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1.  Antiestrogenic Activity of Flavonoid Phytochemicals Mediated via the c-Jun N-terminal Protein Kinase Pathway 
Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling independent of direct receptor binding. Here we demonstrate both chalcone and flavone function as cell type-specific selective ER modulators. In MCF-7 breast carcinoma cells chalcone and flavone suppress ERα activity through stimulation of the stress-activated members of the mitogen-activated protein kinase (MAPK) family: c-Jun N-terminal kinase (JNK)1 and JNK2. The use of dominant-negative mutants of JNK1 or JNK2 in stable transfected cells established that the antiestrogenic effects of chalcone and flavone required intact JNK signaling. We further show that constitutive activation of the JNK pathway partially suppresses estrogen (E2)-mediated gene expression in breast, but not endometrial carcinoma cells. Our results demonstrate a role for stress-activated MAPKs in the cell type-specific regulation of ERα function.
PMCID: PMC4083692  PMID: 22634477
flavonoids; phytoestrogens; estrogen receptor; mitogen-activated protein kinase; antiestrogens; c-Jun N-terminal kinase (JNK)
2.  Vitamin D Metabolism and Action in Human Marrow Stromal Cells: Effects of Chronic Kidney Disease 
Human marrow stromal cells (hMSCs) are targets of 1! ,25-dihydroxyvitamin D [1! ,25(OH)2D3] action to promote their differentiation to osteoblasts, but they also participate in vitamin D metabolism by converting 25-dihydroxyvitamin D3 [25(OH)D3] to 1! ,25(OH)2D3 by 1α-hydroxylase (CYP27B1). Chronic kidney disease (CKD) is associated with impaired renal biosynthesis of 1! ,25(OH)2D, low bone mass, and increased fracture risk. We tested whether CKD influences hMSCs' responses to vitamin D3 metabolites. The hMSCs were obtained from tissues discarded during arthroplasty for hip osteoarthrosis, including a subject who had been undergoing hemodialysis for 2+ years. There was a significant positive correlation between in vitro stimulation of osteoblastogenesis (alkaline phosphatase activity) by 1! ,25(OH)2D3 and subjects' estimated glomerular filtration rate (eGFR, r=0.47, p=0.015, n=26, 56–83 years of age). Osteoblastogenesis was stimulated in hMSCs from both the hemodialysis and control subjects by 1! ,25(OH)2D3 (10 ! M), 25(OH)D3 (100 ! M), or D3 (1000 ! M). Thus, vitamin D metabolism may play an autocrine/paracrine role in osteoblast differentiation of hMSCs. These findings suggest that in CKD patients 25(OH)D-sufficiency may play an important role in skeletal health; osteoblastic bone formation in CKD patients may not be optimal unless there is sufficient serum 25(OH)D substrate for the MSCs to synthesize and respond to local 1! ,25(OH)2D.
PMCID: PMC3535685  PMID: 22989482
Human Marrow Stromal Cells; Osteoblastogenesis; Chronic Kidney Disease; eGFR; vitamin D metabolites
3.  Equivalent anticancer activities of dietary vitamin D and calcitriol in an animal model of breast cancer: importance of mammary CYP27B1 for treatment and prevention 
Calcitriol [1,25(OH)2D3], the hormonally active form of vitamin D exerts anti-proliferative, pro-apoptotic, anti-inflammatory effects and other anticancer actions in breast cancer (BCa) cell cultures and animal models of BCa. Our research is focused on investigating the potential beneficial effects of dietary vitamin D3 compared to calcitriol and the underlying mechanisms in BCa treatment and chemoprevention. We recently found that dietary vitamin D3 exhibits significant tumor inhibitory effects in xenograft models of BCa that are equivalent to those elicited by the administration of the active hormone calcitriol. At the easily achievable dose tested in our studies, dietary vitamin D3 exhibited substantial tumor inhibitory activity and, unlike calcitriol, did not cause hypercalcemia demonstrating its relative safety. We found elevations in circulating calcitriol as well as increased CYP27B1 expression in the tumor and the intestine in tumor-bearing mice ingesting a vitamin D3-supplemented diet. We hypothesize that the elevation in circulating 25(OH)D induced by dietary vitamin D3 supplements stimulates local synthesis of calcitriol in the mammary tumor microenvironment and the ensuing paracrine/autocrine actions play a major role in the anticancer activity of dietary vitamin D3. Our findings suggest that the endocrine activity of calcitriol derived from tumor and other extra-renal sources such as the intestine, probably also plays a role in mediating the anticancer effects of dietary vitamin D3. Thus it appears that multiple sites of 1α-hydroxylation contribute to the anticancer effects of dietary vitamin D3. Our data strongly suggest that dietary vitamin D will be useful in the chemoprevention and treatment of BCa since it is a safe, economical and easily available nutritional agent that is equivalent to calcitriol in exerting anticancer effects, at least in mouse models. Furthermore, adequate vitamin D nutrition and avoidance of vitamin D deficiency appear to be important in reducing BCa risk. These findings warrant clinical trials in BCa patients and in women at high risk for BCa to evaluate the benefits of dietary vitamin D3 supplementation.
