D3 Pretreatment Research Articles

Comparison of Bone Mineral Density and Markers of Bone Turnover in Osteoporotic Women after 6-Month Treatment with Alendronate or Bazedoxifene: A Randomized Controlled Trial.

BackgroundIn a randomized controlled trial, we compared the bone mineral densities (BMDs) and blood markers of bone turnover during short-term treatment of osteoporotic women with bisphosphonate alendronate or bazedoxifene, a selective estrogen receptor modulator.MethodsTen and eleven patients were randomized to the alendronate and bazedoxifene groups, respectively. BMDs were measured before and after 6 months of treatment. Blood tests were used to measure the levels of osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX), vitamin D3, and parathyroid hormone pretreatment and after 3 and 6 months of treatment. The variables were compared statistically.ResultsThe alendronate group showed decreases in blood levels of both OC and CTX during the study period (P<0.001 and P=0.002, respectively), while the bazedoxifene group had a decrease only in OC levels (P=0.012). After 6 months of treatment, BMDs significantly increased in the alendronate group at multiple bone sites, including the L1–4 lumbar vertebrae, femur trochanter, and total femur. However, there was no significant increase in BMD in the bazedoxifene group. BMDs were not significantly different between the 2 groups.ConclusionsPatients treated with alendronate showed more rapid suppression of markers of bone turnover and higher BMD than those treated with bazedoxifene during a short-term regime. Considering the effects and complications of each medication, the relationship between bone turnover rate and bone quality will need to be investigated in future studies.

Vitamin D3 Controls TLR4- and TLR2-Mediated Inflammatory Responses of Endometrial Cells

Objectives: Vitamin D has potent immunoregulatory features and modulates innate and adaptive immune responses. There is a significant association between intrauterine infection-associated inflammatory responses and pregnancy complications such as abortion and preterm labor. Here, we investigated how 1,25 (OH)2 D3 could modulate inflammatory responses of endometrial cells. Design: This is an in vitro experimental study. Endometrial stromal cells (ESCs) and whole endometrial cells (WECs) were collected from 15 apparently normal women, and the immunomodulatory effects of 1,25 (OH)2 D3 on lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated ESCs and WECs were investigated. Participants/Materials, Setting, and Methods: Women with no history of abortion, infertility, endometriosis, or sign of vaginal infection were enrolled in this study. Endometrial samples were collected by gynecologists using a Pipelle pipette in the proliferative phase of the menstrual cycle. WECs and ESCs were collected and treated with either LPS or LTA. The levels of IL-6, IL-8, and TNF-α in culture supernatants were quantified using the ELISA technique. TLR2, TLR4, and MyD88 expressions were assessed by RT-qPCR. TLR4 expression at the protein level was studied by the Western blot technique. Results: 1,25 Dihydroxycholecalciferol (1,25 (OH)2 D3) significantly reduced TNF-α production in LPS-activated ESCs and TNF-α and IL-6 production by LTA-stimulated WECs. In contrast, 1,25 (OH)2 D3 pretreatment increased the production of IL-8 by LPS- and LTA-stimulated endometrial cells. 1,25 (OH)2 D3 pretreatment markedly reduced LPS-induced TLR4 protein expression by ESCs. LPS treatment of ESCs significantly induced MyD88 gene expression. This effect was reversed when these cells were pretreated with 1,25 (OH)2 D3 before stimulation with LPS. Limitations: Because of the small size of samples, doing experiments all together on some samples was not feasible. Confirmation of the results obtained here needs well-designed in vivo studies. Conclusions: 1,25 (OH)2 D3 is an immunomodulatory molecule essential for maintaining endometrial immune homeostasis by controlling potentially harmful inflammatory responses associated with female reproductive tract infections.

