Abstract We tested whether lifelong modification of vitamin D signaling can alter the progression of early prostate carcinogenesis in studies using mice that develop high-grade prostatic intraepithelial neoplasia that is similar to humans. Two tissue-limited models showed that prostate vitamin D receptor (VDR) loss increased prostate carcinogenesis. In another study, we fed diets with three vitamin D3 levels (inadequate = 25 IU/kg diet, adequate for bone health = 150 IU/kg, or high = 1,000 IU/kg) and two calcium levels (adequate for bone health = 0.5% and high = 1.5%). Dietary vitamin D caused a dose-dependent increase in serum 25-hydroxyvitamin D levels and a reduction in the percentage of mice with adenocarcinoma but did not improve bone mass. In contrast, high calcium suppressed serum 1,25-dihydroxyvitamin D levels and improved bone mass but increased the incidence of adenocarcinoma. Analysis of the VDR cistrome in RWPE1 prostate epithelial cells revealed vitamin D–mediated regulation of multiple cancer-relevant pathways. Our data support the hypothesis that the loss of vitamin D signaling accelerates the early stages of prostate carcinogenesis, and our results suggest that different dietary requirements may be needed to support prostate health or maximize bone mass. Significance: This work shows that disrupting vitamin D signaling through diet or genetic deletion increases early prostate carcinogenesis through multiple pathways. Higher-diet vitamin D levels are needed for cancer than bone.

Abstract We tested whether lifelong modification of vitamin D signaling can alter the progression of early prostate carcinogenesis in studies using mice that develop high-grade prostatic intraepithelial neoplasia that is similar to humans. Two tissue-limited models showed that prostate vitamin D receptor (VDR) loss increased prostate carcinogenesis. In another study, we fed diets with three vitamin D3 levels (inadequate = 25 IU/kg diet, adequate for bone health = 150 IU/kg, or high = 1,000 IU/kg) and two calcium levels (adequate for bone health = 0.5% and high = 1.5%). Dietary vitamin D caused a dose-dependent increase in serum 25-hydroxyvitamin D levels and a reduction in the percentage of mice with adenocarcinoma but did not improve bone mass. In contrast, high calcium suppressed serum 1,25-dihydroxyvitamin D levels and improved bone mass but increased the incidence of adenocarcinoma. Analysis of the VDR cistrome in RWPE1 prostate epithelial cells revealed vitamin D–mediated regulation of multiple cancer-relevant pathways. Our data support the hypothesis that the loss of vitamin D signaling accelerates the early stages of prostate carcinogenesis, and our results suggest that different dietary requirements may be needed to support prostate health or maximize bone mass. Significance: This work shows that disrupting vitamin D signaling through diet or genetic deletion increases early prostate carcinogenesis through multiple pathways. Higher-diet vitamin D levels are needed for cancer than bone.

Objective: To investigate the correlation between psychological stress and masseter muscle (MM) alterations, and explore the therapeutic agents for restoring the impaired masticatory muscle.Design: We established a chronic unpredictable mild stress (GUMS) animal model and observed the changes of ultrastructure, redox homeostasis and energy metabolism in MM in rats with and without curcumin treatment.Results: The depressive-like behavior in stressed rats was confirmed by the evidences of altered behaviors in sucrose preference test and open field test; while these phenomena were eased by curcumin. Except for the pathological changes in ultrastructure, decreased SOD, GSH-Px, CAT, Na+-K(+)ATPase, and Ca2+-Mg2+ ATPase activities as well as increased MDA and LD content and LDH activity were also observed in MM in stressed rats. However, curcumin was capable of reversing GUMS-induced MM disorder by improving the activities of the examined anti-oxidant enzymes and energy metabolism enzymes. Additionally, the increased MDA content, LD content, and LDH activity in stressed rats were reduced by curcumin.Conclusion: All the findings indicate the adverse effects of GUMS on MM function in rats, and raise the possibility of developing curcumin as a potential therapeutic agent for psychological stress-induced masseter dysfunction. (C) 2013 Elsevier Ltd. All rights reserved.

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an apical membrane calcium (Ca) channel in the small intestine proposed to be essential for vitamin D-regulated intestinal Ca absorption. Recent studies have challenged the proposed role for TRPV6 in Ca absorption. We directly tested intestinal TRPV6 function in Ca and bone metabolism in wild-type (WT) and vitamin D receptor knockout (VDRKO) mice. TRPV6 transgenic mice (TG) were made with intestinal epithelium-specific expression of a 3X Flag-tagged human TRPV6 protein. TG and VDRKO mice were crossed to make TG-VDRKO mice. Ca and bone metabolism was examined in WT, TG, VDRKO, and TG-VDRKO mice. TG mice developed hypercalcemia and soft tissue calcification on a chow diet. In TG mice fed a 0.25% Ca diet, Ca absorption was more than three-fold higher and femur bone mineral density (BMD) was 26% higher than WT. Renal 1α hydroxylase (CYP27B1) mRNA and intestinal expression of the natural mouse TRPV6 gene were reduced to <10% of WT but small intestine calbindin-D(9k) expression was elevated >15 times in TG mice. TG-VDRKO mice had high Ca absorption that prevented the low serum Ca, high renal CYP27B1 mRNA, low BMD, and abnormal bone microarchitecture seen in VDRKO mice. In addition, small intestinal calbindin D(9K) mRNA and protein levels were elevated in TG-VDRKO. Transgenic TRPV6 expression in intestine is sufficient to increase Ca absorption and bone density, even in VDRKO mice. VDR-independent upregulation of intestinal calbindin D(9k) in TG-VDRKO suggests this protein may buffer intracellular Ca during Ca absorption. © 2012 American Society for Bone and Mineral Research.