PMCID: PMC3554854  PMID: 22939886
4.  Cholecalciferol attenuates perseverative behavior associated with developmental alcohol exposure in rats in a dose-dependent manner 
Alcohol is a known teratogen that is estimated to affect 2–5% of the births in the U.S. Prenatal alcohol exposure can produce physical features such as facial dysmorphology, physiological alterations such as cell loss in the central nervous system (CNS), and behavioral changes that include hyperactivity, cognitive deficits, and motor dysfunction. The range of effects associated with prenatal alcohol exposure is referred to as fetal alcohol spectrum disorders (FASD). Despite preventative measures, some women continue to drink while pregnant. Therefore, identifying interventions that reduce the severity of FASD is critical. This study investigated one such potential intervention, vitamin D3, a nutrient that exerts neuroprotective properties. The present study determined whether cholecalciferol, a common vitamin D3 nutritional supplement, could serve as a means of mitigating alcohol-related learning deficits. Using a rat model of FASD, cholecalciferol was given before, during, and after 3rd trimester equivalent alcohol exposure. Three weeks after cholecalciferol treatment, subjects were tested on a serial spatial discrimination reversal learning task. Animals exposed to ethanol committed significantly more errors compared to controls. Cholecalciferol treatment reduced perseverative behavior that is associated with developmental alcohol exposure in a dose-dependent manner. These data have important implications for the treatment of FASD and suggest that cholecalciferol may reduce some aspects of FASD.
PMCID: PMC3586753  PMID: 23104117
fetal alcohol; vitamin D3; behavior; treatment; nutrition; ethanol
5.  The Effect Of Vitamin D Supplementation On Serum 25OHD In Thin And Obese Women 
Obese people are known to have lower serum 25OHD levels compared to non-obese people. It is not known whether it is due to storage of vitamin D in fat, inadequate input from sunlight, diet or other unknown factors. We examined the relationship at study baseline of serum 25OHD, PTH, 1,25(OH)2D with body composition measurements using Dual energy X-ray Absorptiometry. The results showed a significant inverse relation between total body fat mass and serum 25OHD (p<0.0001) and serum 1,25(OH2)D (p=034) and an independent positive correlation between serum PTH and total body fat mass (p<0.0001). In a randomized controlled study of seven doses of vitamin D (400-4800IU/d) the increase in serum 25OHD levels was compared in women with a normal body mass index to obese women. The response to the low doses of vitamin D (400-800IU/d) was significantly less than that of the medium (1600-2400IU/d) and high doses groups (3200-4800IU) (p<0.0001) in all BMI categories. The increase in serum 25OHD in the medium and high dose groups was not significantly different with increasing level of obesity. But thinner women with a normal BMI (< 25 kg/m2) showed a much higher response to vitamin D at any dose level compared to other BMI groups. There was no significant change in total body fat mass after treatment with vitamin D or calcitriol in our randomized trials. In summary, the response to vitamin D is dependent on body weight. Women with BMI <25 kg/m2 develop much higher levels of serum 25OHD after vitamin D supplementation compared to those with BMI of >25 kg/m2. The differences in serum 25OHD levels between normal and obese women may be due to differences in volume dilution. After vitamin D supplementation, all obese women reach adequate levels of serum 25OHD but normal women (BMI<25 kg/m2) reach much higher levels of 25OHD and in this group smaller doses of vitamin D used should be used.
PMCID: PMC3686842  PMID: 23246640
Vitamin D; 25(OH) D; 1,25(OH) 2D; PTH; Obesity; Fat; Postmenopausal Women
6.  24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics 
The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linking the incidence and prognosis of certain cancers to low serum 25 (OH)D3 levels and high expression of vitamin D 24-hydroxylase supporting the idea that elevated CYP24A1 expression may stimulate degradation of vitamin D metabolites including 25-(OH)D3 and 1,25(OH)2D3. The over expression of CYP24A1 in cancer cells may be a factor affecting 1,25(OH)2D3 bioavailability and anti-proliferative activity pre-clinically and clinically. The combination of 1,25(OH)2D3 with CYP24A1 inhibitors enhances 1,25(OH)2D3 mediated signaling and anti-proliferative effects and may be useful in overcoming effects of aberrant CYP24 expression.