The effect of vitamin D3 and paricalcitol on penicillin-induced epileptiform activity in rats

AimEpilepsy is a disease characterized by seizures which impair human life considerably. Vitamin D is of different systemic effects on metabolism and its deficiency is known to have a high prevalence among epilepsy patients. Paricalcitol, a vitamin D receptor agonist, has relatively fewer side effects. This study aimed to investigate the anticonvulsant effect of vitamin D3 (cholecalciferol) and paricalcitol on penicillin-induced epileptiform activity. Method21 male Wistar rat weighing 180−240 g were used. After anesthetized by 1.25 g/kg urethane intraperitoneally (i.p.), rats were placed in the stereotaxic frame and tripolar electrodes were placed on the skull. The single microinjection of penicillin (2.5 μl, 500 IU, i.c.) into left sensorimotor cortex induced epileptiform activity. A single dose of 60.000 IU/kg (i.p.) vitamin D3 was administered 14 days before intracortical penicillin (500 IU) injection. Paricalcitol (10 μg/kg, i.p.) was administered 30 min before intracortical penicillin (500 IU) administration and recorded for the following 180 min. ResultsVitamin D3 pretreatment and paricalcitol diminished the frequency of epileptiform activity (p < 0.001) without changing the amplitude (p > 0.05) compared to the penicillin-injected group. Vitamin D3 pretreatment and paricalcitol led to an important delay in the onset of penicillin-induced epileptiform activity (p < 0.001 and p < 0.05, respectively). Vitamin D3 increased the latency of penicillin-induced epileptic activity compared to paricalcitol group (p < 0.001). ConclusionResults indicate that vitamin D3 and paricalcitol decreased the frequency and increased the latency of the penicillin-induced epileptic activity. Vitamin D3 was more effective than paricalcitol.

1,25-Vitamin D3 protects against cooking oil fumes-derived PM2.5-induced cell damage through its anti-inflammatory effects in cardiomyocytes

The functional role of 1,25-vitamin D3 in cooking oil fumes (COFs)-derived PM2.5-induced cell damage is largely unexplored. The present study investigated the protective role of 1,25-vitamin D3 against cell injury by possible involvement of JAK/STAT and NF-κB signaling pathways in cardiomyocytes. Cell viability was measured using CCK-8 assay, and cell apoptosis was analyzed by flow cytometry, qRT-PCR and Western blot in cultured rat neonatal cardiomyocytes treated with 1,25-vitamin D3 and COFs-derived PM2.5. Expressions of JAK/STAT and NF-κB signaling pathway were measured by Western blot. The results suggested that treatment with COFs-derived PM2.5 significantly decreased cell viability and increased apoptosis and oxidative stress in cultured rat neonatal cardiomyocytes. 1,25-vitamin D3 pretreatment alleviated the cell injury by increasing cell viability and decreasing apoptosis in the cardiomyocytes. 1,25-vitamin D3 pretreatment also decreased the ROS level and inflammation in the cardiomyocytes. Furthermore, 1,25-vitamin D3 pretreatment alleviated COFs-derived PM2.5-evoked elevation of JAK/STAT and NF-κB signaling pathways. Our study showed that 1,25-vitamin D3 pretreatment protected cardiomyocytes from COFs-derived PM2.5-induced injury by decreasing ROS, apoptosis and inflammation level via activations of the JAK/STAT and NF-κB signaling pathways.

Vitamin D3 enhances the response to cisplatin in bladder cancer through VDR and TAp73 signaling crosstalk.

BackgroundVitamin D3 (VitD) deficiency is linked to increased incidence and worse survival in bladder cancer (BCa). In addition to cystectomy, patients are treated with cisplatin‐based chemotherapy, however 30%‐50% of patients do not benefit from this treatment. The effects of VitD deficiency on response to chemotherapy remain unknown.MethodsTo test effects of VitD supplementation on the response to cisplatin we analyzed patient serum VitD levels and correlated that with survival. In vivo, VitD deficient mice were treated with cisplatin, with or without pretreatment with the active VitD metabolite, 1,25 dihydroxyvitamin D3 (1,25D3). Lastly, using BCa cell lines, T24 and RT‐112, the mechanism of action of 1,25D3 and cisplatin combination treatment was determined by apoptosis assays, as well as western blot and RT‐PCR.ResultsIn this study, we determined that low serum 25 hydroxyvitamin D3 (25D3) levels was significantly associated with worse response to cisplatin. Pretreating deficient mice with 1,25D3, reduced tumor volume compared to cisplatin monotherapy. In vitro, 1,25D3 pretreatment increased the apoptotic response to cisplatin. 1,25D3 pretreatment increased expression of TAp73 and its pro‐apoptotic targets, in a VDR dependent manner. VDR and its transcriptional targets were induced after 1,25D3 treatment and further increased after the combination of 1,25D3 and cisplatin in a TAp73 dependent manner.ConclusionsOur data suggest that VitD deficiency could be a biomarker for poor response to cisplatin, and pretreating with VitD can increase the apoptotic response to cisplatin through VDR and TAp73 signaling crosstalk.

