitol did not. The difference may depend on their affinities to ERp57 through which extracellular Ca(2+) influx is induced.

  1. The generation of osteoclasts from RAW 264.7 precursors in defined, serum-free conditions.

    PubMed

    Vincent, Cristina; Kogawa, Masakazu; Findlay, David M; Atkins, Gerald J

    2009-01-01

    Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete alpha-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1alpha,25-dihydroxyvitamin D(3) (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.

  2. Meningioma 1 is required for appropriate osteoblast proliferation, motility, differentiation, and function.

    PubMed

    Zhang, Xiaoxue; Dowd, Diane R; Moore, Meika C; Kranenburg, Tanya A; Meester-Smoor, Magda A; Zwarthoff, Ellen C; MacDonald, Paul N

    2009-07-03

    The vitamin D endocrine system is essential for calcium and phosphate homeostasis and skeletal mineralization. The 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) hormone binds to the vitamin D receptor (VDR) to regulate gene expression. These gene products in turn mediate the actions of 1,25(OH)(2)D(3) in mineral-regulating target cells such as the osteoblast. We showed previously that meningioma 1 (MN1) is a novel target of 1,25(OH)(2)D(3) in MG-63 osteoblastic cells and that it is a coactivator for VDR-mediated transcription (Sutton, A. L., Zhang, X., Ellison, T. I., and MacDonald, P. N. (2005) Mol. Endocrinol. 19, 2234-2244). However, the functional significance of MN1 in osteoblastic cell biology is largely unknown. Here, we demonstrate that MN1 expression is increased dramatically during differentiation of primary osteoblastic cells. Using calvarial osteoblasts derived from wild-type and MN1 knock-out mice, we provide data supporting an essential role of MN1 in maintaining appropriate osteoblast proliferation, differentiation, and function. MN1 knock-out osteoblasts displayed altered morphology, decreased growth rate, impaired motility, and attenuated 1,25(OH)(2)D(3)/VDR-mediated transcription as well as reduced alkaline phosphatase activity and mineralized nodule formation. MN1 null osteoblasts were also impaired in supporting osteoclastogenesis in co-culture studies presumably because of marked reduction in the RANKL:OPG ratio in the MN1 null cells. Mechanistic studies supported a transcriptional role for MN1 in controlling RANKL gene expression through activation of the RANKL promoter. Cumulatively, these studies indicate an important role for MN1 in maintaining the appropriate maturation and function of calvarial osteoblasts.

  3. The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction

    PubMed Central

    Shimo, Tsuyoshi; Matsumoto, Kenichi; Takabatake, Kiyofumi; Aoyama, Eriko; Takebe, Yuichiro; Ibaragi, Soichiro; Okui, Tatsuo; Kurio, Naito; Takada, Hiroyuki; Obata, Kyoichi; Pang, Pai; Iwamoto, Masahiro; Nagatsuka, Hitoshi; Sasaki, Akira

    2016-01-01

    Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b+ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment. PMID:27007126

  4. Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1

    PubMed Central

    Chen, Yueqi; Sun, Jingjing; Dou, Ce; Li, Nan; Kang, Fei; Wang, Yuan; Cao, Zhen; Yang, Xiaochao; Dong, Shiwu

    2016-01-01

    The healthy skeleton requires a perfect coordination of the formation and degradation of bone. Metabolic bone disease like osteoporosis is resulted from the imbalance of bone formation and/or bone resorption. Osteoporosis also reflects lower level of bone matrix, which is contributed by up-regulated osteoclast-mediated bone resorption. It is reported that monocytes/macrophage progenitor cells or either hematopoietic stem cells (HSCs) gave rise to multinucleated osteoclasts. Thus, inhibition of osteoclastic bone resorption generally seems to be a predominant therapy for treating osteoporosis. Recently, more and more natural compounds have been discovered, which have the ability of inhibiting osteoclast differentiation and fusion. Alliin (S-allyl-l-cysteine sulfoxides, SACSO) is the major component of aged garlic extract (AGE), bearing broad-spectrum natural antioxidant properties. However, its effects on bone health have not yet been explored. Hence, we designed the current study to explore its effects and role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast fusion and differentiation. It was revealed that alliin had an inhibitory effect in osteoclasteogenesis with a dose-dependent manner via blocking the c-Fos-NFATc1 signaling pathway. In addition, alliin decreased the generation of reactive oxygen species (ROS) and down-regulated the expression of NADPH oxidase 1 (Nox1). The overall results revealed that alliin could be a potential therapeutic agent in the treatment of osteoporosis. PMID:27657047

  5. Osteocyte Alterations Induce Osteoclastogenesis in an In Vitro Model of Gaucher Disease

    PubMed Central

    Bondar, Constanza; Ormazabal, Maximiliano; Crivaro, Andrea; Ferreyra-Compagnucci, Malena; Delpino, María Victoria; Rozenfeld, Paula Adriana; Mucci, Juan Marcos

    2017-01-01

    Gaucher disease (GD) is caused by mutations in the glucosylceramidase β (GBA 1) gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to the accumulation of the glycolipid glucocerebroside in the lysosomes of cells, mainly in the monocyte/macrophage lineage. Its mildest form is Type I GD, characterized by non-neuronopathic involvement. Bone compromise is the most disabling aspect of the Gaucher disease. However, the pathophysiological aspects of skeletal alterations are not yet fully understood. The bone tissue homeostasis is maintained by a balance between resorption of old bone by osteoclasts and new bone formation by osteoblasts. A central player in this balance is the osteocyte as it controls both processes. We studied the involvement of osteocytes in an in vitro chemical model of Gaucher disease. The osteocyte cell line MLO-Y4 was exposed to conduritol-β-epoxide (CBE), an inhibitor of GCase, for a period of 7, 14 and 21 days. Conditioned media from CBE-treated osteocytes was found to induce osteoclast differentiation. GCase inhibition caused alterations in Cx43 expression and distribution pattern and an increase in osteocyte apoptosis. Osteoclast differentiation involved osteocyte apoptotic bodies, receptor activator of nuclear factor κ-B ligand (RANKL) and soluble factors. Thus, our results indicate that osteocytes may have a role to play in the bone pathophysiology of GD. PMID:28098793

  6. Signaling pathways in osteogenesis and osteoclastogenesis: Lessons from cranial sutures and applications to regenerative medicine

    PubMed Central

    Maxhimer, Justin B.; Bradley, James P.; Lee, Justine C.

    2015-01-01

    One of the simplest models for examining the interplay between bone formation and resorption is the junction between the cranial bones. Although only roughly a quarter of patients diagnosed with craniosynostosis have been linked to known genetic disturbances, the molecular mechanisms elucidated from these studies have provided basic knowledge of bone homeostasis. This work has translated to methods and advances in bone tissue engineering. In this review, we examine the current knowledge of cranial suture biology derived from human craniosynostosis syndromes and discuss its application to regenerative medicine. PMID:25961069

  7. OCLI-023, a Novel Pyrimidine Compound, Suppresses Osteoclastogenesis In Vitro and Alveolar Bone Resorption In Vivo

    PubMed Central

    Kim, Ju Ang; Lee, Doohyun; Kim, Nam Doo; Shin, Hong-In; Bae, Yong Chul; Park, Eui Kyun

    2017-01-01

    An abnormal increase in osteoclast differentiation and activation results in various bone-resorptive diseases, including periodontitis, rheumatoid arthritis, and osteoporosis. Chemical compounds containing pyrimidine ring have been shown to regulate a variety of biological processes. Therefore, in order to identify an antiresorptive agent, we synthesized a series of pyrimidine ring-containing chemical compounds, and found that OCLI-023 suppressed the differentiation and activation of osteoclasts in vitro. OCLI-023 directly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced differentiation of bone marrow macrophages into osteoclasts, without a cytotoxic response. OCLI-023 also downregulated the RANKL-induced mRNA expression of osteoclast markers as well as inhibited the formation of actin rings and resorption pits. OCLI-023 attenuated the RANKL-induced activation of c-Jun N-terminal kinase and nuclear factor kappa-light-chain-enhancer of activated B cell signaling pathways. In a mouse model of periodontitis, ligature induced an increase of distance between cementoenamel junction (CEJ) and alveolar bone crest (ABC) in the second molar, and OCLI-023 significantly reduced it. Histological analysis showed ligature-induced increase of osteoclast numbers was also significantly reduced by OCLI-023. These data demonstrated the inhibitory effect of OCLI-023 on osteoclast differentiation and activity of osteoclasts in vitro, as well as on ligature-induced bone loss in vivo, and OCLI-023 can be proposed as a novel anti-resorptive compound. PMID:28085946

  8. Osteoblast-specific expression of MEF induces osteopenia through downregulation of osteoblastogenesis and upregulation of osteoclastogenesis.

    PubMed

    Seul, Keyung-Jo; Cho, Hye-Sim; Heo, Sun-Hee; Baek, Wook-Young; Kim, Jung-Eun; Park, Eui Kyun; Choi, Je-Yong; Ryoo, Hyun-Mo; Cho, Je-Yoel

    2011-02-01

    In bone remodeling, various transcriptional factors are involved, and the deficiency or overexpression of some of these factors results in bone defects. Myeloid elf-1-like factor (MEF) is one of the Ets transcription factors that control the expression of genes that are critical for biologic processes such as cell proliferation, differentiation, and death. Previously, we reported that MEF promotes cell proliferation and functions as a negative regulator of osteogenic differentiation by interacting directly with Runx2 and suppressing its transcriptional activity. To investigate the in vivo function of MEF in bone formation and bone remodeling in vivo, we generated transgenic mice that overexpress MEF in osteoblasts under the control of the 2.3-kb Col1α1 promoter, named Col1α1-MEF. Femoral bone in Col1α1-MEF transgenic mice exhibited low bone mass with fewer trabecular bones and thinner and less developed cortical bones. The mineralized volume fraction (BV/TV) and bone-forming rate (BFR) were remarkably decreased to about 63% and 40%, respectively, in 6-week-old MEF transgenic mice compared with wild-type mice. In addition, reduced bone mineral density was observed in lumbar vertebrae of Col1α1-MEF transgenic mice. The number of TRACP(+) osteoclasts was increased in Col1α1-MEF transgenic mice and MEF-overexpressing MC3T3-E1 cells. All these in vivo results suggest that MEF suppresses bone formation by osteoblasts and facilitates bone resorption by activating osteoclasts indirectly.

  9. Screening of Korean medicinal plants for possible osteoclastogenesis effects in vitro

    PubMed Central

    Youn, Yu Na; Lim, Erang; Lee, Nari; Kim, Young Seop; Koo, Min Seon

    2007-01-01

    Bone undergoes continuous remodeling through bone formation and resorption, and maintaining the balance for skeletal rigidity. Bone resorption and loss are generally attributed to osteoclasts. Differentiation of osteoclasts is regulated by receptor activator of nuclear factor NF-kB ligand (RANKL), a member of tumor necrosis factor family. When the balance is disturbed, pathological bone abnormality ensues. Through the screening of traditional Korean medicinal plants, the effective molecules for inhibition and stimulation of RANKL-induced osteoclast differentiation in mouse bone marrow macrophages were identified. Among 222 methanol extracts, of medicinal plants, 10 samples exhibited ability to induce osteoclast differentiation. These include Dryobalanops aromatica, Euphoria longana, Lithospermum erythrorhizon, Prunus mume, Prunus nakaii, and Polygonatum odoratum. In contrast, Ailanthus altissima, Curcuma longa, Solanum nigrum, Taraxacum platycarpa, Trichosanthes kirilowii, and Daphne genkwa showed inhibitory effects in RANKL-induced osteoclast differentiation. PMID:18850234

  10. Heptamethoxyflavone, a citrus flavonoid, suppresses inflammatory osteoclastogenesis and alveolar bone resorption.

    PubMed

    Matsumoto, Chiho; Inoue, Hiroki; Tominari, Tsukasa; Watanabe, Kenta; Hirata, Michiko; Miyaura, Chisato; Inada, Masaki

    2015-01-01

    We examined the effects of heptamethoxyflavone (HMF), a citrus flavonoid on inflammatory bone resorption. HMF suppressed the osteoclast formation and PGE2 production induced by IL-1. In mouse calvarial organ cultures, HMF attenuated the bone resorption elicited by LPS. HMF suppressed bone resorption in the mandibular alveolar bone. HMF may protect against inflammatory bone loss such as periodontal disease.

  11. Effects of Osteoporosis-Inducing Drugs on Vitamin D-Related Gene Transcription and Mineralization in MG-63 and Saos-2 Cells.

    PubMed

    Wegler, Christine; Wikvall, Kjell; Norlin, Maria

    2016-11-01

    Vitamin D3 is important for calcium and phosphate homeostasis. To exert its effects, vitamin D3 has to be enzymatically activated into 1,25D3 (1,25-dihydroxyvitamin D3 ). Regulation by endogenous vitamin D metabolites of the activation and inactivation of 1,25D3 is important to maintain adequate amounts of active vitamin D3 . Vitamin D deficiency and low bone mineral density have been linked to treatments with antiretroviral drugs and glucocorticoids. However, the causes of drug-induced osteoporosis remain unclear. The antiretroviral drugs efavirenz and ritonavir as well as the glucocorticoid dexamethasone were included in this study. Their effects on transcription of vitamin D-regulating enzymes in MG-63 cells were investigated. Ritonavir and dexamethasone both induced transcription of CYP27B1, the enzyme responsible for the formation of 1,25D3 . Efavirenz, however, suppressed CYP27B1 expression. When administered together with endogenous vitamin D metabolites, dexamethasone and efavirenz counteracted the 1,25D3 -mediated up-regulation of CYP24A1, which inactivates 1,25D3 . This suggests that the drugs may interfere with local regulation of the vitamin D metabolizing system in osteoblasts. Studies on mineralization were performed in MG-63 cells and Saos-2 cells by measuring calcium concentrations accumulated over time. The effects of efavirenz, ritonavir and dexamethasone and/or vitamin D metabolites were examined. 1,25D3 induced mineralization in both cell lines. Efavirenz administered alone did not affect mineralization but suppressed the inducing effects of 1,25D3 on mineralization in both MG-63 cells and Saos-2 cells. In summary, the results suggest that antiretroviral drugs and glucocorticoids may adversely affect bone by interference with the vitamin D system in osteoblasts.

  12. Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.

    PubMed

    Lee, Seong Min; Bishop, Kathleen A; Goellner, Joseph J; O'Brien, Charles A; Pike, J Wesley

    2014-06-01

    The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

  13. Dietary Influences on Alpha-Methylacyl-CoA Racemase (AMACR) Expression in the Prostate

    DTIC Science & Technology

    2007-04-01

    activities of vitamin D. Up-regulation of MKP5 mRNA by 1,25- dihydroxyvitamin- D3 (1,25D) was dependent on the vitamin D receptor. We also identified a...serum levels of vitamin D metabolites to PCa incidence. Low serum levels of 1,25- dihydroxyvitamin D3 (1,25D), the active vitamin D metabolite, are...Cytogenet Cell Genet 2000;90:71–4. 25. Kim S, Shevde NK, Pike JW. 1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA

  14. Molecular Mechanism for Prostate Cancer Resistance to the Anti-tumor Activity of Vitamin D

    DTIC Science & Technology

    2006-11-01

    of MKP5 mRNA by 1,25- dihydroxyvitamin- D3 (1,25D) was dependent on the vitamin D receptor. We also identified a putative positive vitamin D response...to PCa incidence. Low serum levels of 1,25- dihydroxyvitamin D3 (1,25D), the active vitamin D metabolite, are associated with increased risk of PCa...Kim S, Shevde NK, Pike JW. 1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and

  15. Regulators of G protein signaling 12 (Rgs12) promotes osteoclastogenesis in bone remodeling and pathologic bone loss

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium (Ca2+) signaling plays a pivotal role in controlling various cellular processes such as secretion, differentiation, proliferation, motility, and cell death through the release of Ca2+ from internal stores and entry from extracellular fluid. In bone, receptor activator of NF-kB ligand (RANKL)...

  16. High-fat diet causes bone loss in young mice by promoting osteoclastogenesis through alteration of the bone marrow environment.

    PubMed

    Shu, Lei; Beier, Eric; Sheu, Tzong; Zhang, Hengwei; Zuscik, Michael J; Puzas, Edward J; Boyce, Brendan F; Mooney, Robert A; Xing, Lianping

    2015-04-01

    Obesity is a severe health problem in children, afflicting several organ systems including bone. However, the role of obesity on bone homeostasis and bone cell function in children has not been studied in detail. Here we used young mice fed a high-fat diet (HFD) to model childhood obesity and investigate the effect of HFD on the phenotype of cells within the bone marrow environment. Five-week-old male mice were fed a HFD for 3, 6, and 12 weeks. Decreased bone volume was detected after 3 weeks of HFD treatment. After 6 and 12 weeks, HFD-exposed mice had less bone mass and increased osteoclast numbers. Bone marrow cells, but not spleen cells, from HFD-fed mice had increased osteoclast precursor frequency, elevated osteoclast formation, and bone resorption activity, as well as increased expression of osteoclastogenic regulators including RANKL, TNF, and PPAR-gamma. Bone formation rate and osteoblast and adipocyte numbers were also increased in HFD-fed mice. Isolated bone marrow cells also had a corresponding elevation in the expression of positive regulators of osteoblast and adipocyte differentiation. Our findings indicate that in juvenile mice, HFD-induced bone loss is mainly due to increased osteoclast bone resorption by affecting the bone marrow microenvironment. Thus, targeting osteoclast formation may present a new therapeutic approach for bone complications in obese children.