PMCID: PMC3686893  PMID: 23059474
CYP24; 24-hydroxylase; calcitriol and anti-tumor
7.  The sum of many small changes: microRNAs are specifically and potentially globally altered by vitamin D3 metabolites 
Vitamin D3 deficiency is rampant which may contribute to increased risk of many diseases including cancer, cardiovascular disease and autoimmune disorders. Genomic activity of the active metabolite 1,25-dihydroxyvitamin D (1,25D) mediates most vitamin D3's actions and many gene targets of 1,25D have been characterized. As the importance of non-coding RNAs has emerged, the ability of vitamin D3 via 1,25D to regulate microRNAs (miRNAs) has been demonstrated in several cancer cell lines, patient tissue and sera. In vitamin D3 intervention patient trials, significant differences in miRNAs are observed between treatment groups and/or between baseline and followup. In patient sera from population studies, specific miRNA differences associate with serum levels of 25D. The findings thus far indicate that dietary vitamin D3 in patients and 1,25D in vitro not only regulate specific miRNA(s), but may also globally upregulate miRNA levels.
This article is part of a Special Issue entitled ‘Vitamin D Workshop’.
PMCID: PMC3686905  PMID: 23333596
Vitamin D3; microRNAs
8.  1,25(OH)2 vitamin D suppresses macrophage migration and reverses atherogenic cholesterol metabolism in type 2 diabetic patients 
Reduced monocyte infiltration into the vessel wall and increased macrophage cholesterol efflux are critical components in atherosclerotic plaque regression. During inflammation, monocyte chemotactic protein 1 (MCP-1) signaling activation and cholesterol deposition in macrophages induce endoplasmic reticulum (ER) stress, which promotes an increased inflammatory response. Increased macrophage ER stress shifts macrophages into an M2 macrophage phenotype with increased cholesterol uptake and deposition. In type 2 diabetes, a population with elevated baseline risk of cardiovascular disease (CVD), vitamin D deficiency doubles that risk. We have found that 1,25-dihydroxy vitamin D [1,25(OH)2D] prevents foam cell formation during macrophage differentiation by suppressing ER stress. However, it is unknown whether suppression of ER stress by 1,25(OH)2D decreases monocyte infiltration and reverses atherogenic cholesterol metabolism in previously-differentiated, vitamin D-deplete macrophages. We collected peripheral monocytes from type 2 diabetic patients and differentiated them into macrophages under vitamin D-deplete or 1,25(OH)2D-supplemented conditions. 1, 25(OH)2D supplementation suppressed macrophage migration in response to MCP-1 and mRNA expression of chemokine (C-C motif) receptor 2 (CCR2), the MCP-1 receptor, compared to vitamin D-deplete cells. Furthermore, inhibition of ER stress with phenyl butyric acid resulted in similar effects even in vitamin D-deplete cells, while induction of ER stress with Thapsigargin under 1,25(OH)2D-supplemented conditions increased macrophage migration and CCR2 expression, suggesting that the effects of vitamin D on migration are mediated through ER stress suppression. To determine whether the detrimental pattern of macrophage cholesterol metabolism in vitamin D depletion is reversible, we assessed cholesterol uptake in macrophages differentiated under vitamin D-deplete conditions as described above, then supplemented with 1,25(OH)2D or maintained in vitamin D-deplete conditions. Cholesterol uptake was decreased in 1,25(OH)2D-supplemented compared to vitamin D-deplete cells, suggesting slowed cholesterol deposition with active vitamin D. 1,25(OH)2D supplementation also suppressed cholesteryl ester formation and enhanced cholesterol efflux in M2 macrophages compared to vitamin D-deplete cells, suggesting facilitation of cholesterol egress in the presence of 1,25(OH)2D. We thus provide further evidence that active vitamin D is an ER stress reliever that may have a role in atherosclerotic plaque regression.
PMCID: PMC3687029  PMID: 23333932
Vitamin D; macrophage phenotype; migration; cholesterol metabolism; ER stress; diabetes
9.  Corepressors (NCoR and SMRT) as well as Coactivators are Recruited to Positively Regulated 1α,25-Dihydroxyvitamin D3-Responsive Genes 
Transcription factors require coactivators and corepressors to modulate transcription in mammalian cells. The vitamin D receptor (VDR) utilizes coactivators and corepressors to gain tight control over the activity of a diverse set of genes that can regulate calcium transport, slow proliferation and promote immune responses. We have recently established the VDR/RXR cistrome in human colon cancer cells and have linked these binding sites to the genes that are regulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In additional studies described herein, we demonstrate that the coactivators SRC1, CBP and MED1 are recruited to upregulated genes to facilitate transcription as expected. SRC1 was the most highly correlated to VDR/RXR binding (50%). However, we also found that corepressor molecules such as NCoR and SMRT were present along with SRC1, CBP or MED1 at these 1,25(OH)2D3 activated gene enhancers. Interestingly, genome-wide NCoR binding mimicked VDR binding by increasing its association with VDR binding in response to 1,25(OH)2D3 treatment. Overall, these data indicate a complex role for corepressor and coactivator complexes in the activation or active repression of 1,25(OH)2D3 responsive genes.