1,25-Dihydroxyvitamin D3 modulates the effects of sublethal BPA on mitochondrial function via activating PI3K-Akt pathway and 17β-estradiol secretion in rat granulosa cells

Bisphenol A (BPA), an endocrine-disrupting chemical, is capable of producing reproductive toxicity. BPA results in mitochondrial DNA (mtDNA) deletion and mitochondrial dysfunction; however, the effect of BPA on the mitochondria of ovarian granulosa cells is not clear. Further, 1,25-dihydroxyvitamin D3 (1,25D3) may play a role in reproduction, because its receptor, VDR, contributes to the inhibition of oxidative stress and predominantly exists in the nuclei of granulosa cells. Hence, the role of 1,25D3 in BPA-mediated effects on mitochondrial function was examined in this study. Primary rat granulosa cells treated with BPA, 1,25D3, or both were subjected to molecular/biochemical assays to measure cell survival, mtDNA content, mtDNA deletion, superoxide dismutase activity, levels of proteins related to mitochondrial biogenesis, and mitochondrial function. We found that cell viability was dose-dependently reduced and reactive oxygen species (ROS) levels were increased by BPA treatment. BPA administration elevated Mn-superoxide dismutase (MnSOD) expression but negatively regulated total SOD activity. 1,25D3 treatment alone increased 17β-estradiol secretion, ATP production, and cellular oxygen consumption. In cells treated with both agents, 1,25D3 enhanced BPA-induced MnSOD protein upregulation and blocked the BPA-mediated decline in total SOD activity. Furthermore, 1,25D3 attenuated BPA-mediated mtDNA deletion but showed no effect on BPA-induced increases in mtDNA content. Although BPA had no influence on the levels of peroxisome proliferator-activated receptor-γ coactivator-1 α, nuclear respiratory factor-1, mitochondrial transcription factor A, or cytochrome c oxidase subunit IV, 1,25D3 plus BPA markedly increased mitochondrial biogenesis-related protein expression via the PI3K-Akt pathway. Moreover, BPA-mediated negative regulation of cytochrome c oxidase subunit I levels and 17β-estradiol secretion was attenuated by 1,25D3 pre-treatment. Our results suggest that 1,25D3 attenuates BPA-induced decreases in 17β-estradiol and that treatment with 1,25D3 plus BPA regulates granulosa cell mitochondria by elevating mitochondrial biogenesis-related protein levels.

Effects of Vitamin D3 on the NADPH Oxidase and Matrix Metalloproteinase 9 in an Animal Model of Global Cerebral Ischemia.

Decreased blood flow in the brain leads to a rapid increase in reactive oxygen species (ROS). NADPH oxidase (NOX) is an enzyme family that has the physiological function to produce ROS. NOX2 and NOX4 overexpression is associated with aggravated ischemic injury, while NOX2/4-deficient mice had reduced stroke size. Dysregulation of matrix metalloproteinases (MMPs) contributes to tissue damage. The active form of vitamin D3 expresses neuroprotective, immunomodulatory, and anti-inflammatory effects in the CNS. The present study examines the effects of the vitamin D3 pretreatment on the oxidative stress parameters and the expression of NOX subunits, MMP9, microglial marker Iba1, and vitamin D receptor (VDR), in the cortex and hippocampus of Mongolian gerbils subjected to ten minutes of global cerebral ischemia, followed by 24 hours of reperfusion. The ischemia/reperfusion procedure has induced oxidative stress, changes in the expression of NOX2 subunits and MMP9 in the brain, and increased MMP9 activity in the serum of experimental animals. Pretreatment with vitamin D3 was especially effective on NOX2 subunits, MMP9, and the level of malondialdehyde and superoxide anion. These results outline the significance of the NOX and MMP9 investigation in brain ischemia and the importance of adequate vitamin D supplementation in ameliorating the injury caused by I/R.