  17. Retinoic acid therapy resistance progresses from unilineage to bilineage in HL-60 leukemic blasts.

    PubMed

    Jensen, Holly A; Bunaciu, Rodica P; Ibabao, Christopher N; Myers, Rebecca; Varner, Jeffrey D; Yen, Andrew

    2014-01-01

    Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10-20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or "precommitment" phase) or subsequently (the late or "lineage-commitment" phase). HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness (unilineage

  18. Reciprocal role of vitamin D receptor on β-catenin regulated keratinocyte proliferation and differentiation.

    PubMed

    Hu, Lizhi; Bikle, Daniel D; Oda, Yuko

    2014-10-01

    The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), suppresses the proliferation while promoting the differentiation of keratinocytes through the vitamin D receptor (VDR). β-Catenin, on the other hand, promotes proliferation and blocks epidermal differentiation, although it stimulates hair follicle differentiation. In intestinal epithelia VDR binds β-catenin and blocks its proliferative effects. In this study we investigated the role of 1,25(OH)2D3/VDR on β-catenin regulated gene transcription during keratinocyte proliferation and differentiation. 1,25(OH)2D3 suppressed promoter reporter activity driven by synthetic and natural TCF/β-catenin response elements. Over-expression of VDR further suppressed these TCF/β-catenin promoter activities. 1,25(OH)2D3 also suppressed the mRNA expression of the β-catenin regulated gene Gli1 through VDR. These data were consistent with our previous observations that VDR silencing resulted in keratinocyte hyperproliferation with increased expression of Gli1 in vitro, whereas VDR null skin showed hyperproliferation in vivo. In contrast, 1,25(OH)2D3 induced expression of another β-catenin regulated gene, PADI1, important for both epidermal and hair follicle differentiation. Deletion of VDR resulted in defects in hair differentiation in vivo, with decreased expression of β-catenin regulated hair differentiation genes such as PADI1, hair keratin KRT31 and calcium binding protein S100a3. These genes possess vitamin D response elements (VDRE) adjacent to TCF/β-catenin response elements and are regulated by both VDR and β-catenin signaling. Therefore, we propose that VDR and β-catenin interact reciprocally to promote VDR stimulation of genes involved with differentiation that contain both VDR and β-catenin response elements while inhibiting β-catenin stimulation of genes involved with proliferation. Thus the major finding of this study is that while 1,25(OH)2D3/VDR inhibits the actions of β-catenin to

  19. Metabolic profiling of major vitamin D metabolites using Diels-Alder derivatization and ultra-performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Aronov, Pavel A; Hall, Laura M; Dettmer, Katja; Stephensen, Charles B; Hammock, Bruce D

    2008-07-01

    Biologically active forms of vitamin D are important analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous metabolic profiling of entire biochemical pathways. The impediment to the metabolic profiling of vitamin D metabolites is the low level of 1alpha,25-dihydroxyvitamin D(3) in human serum (15-60 pg/mL). Here, we demonstrate that liquid-liquid or solid-phase extraction of vitamin D metabolites in combination with Diels-Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)-electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1alpha,25-dihydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(2), 24R,25-dihydroxyvitamin D(3), 25-hydroxyvitamin D(3) and 25-hydroxyvitamin D(2) in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6-4.8 % and 5-16 % for 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3), respectively, using solid-phase extraction.

  20. Effect of various vitamin D metabolites on serum calcium and inorganic phosphate in the freshwater snake Natrix piscator.

    PubMed

    Srivastav, A K; Srivastav, S K; Singh, S; Norman, A W

    1995-10-01

    Vitamin D3 (650 pmol and 6.50 nmol/100 g body wt), 25-hydroxyvitamin D (650 pmol and 6.50 nmol/100 g body wt), and 1,25-dihydroxyvitamin D (65 pmol and 650 pmol/100 g body wt) were administered daily to the freshwater snake Natrix piscator for 15 days. Both serum calcium and inorganic phosphate levels were increased significantly in all of the treated groups. This is the first report of hypercalcemia and hyperphosphatemia in reptiles induced by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

  1. Dietary Influences on Alpha-Methylacyl-CoA Racemase (AMACR) Expression in the Prostate

    DTIC Science & Technology

    2008-04-01

    regulation of MKP5 mRNA by 1,25- dihydroxyvitamin- D3 (1,25D) was dependent on the vitamin D receptor. We also identified a putative positive vitamin D...metabolites to PCa incidence. Low serum levels of 1,25- dihydroxyvitamin D3 (1,25D), the active vitamin D metabolite, are associated with increased risk of...Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and histone acetylation in intact

  2. Metabolic alkalosis transition in renal proximal tubule cells facilitates an increase in CYP27B1, while blunting responsiveness to PTH

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Parathyroid hormone (PTH) is the central activator of renal proximal 1-alpha-hydroxylase (CYP27B1), the enzyme responsible for synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Past studies have documented a disruption of CYP27B1 activity in chronic metabolic acidosis in vivo, while simulated ac...

  3. Syntheses of 24R,25-dihydroxy-(6,19,19-3H)vitamin D3 and 24R,25-dihydroxy-(6,19,19-2H)vitamin D3

    SciTech Connect

    Yamada, S.; Shimizu, M.; Fukushima, K.; Niimura, K.; Maeda, Y. )

    1989-08-01

    24R,25-Dihydroxy-(6,19,19-3H)vitamin D3 with a specific activity of 54 Ci/mmol and 24R,25-dihydroxy-(6,19,19-2H)vitamin D3 with 2.6 deuterium atoms/mol were synthesized in four steps starting from 24R,25-Dihydroxyvitamin D3 via its sulfur dioxide adduct.

  4. Novel vitamin D receptor ligands having a carboxyl group as an anchor to arginine 274 in the ligand-binding domain.

    PubMed

    Fujishima, Toshie; Tsuji, Genichiro; Tanaka, Chika; Harayama, Hiroshi

    2010-07-01

    Vitamin D3 is metabolized into the hormonally active form, 1alpha,25-dihydroxyvitamin D3 (1), via 25-hydroxyvitamin D3 (2) which is the most abundant circulating metabolite. Introduction of the 1alpha-hydroxyl group into 25-hydroxyvitamin D3 (2) to produce 1alpha,25-dihydroxyvitamin D3 (1) increases the VDR binding affinity by approximately 1000-fold. The X-ray crystal structure of human VDR in complex with 1alpha,25-dihydroxyvitamin D3 (1) shows that, together with Ser-237, the 1alpha-hydroxyl group of 1alpha,25-dihydroxyvitamin D3 (1) makes hydrogen bonds with Arg-274, single mutation of which results in impaired ligand recognition. In 2002, lithocholic acid, which possesses a carboxyl group at position C24, was demonstrated to be a weak VDR ligand. We speculated that the carboxylic acid of lithocholic acid could be recognized by Arg-274 in the ligand-binding domain of VDR. In view of the significance of Arg-274 to direct the 1alpha-hydroxyl group, as well as the results with lithocholic acid and its derivatives, we designed the C2 modified analogues of 25-hydroxylvitamin D3 (2) having a carboxyl group, instead of the 1-hydroxyl group, for better electrostatic interaction to the guanidinium side-chain of arginine.

  5. Vitamin D receptor in the paraventricular nucleus of the hypothalamus is necessary for beneficial effects of 1,25D[3] on peripheral glucose levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While a wide range of data correlates low vitamin D levels with type 2 diabetes, few studies examine potential mechanisms by which vitamin D might impact key aspects of metabolism. The active form of 1alpha,25-dihydroxyvitamin D[3] (1,25D[3]; calcitriol) is hydroxylated in the liver and kidney from ...

  6. The Role of Vitamin D Stimulation of Mullerian Inhibiting Substance (MIS) in Prostate Cancer Therapy

    DTIC Science & Technology

    2007-12-01

    Interestingly, in one VDR knockout mouse model uterine hypoplasia with impaired folliculogenesis was found in female reproductive organs but not vitamin...uterine hypoplasia and growth retardation after weaning. Nat Genet 16:391-396 4. Malloy PJ, Pike JW, Feldman D 2005 Hereditary 1,25-dihydroxyvitamin D

  7. The effect of vitamin D2 and vitamin D3 on intestinal calcium absorption in Nigerian children with rickets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Children with calcium-deficiency rickets have high 1,25-dihydroxyvitamin D values. The objective of the study was to determine whether vitamin D increased calcium absorption. This was an experimental study. The study was conducted at a teaching hospital. Participants included 17 children with nutrit...

  8. EB 1089, a novel vitamin D analog with strong antiproliferative and differentiation-inducing effects on target cells.

    PubMed

    Hansen, C M; Mäenpää, P H

    1997-12-01

    The physiologically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3, plays an important role not only in the establishment and maintenance of calcium metabolism, but also in regulating cell growth and differentiation. As the clinical usefulness of 1alpha,25-dihydroxyvitamin D3 is limited by its tendency to cause hypercalcemia, new analogs with a better therapeutic profile have been synthesized. One of these new synthetic vitamin D analogs is EB 1089, which is characterized by an altered side chain structure featuring 26,27-dimethyl groups and two double bonds. This analog has been shown to be more potent than 1,25-dihydroxyvitamin D3 in inhibiting proliferation, stimulating differentiation, and inducing apoptosis in a number of different cell types, including cancer cells. Despite being more potent than 1alpha,25-dihydroxyvitamin D3 with respect to its cell regulatory effects, EB 1089 displays weaker calcemic side-effects. These characteristics make EB 1089 a potentially useful compound for the treatment of a diversity of clinical disorders, including cancer and metabolic bone diseases. A promising phase I study with EB 1089 in patients with advanced breast and colon cancer has already been carried out, and more clinical trials evaluating the clinical effectiveness of EB 1089 in other types of cancer are in progress.

  9. Bad to the Bone: On In Vitro and Ex Vivo Microbial Biofilm Ability to Directly Destroy Colonized Bone Surfaces without Participation of Host Immunity or Osteoclastogenesis

    PubMed Central

    Junka, Adam; Szymczyk, Patrycja; Ziółkowski, Grzegorz; Karuga-Kuzniewska, Ewa; Smutnicka, Danuta; Bil-Lula, Iwona; Bartoszewicz, Marzenna; Mahabady, Susan; Sedghizadeh, Parish Paymon

    2017-01-01

    Bone infections are a significant public health burden associated with morbidity and mortality in patients. Microbial biofilm pathogens are the causative agents in chronic osteomyelitis. Research on the pathogenesis of osteomyelitis has focused on indirect bone destruction by host immune cells and cytokines secondary to microbial insult. Direct bone resorption by biofilm pathogens has not yet been seriously considered. In this study, common osteomyelitis pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans) were grown as biofilms in multiple in vitro and ex vivo experiments to analyze quantitative and qualitative aspects of bone destruction during infection. Pathogens were grown as single or mixed species biofilms on the following substrates: hydroxyapatite, rat jawbone, or polystyrene wells, and in various media. Biofilm growth was evaluated by scanning electron microscopy and pH levels were monitored over time. Histomorphologic and quantitative effects of biofilms on tested substrates were analyzed by microcomputed tomography and quantitative cultures. All tested biofilms demonstrated significant damage to bone. Scanning electron microscopy indicated that all strains formed mature biofilms within 7 days on all substrate surfaces regardless of media. Experimental conditions impacted pH levels, although this had no impact on biofilm growth or bone destruction. Presence of biofilm led to bone dissolution with a decrease of total volume by 20.17±2.93% upon microcomputed tomography analysis, which was statistically significant as compared to controls (p <0.05, ANOVA). Quantitative cultures indicated that media and substrate did not impact biofilm formation (Kruskall-Wallis test, post-hoc Dunne’s test; p <0.05). Overall, these results indicate that biofilms associated with osteomyelitis have the ability to directly resorb bone. These findings should lead to a more complete understanding of the etiopathogenesis of osteomyelitis, where direct bone resorption by biofilm is considered in addition to the well-known osteoclastic and host cell destruction of bone. PMID:28076372

  10. The blocking of uPAR suppresses lipopolysaccharide‐induced inflammatory osteoclastogenesis and the resultant bone loss through attenuation of integrin β3/Akt pathway

    PubMed Central

    Ishisaki, Akira; Miyashita, Mei; Matsuo, Osamu

    2016-01-01

    Abstract Introduction Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis, cause the bone destruction by promotion of the differentiation of monocyte/macrophage lineage cells into mature osteoclasts (OCs) with active bone‐resorbing character. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated the role of urokinase plasminogen activator receptor (uPAR) in the bone destruction caused by chronic inflammation. Methods We investigated that the effect of uPAR on inflammatory OC formation induced by lipopolysaccharide (LPS) in inflammatory diseases. Results We found that the LPS more weakly induced OC formation and the resultant bone loss in uPAR‐deficient mice than in wild‐type mice. Additionally, we demonstrated that uPAR significantly potentiated LPS‐induced OC formation of RAW264.7 mouse monocyte/macrophage linage cells in integrin β3/Akt‐dependent manner. Moreover, we showed that the blocking of uPAR function by the administration of anti‐uPAR neutralizing antibody significantly attenuated the LPS‐induced OC formation and the resultant bone loss in mice. Conclusions These results strongly suggest that uPAR negatively regulates the LPS‐induced inflammatory OC formation and the resultant bone loss mediated through the integrin β3/Akt pathway. Our findings partly clarify the molecular mechanisms underlying bone destruction caused by chronic inflammatory diseases, and would benefit research on identifying antibody therapy for the treatment of these diseases. PMID:27621816

  11. Insulin enhances RANKL-induced osteoclastogenesis via ERK1/2 activation and induction of NFATc1 and Atp6v0d2.

    PubMed

    Oh, Ju Hee; Lee, Jae Yoon; Joung, Seung Hee; Oh, Yoon Taek; Kim, Hong Sung; Lee, Na Kyung

    2015-12-01

    Insulin is one of the main factors affecting bone and energy metabolism, however, the direct effect of insulin on osteoclast differentiation remains unclear. Thus, in order to help elucidate that puzzle, the authors investigated the roles and regulatory mechanisms of insulin on osteoclasts differentiation. Co-stimulation with insulin and RANKL significantly enhanced the number of larger (>100 μm) osteoclastic cells and of TRAP-positive multinucleated cells compared with treatment by RANKL alone. Conversely, the insulin receptor shRNA markedly decreased osteoclast differentiation induced by insulin and RANKL. Insulin treatment significantly activated ERK1/2 MAP kinase as well as markedly induced the expression of NFATc1, an osteoclast marker gene, and Atp6v0d2, an osteoclast fusion-related gene. The pretreatment of PD98059, an ERK1/2 inhibitor, or insulin receptor shRNA effectively suppressed osteoclast differentiation and, in addition, blocked the expression of NFATc1 and Atp6vod2 induced by insulin stimulation. These data reveal insights into the regulation of osteoclast differentiation and fusion through ERK1/2 activation and the induction of NFATc1 and Atp6v0d2 by insulin.

  12. Rebamipide Attenuates Mandibular Condylar Degeneration in a Murine Model of TMJ-OA by Mediating a Chondroprotective Effect and by Downregulating RANKL-Mediated Osteoclastogenesis

    PubMed Central

    Izawa, Takashi; Mori, Hiroki; Shinohara, Tekehiro; Mino-Oka, Akiko; Hutami, Islamy Rahma; Iwasa, Akihiko; Tanaka, Eiji

    2016-01-01

    Temporomandibular joint osteoarthritis (TMJ-OA) is characterized by progressive degradation of cartilage and changes in subchondral bone. It is also one of the most serious subgroups of temporomandibular disorders. Rebamipide is a gastroprotective agent that is currently used for the treatment of gastritis and gastric ulcers. It scavenges reactive oxygen radicals and has exhibited anti-inflammatory potential. The aim of this study was to investigate the impact of rebamipide both in vivo and in vitro on the development of cartilage degeneration and osteoclast activity in an experimental murine model of TMJ-OA, and to explore its mode of action. Oral administration of rebamipide (0.6 mg/kg and 6 mg/kg) was initiated 24 h after TMJ-OA was induced, and was maintained daily for four weeks. Rebamipide treatment was found to attenuate cartilage degeneration, to reduce the number of apoptotic cells, and to decrease the expression levels of matrix metalloproteinase-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in TMJ-OA cartilage in a dose-dependent manner. Rebamipide also suppressed the activation of transcription factors (e.g., NF-κB, NFATc1) and mitogen-activated protein kinases (MAPK) by receptor activator of nuclear factor kappa-B ligand (RANKL) to inhibit the differentiation of osteoclastic precursors, and disrupted the formation of actin rings in mature osteoclasts. Together, these results demonstrate the inhibitory effects of rebamipide on cartilage degradation in experimentally induced TMJ-OA. Furthermore, suppression of oxidative damage, restoration of extracellular matrix homeostasis of articular chondrocytes, and reduced subchondral bone loss as a result of blocked osteoclast activation suggest that rebamipide is a potential therapeutic strategy for TMJ-OA. PMID:27123995

  13. Decreased osteoclastogenesis and high bone mass in mice with impaired insulin clearance due to liver-specific inactivation to CEACAM1.