PMCID: PMC3548980  PMID: 22944139
VDR; ChIP-seq; corepressor; coactivator; coregulator; vitamin D
10.  Interplay between Vitamin D and the Drug Metabolizing Enzyme CYP3A4 
Cytochrome P450 3A4 (CYP3A4) is a multifunctional enzyme involved in both xenobiotic and endobiotic metabolism. This review focuses on two aspects: regulation of CYP3A4 expression by vitamin D and metabolism of vitamin D by CYP3A4. Enterohepatic circulation of vitamin D metabolites and their conjugates will be also discussed. The interplay between vitamin D and CYP3A4 provides new insights into our understanding of how enzyme induction can contribute to vitamin D deficiency.
PMCID: PMC3549031  PMID: 22985909
Cytochrome P450 3A4; Vitamin D; Drug metabolism; Enterohepatic circulation; Osteomalacia
11.  Protective Role of Vitamin D Signaling in Skin Cancer Formation 
Vitamin D sufficiency is associated with protection against malignancy in a number of tissues clinically, and a strong body of evidence from animal and cell culture studies supports this protective role. Cancers in the skin differ, however, in that higher serum levels of 25OHD are associated with increased basal cell carcinomas (BCC), the most common form of epidermal malignancy. This result may be interpreted as indicating the role of UVR (spectrum 280–320) in producing vitamin D in the skin as well as causing those DNA mutations and proliferative changes that lead to epidermal malignancies. Recent animal studies have shown that mice lacking the vitamin D receptor (VDR) are predisposed to developing skin tumors either from chemical carcinogens such as 7,12 dimethylbenzanthracene (DMBA) or chronic UVR exposure. Such studies suggest that vitamin D production and subsequent signaling through the VDR in the skin may have evolved in part as a protective mechanism against UVR induced epidermal cancer formation. In this manuscript we provide evidence indicating that vitamin D signaling protects the skin from cancer formation by controlling keratinocyte proliferation and differentiation, facilitating DNA repair, and suppressing activation of the hedgehog (Hh) pathway following UVR exposure.
PMCID: PMC3596439  PMID: 23059470
vitamin D receptor; 1,25 dihydroxyvitamin D; keratinocyte; UVR; DNA damage repair; sonic hedgehog
12.  Differential response to 1α, 25-dihydroxyvitamin D3 (1α,25(OH)2D3) in non-small cell lung cancer cells with distinct oncogene mutations1 
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 identify a lung cancer subset which remains responsive to and is likely to benefit from 1,25(OH)2D3 administration.
PMCID: PMC3674199  PMID: 23026510
non-small cell lung cancer; vitamin D receptor; 1,25-dihydroxyvitamin D3; epidermal growth factor receptor; K-ras
13.  Histone Deacetylation Mediates the Rejuvenation of Osteoblastogenesis by the Combination of 25(OH)D3 and Parathyroid Hormone in MSCs from Elders 
Vitamin D metabolites are important effectors of bone and mineral homeostasis. Human bone marrow stromal cells (hMSCs) are targets of 1α,25-dihydroxyvitamin D [1α, 25(OH)2D] action to promote their differentiation to osteoblasts. Osteoblastogenesis is also stimulated by 25-hydroxyvitamin D [25(OH)D], an effect that requires conversion to 1α, 25(OH)2D3 by 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1). These findings support an autocrine/paracrine role of vitamin D metabolism in osteoblastogenesis of hMSCs. In this study, we assessed whether and by what mechanisms osteoblastogenesis could be rejuvenated with hMSCs from elders. First, knockdown studies with VDR-siRNA showed that both the pro-differentiation and anti-proliferative effects of 1α, 25(OH)2D3 required VDR. Second, 100 nM 25(OH)D3 (p<0.01 vs. control, ANOVA) and 100 nM PTH1-34 (p<0.05) significantly stimulated alkaline phosphatase activity (a measure of osteoblastogenesis), with a synergistic effect when combined (p<0.001). Scriptaid, an inhibitor of histone deacetylase, blocked the effect of 25(OH)D3 and PTH on osteoblastogenesis. Scriptaid alone downregulated VDR in hMSCs. These data demonstrate that histone deacetylation is required for the synergistic effect of 25(OH)D3 and PTH on osteoblastogenesis in hMSCs. Both VDR siRNA and Scriptaid dowregulated VDR mRNA and inhibited osteoblastogenesis. Thus, epigenetic regulation of the VDR may be central to rejuvenating osteoblastogenesis in hMSCs from elders.