Vitamin D3 attenuates cognitive deficits and neuroinflammatory responses in ICV-STZ induced sporadic Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by intracellular neurofibrillary tangles and extracellular Aβ deposition. Growing experimental evidence indicate diverse biological effects of vitamin D3 including antioxidant, neuroprotective, anti-inflammatory and cardiovascular benefits. However, the underlying neuroprotective mechanism of vitamin D3 is still largely elusive. Therefore, the present study was aimed to investigate the neuroprotective effects of vitamin D3 on ICV-STZ induced sporadic AD. Our study demonstrated that vitamin D3 pretreatment significantly improved spatial learning and memory functions and effectively mitigated ICV-STZ mediated neuronal oxidative stress, mitochondrial aberrations and improved cholinergic functions. Moreover, vitamin D3 attenuated hippocampal neuroinflammatory response and reduced neuronal death in cortex and hippocampus. Our findings indicated that prophylactic vitamin D3 supplementation ameliorated ICV-STZ mediated neurobehavioral alterations, oxidative stress and neuroinflammation thereby improving cholinergic functions and reversed degenerative changes in brain. Thus, our study further provides evidence for its therapeutic supplementation for various neurodegenerative disorders including AD.

Vitamin D3 pretreatment protects against lipopolysaccharide-induced early embryo loss through its anti-inflammatory effects.

Increasing evidence demonstrates that inflammatory cytokines are involved in LPS-induced adverse pregnant outcomes including early embryo loss. Vitamin D3 (VitD3) has anti-inflammatory activity. We aimed to investigate the effects of vitamin D3 (VitD3) on LPS-induced early embryo loss in mice. All pregnant mice except controls were intraperitoneally (ip) injected with LPS on GD7. In VitD3 alone and LPS+VitD3 groups, pregnant mice were pretreated with VitD3 by gavage daily from GD5 to GD7. LPS caused 62.5% pregnant mice with early embryo loss. Interestingly, the rate of abortion dropped to 14.3% when pregnant mice were pretreated with VitD3. Additional experiment showed that VitD3 significantly attenuated LPS-evoked elevation on TNF-α, IFN-γ, MIP-2, and nitrate plus nitrite in maternal serum. In addition, VitD3 alleviated LPS-induced COX-2 expression in the decidua and attenuated the elevation of PGF2α in maternal serum. Although VitD3 had no effect on IL-10 in maternal serum, it induced further elevation of serum IL-10 level in LPS-treated mice. Further analysis showed that VitD3 activated VDR signaling, simultaneously inhibited LPS-induced nuclear translocation of NF-κB p65 subunits in the decidua. VitD3 protects mice from LPS-induced early embryo loss at least partially through its anti-inflammatory effects.

Vitamin D3 pretreatment regulates renal inflammatory responses during lipopolysaccharide-induced acute kidney injury.

Vitamin D receptor (VDR) is highly expressed in human and mouse kidneys. Nevertheless, its functions remain obscure. This study investigated the effects of vitamin D3 (VitD3) pretreatment on renal inflammation during lipopolysaccharide (LPS)-induced acute kidney injury. Mice were intraperitoneally injected with LPS. In VitD3 + LPS group, mice were pretreated with VitD3 (25 μg/kg) at 48, 24 and 1 h before LPS injection. As expected, an obvious reduction of renal function and pathological damage was observed in LPS-treated mice. VitD3 pretreatment significantly alleviated LPS-induced reduction of renal function and pathological damage. Moreover, VitD3 pretreatment attenuated LPS-induced renal inflammatory cytokines, chemokines and adhesion molecules. In addition, pretreatment with 1,25(OH)2D3, the active form of VitD3, alleviated LPS-induced up-regulation of inflammatory cytokines and chemokines in human HK-2 cells, a renal tubular epithelial cell line, in a VDR-dependent manner. Further analysis showed that VitD3, which activated renal VDR, specifically repressed LPS-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65 subunit in the renal tubules. LPS, which activated renal NF-κB, reciprocally suppressed renal VDR and its target gene. Moreover, VitD3 reinforced the physical interaction between renal VDR and NF-κB p65 subunit. These results provide a mechanistic explanation for VitD3-mediated anti-inflammatory activity during LPS-induced acute kidney injury.