    PubMed

    Huang, S; Kaw, M; Harris, M T; Ebraheim, N; McInerney, M F; Najjar, S M; Lecka-Czernik, B

    2010-04-01

    Type 2 diabetes is associated with normal-to-higher bone mineral density (BMD) and increased rate of fracture. Hyperinsulinemia and hyperglycemia may affect bone mass and quality in the diabetic skeleton. In order to dissect the effect of hyperinsulinemia from the hyperglycemic impact on bone homeostasis, we have analyzed L-SACC1 mice, a murine model of impaired insulin clearance in liver causing hyperinsulinemia and insulin resistance without fasting hyperglycemia. Adult L-SACC1 mice exhibit significantly higher trabecular and cortical bone mass, attenuated bone formation as measured by dynamic histomorphometry, and reduced number of osteoclasts. Serum levels of bone formation (BALP) and bone resorption markers (TRAP5b and CTX) are decreased by approximately 50%. The L-SACC1 mutation in the liver affects myeloid cell lineage allocation in the bone marrow: the (CD3(-)CD11b(-)CD45R(-)) population of osteoclast progenitors is decreased by 40% and the number of (CD3(-)CD11b(-)CD45R(+)) B-cell progenitors is increased by 60%. L-SACC1 osteoclasts express lower levels of c-fos and RANK and their differentiation is impaired. In vitro analysis corroborated a negative effect of insulin on osteoclast recruitment, maturation and the expression levels of c-fos and RANK transcripts. Although bone formation is decreased in L-SACC1 mice, the differentiation potential and expression of the osteoblast-specific gene markers in L-SACC1-derived mesenchymal stem cells (MSC) remain unchanged as compared to the WT. Interestingly, however, MSC from L-SACC1 mice exhibit increased PPARgamma2 and decreased IGF-1 transcript levels. These data suggest that high bone mass in L-SACC1 animals results, at least in part, from a negative regulatory effect of insulin on bone resorption and formation, which leads to decreased bone turnover. Because low bone turnover contributes to decreased bone quality and an increased incidence of fractures, studies on L-SACC1 mice may advance our understanding of altered bone homeostasis in type 2 diabetes.

  14. Fisetin inhibits osteoclastogenesis through prevention of RANKL-induced ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes.

    PubMed

    Sakai, Eiko; Shimada-Sugawara, Megumi; Yamaguchi, Yu; Sakamoto, Hiroshi; Fumimoto, Reiko; Fukuma, Yutaka; Nishishita, Kazuhisa; Okamoto, Kuniaki; Tsukuba, Takayuki

    2013-01-01

    Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor. We recently demonstrated that regulation of heme-oxygenase 1 (HO-1), a stress-induced cytoprotective enzyme, also functions in OCL differentiation. In this study, we investigated effects of fisetin, a natural bioactive flavonoid that has been reported to induce HO-1 expression, on the differentiation of macrophages into OCLs. Fisetin inhibited the formation of OCLs in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Moreover, fisetin-treated OCLs showed markedly decreased phosphorylation of extracellular signal-regulated kinase, Akt, and Jun N-terminal kinase, but fisetin did not inhibit p38 phosphorylation. Fisetin up-regulated mRNA expression of phase II antioxidant enzymes including HO-1 and interfered with RANKL-mediated reactive oxygen species (ROS) production. Studies with RNA interference showed that suppression of NF-E2-related factor 2 (Nrf2), a key transcription factor for phase II antioxidant enzymes, rescued fisetin-mediated inhibition of OCL differentiation. Furthermore, fisetin significantly decreased RANKL-induced nuclear translocation of cFos and nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a transcription factor critical for osteoclastogenic gene regulation. Therefore, fisetin inhibits OCL differentiation through blocking RANKL-mediated ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes.

  15. Bad to the Bone: On In Vitro and Ex Vivo Microbial Biofilm Ability to Directly Destroy Colonized Bone Surfaces without Participation of Host Immunity or Osteoclastogenesis.

    PubMed

    Junka, Adam; Szymczyk, Patrycja; Ziółkowski, Grzegorz; Karuga-Kuzniewska, Ewa; Smutnicka, Danuta; Bil-Lula, Iwona; Bartoszewicz, Marzenna; Mahabady, Susan; Sedghizadeh, Parish Paymon

    2017-01-01

    Bone infections are a significant public health burden associated with morbidity and mortality in patients. Microbial biofilm pathogens are the causative agents in chronic osteomyelitis. Research on the pathogenesis of osteomyelitis has focused on indirect bone destruction by host immune cells and cytokines secondary to microbial insult. Direct bone resorption by biofilm pathogens has not yet been seriously considered. In this study, common osteomyelitis pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans) were grown as biofilms in multiple in vitro and ex vivo experiments to analyze quantitative and qualitative aspects of bone destruction during infection. Pathogens were grown as single or mixed species biofilms on the following substrates: hydroxyapatite, rat jawbone, or polystyrene wells, and in various media. Biofilm growth was evaluated by scanning electron microscopy and pH levels were monitored over time. Histomorphologic and quantitative effects of biofilms on tested substrates were analyzed by microcomputed tomography and quantitative cultures. All tested biofilms demonstrated significant damage to bone. Scanning electron microscopy indicated that all strains formed mature biofilms within 7 days on all substrate surfaces regardless of media. Experimental conditions impacted pH levels, although this had no impact on biofilm growth or bone destruction. Presence of biofilm led to bone dissolution with a decrease of total volume by 20.17±2.93% upon microcomputed tomography analysis, which was statistically significant as compared to controls (p <0.05, ANOVA). Quantitative cultures indicated that media and substrate did not impact biofilm formation (Kruskall-Wallis test, post-hoc Dunne's test; p <0.05). Overall, these results indicate that biofilms associated with osteomyelitis have the ability to directly resorb bone. These findings should lead to a more complete understanding of the etiopathogenesis of osteomyelitis, where direct bone resorption by biofilm is considered in addition to the well-known osteoclastic and host cell destruction of bone.

  16. The Vitamin D Deficiency Pandemic and Consequences for Nonskeletal Health: Mechanisms of Action

    PubMed Central

    Holick, Michael F.

    2008-01-01

    Vitamin D, the sunshine vitamin, is important for childhood bone health. Over the past two decades, it is now recognized that vitamin D not only is important for calcium metabolism and maintenance of bone health throughout life, but also plays an important role in reducing risk of many chronic diseases including type I diabetes, multiple sclerosis, rheumatoid arthritis, deadly cancers, heart disease and infectious diseases. How vitamin D is able to play such an important role in health is based on observation that all tissues and cells in the body have a vitamin D receptor, and, thus, respond to its active form 1,25-dihydroxyvitamin D. However, this did not explain how living at higher latitudes and being at risk of vitamin D deficiency increased risk of these deadly diseases since it was also known that the 1,25-dihydroxyvitamin D levels are normal or even elevated when a person is vitamin D insufficient. Moreover, increased intake of vitamin D or exposure to more sunlight will not induce the kidneys to produce more 1,25-dihydroxyvitamin D. The revelation that the colon, breast, prostate, macrophages and skin among other organs have the enzymatic machinery to produce 1,25-dihydroxyvitamin D provides further insight as to how vitamin D plays such an essential role for overall health and well being. This review will put into perspective many of the new biologic actions of vitamin D and on how 1,25-dihydroxyvitamin D is able to regulate directly or indirectly more than 200 different genes that are responsible for a wide variety of biologic processes. PMID:18801384

  17. Thiazide diuretics affect osteocalcin production in human osteoblasts at the transcription level without affecting vitamin D3 receptors.

    PubMed

    Lajeunesse, D; Delalandre, A; Guggino, S E

    2000-05-01

    Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1-100 microM) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced OC release by these cells (maximal effect, -40-50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (-47.2+/-4.0%; p < 0.0001 by paired ANOVA with 100 microM 13.6+/-0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, -22.5+/-6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1-100 microM) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed

  18. Effect of exchange transfusions with citrated blood on plasma concentrations of vitamin D metabolites in neonates.

    PubMed

    Markestad, T; Aksnes, L; Finne, P H; Aarskog, D

    1984-05-01

    The plasma concentrations of 25-hydroxyvitamin D (25-OHD), 1,25-dihydroxyvitamin D (1,25-(OH)2D), and 24,25-dihydroxyvitamin D (24,25-(OH)2D) were determined pre- and postexchange , and in donors' blood in 10 blood exchange transfusions with citrated blood for neonatal hyperbilirubinemia. The postexchange concentrations of 25-OHD and 24,25-(OH)2D were intermediate between the levels before exchange and in donors' blood. Before therapy, the 1,25-(OH)2D concentrations were higher in the infants' than in donors' blood, and the pre-exchange levels were re-established during the procedure. The results suggest that postexchange concentrations of 25-OHD and 24,25-(OH)2D could be explained on the basis of redistribution of the metabolites between plasma and extravascular pools, whereas de novo synthesis was the most likely cause for the restoration of 1,25-(OH)2D levels.

  19. Vitamin D metabolites in idiopathic infantile hypercalcaemia.

    PubMed Central

    Martin, N D; Snodgrass, G J; Cohen, R D; Porteous, C E; Coldwell, R D; Trafford, D J; Makin, H L

    1985-01-01

    Metabolites of vitamin D were measured in plasma from 83 patients with idiopathic infantile hypercalcaemia syndrome who were mentally handicapped but had normal calcium values at the time of the study. No significant difference was detected in the mean plasma concentrations of 25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D3, or 25,26-dihydroxyvitamin D3 between patients and age matched controls. The mean plasma concentration of 25-hydroxyvitamin D3 was significantly lower in patients than controls but this may be a secondary phenomenon related to less sunlight exposure. In addition, two hypercalcaemic patients with this syndrome were studied during the first year of life, and were found to have normal concentrations of vitamin D metabolites. These findings do not support a role for abnormal vitamin D metabolism in the pathogenesis of this syndrome. PMID:3879160

  20. Bone mineralization is regulated by signaling cross talk between molecular factors of local and systemic origin: the role of fibroblast growth factor 23.

    PubMed

    Sapir-Koren, Rony; Livshits, Gregory

    2014-01-01

    Body phosphate homeostasis is regulated by a hormonal counter-balanced intestine-bone-kidney axis. The major systemic hormones involved in this axis are parathyroid hormone (PTH), 1,25-dihydroxyvitamin-D, and fibroblast growth factor-23 (FGF23). FGF23, produced almost exclusively by the osteocytes, is a phosphaturic hormone that plays a major role in regulation of the bone remodeling process. Remodeling composite components, bone mineralization and resorption cycles create a continuous influx-efflux loop of the inorganic phosphate (Pi) through the skeleton. This "bone Pi loop," which is formed, is controlled by local and systemic factors according to phosphate homeostasis demands. Although FGF23 systemic actions in the kidney, and for the production of PTH and 1,25-dihydroxyvitamin-D are well established, its direct involvement in bone metabolism is currently poorly understood. This review presents the latest available evidence suggesting two aspects of FGF23 bone local activity: (a) Regulation of FGF23 production by both local and systemic factors. The suggested local factors include extracellular levels of Pi and pyrophosphate (PPi), (the Pi/PPi ratio), and another osteocyte-derived protein, sclerostin. In addition, 1,25-dihydroxyvitamin-D, synthesized locally by bone cells, may contribute to regulation of FGF23 production. The systemic control is achieved via PTH and 1,25-dihydroxyvitamin-D endocrine functions. (b) FGF23 acts as a local agent, directly affecting bone mineralization. We support the assumption that under balanced physiological conditions, sclerostin, by para- autocrine signaling, upregulates FGF23 production by the osteocyte. FGF23, in turn, acts as a mineralization inhibitor, by stimulating the generation of the major mineralization antagonist-PPi.

  1. [Current Topics on Vitamin D. Cross talks among vitamin D endocrine system, PTH and FGF23].

    PubMed

    Fukumoto, Seiji

    2015-03-01

    1,25-dihydroxyvitamin D and parathyroid hormone have been known to be essential for maintaining serum calcium and phosphate levels. In addition, fibroblast growth factor 23 was shown to work as a phosphotropic hormone. These hormones work through activating different intracellular signaling pathways, but regulate their productions each other directly or indirectly via changes in serum mineral levels. Fine regulation of these hormone actions seems to be essential for maintaining serum mineral levels.

  2. Dahl salt-sensitive rats develop hypovitaminosis D and hyperparathyroidism when fed a standard diet

    NASA Technical Reports Server (NTRS)

    Thierry-Palmer, Myrtle; Cephas, Stacy; Sayavongsa, Phouyong; Doherty, Akins; Arnaud, Sara B.

    2005-01-01

    The Dahl salt-sensitive rat (S), a model for salt-sensitive hypertension, excretes protein-bound 25-hydroxyvitamin D (25-OHD) into urine when fed a low salt diet. Urinary 25-OHD increases during high salt intake. We tested the hypothesis that continuous loss of 25-OHD into urine would result in low plasma 25-OHD concentration in mature S rats raised on a standard diet. Dahl S and salt-resistant (R) male rats were raised to maturity (12-month-old) on a commercial rat diet (1% salt) and switched to 0.3% (low) or 2% (high) salt diets 3 weeks before euthanasia. Urine (24 h) was collected at the end of the dietary treatments. Urinary 25-OHD and urinary 25-OHD binding activity of S rats were three times that of R rats, resulting in lower plasma 25-OHD and 24,25-dihydroxyvitamin D concentrations in S rats than in R rats (P < 0.001). Plasma parathyroid hormone concentrations of S rats were twice that of R rats. S rats fed 2% salt had higher plasma 1,25-dihydroxyvitamin D concentrations than those fed 0.3% salt (P = 0.002). S rats excreted more calcium into urine than R rats (P < 0.001) and did not exhibit the expected calciuric response to salt. Proteinuria of the S rats was three times that of the R rats, suggesting kidney damage in the S rats. Low plasma 25-OHD and 24,25-dihydroxyvitamin D and high plasma 1,25-dihydroxyvitamin D and PTH concentrations seen in the mature S rats have also been reported for elderly patients with low-renin (salt-induced) hypertension. An implication of this study is that low vitamin D status may occur with age in salt-sensitive individuals, even when salt intake is normal.

  3. Sunlight and Vitamin D

    PubMed Central

    Wacker, Matthias; Holick, Michael F.

    2013-01-01

    Vitamin D is the sunshine vitamin that has been produced on this earth for more than 500 million years. During exposure to sunlight 7-dehydrocholesterol in the skin absorbs UV B radiation and is converted to previtamin D3 which in turn isomerizes into vitamin D3. Previtamin D3 and vitamin D3 also absorb UV B radiation and are converted into a variety of photoproducts some of which have unique biologic properties. Sun induced vitamin D synthesis is greatly influenced by season, time of day, latitude, altitude, air pollution, skin pigmentation, sunscreen use, passing through glass and plastic, and aging. Vitamin D is metabolized sequentially in the liver and kidneys into 25-hydroxyvitamin D which is a major circulating form and 1,25-dihydroxyvitamin D which is the biologically active form respectively. 1,25-dihydroxyvitamin D plays an important role in regulating calcium and phosphate metabolism for maintenance of metabolic functions and for skeletal health. Most cells and organs in the body have a vitamin D receptor and many cells and organs are able to produce 1,25-dihydroxyvitamin D. As a result 1,25-dihydroxyvitamin D influences a large number of biologic pathways which may help explain association studies relating vitamin D deficiency and living at higher latitudes with increased risk for many chronic diseases including autoimmune diseases, some cancers, cardiovascular disease, infectious disease, schizophrenia and type 2 diabetes. A three-part strategy of increasing food fortification programs with vitamin D, sensible sun exposure recommendations and encouraging ingestion of a vitamin D supplement when needed should be implemented to prevent global vitamin D deficiency and its negative health consequences. PMID:24494042

  4. Vitamin D-Prostaglandin Interactions and Effects in Prostate Cancer

    DTIC Science & Technology

    2006-10-01

    Abstract 1,25-dihydroxyvitamin D3 (calcitriol), the active form of vitamin D3 is a hormone with anti-proliferative and pro- differentiation...use of reduced doses of both drugs that still resulted in enhanced anti-proliferative activity . The actions of calcitriol to reduce COX-2 expression...and to induce 15-PGDH availability would potentially constitute a pathway to reduce and/or remove active PGs thereby diminishing PCa proliferation

  5. CYP24A1 — EDRN Public Portal

    Cancer.gov

    The CYP24A1 protein is a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. CYP24A1 is involved in maintaining calcium homeostasis and catalyzes the NADPH-dependent 24-hydroxylation of calcidiol (25-hydroxyvitamin D3) and calcitriol (1-alpha,25-dihydroxyvitamin D3). Several different isoforms exist, encoded by alternatively spliced transcript variants.

  6. Quantitative analytical method to evaluate the metabolism of vitamin D.

    PubMed

    Mena-Bravo, A; Ferreiro-Vera, C; Priego-Capote, F; Maestro, M A; Mouriño, A; Quesada-Gómez, J M; Luque de Castro, M D

    2015-03-10

    A method for quantitative analysis of vitamin D (both D2 and D3) and its main metabolites - monohydroxylated vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) and dihydroxylated metabolites (1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3) in human serum is here reported. The method is based on direct analysis of serum by an automated platform involving on-line coupling of a solid-phase extraction workstation to a liquid chromatograph-tandem mass spectrometer. Detection of the seven analytes was carried out by the selected reaction monitoring (SRM) mode, and quantitative analysis was supported on the use of stable isotopic labeled internal standards (SIL-ISs). The detection limits were between 0.3-75pg/mL for the target compounds, while precision (expressed as relative standard deviation) was below 13.0% for between-day variability. The method was externally validated according to the vitamin D External Quality Assurance Scheme (DEQAS) through the analysis of ten serum samples provided by this organism. The analytical features of the method support its applicability in nutritional and clinical studies targeted at elucidating the role of vitamin D metabolism.

  7. Inhibition of poly(adenosine diphosphate-ribose) polymerase by the active form of vitamin D

    PubMed Central

    MABLEY, JON G.; WALLACE, REBECCA; PACHER, PÁL; MURPHY, KANNEGANTI; SZABÓ, CSABA

    2008-01-01

    Vitamin D is well characterized for its role in mineral homeostasis and maintenance of normal skeletal architecture. Vitamin D has been demonstrated to exert anti-inflammatory effects in a variety of disease states including diabetes, arthritis and inflammatory bowel disease. In these diseases poly[adenosine diphosphate (ADP)-ribose] polymerase (PARP) inhibitors have also proved effective as anti-inflammatory agents. Here we present data demonstrating that the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D3, is a PARP inhibitor. UV irradiation-mediated PARP activation in human keratinocytes can be inhibited by treatment with vitamin D, 7-dehydrocholesterol or 1α,25-dihydroxyvitamin D3. Inhibition of cytochrome P450 reversed the PARP inhibitory action of vitamin D and 7-dehydrocholesterol, indicating that conversion to 1α,25-dihydroxyvitamin D3 mediates their PARP inhibitory action. Vitamin D may protect keratinocytes against over-activation of PARP resulting from exposure to sunlight. PARP inhibition may contribute to the pharmacological and anti-inflammatory effects of vitamin D. PMID:17487428

  8. Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture.