PMCID: PMC3548026  PMID: 22982627
Vitamin D; Vitamin D Receptor; Parathyroid Hormone; Epigenetic Regulation; Osteoblastogenesis; Human Marrow Stromal Cells
14.  Chlamydial infection in vitamin D receptor knockout mice is more intense and prolonged than in wild-type mice 
Vitamin D hormone (1,25-dihydroxyvitamin D) is involved in innate immunity and induces host defense peptides in epithelial cells, suggesting its involvement in mucosal defense against infections. Chlamydia trachomatis is a major cause of bacterial sexually transmitted disease worldwide. We tested the hypothesis that the vitamin D endocrine system would attenuate chlamydial infection. Vitamin D receptor knock-out mice (VDR−/−) and wild-type mice (VDR+/+) were infected with 103 inclusion forming units of Chlamydia muridarum and cervical epithelial cells (HeLa cells) were infected with C. muridarum at multiplicity of infection 5:1 in the presence and absence of 1,25-dihydroxyvitamin D3.VDR−/− mice exhibited significantly higher bacterial loading than wild-type VDR+/+ mice (P<0.01) and cleared the chlamydial infection in 39 days, compared with 18 days for VDR+/+ mice. Monocytes and neutrophils were more numerous in the uterus and oviduct of VDR−/− mice than in VDR+/+ mice (P< 0.05) at d 45 after infection. Pre-treatment of HeLa cells with 10nM or 100nM 1,25-dihydroxyvitamin D3 decreased the infectivity of C. muridarum (P< 0.001). Several differentially expressed protein spots were detected by proteomic analysis of chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D3. Leukocyte elastase inhibitor (LEI), an anti-inflammatory protein, was up-regulated. Expression of LEI in the ovary and oviduct of infected VDR+/+ mice was greater than that of infected VDR−/− mice. We conclude that the vitamin D endocrine system reduces the risk for prolonged chlamydial infections through regulation of several proteins and that LEI is involved in its anti-inflammatory activity.
PMCID: PMC4065015  PMID: 23201171
Chlamydial infection; 1,25-Dihydroxyvitamin D3; Vitamin D receptor knock out mouse; HeLa cells; Leukocyte elastase inhibitor
15.  Integration of steroid research: Perspectives on environment factors, homeostasis in health, and disease treatment 
PMCID: PMC4008313  PMID: 21558003
Endocrine disruptors; Steroids; Breast cancer; Prostate cancer; Obesity; Diabetes; Mice model; Chemistry of synthesis
16.  APRIN is a Unique Pds5 Paralog with Features of a Chromatin Regulator in Hormonal Differentiation 
Activation of steroid receptors results in global changes of gene expression patterns. Recent studies showed that steroid receptors control only a portion of their target genes directly, by promoter binding. The majority of the changes are indirect, through chromatin rearrangements. The mediators that relay the hormonal signals to large-scale chromatin changes are, however, unknown. We report here that APRIN, a novel hormone-induced nuclear phosphoprotein has the characteristics of a chromatin regulator and may link endocrine pathways to chromatin. We showed earlier that APRIN is involved in the hormonal regulation of proliferative arrest in cancer cells. To investigate its function we cloned and characterized APRIN orthologs and performed homology and expression studies. APRIN is a paralog of the cohesin-associated Pds5 gene lineage and arose by gene-duplication in early vertebrates. The conservation and domain differences we found suggest, however, that APRIN acquired novel chromatin-related functions (e.g. the HMG-like domains in APRIN, the hallmarks of chromatin regulators, are absent in Pds5). We show that in interphase nuclei APRIN localizes in the euchromatin/heterochromatin interface and we also identified its DNA-binding and nuclear import signal domains. Our results indicate that APRIN, in addition to its Pds5 similarity, has the features and localization of a hormone-induced chromatin regulator.
PMCID: PMC3966471  PMID: 17997301
APRIN; hormonal regulation; proliferative arrest; chromatin; cancer; HMG domain; AT-hook; Pds 5
17.  17β-Estradiol-Mediated Increase in Cu/Zn Superoxide Dismutase Expression in the Brain: A Mechanism to Protect Neurons from Ischemia 
A number of studies have demonstrated that 17β-estradiol (E2) protects the brain from ischemia and yet, the mechanism by which this hormone brings about its protective effect is unclear. Interestingly, like E2, overexpression of the oxidative stress response protein Cu/Zn superoxide dismutase (SOD1), which plays a critical role in regulating reactive oxygen species, also protects the brain from ischemia. Because we previously showed that E2 treatment of cultured mammary cells increases SOD1 expression, we hypothesized that E2 might increase SOD1 expression in the brain and that this E2-mediated increase in SOD1 expression might help to protect the brain from ischemia. We now show that SOD1 is expressed in cortical neurons, that SOD1 expression is increased by exposure of brain slice cultures to E2, and that the E2–mediated increase in SOD1 expression is further augmented by exposure of brain slice cultures to increased superoxide levels or oxygen and glucose deprivation. Importantly, when cortical neurons are exposed to increased superoxide levels and markers of protein and DNA damage, nitrotyrosine and 8-oxoguanine, respectively, are measured, both protein and DNA damage is reduced. In fact, E2 reduces nitrotyrosine and 8-oxoguanine levels in brain slice cultures regardless of whether they have or have not been exposed to increased superoxide levels. Likewise, when brain slice cultures are treated with E2 and deprived of oxygen and glucose, 8-oxoguanine levels are reduced. Taken together, these studies provide a critical link between E2 treatment, SOD1 expression, and neuroprotection and help to define a mechanism through which E2– mediated neuroprotection may be conferred.