Abstract LB-039: 1,25 dihydroxyvitamin D3 and cisplatin combination modulate p73 in bladder cancer

Abstract Cisplatin-based combination chemotherapy is the standard approach to therapy of advanced bladder cancer. While there is a substantial initial response rate and occasional complete response, long term control is not common. There is a need to develop new therapeutic approaches to bladder cancer. 1,25 dihydroxyvitamin D3 (1,25D3), the active metabolite of vitamin D, enhances the anti-tumor effects of cisplatin in preclinical bladder cancer models. We evaluated the mechanism of 1,25D3 potentiation of cisplatin cytotoxicity through in vitro studies in bladder cancer cell lines. Our studies suggest that enhanced cytotoxicity is mediated through modulation of p73. The ratio of the pro-apoptotic TAp73 isoform to the anti-apoptotic ΔNp73 isoform is important in determining cellular response to cisplatin. Our studies demonstrate that pretreatment with 1,25D3 (0.5 uM) for 24 hrs followed by cisplatin (0.75 ug/ml) for 48 hrs increases the ratio of TA/ΔNp73 mRNA transcripts 2-fold in the T24 bladder cancer cell line, and increases TAp73's transcriptional target, BAX approximately 3-fold compared to cisplatin alone. Protein levels of p73 and BAX, are also increased with 1,25D3 pretreatment followed by cisplatin in T24 cells as determined by western blot. Using a TransAm p53 binding assay after p53 immunodepletion, cisplatin treatment was found to decrease TAp73 functional ability to bind DNA by approximately half, although this has not yet been determined to be statistically significant. 1,25D3 pretreatment followed by cisplatin prevents the decrease in TAp73 DNA binding found after cisplatin alone. These data suggest an increase in TAp73 pro-apoptotic functional abilities resulting from 1,25D3 pretreatment followed by cisplatin in T24 cells. These findings suggest that 1,25D3 may have potential to be used in combination with cisplatin to increase the apoptotic response. Further studies are being performed to determine the requirement of TAp73 in 1,25D3 potentiation of cisplatin cytotoxicity by using T24 cells transfected with TAp73 shRNA in assays previously used to determine synergism, ie. MTT, clonogenic, and apoptosis assays. Supported by NCI grants CA067267 and CA016056. Citation Format: Brittany L. Bunch, Anna Woloszynska-Read, Donald L. Trump, Candace S. Johnson. 1,25 dihydroxyvitamin D3 and cisplatin combination modulate p73 in bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-039. doi:10.1158/1538-7445.AM2015-LB-039

Vitamin D3 pretreatment alleviates renal oxidative stress in lipopolysaccharide-induced acute kidney injury

Increasing evidence demonstrates that reactive oxygen species plays important roles in sepsis-induced acute kidney injury. This study investigated the effects of VitD3 pretreatment on renal oxidative stress in sepsis-induced acute kidney injury. Mice were intraperitoneally injected with lipopolysaccharide (LPS, 2.0mg/kg) to establish an animal model of sepsis-induced acute kidney injury. In VitD3+LPS group, mice were orally pretreated with three doses of VitD3 (25μg/kg) at 1, 24 and 48h before LPS injection. As expected, oral pretreatment with three daily recommended doses of VitD3 markedly elevated serum 25(OH)D concentration and efficiently activated renal VDR signaling. Interestingly, LPS-induced renal GSH depletion and lipid peroxidation were markedly alleviated in VitD3-pretreated mice. LPS-induced serum and renal nitric oxide (NO) production was obviously suppressed by VitD3 pretreatment. In addition, LPS-induced renal protein nitration, as determined by 3-nitrotyrosine residue, was obviously attenuated by VitD3 pretreatment. Further analysis showed that LPS-induced up-regulation of renal inducible nitric oxide synthase (inos) was repressed in VitD3-pretreated mice. LPS-induced up-regulation of renal p47phox and gp91phox, two NADPH oxidase subunits, were normalized by VitD3 pretreatment. In addition, LPS-induced down-regulation of renal superoxide dismutase (sod) 1 and sod2, two antioxidant enzyme genes, was reversed in VitD3-pretreated mice. Finally, LPS-induced tubular epithelial cell apoptosis, as determined by TUNEL, was alleviated by VitD3 pretreatment. Taken together, these results suggest that VitD3 pretreatment alleviates LPS-induced renal oxidative stress through regulating oxidant and antioxidant enzyme genes.