The 1,25 dihydroxyvitamin D (1,25D) activates the VDR to bind DNA and initiate gene transcription. While microarray studies show 1,25D regulates expression of many transcripts, few of these genes have been examined for VDR binding. Using ChIP‐seq, we mapped VDR binding sites in DNA from the human PEC line, RWPE1. Cells were treated with vehicle or 1,25D (10 nM, 3 h) and subjected to ChIP using either VDR antibody or IgG. Using IgG as the reference, 1236 and 1175 VDR binding peaks were found in 1,25D and vehicle‐treated cells, respectively (0.1 FDR, 200 bp window, Cisgenome). Genes with known VDREs were confirmed e.g. CYP24 and IGFBP3. Correlation between ChIP‐seq peaks and a previous microarray study found that only 39% were associated with 1,25D‐regulated transcripts, including novel peaks close to the transcription factors (TF) JunB, FOS and TBP. Also only 5% of VDR peaks were found within 5 kb of the transcription start site of a gene and the known DR3 VDR binding motif was found in just 15% of the peaks. However, several other DNA‐binding motifs were enriched under the peaks suggesting indirect DNA association of VDR through other TF. Our data suggest the relationship between VDR binding and transcription is more complex that previously reported.Grant Funding Source: Showalter Trust to JCF

We studied the effect of prolonged activation of mitogen‐activated protein kinase (MAPK) signaling on 1,25 dihydroxyvitamin D (1,25(OH) 2 D 3 ) action in the immortalized human prostate epithelial cell line RWPE1 and its Ki‐Ras transformed clone RWPE2. 1,25(OH) 2 D 3 ‐treatment caused growth arrest and induced gene expression in both cell lines but the response was blunted in RWPE2 cells. Vitamin D receptor (VDR) levels were lower in RWPE2 cells but VDR over‐expression did not increase vitamin‐D‐mediated gene transcription in either cell line. In contrast, MAPK inhibition restored normal vitamin D transcriptional responses in RWPE2 cells and MAPK activation with constitutively active MEK1R4F reduced vitamin‐D‐regulated transcription in RWPE1 cells. 1,25(OH) 2 D 3 ‐mediated transcription depends upon the VDR and its heterodimeric partner the retinoid X receptor (RXR) so we studied whether changes in the VDR–RXR transcription complex occur in response to MAPK activation. Mutation of putative phosphorylation sites in the activation function 1 (AF‐1) domain (S32A, T82A) of RXRα restored 1,25(OH) 2 D 3 ‐mediated transactivation in RWPE2 cells. Mammalian two‐hybrid and co‐immunoprecipitation assays revealed a vitamin‐D‐independent interaction between steroid receptor co‐activator‐1 (SRC‐1) and RXRα that was reduced by MAPK activation and was restored in RWPE2 cells by mutating S32 and T82 in the RXRα AF‐1 domain. Our data show that a common contributor to cancer development, prolonged activation of MAPK signaling, impairs 1,25(OH) 2 D 3 ‐mediated transcription in prostate epithelial cells. This is due in part to the phosphorylation of critical amino acids in the RXRα AF‐1 domain and impaired co‐activator recruitment. J. Cell. Physiol. 224: 433–442, 2010. © 2010 Wiley‐Liss, Inc.

Background Prostate cancer is the second leading cause of cancer mortality among US men. Epidemiological evidence suggests that high vitamin D status protects men from prostate cancer and the active form of vitamin D, 1α,25 dihydroxyvitamin D 3 (1,25(OH) 2 D) has anti-cancer effects in cultured prostate cells. Still, the molecular mechanisms and the gene targets for vitamin D-mediated prostate cancer prevention are unknown. Results We examined the effect of 1,25(OH) 2 D (+/- 100 nM, 6, 24, 48 h) on the transcript profile of proliferating RWPE1 cells, an immortalized, non-tumorigenic prostate epithelial cell line that is growth arrested by 1,25(OH) 2 D (Affymetrix U133 Plus 2.0, n = 4/treatment per time and dose). Our analysis revealed many transcript level changes at a 5% false detection rate: 6 h, 1571 (61% up), 24 h, 1816 (60% up), 48 h, 3566 (38% up). 288 transcripts were regulated similarly at all time points (182 up, 80 down) and many of the promoters for these transcripts contained putative vitamin D response elements. Functional analysis by pathway or Gene Set Analysis revealed early suppression of WNT, Notch, NF-kB, and IGF1 signaling. Transcripts related to inflammation were suppressed at 6 h (e.g. IL-1 pathway) and suppression of proinflammatory pathways continued at later time points (e.g. IL-17 and IL-6 pathways). There was also evidence for induction of anti-angiogenic pathways and induction of transcripts for protection from oxidative stress or maintenance of cell redox homeostasis at 6 h. Conclusions Our data reveal of large number of potential new, direct vitamin D target genes relevant to prostate cancer prevention. In addition, our data suggests that rather than having a single strong regulatory effect, vitamin D orchestrates a pattern of changes within prostate epithelial cells that limit or slow carcinogenesis.