PMCID: PMC3901640  PMID: 21704159
estrogen; estrogen receptor; superoxide dismutase; reactive oxygen species; ischemia; neuroprotection
18.  1,25(OH)2vitamin D3 Enhances Myogenic Differentiation by Modulating the Expression of Key Angiogenic Growth factors and Angiogenic Inhibitors in C2C12 Skeletal Muscle Cells 
Vitamin D is mostly recognized for its regulation of calcium homeostasis in relation to the intestine, kidney, and bone. Although clinical studies have linked vitamin D with increased muscle function and strength, little is known of its underlying molecular mechanism. We recently demonstrated that 1,25-D3 exerts a direct pro-myogenic effect on skeletal muscle cells; this has provoked our investigation of 1,25-D’s effect on angiogenesis, a vital process for new capillary development and tissue repair. In this study, we examined the mechanism by which 1,25-D3 modulates key angiogenic growth factors and angiogenic inhibitors. C2C12 myoblasts were incubated with 100 nM 1,25-D3 or placebo for 1, 4 and 10 days. At the end of the respective incubation time, total RNA was isolated for PCR arrays and for qRT-PCR. Total proteins were isolated for western blots and proteome profiler arrays. The addition of 1,25-D3 to C2C12 myoblasts increased VEGFa and FGF-1: two pro-angiogenic growth factors that promote neo-vascularization and tissue regeneration, and decreased FGF-2 and TIMP-3: two myogenic and/or angiogenic inhibitors. Our previous study demonstrated that 1,25-D3 altered IGF-I/II expression, consistent with the observed changes in VEGFa and FGF-2 expression. These results extend our previous findings and demonstrate the modulation of angiogenesis which may be an additional mechanism by which 1,25-D3 promotes myogenesis. This study supports the mechanistic rationale for assessing the administration of vitamin D and/or vitamin D analogues to treat select muscle disorders and may also provide an alternative solution for therapies that directly manipulate VEGF and FGF’s to promote angiogenesis.
PMCID: PMC3513642  PMID: 22982629
19.  Regulation of hepatic sulfotransferase (SULT) 1E1 expression and effects on estrogenic activity in cystic fibrosis (CF)☆ 
The Journal of steroid biochemistry and molecular biology  2009;114(0):10.1016/j.jsbmb.2009.02.009.
Cystic fibrosis (CF) is a major genetic disease in Caucasians affecting 1 in 2500 newborns. Hepatobiliary pathology is a major cause of morbidity and mortality in CF second only to pulmonary disease. SULT1E1 activity is significantly elevated, generally 20–30-fold, in hepatocytes of mouse models of CF. SULT1E1 is responsible for the inactivation of β-estradiol (E2) at physiological concentrations via conjugation with sulfonate. The increase in SULT1E1 activity results in the alteration of E2-regulated protein expression in CF mouse liver. To investigate the mechanism by which the absence of CFTR in human cholangiocytes induces SULT1E1 expression in hepatocytes, a membrane-separated human MMNK-1 cholangiocyte and human HepG2 hepatocyte co-culture system was developed. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in bile duct cholangiocytes but not hepatocytes, whereas SULT1E1 is expressed in hepatocytes but not cholangiocytes. CFTR expression in MMNK-1 cells was inhibited with siRNA by >90% as determined by immunoblot and immunohistochemical analysis. Control and CFTR-siRNA-MMNK-1 cells were co-cultured with HepG2 cells in a Transwell membrane-separated system. After 8 h of co-culture, HepG2 cells were removed from exposure to MMNK-1 cells and placed in fresh medium. After 24–48 h, expression of SULT1E1 and selected E2-regulated proteins was analyzed in the HepG2 cells. Results demonstrated that SULT1E1 message and activity were selectively induced in HepG2 cells co-cultured with CFTR-deficient MMNK-1 cells. The expression of E2-regulated proteins (IGF-1, GST-P1 and carbonic anhydrase II) was also altered in response to decreased E2 levels. Thus, the loss of CFTR activity in cholangiocytes stimulates the expression of SULT1E1 in hepatocytes by a paracrine mechanism. SULT1E1 expression in HepG2 cells is inducible by sterol mediated liver-X-receptor (LXR) activation although not by progestins that induce SULT1E1 in the endometrium. SULT1E1 induction in the human cholangiocyte/hepatocyte co-culture system is consistent with and supports the results observed in CF mice. The changes in hepatocyte gene expression affect liver biochemistry and may facilitate the development of CF liver disease.