1,25D3 Enhances antitumor activity of gemcitabine and cisplatin in human bladder cancer models

1,25 dihydroxyvitamin D3 (1,25D3) potentiates the cytotoxic effects of several common chemotherapeutic agents. The combination of gemcitabine and cisplatin is a current standard chemotherapy regimen for bladder cancer. The authors investigated whether 1,25D3 could enhance the antitumor activity of gemcitabine and cisplatin in bladder cancer model systems. Human bladder cancer T24 and UMUC3 cells were pretreated with 1,25D3 followed by gemcitabine and cisplatin. Apoptosis was assessed by annexin V staining. Caspase activation was examined by immunoblot analysis and substrate-based caspase activity assay. The cytotoxic effects were examined by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and in vitro clonogenic assay. p73 protein levels were assessed by immunoblot analysis. Knockdown of p73 was achieved by siRNA. The in vivo antitumor activity was assessed by in vivo excision clonogenic assay and tumor regrowth delay in the T24 xenograft model. 1,25D3 pretreatment enhanced gemcitabine and cisplatin-induced apoptosis and the activities of caspases 8, 9, and 3 in T24 and UMUC3 cells. 1,25D3 synergistically reduced gemcitabine and cisplatin-suppressed surviving fraction in T24 cells. 1,25D3, gemcitabine, or cisplatin induced p73 accumulation, which was enhanced by gemcitabine and cisplatin or 1,25D3 and gemcitabine and cisplatin. p73 expression was lower in human primary bladder tumor tissue compared with adjacent normal tissue. Knockdown of p73 increased clonogenic capacity of T24 cells treated with 1,25D3, gemcitabine and cisplatin, or 1,25D3 and gemcitabine and cisplatin. 1,25D3 and gemcitabine and cisplatin combination enhanced tumor regression compared with 1,25D3 or gemcitabine and cisplatin alone. 1,25D3 potentiates gemcitabine and cisplatin-mediated growth inhibition in human bladder cancer models in vitro and in vivo, which involves p73 induction and apoptosis.

Metallothionein, an Endogenous Antioxidant, Protects against Retinal Neuron Damage in Mice

To clarify the functional role of metallothionein (MT) in retinal damage in mice deficient in both MT-I and -II (MT-I/-II-deficient mice [C57BL/6J background]) and wild-type (C57BL/6J) mice and MT induction (zinc sulfate [ZnSO4] and 1alpha, 25-dihydroxyvitamin D3 [Vit. D3]). Retinal, cell damage was induced by intravitreous injection of N-methyl-D-aspartate (NMDA; 40 nmol/eye). Retinal MT-I, -II, and -III mRNA expression was monitored by real-time reverse-transcription-PCR of total retinal RNA from eyes injected or not injected with NMDA. In wild-type mice, MT-I and -II immunohistochemistry was performed (with antibody that recognizes both proteins) 12 and 24 hours after intravitreous NMDA injection. To examine the involvement of induced retinal MT, ZnSO4 (10 nmol/eye) or Vit. D3 (0.2 or 2 ng/eye) was intravitreously injected 24 hours before NMDA injection in wild-type or MT-I/-II-deficient mice, and ganglion cell layer (GCL) cell loss and inner plexiform layer (IPL) thinning were evaluated 7 days after the NMDA injection. The protective effect of Vit. D3 was assessed against the RGC-5 cell death induced by oxidative stress (using buthionine sulfoximine [BSO] to deplete glutathione in combination with glutamate to inhibit cystine uptake). In wild-type mice, MT-II mRNA expression was time-dependently elevated by NMDA (5.9 and 7.4 times versus the nontreated control at 4 and 12 hours, respectively, after injection), with the normal level being regained within 24 hours. In contrast, MT-I and -III showed persistent decreases (to <50% control) from 4 to 24 hours. In wild-type mice, MT-like immunoreactivity was increased in the inner retina (GCL and IPL) 12 and 24 hours after NMDA injection. At 7 days after NMDA injection in MT-I/-II-deficient mice (versus wild-type mice), GCL cell loss was increased, but IPL thickness was not different. Pretreatment with ZnSO4 or Vit. D3 increased inner retinal MT-like immunoreactivity 24 hours after NMDA injection and significantly attenuated NMDA-induced GCL cell loss in wild-type mice, but ZnSO4 pretreatment did not protect against such cell loss in MT-I/-II-deficient mice. In vitro, Vit. D3 pretreatment (100 nM) reduced BSO+glutamate-induced RGC-5 cell death. These findings suggest that MT, especially MT-II, protects against retinal neuron damage, by acting as an endogenous antioxidant.