    PubMed

    Ritchie, H H; Park, H; Liu, J; Bervoets, T J M; Bronckers, A L J J

    2004-09-17

    Vitamin A, 1,25-dihydroxyvitamin D3 and dexamethasone are well-characterized hydrophobic molecules whose biological actions are mediated via different members of the nuclear hormone receptor family. We report here their actions on tooth formation at the molecular level. We have tested the effects of these compounds on osteopontin (OPN), dentin sialoprotein (DSP-PP), and collagen type I expression in pre-mineralization and mineralization stage rat tooth organ cultures which mirror in vivo developmental patterns. These proteins are all believed to participate in the mineralization of dentin. 1,25-Dihydroxyvitamin D3 up-regulated OPN, but had no effect on DSP-PP mRNA expression. Vitamin A up-regulated DSP-PP expression as did dexamethasone. Dexamethasone also up-regulated collagen type I expression. Our results suggest that 1,25-dihydroxyvitamin D3 does not modulate dentin mineralization by directly affecting DSP-PP expression. Vitamin A likely contributes to dentin mineralization by up-regulating DSP-PP expression. Finally, the up-regulation of DSP-PP expression in tooth germ cultures treated with dexamethasone suggests that its application to patient's dental pulp might promote increased extracellular matrix synthesis and mineralization in the pulp and may explain the narrowing of the dental pulp cavity in patients undergoing long-term dexamethasone administration.

  9. Two-dimensional liquid chromatography coupled to tandem mass spectrometry for vitamin D metabolite profiling including the C3-epimer-25-monohydroxyvitamin D3.

    PubMed

    Mena-Bravo, A; Priego-Capote, F; Luque de Castro, M D

    2016-06-17

    A method based on automated on-line solid phase extraction coupled to two-dimensional liquid chromatography with tandem mass spectrometry detection (SPE-2DLC-MS/MS) is here reported for vitamin D metabolite profiling in human serum with absolute quantitation. Two-dimensional LC was configured with two complementary analytical columns, pentafluorophenyl (PFP) and C18 phases, for determination of 25 hydroxyvitamin D3 epimers and the resting bioactive metabolites of vitamin D (D3 and D2)-25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D2 and 24,25-dihydroxyvitamin D3. Quantitative determination was supported on the use of a stable isotopic labelled internal standard for each analyte and the resulting method was validated by analysis of a standard reference material certified by the National Institute of Standards & Technology (NIST-972a) and 5 samples provided by the vitamin D External Quality Assurance Scheme (DEQAS). The limits of detection were between 9 and 90pg/mL for the eight analytes, and precision, expressed as relative standard deviation, was lower than 11.6%. Two-dimensional LC has shown to be the key to discriminate between 25 hydroxyvitamin D3 epimers in a quantitative analysis also involving dihydroxyvitamin D metabolites.

  10. Coupling energy metabolism with a mechanism to support brain-derived neurotrophic factor-mediated synaptic plasticity.

    PubMed

    Vaynman, S; Ying, Z; Wu, A; Gomez-Pinilla, F

    2006-01-01

    Synaptic plasticity and behaviors are likely dependent on the capacity of neurons to meet the energy demands imposed by neuronal activity. We used physical activity, a paradigm intrinsically associated with energy consumption/expenditure and cognitive enhancement, to study how energy metabolism interacts with the substrates for neuroplasticity. We found that in an area critical for learning and memory, the hippocampus, exercise modified aspects of energy metabolism by decreasing oxidative stress and increasing the levels of cytochrome c oxidase-II, a specific component of mitochondrial machinery. We infused 1,25-dihydroxyvitamin D3, a modulator of energy metabolism, directly into the hippocampus during 3 days of voluntary wheel running and measured its effects on brain-derived neurotrophic factor-mediated synaptic plasticity. Brain-derived neurotrophic factor is a central player for the effects of exercise on synaptic and cognitive plasticity. We found that 25-dihydroxyvitamin D3 decreased exercise-induced brain-derived neurotrophic factor but had no significant effect on neurotrophin-3 levels, thereby suggesting a level of specificity for brain-derived neurotrophic factor in the hippocampus. 25-Dihydroxyvitamin D3 injection also abolished the effects of exercise on the consummate end-products of brain-derived neurotrophic factor action, i.e. cyclic AMP response element-binding protein and synapsin I, and modulated phosphorylated calmodulin protein kinase II, a signal transduction cascade downstream to brain-derived neurotrophic factor action that is important for learning and memory. We also found that exercise significantly increased the expression of the mitochondrial uncoupling protein 2, an energy-balancing factor concerned with ATP production and free radical management. Our results reveal a fundamental mechanism by which key elements of energy metabolism may modulate the substrates of hippocampal synaptic plasticity.

  11. An Inducible Cytochrome P450 3A4-Dependent Vitamin D Catabolic Pathway

    PubMed Central

    Wang, Zhican; Lin, Yvonne S.; Zheng, Xi Emily; Senn, Tauri; Hashizume, Takanori; Scian, Michele; Dickmann, Leslie J.; Nelson, Sidney D.; Baillie, Thomas A.; Hebert, Mary F.; Blough, David; Davis, Connie L.

    2012-01-01

    Vitamin D3 is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D3 (25OHD3), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD3 was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD3 hydroxylation by human liver microsomes, with the formation of 4β,25-dihydroxyvitamin D3 [4β,25(OH)2D3] dominating (Vmax/Km = 0.85 ml · min−1 · nmol enzyme−1). 4β,25(OH)2D3 was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D3, and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4β,25(OH)2D3 in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4β,25(OH)2D3, but not CYP24A1-dependent 24R,25-dihydroxyvitamin D3 formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD3 metabolism may play an important role in the regulation of vitamin D3 in vivo and in the etiology of drug-induced osteomalacia. PMID:22205755

  12. Characterization of the metabolic pathway of 1,25-dihydroxy-16-ene vitamin D3 in rat kidney by on-line high performance liquid chromatography-electrospray tandem mass spectrometry.

    PubMed

    Yeung, B; Vouros, P; Siu-Caldera, M L; Reddy, G S

    1995-04-18

    1,25-Dihydroxy-16-ene vitamin D3 is a synthetic analog of 1,25-dihydroxyvitamin D3, the most physiologically active metabolite of vitamin D3. The renal metabolism of 1,25-dihydroxy-16-ene vitamin D3 had been studied previously using a perfused rat kidney system [Reddy et al., Bioorg Med Chem Lett 3: 1879-1884, 1993], and its C-24 oxidative metabolic pathway had been found to be different from that of 1,25-dihydroxyvitamin D3 by HPLC. To further delineate the differences between the C-24 oxidative metabolic pathways of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxy-16-ene vitamin D3 in this present study we investigated the C-24 oxidation pathway of 1,25-dihydroxy-16-ene vitamin D3 using a novel detection approach based on on-line capillary liquid chromatography coupled to electrospray tandem mass spectrometry. Two types of tandem mass spectrometric detection were employed to characterize the metabolites in the kidney perfusate: (a) the preliminary screening of metabolites by parent scan, which led to the tentative discovery of the production of 1,23,25-trihydroxy-24-oxo-16-ene vitamin D3, a new metabolite of 1,25-dihydroxy-16-ene vitamin D3, and (b) the pharmacokinetic studies of the substrate, 1,25-dihydroxy-16-ene vitamin D3 and its metabolites by multiple reaction monitoring. In the latter, the mass spectrometric sensitivity for quantification was found to be about 20-fold better than UV detection. The current work concluded that the C-24 oxidative metabolic pathway of 1,25-dihydroxy-16-ene vitamin D3 closely mimicked that of its natural counterpart. Furthermore, the use of mass spectrometry permitted the clearance rate of the starting substrate to be studied at a more physiological level (ng/mL or submicromolar level), which had not been possible previously by HPLC-UV detection.

  13. The determination of serum concentrations of osteocalcin in growing pigs and its relationship to end-measures of bone mineralization.

    PubMed

    Carter, S D; Cromwell, G L; Combs, T R; Colombo, G; Fanti, P

    1996-11-01

    Osteocalcin, a 49-amino acid, gamma-carboxyglutamic acid-containing protein produced by the osteoblast, has been shown in laboratory animals to be a better marker of bone turnover than alkaline phosphatase. To determine serum osteocalcin levels in growing pigs, we isolated pure porcine osteocalcin and developed a double-antibody RIA. To evaluate the effects of dietary Ca and P levels on serum osteocalcin, 36 individually penned crossbred pigs (19.5 kg initial BW) were fed fortified corn-soybean meal diets (.95% lysine) containing four levels of Ca (.42, .66, .90, 1.14%) and P (.35, .55, .75, .95%) in a 30-d test. Increasing dietary Ca and P improved body weight gain quadratically (P < .02). Most bone traits improved quadratically (P < .05) with increasing Ca and P. Pigs were bled on d 0, 10, 20, and 30 to determine serum levels of alkaline phosphatase, 1,25-dihydroxyvitamin D3, and osteocalcin. Osteocalcin decreased (P < .02) linearly with increasing Ca and P on d 10, 20, and 30. However, this effect was much more pronounced on d 20 and 30. Alkaline phosphatase decreased with the first incremental increase in dietary Ca and P, but was not affected by higher levels on any day measured. Osteocalcin was inversely correlated with growth rate (r = -.54, P < .01), bone strength (r = -.57, P < .01), metacarpal ash (r = -.29, P < .10), femur ash (r = -.60, P < .01), and femur ash weight (r = -.65, P < .01). Similar results were found for 1,25-dihydroxyvitamin D3. Alkaline phosphatase was not correlated with performance or most bone traits on d 30. Based on this model, these results suggest that serum osteocalcin and 1,25-dihydroxyvitamin D3 are better predictors of bone mineralization and(or) turnover in pigs than serum alkaline phosphatase.

  14. Use of surface plasmon resonance in the binding study of vitamin D, metabolites and analogues with vitamin D binding protein.

    PubMed

    Canoa, Pilar; Rivadulla, Marcos L; Popplewell, Jonathan; van Oosten, René; Gómez, Generosa; Fall, Yagamare

    2017-04-01

    Vitamin D3 and its metabolites are lipophilic molecules with low aqueous solubility and must be transported bound to plasma carrier proteins, primarily to vitamin D binding protein (DBP). The biological functions of vitamin D3 metabolites are intimately dependent on the protein, hence the importance of determining their affinity for DBP. In this study, we developed a novel procedure for measuring the kinetic and equilibrium constants of human-DBP with vitamin D3 and three metabolites: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 25-hydroxyvitamin D3 (25OHD3) and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] by surface plasmon resonance (SPR). At the same time, five different analogues, synthetized in our laboratory, were evaluated in order to compare the affinity values with the metabolites. Real-time SPR measurements showed that 25OHD3 and 24,25(OH)2D3 had higher affinity (0.3 μM) than 1,25(OH)2D3 (5 μM), with the higher affinity of 25OHD3 and 24,25(OH)2D3 due to dissociation constants 1 order of magnitude slower. In the case of the analogues, the affinity values were lower, with 1-hydroxy-25-nitro-vitamin D3 (NO2-446), structurally closer to 1,25(OH)2D3, showing the highest value with a K D of 50 μM. (24R)-1,25-dihydroxyvitamin-24-buthyl-28-norvitamin D2 (Bu-471) and (24R)-1,25-dihydroxyvitamin-24-phenyl-28-norvitamin D2 (Ph-491), structurally similar, had affinities of 180 and 170 μM, respectively. (22R,23E)-1-hydroxy-22-ethenyl-25-methoxy-23-dehydrovitamin D3 (MeO-455) and 1-hydroxy-20(R)-[5(S)-(2,2-dimethyltetrahydropyran-5-yl)]-22,23-dinor vitamin D3 (Oxan-429) had affinities of 310 and 100 μM, respectively. The binding of the metabolites and analogues was reversible allowing the rapid capture of data for replicates. The kinetic and equilibrium data for both the metabolites and the analogues fitted to the Langmuir model describing a 1:1 interaction. Graphical Abstract Label-free, real time binding study between vitamin D binding protein immmobilized on the

  15. Severe hypophosphatemic osteomalacia with Fanconi syndrome, renal tubular acidosis, vitamin D deficiency and primary biliary cirrhosis.

    PubMed

    Bando, Hironori; Hashimoto, Naoko; Hirota, Yushi; Sakaguchi, Kazuhiko; Hisa, Itoko; Inoue, Yoshifumi; Imanishi, Yasuo; Seino, Susumu; Kaji, Hiroshi

    2009-01-01

    A 49-year-old woman was admitted to our hospital for back pain with marked thoracic and extremity deformities leading to bed-rest for three years. She was diagnosed with hypophosphatemic osteomalacia based on her symptoms, X-ray and bone scintigram, high serum alkaline phosphatase level, and low serum levels of both phosphorus and 1,25 dihydroxyvitamin D(3) with inhibition of phosphorus reabsorption. Fanconi syndrome with renal tubular acidosis, vitamin D deficiency and primary biliary cirrhosis were related to the pathogenesis of osteomalacia in this case. Several causal diseases may be concomitantly responsible for acceleration of the severity of osteomalacia in this patient.

  16. Interactions Between Vitamin D and Breast Cancer

    DTIC Science & Technology

    2009-09-01

    654384 NM_000103 CYP19A1 Cytochrome P450, family 19, subfamily A, polypeptide 1 A10 Hs.162807 NM_003225 TFF1 Trefoil factor 1 A11 Hs.446352 NM_004448...Vitamin D (1,25- dihydroxyvitamin D3) receptor C01 Hs.89663 NM_000782 CYP24A1 Cytochrome P450, family 24, subfamily A, polypeptide 1 C02 Hs.524528...NM_000785 CYP27B1 Cytochrome P450, family 27, subfamily B, polypeptide 1 C03 Hs.272367 NM_018411 HR Hairless homolog (mouse) C04 Hs.360174 NM_003068

  17. Design, synthesis and X-ray crystallographic study of new nonsecosteroidal vitamin D receptor ligands.

    PubMed

    Demizu, Yosuke; Takahashi, Takeo; Kaneko, Fumiya; Sato, Yukiko; Okuda, Haruhiro; Ochiai, Eiji; Horie, Kyohei; Takagi, Ken-Ichiro; Kakuda, Shinji; Takimoto-Kamimura, Midori; Kurihara, Masaaki

    2011-10-15

    We designed and synthesized nonsecosteroidal vitamin D receptor (VDR) ligands that formed H-bonds with six amino acid residues (Tyr143, Ser233, Arg270, Ser274, His301 and His393) of the VDR ligand-binding domain. The ligand YR335 exhibited potent transcriptional activity, which was comparable to those of 1α,25-dihydroxyvitamin D(3) and YR301. The crystal structure of the complex formed between YR335 and the VDR ligand-binding domain was solved, which revealed that YR335 formed H-bonds with the six amino acid residues mentioned above.

  18. Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide.

    PubMed

    Vanhooke, Janeen L; Benning, Matthew M; Bauer, Cary B; Pike, J Wesley; DeLuca, Hector F

    2004-04-13

    We have determined the crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor in ternary complexes with a synthetic LXXLL-containing peptide and the following four ligands: 1alpha,25-dihydroxyvitamin D(3); 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD); 1alpha-hydroxy-2-methylene-19-nor-(20S)-bishomopregnacalciferol (2MbisP), and 2alpha-methyl-19-nor-1alpha,25-dihydroxyvitamin D(3) (2AM20R). The conformation of the LBD is identical in each complex. Binding of the 2-carbon-modified analogues does not change the positions of the amino acids in the ligand binding site and has no effect on the interactions in the coactivator binding pocket. The CD ring of the superpotent analogue, 2MD, is tilted within the binding site relative to the other ligands in this study and to (20S)-1alpha,25-dihydroxyvitamin D(3) [Tocchini-Valentini et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 5491-5496]. The aliphatic side chain of 2MD follows a different path within the binding site; nevertheless, the 25-hydroxyl group at the end of the chain occupies the same position as that of the natural ligand, and the hydrogen bonds with histidines 301 and 393 are maintained. 2MbisP binds to the receptor despite the absence of the 25-hydroxyl group. A water molecule is observed between His 301 and His 393 in this structure, and it preserves the orientation of the histidines in the binding site. Although the alpha-chair conformer is highly favored in solution for the A ring of 2AM20R, the crystal structures demonstrate that this ring assumes the beta-chair conformation in all cases, and the 1alpha-hydroxyl group is equatorial. The peptide folds as a helix and is anchored through hydrogen bonds to a surface groove formed by helices 3, 4, and 12. Electrostatic and hydrophobic interactions between the peptide and the LBD stabilize the active receptor conformation. This stablization appears necessary for crystal growth.

  19. Functions of key residues in the ligand-binding pocket of vitamin D receptor: Fragment molecular orbital interfragment interaction energy analysis

    NASA Astrophysics Data System (ADS)

    Yamagishi, Kenji; Yamamoto, Keiko; Yamada, Sachiko; Tokiwa, Hiroaki

    2006-03-01

    Fragment molecular orbital-interfragment interaction energy calculations of the vitamin D receptor (VDR)/1α,25-dihydroxyvitamin D 3 complex were utilized to assign functions of key residues of the VDR. Only one residue forms a significant interaction with the corresponding hydroxy group of the ligand, although two residues are located around each hydroxy group. The degradation of binding affinity for derivatives upon removal of a hydroxy group is closely related to the trend in the strength of the hydrogen bonds. Type II hereditary rickets due to an Arg274 point mutation is caused by the lack of the strongest hydrogen bond.