PMCID: PMC3855421  PMID: 19429440
Cystic fibrosis; Liver disease; Cystic fibrosis transmembrane conductance; regulator; Sulfotransferase; SULT1E1; β-Estradiol; Hepatocytes; Cholangiocytes; Oxysterols; Liver-X-receptor
20.  Insulin and hyperandrogenism in women with polycystic ovary syndrome 
The Journal of steroid biochemistry and molecular biology  2009;122(0):10.1016/j.jsbmb.2009.12.010.
Polycystic ovary syndrome (PCOS) is a very common endocrine disorder characterized by chronic anovulation, clinical and/or biochemical hyperandrogenism, and/or polycystic ovaries. But most experts consider that hyperandrogenism is the main characteristic of PCOS. Several theories propose different mechanisms to explain PCOS manifestations: (1) a primary enzymatic default in the ovarian and/or adrenal steroidogenesis; (2) an impairment in gonadotropin releasing hormone (GnRH) secretion that promotes luteal hormone (LH) secretion; or (3) alterations in insulin actions that lead to insulin resistance with compensatory hyperinsulinemia. However, in the past 20 years there has been growing evidence supporting that defects in insulin actions or in the insulin signalling pathways are central in the pathogenesis of the syndrome. Indeed, most women with PCOS are metabolically insulin resistant, in part due to genetic predisposition and in part secondary to obesity. But some women with typical PCOS do not display insulin resistance, which supports the hypothesis of a genetic predisposition specific to PCOS that would be revealed by the development of insulin resistance and compensatory hyperinsulinemia in most, but not all, women with PCOS. However, these hypotheses are not yet appropriately confirmed, and more research is still needed to unravel the true pathogenesis underlying this syndrome. The present review thus aims at discussing new concepts and findings regarding insulin actions in PCOS women and how it is related to hyperandrogenemia.
PMCID: PMC3846536  PMID: 20036327 CAMSID: cams3752
Polycystic ovary syndrome; Hyperandrogenism; Insulin; Insulin signalling pathways; Insulin resistance; Free fatty acids
21.  Cholesterol metabolite, 5-cholesten-3beta, 25-diol 3-sulfate, promotes hepatic proliferation in mice 
Oxysterols are well known as physiological ligands of Liver X receptors (LXRs). Oxysterols, 25-Hydroxycholesterol (25HC) and 27-hydroxycholesterol as endogenous ligands of LXRs, suppress cell proliferation via LXRs signaling pathway. Recent reports have shown that sulfated oxysterol, 5-cholesten-3β, 25-diol 3-sulfate (25HC3S) as LXRs antagonist, plays an opposite direction to oxysterols in lipid biosynthesis. The present report was to explore the effect and mechanism of 25HC3S on hepatic proliferation in vivo. Following administration, 25HC3S had a 48 h half life in the circulation and widely distributed in mouse tissues. Profiler™ PCR array and RTqPCR analysis showed that either exogenous or endogenous 25HC3S generated by overexpression of oxysterol sulfotransferase (SULT2B1b) plus administration of 25HC significantly up-regulated the proliferation gene expression of Wt1, Pcna, cMyc, cyclin A, FoxM1b, and CDC25b in a dose-dependent manner in liver while substantially down-regulating the expression of cell cycle arrest gene Chek2 and apoptotic gene Apaf1. Either exogenous or endogenous administration of 25HC3S significantly induced hepatic DNA replication as measured by immunostaining of the PCNA labeling index and was associated with reduction in expression of LXR response genes, such as ABCA1 and SREBP-1c. Synthetic LXR agonist T0901317 effectively blocked 25HC3S-induced hepatic proliferation. Conclusions: 25HC3S may be a potent regulator of hepatocyte proliferation and oxysterol sulfation may represent a novel regulatory pathway in liver proliferation via inactivating LXR signaling.