1,25-Dihydroxyvitamin D3 inhibits lipopolysaccharide-induced immune activation in human endothelial cells

In addition to its well-known role in mineral and skeletal homeostasis, 1,25-dihydroxyvitamin D3 [1,25-(OH)2, D3] regulates the differentiation, growth and function of a broad range of immune system cells, including monocytes, dendritic cells, T and B lymphocytes. Vascular endothelial cells play a major role in the innate immune activation during infections, sepsis and transplant rejection; however, currently there are no data on the effect of 1,25-(OH)2 D3 on microbial antigen-induced endothelial cell activation. Here we show that 1,25-(OH)2 D3 pretreatment of human microvessel endothelial cells (HMEC) inhibited the enteric gram-negative bacterial lipopolysaccharide (LPS) activation of transcription factor NF-kappaB and interleukin (IL)-6, IL-8 and regulated upon activation normal T cell exposed and secreted (RANTES) release. The effect of 1,25-(OH)2 D3 was not due to increased cell death or inhibition of endothelial cell proliferation. 1,25-(OH)2 D3 pretreatment of HMEC did not block MyD88-independent LPS-induced interferon (IFN)-beta promoter activation. 1,25-(OH)2 D3 pretreatment of HMEC did not modulate Toll-like receptor 4 (TLR4) or MD-2 expression. These data suggest that 1,25-(OH)2 D3 may play a role in LPS-induced immune activation of endothelial cells during gram-negative bacterial infections, and a suggest a potential role for 1,25-(OH)2 D3 and its analogues as an adjuvant in the treatment of gram-negative sepsis.

Effects of 1α,25-dihydroxy-vitamin D3 pretreatment and MAP kinase inhibitor PD 98059 on response of osteoblasts to prostate-derived osteoblastic factors

Prostate carcinoma-derived factors induce a proliferative response in osteoblasts. The present study investigated the involvement of MAP kinase in the osteoblastic reaction of osteocytes and the response of 1alpha,25-hydroxy-vitamin D3 (1,25-vitD3)-pretreated osteoblasts. Conditioned media (CM) from prostate, colon, pancreatic, renal cell and breast cancer cell lines were tested on their proliferative activity using murine osteoblast-like MC3T3-E1 cells, MG63 human osteosarcoma cells and immortalized human osteoblasts (AHTO-7). Changes in osteoblastic activities of the supernantants were measured in the presence of MAP kinase inhibitors and following 1,25-vitD3-induced differentiation of the target osteoblasts. Supernatants of prostate cancer cells stimulated proliferation of osteoblasts in all three indicator cell lines, with AHTO-7 exhibiting the most significant correlation to human primary osteoblast cultures. 1,25-vitD3 induced the differentiation marker alkaline phosphatase (ALP) in MC3T3-E1 and AHTO-7, but only to a minor degree in MG63 cells. 1,25-vitD3-induced differentiation reduced the proliferative response to CM from several cell lines in MC3T3-E1 and MG63 to a minor degree, whereas in AHTO-7 cells the osteoblastic reaction was reduced for 2/4 pancreatic, 3/3 colon and 1/1 renal cancer CMs, however not for 3/3 prostate cancer CMs. Stimulation of AHTO-7 cells by CM from prostate cancer lines is inhibited significantly by MEK1 kinase inhibitor PD 98059 in contrast to CMs derived from other carcinomas, except ACHN renal cancer cells. The findings in the present study demonstrate that human AHTO-7 cells seem to represent a valid human system to monitor osteoblastic activity, especially in respect to 1,25-vitD3-induced differentiation. Vitamin D3-induced differentiation has no direct effect on prostate cancer-derived osteoblastic activity in the same cell line in vitro, which however, could be reversed by disruption of the signal transduction at the MAP kinase level, revealing a new target for the inhibition of prostate cancer-associated bone formation.