  20. Modulation of the Proliferation and Metastasis of Human Breast Tumor Cells by SLUG (IDEA)

    DTIC Science & Technology

    2008-04-01

    transcription. Mol. Cell. Biol. 20, 5087-5095. 4. Tripathi, M. K., Misra, S., Khedkar, S. V., Hamilton, N., Irvin -Wilson, C., Sharan, C., Sealy, L...Narvaez, G. Zinser, J. Welsh , Functions of 1 alpha,25-dihydroxyvitamin D(3) in mammary gland: from normal development to breast cancer, Steroids 66 (2001...301-308. [2] J. Welsh , J.A. Wietzke, G.M. Zinser, S. Smyczek, S. Romu, E. Tribble, J.C. Welsh , B. Byrne, C.J. Narvaez, Impact of the Vitamin D3

  1. Increased disease activity in a patient with sarcoidosis after high dose interleukin 2 treatment for metastatic renal cancer.

    PubMed

    Logan, T F; Bensadoun, E S

    2005-07-01

    Sarcoidosis is a disease of unknown aetiology in which cytokines such as interleukin 2 (IL-2) are thought to play an important role. We present the case history of a 48 year old man with sarcoidosis who received treatment with high dose IL-2 for metastatic renal cell cancer, following which he developed hypercalcaemia characterised by a raised level of 1,25-dihydroxyvitamin D (1,25-(OH)2-D3), a finding consistent with sarcoidosis associated hypercalcaemia. The increased activity in his sarcoidosis following IL-2 treatment provides direct supportive evidence for the role of IL-2 in the pathogenesis of sarcoidosis.

  2. Assays for Identifying Inducers of the Antimicrobial Peptide LL-37.

    PubMed

    Nylén, Frank; Bergman, Peter; Gudmundsson, Gudmundur H; Agerberth, Birgitta

    2017-01-01

    One promising approach to meet the growing problem of antibiotic resistance is to modulate host defense mechanisms, i.e., host-directed therapy (HDT), in the fight against infections. Induction of endogenous antimicrobial peptides (AMPs) via small molecular compounds, such as 1,25-dihydroxyvitamin D3 or phenylbutyrate, could provide one such HDT-based approach.We have developed a cell-based screening assay for the identification of novel compounds with the capacity to induce AMP expression and here follows the detailed protocol.

  3. Effect of space flight on interferon production - mechanistic studies

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, Gerald

    1991-01-01

    Ground-based models were studied for the effects of space flight on immune responses. Most time was spent on the model for the antiorthostatic, hypokinetic, hypodynamic suspension model for rats. Results indicate that suspension is useful for modeling the effects of spaceflight on functional immune responses, such as interferon and interleukin production. It does not appear to be useful for modeling shifts in leukocyte sub-populations. Calcium and 1,25-dihydroxyvitamin D sub 3 appear to play a pivitol role in regulating shifts in immune responses due to suspension. The macrophage appears to be an important target cell for the effects of suspension on immune responses.

  4. Role of megalin and cubilin in the metabolism of vitamin D(3).

    PubMed

    Kaseda, Ryohei; Hosojima, Michihiro; Sato, Hiroyoshi; Saito, Akihiko

    2011-06-01

    Vitamin D deficiency is associated with various medical conditions including musculoskeletal disorders, infection, metabolic diseases, and cardiovascular disease. Megalin and cubilin, endocytic receptors in proximal tubule cells, are involved in the reabsorption of vitamin D binding protein from glomerular filtrates and the subsequent intracellular conversion of 25-hydroxyvitamin D(3) to biologically active 1α,25-dihydroxyvitamin D(3). Dysfunction of these receptors, which is commonly found in patients with diabetic nephropathy, even at early stages, may explain why vitamin D deficiency is often complicated in these patients. Therapeutic strategies to protect the functions of these receptors from injury could be used to prevent vitamin D deficiency and its related disorders.

  5. Susceptibility to Radiation Induced Apoptosis and Senescence in p53 Wild Type and p53 Mutant Breast Tumor Cells

    DTIC Science & Technology

    2006-07-01

    dihydroxyvitamin D3 (1,25(OH)2D3) and vitamin D3 analogs such as EB 1089 potentiate the response to ionizing radiation in breast tumor cells. The current...Appended Page Proofs ………………………………………………………… 5 Introduction 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and vitamin D3 analogs such as EB 1089...analogs and ionizing radiation are the following: 1. The vitamin D analog, EB 1089 ( and by extension, vitamin D3 ), converts an accelerated

  6. [Vitamin D deficiency in adults: to better understand a new presentation of an old disease].

    PubMed

    Premaor, Melissa Orlandin; Furlanetto, Tania Weber

    2006-02-01

    Vitamin D is synthesized in skin through a reaction mediated by sunlight, and it is metabolized to 25-hydroxyvitamin D, in liver, and in 1,25-dihydroxyvitamin D, in kidney. This last reaction has a tight feedback mechanism. 1,25-dihydroxyvitamin D is the active hormone, and its actions are mediated mainly by nuclear receptors. Its major functions are in calcium metabolism and bone mass maintenance. Hypovitaminosis D, as a disease in adult people, manifests itself with hypocalcemia and secondary hyperparathyroidism with subsequent loss of trabecular bone, thinning of cortical bone, and, eventually, a higher risk of fractures. Hypovitaminosis D is a very common condition in Europe, Africa, North America and some South American countries, such as Chile and Argentina. Measurement of serum total 25-hydroxyvitamin D concentration is the gold standard to diagnose vitamin D deficiency. Serum concentrations below 50 nmol/L are associated with an increase in parathyroid hormone concentration, and bone loss. Risk factors for vitamin D deficiency, like poor sunlight exposition, aging skin and factors that interfere with normal vitamin D metabolism, are well established. Oral vitamin D supplementation, an easy and inexpensive treatment, is needed to treat this illness.

  7. Phytoestrogens regulate vitamin D metabolism in the mouse colon: relevance for colon tumor prevention and therapy.

    PubMed

    Kállay, Enikö; Adlercreutz, Herman; Farhan, Hesso; Lechner, Daniel; Bajna, Erika; Gerdenitsch, Waltraud; Campbell, Moray; Cross, Heide S

    2002-11-01

    Soybean products are highly represented in the traditional Asian diet. Major components of soy proteins are phytoestrogens, such as isoflavones. They may be responsible for the extremely low incidence of prostate and mammary tumors and possibly also of colon cancer in countries such as China and Japan. Serum 1,25-dihydroxyvitamin D3 level is inversely related to incidence of some cancers. Levels are determined by skin exposure to ultraviolet light or, to a minor extent, nutritional uptake and by subsequent conversion of the precursor vitamin D to the active hormone by the cytochrome P450 hydroxylases CYP27A1, CYP27B1 (responsible for synthesis) and CYP24 (responsible for catabolism) in liver and kidney. However, vitamin D synthesis is also found in colonocytes and is enhanced during incipient malignancy. This may indicate an autocrine/paracrine role for this differentiation-inducing hormone in defense against progression. We were able to demonstrate that either a single large oral dose of genistein or feeding soy protein for 4 mo elevated CYP27B1 and decreased CYP24 expression in the mouse colon. Our data therefore suggest that an inverse correlation of soy product consumption with colon tumor incidence may be consequent to enhanced colonic synthesis of the antimitotic hormone 1,25-dihydroxyvitamin D3.

  8. Analytical considerations for the biochemical assessment of vitamin D status

    PubMed Central

    Couchman, Lewis; Moniz, Cajetan F

    2017-01-01

    The most widely used and clinically accepted biochemical marker for assessing vitamin D status is the total serum 25-hydroxyvitamin D [25(OH)D] concentration. Despite the analysis of 25(OH)D dating back to the early 1970s, modern analytical techniques still exhibit significant interassay variability due to varying concentrations of other related vitamin D metabolites and sample-to-sample matrix differences. It is important for clinicians requesting 25(OH)D analyses to understand these issues and limitations, and where necessary to confront laboratories for details of analytical methods used. The availability of reference measurement procedures for 25(OH)D based on liquid chromatography and tandem mass spectrometry, whilst not intended for routine clinical sample analysis, should be utilized to improve assay harmonization and reduce interlaboratory variability. Laboratories should also be forthcoming with details of subscriptions to external quality assessment schemes and assay traceability. As well as discussing the reasons for ongoing assay variability for 25(OH)D, this short review will also briefly discuss other assays related to the assessment of vitamin D status, including parathyroid hormone, 24,25-dihydroxyvitamin D, 1,25-dihydroxyvitamin D and vitamin D binding proteins. PMID:28382113

  9. Origin of osteoclasts: Mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells

    SciTech Connect

    Udagawa, Nobuyuki; Takahashi, Naoyuki; Akatsu, Takuhiko; Tanaka, Hirofumi; Sasaki, Takahisa; Suda, Tatsuo ); Nishihara, Tatsuji; Koga, Toshihiko ); Martin, T.J. )

    1990-09-01

    The authors previously reported that osteoclast-like cells were formed in cocultures of a mouse marrow-derived stromal cell line (ST2) with mouse spleen cells in the presence of 1{alpha},25-dihydroxyvitamin D{sub 3} and dexamethasone. In this study, they developed a new coculture system to determine the origin of osteoclasts. When relatively small numbers of mononuclear cells obtained from mouse bone marrow, spleen, thymus, or peripheral blood were cultured for 12 days on the ST2 cell layers, they formed colonies with a linear relationship between the number of colonies formed and the number of hemopoietic cells inoculated. Tartrate-resistant acid phosphatase (TRAPase)-positive monoculear and multinucleated cells appeared in the colonies (TRAPase-positive colonies) in response to 1{alpha},25-dihydroxyvitamin D{sub 3} and dexamethasone. When hemopoietic cells suspended in a collagen-gel solution were cultured on the ST2 cell layers to prevent their movement, TRAPase-positive colonies were similarly formed, indicating that each colony originated from a single cell. Salmon {sup 125}I-labeled calcitonin specifically bound to the TRAPase-positive cells. Resorption lacunae were formed on dentine slices on which cocultures were performed. These results indicate that osteoclasts are also derived from the mature monocytes and macrophages when a suitable microenvironment is provided by bone marrow-derived stromal cells.

  10. Vitamin D and the skin.

    PubMed

    Shahriari, Mona; Kerr, Philip E; Slade, Karren; Grant-Kels, Jane E

    2010-01-01

    Vitamin D is a fat-soluble nutrient that humans obtain through the diet and by synthesis in the skin upon exposure to ultraviolet B. Vitamin D is then converted by the liver to 25-hydroxyvitamin D, its major circulating form. This form is the best indicator of vitamin D nutritional status and is easily measured. Under the influence of parathyroid hormone, the kidney then converts 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D, the biologically active, hormonal form of the nutrient that is important in the metabolism of calcium and phosphorus and is critical in building and maintaining healthy bones. Many cell types outside of the skeletal system, including various cells in the skin, also express the vitamin D receptor. In addition, many cell types convert circulating 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D for local use. This metabolite has been shown to exert potent effects on cellular differentiation, cellular proliferation, and immune regulation. It is theorized that by these mechanisms vitamin D and its analogues are effective treatment options for psoriasis and other skin diseases. Insufficient vitamin D nutritional status has been associated with a host of other diseases, most notably cancer. There is evidence that supplementation with vitamin D reduces the overall incidence of cancer, although current evidence is insufficient to prove a causative effect. Sunscreen use blocks the ability of the skin to photosynthesize vitamin D, although the effect this has on the vitamin D status of the general population is unclear.

  11. Vitamin D Activities for Health Outcomes

    PubMed Central

    2014-01-01

    Reports describing significant health risks due to inadequate vitamin D status continue to generate considerable interest amongst the medical and lay communities alike. Recent research on the various molecular activities of the vitamin D system, including the nuclear vitamin D receptor and other receptors for 1,25-dihydroxyvitamin D and vitamin D metabolism, provides evidence that the vitamin D system carries out biological activities across a wide range of tissues similar to other nuclear receptor hormones. This knowledge provides physiological plausibility of the various health benefits claimed to be provided by vitamin D and supports the proposals for conducting clinical trials. The vitamin D system plays critical roles in the maintenance of plasma calcium and phosphate and bone mineral homeostasis. Recent evidence confirms that plasma calcium homeostasis is the critical factor modulating vitamin D activity. Vitamin D activities in the skeleton include stimulation or inhibition of bone resorption and inhibition or stimulation of bone formation. The three major bone cell types, which are osteoblasts, osteocytes and osteoclasts, can all respond to vitamin D via the classical nuclear vitamin D receptor and metabolize 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D to activate the vitamin D receptor and modulate gene expression. Dietary calcium intake interacts with vitamin D metabolism at both the renal and bone tissue levels to direct either a catabolic action on the bone through the endocrine system when calcium intake is inadequate or an anabolic action through a bone autocrine or paracrine system when calcium intake is sufficient. PMID:24790904

  12. Vitamin D Status: Current Opinion on Critical Levels for Plasma Calcium and Bone Mineral Homeostasis

    PubMed Central

    2011-01-01

    Currently there is an unprecedented level of interest regarding the purported wide-ranging beneficial effects of an adequate vitamin D status translating into marked increases in test requests for clinical laboratories. The well characterised endocrine pathway of vitamin D metabolism and action is solely responsible for vitamin D regulating plasma calcium and phosphate homeostasis. A large body of data confirm that vitamin D exerts activities within each of the major bone cells and that these same cells are capable of synthesising the active metabolite, 1,25-dihydroxyvitamin D from 25-hydroxyvitamin D. Such data arising from in vitro studies, animal models and clinical sources are consistent with a paradigm that local metabolism of vitamin D by bone cells to form 1,25-dihydroxyvitamin D and its consequent local actions within bone cells exerts an anabolic effect to increase bone mineral status. The data reviewed here provide plausible mechanisms for both catabolic and anabolic actions of vitamin D on bone depending on dietary calcium intake. PMID:27683393

  13. 1,25(OH)2D3 Promotes the Efficacy of CD28 Costimulation Blockade by Abatacept

    PubMed Central

    Gardner, David H.; Jeffery, Louisa E.; Soskic, Blagoje; Briggs, Zoe; Hou, Tie Zheng; Raza, Karim

    2015-01-01

    Inhibition of the CD28:CD80/CD86 T cell costimulatory pathway has emerged as an effective strategy for the treatment of T cell–mediated inflammatory diseases. However, patient responses to CD28-ligand blockade by abatacept (CTLA-4-Ig) in conditions such as rheumatoid arthritis are variable and often suboptimal. In this study, we show that the extent to which abatacept suppresses T cell activation is influenced by the strength of TCR stimulation, with high-strength TCR stimulation being associated with relative abatacept insensitivity. Accordingly, cyclosporin A, an inhibitor of T cell stimulation via the TCR, synergized with abatacept to inhibit T cell activation. We also observed that 1,25-dihydroxyvitamin D3 enhanced the inhibition of T cell activation by abatacept, strongly inhibiting T cell activation driven by cross-linked anti-CD3, but with no effect upon anti-CD28 driven stimulation. Thus, like cyclosporin A, 1,25-dihydroxyvitamin D3 inhibits TCR-driven activation, thereby promoting abatacept sensitivity. Vitamin D3 supplementation may therefore be a useful adjunct for the treatment of conditions such as rheumatoid arthritis in combination with abatacept to promote the efficacy of treatment. PMID:26276872

  14. Calcium homeostasis in diabetes mellitus.

    PubMed

    Heath, H; Lambert, P W; Service, F J; Arnaud, S B

    1979-09-01

    Experimentally diabetic rats have low serum 1,25-dihydroxyvitamin D, intestinal malabsorption of calcium, secondary hyperparathyroidism, and bone loss. To examine the hypothesis that abnormalities similar to those in the diabetic rat might explain human diabetic osteopenia, we studied calcium metabolism in 40 healthy control and 82 diabetic patients aged 18--75 yr [47 untreated: fasting plasma glucose (mean +/- SE), 267 +/- 8 mg/dl; 19 treated but hyperglycemic: glucose 305 +/- 24 mg/dl; 16 treated and in better control: glucose, 146 +/- 8 mg/dl]. Serum total calcium, ionic calcium, immunoreactive parathyroid hormone (Arnaud method, GP-1M and CH-12M antisera), 25-hydroxyvitamin D (Haddad method), and 1,25-dihydroxyvitamin D (Lambert method) concentrations were normal in all 3 groups of diabetics and were not significantly different from values in the control group. We determined absorption of calcium from the intestine by a double isotope method (100 mg Ca carrier; normal range, 40--80%) in 11 control and 13 untreated, uncontrolled diabetics (mean plasma glucose, 285 +/- 17 mg/dl). Absorption of calcium in controls was 60 +/- 3% and in diabetics was 56 +/- 3% (not significantly different). We have found no derangement of calcium metabolism in adults with insulin-requiring juvenile- and adult-onset diabetes regardless of treatment status. The experimental diabetic rat model does not appear to be useful for determining the pathogenesis of adult human diabetic osteopenia.