We examined the role of the extracellular signal regulated kinases (ERK) in 1,25‐dihydroxyvitamin D (1,25(OH) 2 D 3 )‐induced gene expression in the differentiated Caco‐2 cells. 1,25(OH) 2 D 3 ‐regulated expression of the 25‐hydroxyvitamin D, 24‐hydroxylase (CYP24) gene (both natural gene and promoter construct) was strongly modulated by altering ERK activity (i.e., reduced by MEK inhibitors and dominant negative (dn) ERK1 and ERK2, activated by epidermal growth factor) but ERK inhibition had no effect on 1,25(OH) 2 D 3 ‐regulated expression of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6). ERK5‐mediated phosphorylation of the transcription factor Ets‐1 enhanced 1,25(OH) 2 D 3 ‐mediated CYP24 gene transcription in proliferating but not differentiated Caco‐2 cells due to reduced levels of ERK5 and Ets‐1 (total and phosphoprotein levels) in differentiated cells. MEK inhibition reduced 1,25(OH) 2 D 3 ‐induced 3X‐VDRE promoter activity but had no impact on the association of vitamin D receptor (VDR) with chromatin suggesting a role for co‐activator recruitment in ERK‐modulation of vitamin D‐regulated CYP24 gene activation. Chromatin immunoprecipitation assays revealed that the ERK1/2 target, mediator 1 (MED1), is recruited to the CYP24, but not the TRPV6, promoter following 1,25(OH) 2 D 3 treatment. MED1 phosphorylation was sensitive to activators and inhibitors of the ERK1/2 signaling and MED1 siRNA reduced 1,25(OH) 2 D 3 ‐regulated human CYP24 promoter activity. This suggests ERK1/2 signaling enhances 1,25(OH) 2 D 3 effects on the CYP24 promoter by MED1‐mediated events. Our data show that there are both promoter‐specific and cell stage‐specific roles for the ERK signaling pathway on 1,25(OH) 2 D 3 ‐mediated gene induction in enterocyte‐like Caco‐2 cells. J. Cell. Physiol. 219: 132–142, 2009. © 2008 Wiley‐Liss, Inc.

We examined 1,25 dihydroxyvitamin D (1,25(OH) 2 D 3 )‐induced expression of 25‐hydroxyvitamin D 3 24‐hydroxylase (CYP24) and apical calcium channel (TRPV6) mRNA levels in 2‐, 9‐, and 15‐day cultures Caco‐2 cells that model proliferating, post‐proliferative, and differentiated enterocytes. 1,25(OH) 2 D 3 ‐induced (10 nM, 8 h) CYP24 and TRPV6 mRNA levels were significantly greater in differentiated and post‐proliferative than proliferating Caco‐2 cells (>16X and >3X, respectively). Neither CYP24 mRNA half‐life nor induction of a −298 bp rat CYP24 promoter‐luciferase reporter construct (10 nM 1,25(OH) 2 D 3 , 24 h) were different between proliferating and post‐proliferating Caco‐2 cells. We next tested whether the blunted response of natural genes to 1,25(OH) 2 D 3 in proliferating Caco‐2 cells is due to altered chromatin remodeling. VDR and coactivator protein levels do not increase with differentiation but the level of the co‐repressor Alien falls by 50% with differentiation. Over‐expression of Alien reduced 1,25(OH) 2 D 3 ‐induced activity of a minimal VDRE containing promoter‐luciferase construct by more than 60% in differentiated Caco‐2 cells while siRNA knockdown of Alien in proliferating Caco‐2 cells increased 1,25(OH) 2 D 3 ‐induced CYP24 mRNA level by 40%. These observations suggest that Alien is a regulator of VDR‐mediated gene transcription in Caco‐2 cells. In addition, we found that 1,25(OH) 2 D 3 ‐induced association of VDR with chromatin and with the CYP24 promoter was lower in proliferating cells. This suggests that decreased recruitment of VDR to vitamin D response elements also contributes to the blunted transcriptional responsiveness to 1,25(OH) 2 D 3 in proliferating Caco‐2 cells. J. Cell. Physiol. 218: 113–121, 2009. © 2008 Wiley‐Liss, Inc.