Vitamin D 3 attenuates 6-hydroxydopamine-induced neurotoxicity in rats

Previous reports have demonstrated that exogeneous administration of glial cell line-derived neurotrophic factor (GDNF) reduces ventral mesencephalic (VM) dopaminergic (DA) neuron damage induced by 6-hydroxydopamine (6-OHDA) lesioning in rats. Recent studies have shown that 1,25-dihydroxyvitamin D 3 (D3) enhances endogenous GDNF expression in vitro and in vivo. The purpose of present study was to investigate if administration of D3 in vivo and in vitro would protect against 6-OHDA-induced DA neuron injury. Adult male Sprague–Dawley rats were injected daily with D3 or with saline for 8 days and then lesioned unilaterally with 6-OHDA into the medial forebrain bundle. Locomotor activity was measured using automated activity chambers. We found that unilateral 6-OHDA lesioning reduced locomotor activity in saline-pretreated animals. Pretreatment with D3 for 8 days significantly restored locomotor activity in the lesioned animals. All animals were sacrificed for neurochemical analysis 6 weeks after lesioning. We found that 6-OHDA administration significantly reduced dopamine (DA), 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanilic acid (HVA) levels in the substantia nigra (SN) on the lesioned side in the saline-treated rats. D3 pretreatment protected against 6-OHDA-mediated depletion of DA and its metabolites in SN. Using primary cultures obtained from the VM of rat embryos, we found that 6-OHDA or H 2O 2 alone caused significant cell death. Pretreatment with D3 (10 −10 M) protected VM neurons against 6-OHDA- or H 2O 2-induced cell death in vitro. Taken together, our data indicate that D3 pretreatment attenuates the hypokinesia and DA neuronal toxicity induced by 6-OHDA. Since both H 2O 2 and 6-OHDA may injure cells via free radical and reactive oxygen species, the neuroprotection seen here may operate via a reversal of such a toxic mechanism.

1,25-dihydroxyvitamin D3 pretreatment limits prostaglandin biosynthesis by cytokine-stimulated adult human osteoblast-like cells

The steroid derivative 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a regulator of bone biology, and there is evidence that 1,25(OH)2D3 modulates arachidonic acid metabolism in osteoblastic cell model systems and in bone organ cultures. In the present studies, 1,25(OH)2D3 decreased prostaglandin (PG) biosynthesis by normal adult human osteoblast-like (hOB) cell cultures by about 30%. The decrease was observed under basal incubation conditions, or in specimens stimulated by transforming growth factor-beta 1 (TGF-beta) or by tumor necrosis factor-alpha (TNF). The inhibition of the TGF-beta-stimulated PG production appeared to reflect a diminished efficiency of arachidonic acid conversion into PGs by the cells, while the efficiency of substrate utilization for PG biosynthesis was unaffected by 1,25(OH)2D3 pretreatment in the unstimulated samples, or in samples stimulated with TNF or with TNF plus TGF-beta. Free arachidonic acid levels were decreased following 1,25(OH)2D3 pretreatment in the TNF stimulated samples. hOB cell phospholipase A2 activity was measured in subcellular fractions, and this activity was decreased by 20-25% in the 1,25(OH)2D3 pretreated samples. The addition of the selective inhibitor AACOCF3 to the phospholipase A2 assays provided evidence that it was the cytoplasmic isoform of the enzyme that was affected by the 1,25(OH)2D3 pretreatment of the hOB cells. Thus, 1,25(OH)2D3 regulation of hOB cell biology includes significant effects on arachidonic acid metabolism. In turn, this could influence the effects of other hormones and cytokines whose actions include the stimulated production of bioactive arachidonic acid metabolites.

1,25‐dihydroxyvitamin D3 pretreatment limits prostaglandin biosynthesis by cytokine‐stimulated adult human osteoblast‐like cells

1,25‐dihydroxyvitamin D3 pretreatment limits prostaglandin biosynthesis by cytokine‐stimulated adult human osteoblast‐like cells

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line.

The biologically active metabolite of vitamin D3, 1,25 (OH)2 D3, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and 125I-interleukin 1 alpha, we show that 1,25 (OH)2 D3 (5,50 nM) enhanced 125I-interleukin 1 alpha binding up to almost 2-fold over control. This 1,25 (OH)2 D3 effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of 125I-interleukin 1 alpha binding data demonstrated that 1,25 (OH)2 D3 enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D3, 25 (OH) D3, also augmented 125I-interleukin 1 alpha binding but at steroid levels 2-3 log orders greater than 1,25 (OH)2 D3. This observation, combined with the presence of high-affinity 3H-1,25 (OH)2 D3 receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)2 D3-enhanced 125I-interleukin 1 alpha binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)2 D3 enhanced interleukin 1 receptor expression. Northern blots hybridized with a 32P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)2 D3 enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)2 D3 pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1 alpha). These findings further support 1,25 (OH)2 D3's role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.