  15. Genistein inhibits vitamin D hydroxylases CYP24 and CYP27B1 expression in prostate cells.

    PubMed

    Farhan, Hesso; Wähälä, Kristiina; Cross, Heide S

    2003-03-01

    In human prostate cancer cells, the availability of the steroid hormone 1,25-dihydroxyvitamin D(3) for antimitotic action is determined through the activity of the two enzymes CYP24 and CYP27B1, viz. 25-hydroxyvitamin D-24-hydroxylase and 25-hydroxyvitamin D-1alpha-hydroxylase. High performance liquid chromatography (HPLC) analysis of [(3)H]25(OH)D(3) metabolism in human prostate cancer DU-145 cells revealed that genistein and other isoflavonoids, such as dihydrogenistein and daidzein, as well as the antiestrogenic compound ICI 182,780, inhibited Vitamin D-metabolizing enzyme activities. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that only in case of genistein this was due to transcriptional inhibition of CYP24 and CYP27B1 gene expressions. In case of CYP27B1, reduction of gene activity involves histone deacetylation because genistein was inactive in the presence of the histone deactylase inhibitor trichostatin A. In contrast, under the same condition, CYP24 gene activity was largely suppressed. In summary, our results suggest that a combined effect of genistein and trichostatin A could increase the responsiveness of human prostate cancer cells to the antiproliferative action of 1,25-dihydroxyvitamin D(3).

  16. Vitamin D3 analogs stimulate hair growth in nude mice.

    PubMed

    Vegesna, Vijaya; O'Kelly, James; Uskokovic, Milan; Said, Jonathan; Lemp, Nathan; Saitoh, Takayuki; Ikezoe, Takayuki; Binderup, Lise; Koeffler, H Phillip

    2002-11-01

    The active form of vitamin D3 can regulate epidermal keratinization by inducing terminal differentiation; and mice lacking the vitamin D receptor display defects leading to postnatal alopecia. These observations implicate the vitamin D3 pathway in regulation of hair growth. We tested the ability of 1,25 dihydroxyvitamin D3 and its synthetic analogs to stimulate hair growth in biege/nude/xid (BNX) nu/nu (nude) mice exhibiting congenital alopecia. Nude mice were treated with different vitamin D3 analogs at doses that we had previously found to be the highest dose without inducing toxicity (hypercalcemia). The mice were monitored for hair growth and were scored according to a defined scale. Skin samples were taken for histological observation of hair follicles and for extraction of RNA and protein. Vitamin D3 analogs dramatically stimulated the hair growth of nude mice, although parental 1,25 dihydroxyvitamin D3 had no effect. Hair growth occurred in a cyclical pattern, accompanied by formation of normal hair follicles and increased expression of certain keratins (Ha7, Ha8, and Hb3). Vitamin D3 analogs seem to act on keratinocytes to initiate hair follicle cycling and stimulate hair growth in mice that otherwise do not grow hair.

  17. Subcellular localisation of BAG-1 and its regulation of vitamin D receptor-mediated transactivation and involucrin expression in oral keratinocytes: Implications for oral carcinogenesis

    SciTech Connect

    Lee, San San; Crabb, Simon J.; Janghra, Nari; Carlberg, Carsten; Williams, Ann C.; Cutress, Ramsey I.; Packham, Graham; Hague, Angela

    2007-09-10

    In oral cancers, cytoplasmic BAG-1 overexpression is a marker of poor prognosis. BAG-1 regulates cellular growth, differentiation and survival through interactions with diverse proteins, including the vitamin D receptor (VDR), a key regulator of keratinocyte growth and differentiation. BAG-1 is expressed ubiquitously in human cells as three major isoforms of 50 kDa (BAG-1L), 46 kDa (BAG-1M) and 36 kDa (BAG-1S) from a single mRNA. In oral keratinocytes BAG-1L, but not BAG-1M and BAG-1S, enhanced VDR transactivation in response to 1{alpha},25-dihydroxyvitamin D{sub 3.} BAG-1L was nucleoplasmic and nucleolar, whereas BAG-1S and BAG-1M were cytoplasmic and nucleoplasmic in localisation. Having identified the nucleolar localisation sequence in BAG-1L, we showed that mutation of this sequence did not prevent BAG-1L from potentiating VDR activity. BAG-1L also potentiated transactivation of known vitamin-D-responsive gene promoters, osteocalcin and 24-hydroxylase, and enhanced VDR-dependent transcription and protein expression of the keratinocyte differentiation marker, involucrin. These results demonstrate endogenous gene regulation by BAG-1L by potentiating nuclear hormone receptor function and suggest a role for BAG-1L in 24-hydroxylase regulation of vitamin D metabolism and the cellular response of oral keratinocytes to 1{alpha},25-dihydroxyvitamin D{sub 3}. By contrast to the cytoplasmic BAG-1 isoforms, BAG-1L may act to suppress tumorigenesis.

  18. Vitamin D, sunlight and cancer connection.

    PubMed

    Holick, Michael F

    2013-01-01

    It has been more than 100 years when it was first appreciated that increased sun exposure reduced risk of dying of cancer. The most beneficial effect of sun exposure is the production of vitamin D in the skin. Recent evidence suggests that most cells in the body not only have a vitamin D receptor but also have the capacity to convert 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. Once formed 1,25-dihydroxyvitamin D can inhibit cellular proliferation, induce cellular maturation, inhibit angiogenesis and ultimately cause apoptosis to prevent malignancy. A multitude of studies have associated improved vitamin D status with decreased risk for developing several deadly cancers including colon, breast, pancreatic and ovarian cancers. Patients with cancer are at high risk for vitamin D deficiency. Sensible sun exposure, vitamin D fortification and vitamin D supplementation should be encouraged to improve the vitamin D status of children and adults not only for bone health but for reducing risk of developing and dying of cancer. The goal is to achieve a blood level of 25-hydroxyvitamin D of 40-60 ng/mL. This can be accomplished by children taking 600-1000 and adults 1500-2000 international units (IU) vitamin D daily from diet and supplements along with sensible sun exposure when the sun is capable of producing vitamin D in the skin.

  19. Sequential hydroxylation of vitamin D2 by a genetically engineered CYP105A1.

    PubMed

    Hayashi, Keiko; Yasuda, Kaori; Yogo, Yuya; Takita, Teisuke; Yasukawa, Kiyoshi; Ohta, Miho; Kamakura, Masaki; Ikushiro, Shinichi; Sakaki, Toshiyuki

    2016-05-13

    Our previous studies revealed that the double variants of CYP105A1- R73A/R84A and R73V/R84A-show high levels of activity with respect to conversion of vitamin D3 to its biologically active form, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study, we found that both the double variants were also capable of converting vitamin D2 to its active form, that is, 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2), via 25(OH)D2, whereas its 1α-hydroxylation activity toward 25(OH)D2 was much lower than that toward 25(OH)D3. Comparison of the wild type and the double variants revealed that the amino acid substitutions remarkably enhanced both 25- and 26-hydroxylation activity toward vitamin D2. After 25-hydroxylation of vitamin D2, further hydroxylation at C26 may occur frequently without the release of 25(OH)D2 from the substrate-binding pocket. Thus, the double variants of CYP105A1 are quite useful to produce 25,26(OH)2D2 that is one of the metabolites of vitamin D2 detected in human serum.

  20. Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

    SciTech Connect

    Franco, Gilson C.N.; Nakanishi, Tadashi; Ohta, Kouji; Rosalen, Pedro L.; Groppo, Francisco C.; Bartlett, John D.; Stashenko, Philip; Taubman, Martin A.; Kawai, Toshihisa

    2011-06-10

    Tetracycline antibiotics, including doxycycli/e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.

  1. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity.

    PubMed

    Li, Binghan; Lu, Dan; Chen, Yuqing; Zhao, Minghui; Zuo, Li

    2016-04-22

    Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin's effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

  2. Cellular and molecular toxicity of lead in bone.

    PubMed Central

    Pounds, J G; Long, G J; Rosen, J F

    1991-01-01

    To fully understand the significance of bone as a target tissue of lead toxicity, as well as a reservoir of systemic lead, it is necessary to define the effects of lead on the cellular components of bone. Skeletal development and the regulation of skeletal mass are ultimately determined by the four different types of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. These cells, which line and penetrate the mineralized matrix, are responsible for matrix formation, mineralization, and bone resorption, under the control of both systemic and local factors. Systemic components of regulation include parathyroid hormone, 1,25-dihydroxyvitamin D3, and calcitonin: local regulators include numerous cytokines and growth factors. Lead intoxication directly and indirectly alters many aspects of bone cell function. First, lead may indirectly alter bone cell function through changes in the circulating levels of those hormones, particularly 1,25-dihydroxyvitamin D3, which modulate bone cell function. These hormonal changes have been well established in clinical studies, although the functional significance remains to be established. Second, lead may directly alter bone cell function by perturbing the ability of bone cells to respond to hormonal regulation. For example, the 1,25-dihydroxyvitamin D3-stimulated synthesis of osteocalcin, a calcium-binding protein synthesized by osteoblastic bone cells, is inhibited by low levels of lead. Impaired osteocalcin production may inhibit new bone formation, as well as the functional coupling of osteoblasts and osteoclasts. Third, lead may impair the ability of cells to synthesize or secrete other components of the bone matrix, such as collagen or bone sialoproteins (osteopontin). Finally, lead may directly effect or substitute for calcium in the active sites of the calcium messenger system, resulting in loss of physiological regulation. The effects of lead on the recruitment and differentiation of bone cells remains to be

  3. Calcium and Phosphorus Regulatory Hormones and Risk of Incident Symptomatic Kidney Stones

    PubMed Central

    Hoofnagle, Andrew N.; Curhan, Gary C.

    2015-01-01

    Background and objectives Calcium and phosphorus regulatory hormones may contribute to the pathogenesis of calcium nephrolithiasis. However, there has been no prospective study to date of plasma hormone levels and risk of kidney stones. This study aimed to examine independent associations between plasma levels of 1,25-dihydroxyvitamin D (1,25[OH]2D), 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, fibroblast growth factor 23 (FGF23), parathyroid hormone, calcium, phosphate, and creatinine and the subsequent risk of incident kidney stones. Design, setting, participants, & measurements This study was a prospective, nested case-control study of men in the Health Professionals Follow-Up Study who were free of diagnosed nephrolithiasis at blood draw. During 12 years of follow-up, 356 men developed an incident symptomatic kidney stone. Using risk set sampling, controls were selected in a 2:1 ratio (n=712 controls) and matched for age, race, and year, month, and time of day of blood collection. Results Baseline plasma levels of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, parathyroid hormone, calcium, phosphate, and creatinine were similar in cases and controls. Mean 1,25(OH)2D and median FGF23 levels were higher in cases than controls but differences were small and statistically nonsignificant (45.7 versus 44.2 pg/ml, P=0.07 for 1,25[OH]2D; 47.6 versus 45.1 pg/ml, P=0.08 for FGF23). However, after adjusting for body mass index, diet, plasma factors, and other covariates, the odds ratios of incident symptomatic kidney stones in the highest compared with lowest quartiles were 1.73 (95% confidence interval, 1.11 to 2.71; P for trend 0.01) for 1,25(OH)2D and 1.45 (95% confidence interval, 0.96 to 2.19; P for trend 0.03) for FGF23. There were no significant associations between other plasma factors and kidney stone risk. Conclusions Higher plasma 1,25(OH)2D, even in ranges considered normal, is independently associated with higher risk of symptomatic kidney stones. Although

  4. Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages

    NASA Astrophysics Data System (ADS)

    Kuo, Chun-Liang; Kao, Chia-Tze; Fang, Hsin-Yuan; Huang, Tsui-Hsien; Chen, Yi-Wen; Shie, Ming-You

    2015-03-01

    Macrophage cells are the important effector cells in the immune reaction which are indispensable for osteoclastogenesis; their heterogeneity and plasticity renders macrophages a primer target for immune system modulation. In recent years, there have been very few studies about the effects of macrophage cells on laser treatment-regulated osteoclastogenesis. In this study, RAW 264.7 macrophage cells were treated with RANKL to regulate osteoclastogenesis. We used a CO2 laser as a model biostimulation to investigate the role of osteoclastogenic. We also evaluated cell viability, cell death and cathepsin K expression. The CO2 laser inhibited a receptor activator of the NF-ĸB ligand (RANKL)-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that irradiation for two times reduced RANKL-enhanced TRAP activity in a dose-dependent manner. Furthermore, CO2 laser-treatment diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and NF-ĸB activation. The current report demonstrates that CO2 laser abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The CO2 laser can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts and the maturation of osteoclasts. Therefore, the current results serve as an improved explanation of the cellular roles of macrophage cell populations in osteoclastogenesis as well as in alveolar bone remodeling by CO2 laser-treatment.

  5. Current evidence on vitamin D deficiency and kidney transplant: What's new?

    PubMed

    Sarno, Gerardo; Nappi, Riccardo; Altieri, Barbara; Tirabassi, Giacomo; Muscogiuri, Emanuele; Salvio, Gianmaria; Paschou, Stavroula A; Ferrara, Aristide; Russo, Enrico; Vicedomini, Daniela; Vincenzo, Cerbone; Vryonidou, Andromachi; Della Casa, Silvia; Balercia, Giancarlo; Orio, Francesco; De Rosa, Paride

    2017-03-09

    Kidney transplant is the treatment of choice for end-stage chronic kidney disease. Kidneys generate 1,25-dihydroxyvitamin D (calcitriol) from 25-hydroxyvitamin D (calcidiol) for circulation in the blood to regulate calcium levels. Transplant patients with low calcidiol levels have an increased risk of metabolic and endocrine problems, cardiovascular disease, type 2 diabetes mellitus, poor graft survival, bone disorders, cancer, and mortality rate. The recommended calcidiol level after transplant is at least 30 ng/mL (75 nmol/L), which could require 1000-3000 IU/d vitamin D3 to achieve. Vitamin D3 supplementation studies have found improved endothelial function and acute rejection episodes. However, since kidney function may still be impaired, raising calcidiol levels may not lead to normal calcitriol levels. Thus, supplementation with calcitriol or an analog, alfacalcidiol, is often employed. Some beneficial effects found include possible improved bone health and reduced risk of chronic allograft nephropathy and cancer.

  6. A possible new role for vitamin D-binding protein in osteoclast control: inhibition of extracellular Ca2+ sensing at low physiological concentrations.

    PubMed

    Adebanjo, O A; Moonga, B S; Haddad, J G; Huang, C L; Zaidi, M

    1998-08-28

    Upon removal of its sialic acid or galactose residue, vitamin D-binding protein (DBP) becomes a potent macrophage-activating factor, DBP-MAF. Here we document a new function of DBP-MAF and its parent molecule, DBP, in osteoclast control. We show that all DBPs potently inhibit extracellular Ca2+ (cation) sensing at low nanomolar concentrations with the following rank order of potency: native DBP = sialidase-treated DBP > beta-galactosidase-treated DBP. This attenuation remains unaffected despite co-incubation either with the native DBP ligand, 1,25-dihydroxyvitamin D3, or with an asialoglycoprotein receptor modulator, asialoorosomucoid. Taken together, the results suggest that circulating DBP may play a role in the systemic control of osteoclastic bone resorption, a hitherto unrecognized action of the protein.

  7. Stable isotope-labeled vitamin D, metabolites and chemical analogs: Synthesis and use in mass spectrometric studies

    SciTech Connect

    Coldwell, R.D.; Trafford, D.J.; Varley, M.J.; Kirk, D.N.; Makin, H.L. )

    1990-10-01

    Methods for the measurement of vitamin D and its metabolites using stable isotope-labeled internal standards and mass spectrometry are reviewed. The synthesis of both labeled and unlabeled standards is illustrated, and details of the synthesis of (26,26,27,27,27(-2)H5)-25,26-dihydroxyvitamin D3 and (28,28,28(-2)H3)-24,25-dihydroxyvitamin D2 are given. The use of in vitro biologic systems for the production of further metabolites of deuterated 25-hydroxyvitamin D3 is discussed. Use of deuterated 25-hydroxydihydrotachysterol3 as a substrate in the isolated perfused rat kidney has provided valuable data for the assignment of structure to a number of metabolites of 25-hydroxydihydrotachysterol3 formed in this system. 51 refs.

  8. Phagocytic cells metabolize 25-hydroxyvitamin D3 in vitro.

    PubMed Central

    Cohen, M S; Gray, T K

    1984-01-01

    Phagocytic cells are widely distributed in tissues known to be important in the metabolism of vitamin D. Incubation of human polymorphonuclear leukocytes and monocytes and resident rat peritoneal macrophages with 3H-labeled 25-hydroxyvitamin D3 leads to the formation of three radioactive peaks. Peak I is most consistent with a lactone derivative of 25-hydroxyvitamin D3, and peak II has been identified as putative 24,25-dihydroxyvitamin D3. Peak III is a novel metabolite of 25-hydroxyvitamin D3 unlike any of the synthetic standards available in our laboratories. Human neutrophils converted more substrate than did the other phagocytes examined. The stimulation of neutrophils by opsonized zymosan or phorbol myristate acetate led to a 4-fold increase in synthesis of the metabolites. These results suggest that vitamin D metabolism by phagocytic cells may play a role in the microenvironmental events that surround bony metabolism and calcium homeostasis. PMID:6322179

  9. The Role of Vitamin D Deficiency in the Incidence, Progression, and Complications of Type 1 Diabetes Mellitus

    PubMed Central

    Chakhtoura, Marlene; Azar, Sami T.

    2013-01-01

    The “nonclassic” role of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently widely recognized. In type 1 diabetes mellitus (T1D), it plays an immunomodulatory role through the vitamin D receptor (VDR) present on pancreatic and immune cells. Specific VDR allelic variants have been associated with T1D in many countries. Furthermore, vitamin D deficiency has been prevalent in T1D, and the seasonal and latitude variability in the incidence of T1D can be partly explained by the related variability in vitamin D level. In fact, retrospective studies of vitamin D supplementation during pregnancy or infancy showed a lower incidence of T1D. We will review the different mechanisms of the vitamin D protective effect against insulitis and present the available data on the role of vitamin D deficiency in the control, progression, and complications of T1D. PMID:23573085

  10. Molecular mechanism of vitamin D in the cardiovascular system.

    PubMed

    Li, Yan Chun

    2011-08-01

    Vitamin D deficiency is a global health problem that has various adverse consequences. Vitamin D is mainly synthesized in the skin by sunlight (UV light) irradiation; therefore, vitamin D status is influenced by geographic locations, seasonal changes, and skin pigmentations. The kidney is involved in the biosynthesis of 1,25-dihydroxyvitamin D and the reuptake of filtered 25-hydroxyvitamin D from the proximal tubules, thus, vitamin D deficiency is highly prevalent in patients with kidney disease who have renal insufficiency. There is a growing body of epidemiological and clinical evidence in the literature that links vitamin D deficiency to cardiovascular disease. The discovery of the vitamin D hormone functioning as an endocrine inhibitor of the renin-angiotensin system provides an explanation for this association. This review will discuss the mechanism underlying the connection between vitamin D and cardiovascular disease and its physiological and therapeutic implications.

  11. Quantitation of circulating hydroxyvitamin D3 in human plasma by a continuous cleanup/concentration procedure prior to HPLC-UV detection.

    PubMed

    Ortiz-Boyer, F; Fernández-Romero, J M; Luque de Castro, M D; Quesada, J M

    1998-06-22

    A method for the determination of hydroxyvitamin D3 metabolites (25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 and 1,25-didydroxyvitamin D3) based on a continuous cleanup/ preconcentration procedure coupled with HPLC and UV-detection is reported here. The method exhibits a linear range between 0.05 and 100 ng/ml (r2 = 0.9917) with CV values lower than 6.5%, and has been checked by applying it to plasma samples from a hospital with acceptable recoveries. The results compare well with those obtained by routine radioimmunoassay (y = 2.784+/-1.37 + 0.333+/-0.05 sigma(yx), r = 0.8233, n = 19 for 25-hydroxyvitamin D3). The sampling frequency was 4 h(-1); 12 analytes h(-1).

  12. Combination Treatment with Progesterone and Vitamin D Hormone May Be More Effective than Monotherapy for Nervous System Injury and Disease

    PubMed Central

    Cekic, Milos; Sayeed, Iqbal; Stein, Donald G.

    2010-01-01

    More than two decades of pre-clinical research and two recent clinical trials have shown that progesterone (PROG) and its metabolites exert beneficial effects after traumatic brain injury (TBI) through a number of metabolic and physiological pathways that can reduce damage in many different tissues and organ systems. Emerging data on 1,25-dihydroxyvitamin D3 (VDH), itself a steroid hormone, have begun to provide evidence that, like PROG, it too is neuroprotective, although some of its actions may involve different pathways. Both agents have high safety profiles, act on many different injury and pathological mechanisms, and are clinically relevant, easy to administer, and inexpensive. Furthermore, vitamin D deficiency is prevalent in a large segment of the population, especially the elderly and institutionalized, and can significantly affect recovery after CNS injury. The combination of PROG and VDH in pre-clinical and clinical studies is a novel and compelling approach to TBI treatment. PMID:19394357

  13. Glycoconjugate histochemistry in the small and large intestine of normal and Solanum glaucophyllum-intoxicated rabbits.

    PubMed

    Zanuzzi, C N; Barbeito, C G; Ortíz, M L; Lozza, F A; Fontana, P A; Portiansky, E L; Gimeno, E J

    2010-10-01

    Vitamin D participates in mineral homeostasis, immunomodulation, cell growth and differentiation. The leaves of Solanum glaucophyllum contain high levels of 1,25-dihydroxyvitamin D3 as glycoside derivatives and their chronic ingestion generates a hypervitaminosis D-like state. We analyzed changes on carbohydrate expression as a cell differentiation indicator on samples of the small and large intestine of S. glaucophyllum-intoxicated rabbits, using conventional and lectin histochemistry. Male New Zealand white rabbits were intoxicated with S. glaucophyllum during two or four weeks and killed the day after. A group of animals ("possibly recovered group") were intoxicated during 15 days and killed at day 45 of the beginning of the experiment. We found changes in the lectin binding pattern in the small and large intestine of the intoxicated rabbits. Some of these changes were reverted in the possibly recovered group. Vitamin D could be a new regulator factor of the intestinal glycosylation process.

  14. Vitamin D insufficiency underlies unexpected hypocalcemia following high dose glucocorticoid therapy.

    PubMed

    Kinoshita, Yuka; Masuoka, Kazuhiro; Miyakoshi, Shigesaburo; Taniguchi, Shuichi; Takeuchi, Yasuhiro

    2008-01-01

    Vitamin D insufficiency is a reemerging and common health problem for skeletal system. Pharmacological application of glucocorticoid inhibits intestinal calcium absorption and stimulates tubular calcium excretion, thus induces severely negative calcium balance. We report a patient presenting symptomatic hypocalcemia following high dose glucocorticoid administration. After a pulse-therapy with methylprednisolone, hypocalcemia with muscle cramp developed in association with hypercalciuria and secondary hyperparathyroidism in the absence of hypomagnesemia. Circulating level of 1,25-dihydroxyvitamin D was in a reference range, while that of 25-hydroxyvitamin D was insufficient. Treatment with alfacalcidol of 1 mug/day promptly improved serum calcium level within a couple of weeks. Vitamin D insufficiency could be a serious problem in patients with high dose glucocorticoid therapy.

  15. High Salt Intake Promotes Urinary Loss of Vitamin D Metabolites by Dahl Salt-Sensitive Rats in a Space Flight Model

    NASA Technical Reports Server (NTRS)

    Thierry-Palmer, M.; Cephas, S.; Sayavongsa, P.; Clark, T.; Arnaud, S. B.

    2004-01-01

    Vitamin D metabolism in the Dahl salt-sensitive (S) rat, a model of salt-induced hypertension, differs from that in the Dahl salt-resistant (R) rat. We have demonstrated that female S rats are more vulnerable than female R rats to decreases in plasma 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)2D) concentrations during hind limb unloading (a space flight model). We report here on the response of the vitamin D endocrine system of S and R rats to hind limb unloading during high salt intake. Dahl female rats (9.7-week-old) were tail-suspended (hind limb unloaded) for 28 days, while fed a diet containing twice the salt in standard rat chow (2 % sodium chloride). Control rats were fed the same diet, but were not hind limb unloaded. Vitamin D metabolites were analyzed by HPLC and radioimmunoassay kits from Diasorin.

  16. EB 1089, a novel vitamin D analogue, has strong antiproliferative and differentiation inducing effects on cancer cells.

    PubMed

    Mathiasen, I S; Colston, K W; Binderup, L

    1993-09-01

    EB 1089 is a novel vitamin D analogue which in vitro strongly inhibits the proliferation of U937 histiocytic lymphoma cells and MCF-7 breast cancer cells, with a potency of 50 to 100 times that of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Studies of c-myc and c-fos expression in MCF-7 cells and of differentiation markers in U937 cells show that growth inhibition by EB 1089 is accompanied by induction of differentiation. The ability of EB 1089 to affect calcium metabolism in vivo in rats is decreased, compared to 1,25(OH)2D3. This low calcemic effect combined with the strong biological effect on cancer cells in vitro, makes EB 1089 an interesting candidate for treatment of cancer.

  17. Vitamin D prevents podocyte injury via regulation of macrophage M1/M2 phenotype in diabetic nephropathy rats.

    PubMed

    Zhang, Xiao-Liang; Guo, Yin-Feng; Song, Zhi-Xia; Zhou, Min

    2014-12-01

    Increasing evidence suggests the heterogeneity of macrophage phenotype and function ultimately determines the outcome of diabetic nephropathy (DN). This study aimed to investigate the effects of vitamin D on macrophage M1/M2 phenotype and its role in preventing podocyte impairment in streptozotocin-induced DN rats. Calcitriol, a bioactive 1,25-dihydroxyvitamin D3, ameliorated proteinuria and renal damage as well as reversed the decline of both nephrin and podocin, crucial structural proteins in podocytes. DN rats showed increased infiltrating macrophages with M1 phenotype characterized by elevated expression of inducible nitric oxide synthase and TNF-α in glomeruli and interstitium, which were inhibited after calcitriol treatment. Interestingly, calcitriol promoted M2 macrophage activation with enhanced expression of CD163, arginase-1, and mannose receptor at week 18 but not at week 8 or 14. The ratio of CD163 to CD68, considered as the proportion of M2 macrophages, was about 2.9-fold higher at week 18 after calcitriol treatment. Furthermore, the protein expression of inducible nitric oxide synthase, a crucial marker of M1 macrophages, was negatively correlated with the expression of either nephrin or podocin, whereas CD163, indicating M2 macrophages, was positively correlated. In vitro, 1,25-dihydroxyvitamin D3 switched high-glucose-induced M1 macrophages toward an M2 phenotype in either U937-derived macrophages or RAW264.7 cells. Our results suggest that vitamin D not only reduces macrophage infiltration and inhibits M1 macrophage activation but also enhances M2 macrophage phenotype to protect against podocyte injury.

  18. Sclerostin alters serum vitamin D metabolite and fibroblast growth factor 23 concentrations and the urinary excretion of calcium

    PubMed Central

    Ryan, Zachary C.; Ketha, Hemamalini; McNulty, Melissa S.; McGee-Lawrence, Meghan; Craig, Theodore A.; Grande, Joseph P.; Westendorf, Jennifer J.; Singh, Ravinder J.; Kumar, Rajiv

    2013-01-01

    Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin (sost) gene was replaced by the β-D-galactosidase (lacZ) gene in the germ line. Knockout (KO) (sost−/−) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice. β-Galactosidase activity was detected in osteocytes of sost KO mice but was undetectable in WT mice. Eight-week-old, male sost KO mice had increased serum 1α,25-dihydroxyvitamin D, decreased 24,25-dihydroxyvitamin D, decreased intact fibroblast growth factor 23, and elevated inorganic phosphorus concentrations compared with age-matched WT mice. 25-Hydroxyvitamin D 1α-hydroxylase cytochrome P450 (cyp27B1) mRNA was increased in kidneys of sost KO mice compared with WT mice. Treatment of cultured proximal tubule cells with mouse recombinant sclerostin decreased cyp27B1 mRNA transcripts. Urinary calcium and renal fractional excretion of calcium were decreased in sost KO mice compared with WT mice. Sost KO and WT mice had similar serum calcium and parathyroid hormone concentrations. The data show that sclerostin not only alters bone mineralization, but also influences mineral metabolism by altering concentrations of hormones that regulate mineral accretion. PMID:23530237

  19. Quality assessment of vitamin D metabolite assays used by clinical and research laboratories.

    PubMed

    Carter, G D; Berry, J; Durazo-Arvizu, R; Gunter, E; Jones, G; Jones, J; Makin, H L J; Pattni, P; Phinney, K W; Sempos, C T; Williams, E L

    2017-03-15

    The Vitamin D External Quality Assessment Scheme (DEQAS) was launched in 1989 and monitors the performance of 25-hydroxyvitamin D (25-OHD) and 1,25- dihydroxyvitamin D (1,25(OH)2D) assays. In April 2015 a pilot scheme for 24,25-dihydroxyvitamin D (24,25(OH)2D) was introduced. The 25-OHD scheme is accuracy - based with target values assigned by the NIST Reference Measurement Procedure (RMP) for 25-OHD2 and 25-OHD3. A similar method is used to assign values for 3-epi-25-OHD. Five samples of human serum are distributed quarterly to over 1000 participants in 58 countries (April 2016) and clinical laboratories are expected to submit results within approximately 5 weeks. Research laboratories with assays run less frequently are not given a deadline. Archived samples with NIST- assigned values are also available. Performance is assessed on the first four samples with the fifth reserved for investigations e.g. recovery experiments or to assess the influence of other serum constituents such as lipids. DEQAS provides rapid feedback, with an on-line preliminary report available immediately after a participant submits results and a comprehensive report soon after the results deadline. In 2015, DEQAS investigations revealed that several 25-OHD immunoassays under-recovered 25-OHD2 and 25-OHD results were falsely low on a sample with a modestly raised triglyceride concentration. An RMP for 1,25 (OH)2D is not yet available and results are judged against the Method Mean. Free advice is available from the DEQAS Advisory Panel which includes experts on methodology and biostatistics. DEQAS collaborates closely with the Vitamin D Standardization Program (VDSP) and both organizations have successfully worked with participants and manufacturers to improve the accuracy of vitamin D assays.

  20. Characterization of the vitamin D endocrine system in human sebocytes in vitro.

    PubMed

    Krämer, Christina; Seltmann, Holger; Seifert, Markus; Tilgen, Wolfgang; Zouboulis, Christos C; Reichrath, Jörg

    2009-01-01

    Sebocytes are sebum-producing cells that form the sebaceous glands. We investigated the role of sebocytes as target cells for vitamin D metabolites and the existence of an enzymatic machinery for the local synthesis and metabolism of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3), calcitriol], the biologically active vitamin D metabolite, in these cell types. Expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25 OHase), 25-hydroxyvitamin D-1alpha-hydroxylase (1 alphaOHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24 OHase) was detected in SZ95 sebocytes in vitro using real time quantitative polymerase chain reaction. Splice variants of 1alphaOHase were identified by nested touchdown polymerase chain reaction. We demonstrated that incubation of SZ95 sebocytes with 1,25(OH)(2)D(3) resulted in a cell culture condition-, time-, and dose-dependent modulation of cell proliferation, cell cycle regulation, lipid content and interleukin-6/interleukin-8 secretion in vitro. RNA expression of VDR and 24 OHase was upregulated along with vitamin D analogue treatment. Although several other splice variants of 1alphaOHase were detected, our findings indicate that the full length product represents the major 1 alphaOHase gene product in SZ95 cells. In conclusion, SZ95 sebocytes express VDR and the enzymatic machinery to synthesize and metabolize biologically active vitamin D analogues. Sebocytes represent target cells for biologically active metabolites. Our findings indicate that the vitamin D endocrine system is of high importance for sebocyte function and physiology. We conclude that sebaceous glands represent potential targets for therapy with vitamin D analogues or for pharmacological modulation of 1,25(OH)(2)D(3) synthesis/metabolism.

  1. A phase I study of the vitamin D analogue EB 1089 in patients with advanced breast and colorectal cancer.

    PubMed Central

    Gulliford, T.; English, J.; Colston, K. W.; Menday, P.; Moller, S.; Coombes, R. C.

    1998-01-01

    Preclinical studies have shown that the vitamin D analogue EB 1089 has significantly less calcaemic activity than its parent compound 1,25-dihydroxyvitamin D (1,25(OH)2D3) and significant anti-tumour activity. This phase I trial was designed to evaluate the calcaemic effect of the drug in patients with advanced cancer. EB 1089 was given to 36 patients with advanced breast and colorectal cancer in doses of between 0.15 and 17.0 microg m(-2) day(-1). Serial serum and urine calcium, urine creatinine and serum parathyroid hormone (PTH) were monitored. Hypercalcaemia was seen in all patients receiving 17.0 microg m(-2) day(-1). Hypercalcaemia attributable to EB 1089 was reversible by discontinuing or reducing EB 1089 therapy. During the first 5 days of treatment, urine calcium (P = 0.0001) and serum-corrected calcium (P = 0.027) were related to EB 1089 dose, whereas serum parathyroid hormone (P = 0.0001) showed an inverse relationship. Twenty-one patients received compassionate treatment for between 10 and 234 days. No complete or partial responses were seen. Six patients on treatment for more than 90 days showed stabilization of disease. EB 1089 was well tolerated and adverse events considered to be caused by EB 1089 were limited to dose-dependent effects on calcium metabolism. The dose estimated to be tolerable for most patients from this study is around 7 microg m(-2) day(1). These data support previous work that has demonstrated EB 1089 to be significantly less calcaemic than 1,25-dihydroxyvitamin D3. PMID:9662243

  2. Free 25(OH)D and the Vitamin D Paradox in African Americans

    PubMed Central

    Mikhail, Mageda; Dhaliwal, Ruban; Shieh, Albert; Usera, Gianina; Stolberg, Alexandra; Ragolia, Louis; Islam, Shahidul

    2015-01-01

    Context: African Americans have a lower total serum 25-hydroxyvitamin D [25(OH)D] but superior bone health. This has been referred to as a paradox. A recent publication found that free serum 25(OH)D is the same in black and white individuals. However, the study was criticized because an indirect method was used to measure free 25(OH)D. A direct method has recently been developed. Objective: We hypothesized that although total serum 25(OH)D is lower in African Americans, free serum 25(OH)D measured directly would not differ between races. Design: White and black healthy postmenopausal women were matched for age and body mass index. Serum total 25(OH)D, PTH, 1,25-dihydroxyvitamin D, vitamin D binding protein (VDBP), and bone density were measured. Measurement of free 25(OH)D was carried out using an ELISA. Setting: The study was conducted at an ambulatory research unit in a teaching hospital. Outcome: A cross-racial comparison of serum free 25(OH)D was performed. Results: A propensity match resulted in the selection of a total of 164 women. Total 25(OH)D was lower in black women (19.5 ± 4.7 vs 26.9 ± 6.4 ng/mL), but a direct measurement of free 25(OH)D revealed almost identical values (5.25 ± 1.2 vs 5.25 ± 1.3 ng/mL) between races. VDBP was significantly lower in blacks when using a monoclonal-based ELISA but higher with a polyclonal-based ELISA. Serum PTH, 1,25-dihydroxyvitamin D, and bone density were higher in African Americans. Conclusions: Free serum 25(OH)D is the same across races despite the lower total serum 25(OH)D in black women. Results comparing VDBP between races using a monoclonal vs a polyclonal assay were discordant. PMID:26161453

  3. Identification of the vitamin D receptor in osteoblasts and chondrocytes but not osteoclasts in mouse bone.

    PubMed

    Wang, Yongji; Zhu, Jinge; DeLuca, Hector F

    2014-03-01

    Bone is clearly a target of vitamin D and as expected, the vitamin D receptor (VDR) is expressed in osteoblasts. However, the presence of VDR in other cells such as osteocytes, osteoclasts, chondroclasts, and chondrocytes is uncertain. Because of difficulties in obtaining sections of undecalcified adult bone, identification of the site of VDR expression in adult bone tissue has been problematic. In addition, the antibodies to VDR used in previous studies lacked specificity, a property crucial for unambiguous conclusions. In the present study, VDR in the various cells from neonatal and adult mouse bone tissues was identified by a highly specific and sensitive immunohistochemistry method following bone decalcification with EGTA. For accurate evaluation of weak immunosignals, samples from Demay VDR knockout mice were used as negative control. Molecular markers were used to identify cell types. Our results showed that EGTA-decalcification of bone tissue had no detectable effect on the immunoreactivity of VDR. VDR was found in osteoblasts and hypertrophic chondrocytes but not in the multinucleated osteoclasts, chondroclasts, and bone marrow stromal cells. Of interest is the finding that immature osteoblasts contain large amounts of VDR, whereas the levels are low or undetectable in mature osteoblasts including bone lining cells and osteocytes. Proliferating chondrocytes appear devoid of VDR, although low levels were found in the hypertrophic chondrocytes. These data demonstrate that osteoblasts and chondrocytes are major targets of 1α,25-dihydroxyvitamin D, but osteoclasts and chondroclasts are minor targets or not at all. A high level of VDR was found in the immature osteoblasts located in the cancellous bone, indicating that they are major targets of 1α,25-dihydroxyvitamin D. Thus, the immature osteoblasts are perhaps responsible for the vitamin D hormone signaling resulting in calcium mobilization and in osteogenesis.

  4. Cellular and molecular toxicity of lead in bone

    SciTech Connect

    Pounds, J.G. ); Long, G.J.; Rosen, J.F. )

    1991-02-01

    To fully understand the significance of bone as a target tissue of lead toxicity, as well as a reservoir of systemic lead, it is necessary to define the effects of lead on the cellular components of bone. Skeletal development and the regulation of skeletal mass are ultimately determined by the four different types of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. These cells, which line and penetrate the mineralized matrix, are responsible for matrix formation, mineralization, and bone resorption, under the control of both systemic and local factors. Systemic components of regulation include parathyroid hormone, 1,25-dihydroxyvitamin D{sub 3}, and calcitonin; local regulators include numerous cytokines and growth factors. Lead intoxication directly alters many aspects of bone cell function. First, lead may indirectly alter bone cell function through changes in the circulating levels of those hormones, particularly 1,25-dihydroxyvitamin D{sub 3}, which modulate bone cell function. Second, lead may directly alter bone cell function by perturbing the ability of bone cells to respond to hormonal regulation. Third, lead may impair the ability of cells to synthesize or secrete other components of the bone matrix, such as collagen or bone sialoproteins (osteopontin). Finally, lead may directly effect of substitute for calcium in the active sites of the calcium messenger system. The effects of lead on the recruitment and differentiation of bone cells remains to be established. Many of the toxic effects of lead on bone cell function may be produced by perturbation of the calcium and cAMP messenger systems in these cells.

  5. Diurnal Rhythms of Bone Turnover Markers in Three Ethnic Groups

    PubMed Central

    Redmond, Jean; Fulford, Anthony J.; Jarjou, Landing; Zhou, Bo; Prentice, Ann

    2016-01-01

    Context: Ethnic groups differ in fragility fracture risk and bone metabolism. Differences in diurnal rhythms (DRs) of bone turnover and PTH may play a role. Objective: We investigated the DRs of plasma bone turnover markers (BTMs), PTH, and 1,25(OH)2D in three groups with pronounced differences in bone metabolism and plasma PTH. Participants: Healthy Gambian, Chinese, and white British adults (ages 60–75 years; 30 per country). Interventions: Observational study with sample collection every 4 hours for 24 hours. Main Outcomes: Levels of plasma C-terminal telopeptide of type I collagen, procollagen type-1 N-propeptide, N-mid osteocalcin, bone alkaline phosphatase, PTH, and 1,25-dihydroxyvitamin D were measured. DRs were analyzed with random-effects Fourier regression and cross-correlation and regression analyses to assess associations between DRs and fasting and 24-hour means of BTMs and PTH. Results: Concentrations of BTMs, PTH, and 1,25-dihydroxyvitamin D were higher in Gambians compared to other groups (P < .05). The DRs were significant for all variables and groups (P < .03) and were unimodal, with a nocturnal peak and a daytime nadir for BTMs, whereas PTH had two peaks. The DRs of BTMs and PTH were significantly cross-correlated for all groups (P < .05). There was a significant positive association between C-terminal telopeptide of type I collagen and PTH in the British and Gambian groups (P = .03), but not the Chinese group. Conclusions: Despite ethnic differences in plasma BTMs and PTH, DRs were similar. This indicates that alteration of rhythmicity and loss of coupling of bone resorption and formation associated with an elevated PTH in other studies may not uniformly occur across different populations and needs to be considered in the interpretation of PTH as a risk factor of increased bone loss. PMID:27294326

  6. Plasma 25-Hydroxyvitamin D3 and Bladder Cancer Risk According to Tumor Stage and FGFR3 Status: A Mechanism-Based Epidemiological Study

    PubMed Central

    2012-01-01

    Background Previous evidence suggests that 25-hydroxyvitamin D3 [25(OH)D3] protects against several cancers. However, little is known regarding urothelial bladder cancer (UBC). We analyzed the association between plasma 25(OH)D3 and overall risk of UBC, as well as according to stage and FGFR3 molecular subphenotypes. Methods Plasma concentrations of 25(OH)D3 in 1125 cases with UBC and 1028 control subjects were determined by a chemiluminescence immunoassay. FGFR3 mutational status and expression in tumor tissue were assessed. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression adjusting for potential confounders. Analyses were further stratified by tumor invasiveness and grade, FGFR3 expression, and smoking status. Cell proliferation was measured in human UBC cell lines cultured with 1α,25-dihydroxyvitamin D3. Results A statistically significantly increased risk of UBC was observed among subjects presenting the lowest concentrations of 25(OH)D3 (ORadj = 1.83; 95% CI = 1.19 to 2.82; P = .006), showing a dose–response effect (P trend = .004). The association was stronger for patients with muscle-invasive tumors, especially among low-FGFR3 expressers (ORadj = 5.94; 95% CI = 1.72 to 20.45; P = .005). The biological plausibility of these associations is supported by the fact that, in vitro, 1α,25-dihydroxyvitamin D3 upregulates FGFR3 expression in UBC cell lines with low levels of wild-type FGFR3. Conclusion These findings support a role of vitamin D in the pathogenesis of UBC and show that 25(OH)D3 levels are associated with FGFR3 expression in the tumor. Because FGFR3 mutation and overexpression are markers of better outcome, our findings suggest that individuals with low levels of plasma 25(OH)D3 may be at high risk of more aggressive forms of UBC. PMID:23108201

  7. Effects of Decreased Vitamin D and Accumulated Uremic Toxin on Human CYP3A4 Activity in Patients with End-Stage Renal Disease

    PubMed Central

    Tsujimoto, Masayuki; Nagano, Yui; Hosoda, Satomi; Shiraishi, Asuka; Miyoshi, Ayaka; Hiraoka, Shima; Furukubo, Taku; Izumi, Satoshi; Yamakawa, Tomoyuki; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2013-01-01

    In patients with end-stage renal disease, not only renal clearance but also hepatic clearance is known to be impaired. For instance, the concentration of erythromycin, a substrate of cytochrome P450 3A4 (CYP3A4), has been reported to be elevated in patients with end-stage renal disease. The purpose of this study is to elucidate the reason for the decrease in hepatic clearance in patients with end-stage renal disease. Deproteinized pooled sera were used to assess the effects of low-molecular-weight uremic toxins on CYP3A4 activity in human liver microsomes and human LS180 cells. Four uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, hippuric acid, indole-3-acetic acid, and 3-indoxyl sulfate) present at high concentrations in uremic serum were also studied. Simultaneous treatment of uremic serum (less than 10%) or uremic toxins did not affect testosterone 6β-hydroxylation in human liver microsomes. On the other hand, pretreatment of each serum activates CYP3A4 in LS180 cells, and the increased CYP3A4 activity in uremic serum-treated cells was smaller than normal serum-treated cells. In addition, CYP3A4 and CYP24A1 mRNA levels also increased in LS180 cells exposed to normal serum, and this effect was reduced in uremic serum-treated cells and in cells exposed to uremic serum added to normal serum. Furthermore, addition of 1,25-dihydroxyvitamin D to uremic serum partially restored the serum effect on CYP3A4 expression. The present study suggests that the decrease of 1,25-dihydroxyvitamin D and the accumulation of uremic toxins contributed to the decreased hepatic clearance of CYP3A4 substrates in patients with end-stage renal disease. PMID:23965431

  8. Analysis of vitamin D and its metabolites using thermospray liquid chromatography/mass spectrometry.

    PubMed

    Watson, D; Setchell, K D; Ross, R

    1991-07-01

    A new method is described for the analysis of vitamin D and its metabolites utilizing thermospray (TSP) mass spectrometry as an on-line detector for high performance liquid chromatography. Ionization conditions were optimized for use with isocratic reversed phase chromatography. TSP mass spectrometry was employed in series with a UV absorbance detector to facilitate comparisons between the two methods of detection. Positive ion TSP mass spectra were recorded for vitamin D2, vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). The spectra contained protonated molecular ions, ammonium adduct ions and fragment ions due to the loss of one or more molecules of water. A comparison of quantitative precision was made by determining UV absorbance and TSP standard curves for vitamin D3 using two different methods: (1) External standard method with post-column (post UV detector) addition of ammonium acetate. (2) As (1) but using the method of internal standards with a closely eluting internal standard (vitamin D2). In each case the quantitative precision (correlation coefficient) for UV absorbance detection was superior owing to intrinsic instability of the TSP ion beam. A stable isotopically labelled internal standard was employed in the development of an assay for 1,25(OH)2D3. The assay was used to quantify in vitro enzymic conversion of 25(OH)D3 to 1,25(OH)2D3 in guinea pig and sheep renal mitochondrial incubations. TSP LC/MS was also applied to analysis of an extract of human blood plasma in which D3 and each of its principal metabolites were identified in a single analysis.

  9. Genetic Variants Associated with Circulating Parathyroid Hormone.

    PubMed

    Robinson-Cohen, Cassianne; Lutsey, Pamela L; Kleber, Marcus E; Nielson, Carrie M; Mitchell, Braxton D; Bis, Joshua C; Eny, Karen M; Portas, Laura; Eriksson, Joel; Lorentzon, Mattias; Koller, Daniel L; Milaneschi, Yuri; Teumer, Alexander; Pilz, Stefan; Nethander, Maria; Selvin, Elizabeth; Tang, Weihong; Weng, Lu-Chen; Wong, Hoi Suen; Lai, Dongbing; Peacock, Munro; Hannemann, Anke; Völker, Uwe; Homuth, Georg; Nauk, Matthias; Murgia, Federico; Pattee, Jack W; Orwoll, Eric; Zmuda, Joseph M; Riancho, Jose Antonio; Wolf, Myles; Williams, Frances; Penninx, Brenda; Econs, Michael J; Ryan, Kathleen A; Ohlsson, Claes; Paterson, Andrew D; Psaty, Bruce M; Siscovick, David S; Rotter, Jerome I; Pirastu, Mario; Streeten, Elizabeth; März, Winfried; Fox, Caroline; Coresh, Josef; Wallaschofski, Henri; Pankow, James S; de Boer, Ian H; Kestenbaum, Bryan

    2016-12-07

    Parathyroid hormone (PTH) is a primary calcium regulatory hormone. Elevated serum PTH concentrations in primary and secondary hyperparathyroidism have been associated with bone disease, hypertension, and in some studies, cardiovascular mortality. Genetic causes of variation in circulating PTH concentrations are incompletely understood. We performed a genome-wide association study of serum PTH concentrations among 29,155 participants of European ancestry from 13 cohort studies (n=22,653 and n=6502 in discovery and replication analyses, respectively). We evaluated the association of single nucleotide polymorphisms (SNPs) with natural log-transformed PTH concentration adjusted for age, sex, season, study site, and principal components of ancestry. We discovered associations of SNPs from five independent regions with serum PTH concentration, including the strongest association with rs6127099 upstream of CYP24A1 (P=4.2 × 10(-53)), a gene that encodes the primary catabolic enzyme for 1,25-dihydroxyvitamin D and 25-dihydroxyvitamin D. Each additional copy of the minor allele at this SNP associated with 7% higher serum PTH concentration. The other SNPs associated with serum PTH concentration included rs4074995 within RGS14 (P=6.6 × 10(-17)), rs219779 adjacent to CLDN14 (P=3.5 × 10(-16)), rs4443100 near RTDR1 (P=8.7 × 10(-9)), and rs73186030 near CASR (P=4.8 × 10(-8)). Of these five SNPs, rs6127099, rs4074995, and rs219779 replicated. Thus, common genetic variants located near genes involved in vitamin D metabolism and calcium and renal phosphate transport associated with differences in circulating PTH concentrations. Future studies could identify the causal variants at these loci, and the clinical and functional relevance of these variants should be pursued.

  10. Studies on the analysis of 25-hydroxyvitamin D{sub 3} by isotope-dilution liquid chromatography–tandem mass spectrometry using enzyme-assisted derivatisation

    SciTech Connect

    Abdel-Khalik, Jonas; Crick, Peter J.; Carter, Graham D.; Makin, Hugh L.; Wang, Yuqin; Griffiths, William J.

    2014-04-11

    Highlights: • New method for the analysis of 25-hydroxyvitamin D{sub 3} exploiting Girard P derivatisation. • Method also applicable to vitamin D{sub 3}, 1α,25- and 24,25-dihydroxyvitamin D{sub 3}. • By modification of the method 3-epi-25-hydroxyvitamin D{sub 3} can also be analysed. - Abstract: The total serum concentration of 25-hydroxyvitamins D (25-hydroxyvitamin D{sub 3} and 25-hydroxyvitamin D{sub 2}) is currently used as an indicator of vitamins D status. Vitamins D insufficiency is claimed to be associated with multiple diseases, thus accurate and precise reference methods for the quantification of 25-hydroxyvitamins D are needed. Here we present a novel enzyme-assisted derivatisation method for the analysis of vitamins D metabolites in adult serum utilising 25-[26,26,26,27,27,27-{sup 2}H{sub 6}]hydroxyvitamin D{sub 3} as the internal standard. Extraction of 25-hydroxyvitamins D from serum is performed with acetonitrile, which is shown to be more efficient than ethanol. Cholesterol oxidase is used to oxidize the 3β-hydroxy group in the vitamins D metabolites followed by derivatisation of the newly formed 3-oxo group with Girard P reagent. 17β-Hydroxysteroid dehydrogenase type 10 is shown to oxidize selectively the 3α-hydroxy group in the 3α-hydroxy epimer of 25-hydroxyvitamin D{sub 3}. Quantification is achieved by isotope-dilution liquid chromatography–tandem mass spectrometry. Recovery experiments for 25-hydroxyvitamin D{sub 3} performed on adult human serum give recovery of 102–106%. Furthermore in addition to 25-hydroxyvitamin D{sub 3}, 24,25-dihydroxyvitamin D{sub 3} and other uncharacterised dihydroxy metabolites, were detected in adult human serum.

  11. High throughput LC-MS/MS method for the simultaneous analysis of multiple vitamin D analytes in serum

    PubMed Central

    Jenkinson, Carl; Adams, John S.; Stewart, Paul M.; Hewison, Martin; Keevil, Brian G.

    2016-01-01

    Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D ‘status’ most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220 μl of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease. PMID:26874878

  12. High throughput LC-MS/MS method for the simultaneous analysis of multiple vitamin D analytes in serum.

    PubMed

    Jenkinson, Carl; Taylor, Angela E; Hassan-Smith, Zaki K; Adams, John S; Stewart, Paul M; Hewison, Martin; Keevil, Brian G

    2016-03-01

    Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D 'status' most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220μL of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease.

  13. A phase I study of the vitamin D analogue EB 1089 in patients with advanced breast and colorectal cancer.

    PubMed

    Gulliford, T; English, J; Colston, K W; Menday, P; Moller, S; Coombes, R C

    1998-07-01

    Preclinical studies have shown that the vitamin D analogue EB 1089 has significantly less calcaemic activity than its parent compound 1,25-dihydroxyvitamin D (1,25(OH)2D3) and significant anti-tumour activity. This phase I trial was designed to evaluate the calcaemic effect of the drug in patients with advanced cancer. EB 1089 was given to 36 patients with advanced breast and colorectal cancer in doses of between 0.15 and 17.0 microg m(-2) day(-1). Serial serum and urine calcium, urine creatinine and serum parathyroid hormone (PTH) were monitored. Hypercalcaemia was seen in all patients receiving 17.0 microg m(-2) day(-1). Hypercalcaemia attributable to EB 1089 was reversible by discontinuing or reducing EB 1089 therapy. During the first 5 days of treatment, urine calcium (P = 0.0001) and serum-corrected calcium (P = 0.027) were related to EB 1089 dose, whereas serum parathyroid hormone (P = 0.0001) showed an inverse relationship. Twenty-one patients received compassionate treatment for between 10 and 234 days. No complete or partial responses were seen. Six patients on treatment for more than 90 days showed stabilization of disease. EB 1089 was well tolerated and adverse events considered to be caused by EB 1089 were limited to dose-dependent effects on calcium metabolism. The dose estimated to be tolerable for most patients from this study is around 7