PMCID: PMC3463675  PMID: 22732306
25-hydroxycholesterol; Cytosolic sulfotransferase 2B1b; Liver X Receptors; oxysterol; oxysterol sulfation; proliferating cell nuclear antigen
22.  Zoledronic Acid Inhibits Aromatase Activity and Phosphorylation: Potential Mechanism for Additive Zoledronic Acid and Letrozole Drug Interaction 
Zoledronic acid (ZA), a bisphosphonate originally indicated for use in osteoporosis, has been reported to exert a direct effect on breast cancer cells, although the mechanism of this effect is currently unknown. Data from the ABCSG-12 and ZO-FAST clinical trials suggest that treatment with the combination of ZA and aromatase inhibitors (AI) result in increased disease free survival in breast cancer patients over AI alone. To determine whether the mechanism of this combination involved inhibition of aromatase, AC-1 cells (MCF-7 human breast cancer cells transfected with an aromatase construct) were treated simultaneously with combinations of ZA and AI letrozole for 72 hours. This combination significantly increased inhibition of aromatase activity of AC-1 cells by compared to letrozole alone. Combination treatment of 1nM letrozole and 1μM and 10μM zoledronic acid resulted in an additive drug interaction on inhibiting cell viability, as measured by MTT assay. Treatment with ZA was found to inhibit phosphorylation of aromatase on serine 473. Zoledronic acid was also shown to be more effective in inhibiting cell viability in aromatase transfected AC-1 cells when compared to inhibition of cell viability observed in non-transfected MCF-7. Estradiol was able to partially rescue the effect of 1μM and 10μM ZA on cell viability following treatment for 72 hours, as shown by a shift to the right in the estradiol dose response curve. In conclusion, these results indicate that the combination of ZA and letrozole results in an additive inhibition of cell viability. Furthermore, ZA alone can inhibit aromatase activity through inhibition of serine phosphorylation events important for aromatase enzymatic activity and contributes to inhibition of cell viability.
PMCID: PMC3463743  PMID: 22659283
zoledronic acid; letrozole; aromatase enzyme
23.  Novel therapeutic approaches for the treatment of castration-resistant prostate cancer☆ 
•New drugs approved for treatment of castration resistant prostate cancer.•Prime targets: androgen receptor, bone cells, cell division, immune system.•Several promising drugs disappointed in clinical trials.•Further efforts necessary to optimize the sequence and combinations of drugs.•New biomarkers required for stratification of patient and therapy selection.
Prostate cancer is a leading cause of cancer death in men in developed countries. Once the tumor has achieved a castration-refractory metastatic stage, treatment options are limited with the average survival of patients ranging from two to three years only. Recently, new drugs for treatment of castration-resistant prostate cancer (CRPC) have been approved, and others are in an advanced stage of clinical testing. In this review we provide an overview of the new therapeutic agents that arrived in the clinical praxis or are tested in clinical studies and their mode of action including hormone synthesis inhibitors, new androgen receptor blockers, bone targeting and antiangiogenic agents, endothelin receptor antagonists, growth factor inhibitors, novel radiotherapeutics and taxanes, and immunotherapeutic approaches. Results and limitations from clinical studies as well as future needs for improvement of CRPC treatments are critically discussed.
PMCID: PMC3834152  PMID: 23792785
AR, androgen receptor; CRPC, castration-resistant prostate cancer; ET, endothelin; IGF, insulin-like growth factor; OS, overall survival; PCa, prostate cancer; PDGFR, platelet-derived growth factor receptor; PFS, progression free survival; PSA, prostate-specific antigen; RANK-L, RANK ligand; SD, stable disease; TKI, tyrosine kinase inhibitor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; Castration-resistant prostate cancer; Androgen receptor; Bone metastasis angiogenesis; Immunotherapy; Radiotherapy; Chemotherapy; Growth factor receptor inhibitors
24.  Evaluation of serum estrogen-DNA adducts as potential biomarkers for breast cancer risk 
This study was conducted to determine whether the ratio of estrogen-DNA adducts to their respective metabolites and conjugates in serum differed between women with early-onset breast cancer and those with average or high risk of developing breast cancer.
Serum samples from women at average risk (n = 63) or high risk (n = 80) for breast cancer (using Gail model) and women newly diagnosed with early breast cancer (n = 79) were analyzed using UPLC-MS/MS. Adduct ratios were statistically compared among the three groups, and the Area Under the Receiver Operating Characteristic Curve (AUC) was used to identify a diagnostic cut-off point.
The median adduct ratio in the average-risk group was significantly lower than that of both the high-risk group and the breast cancer group (p values <0.0001), and provided good discrimination between those at average versus high risk of breast cancer (AUC = 0.84, 95% CI 0.77–0.90). Sensitivity and specificity were maximized at an adduct ratio of 77. For women in the same age and BMI group, the odds of being at high risk for breast cancer was 8.03 (95% CI 3.46–18.7) times higher for those with a ratio of at least 77 compared to those with a ratio less than 77.
The likelihood of being at high risk for breast cancer was significantly increased for those with a high adduct ratio relative to those with a low adduct ratio. These findings suggest that estrogen-DNA adducts deserve further study as potential biomarkers for risk of developing breast cancer.
PMCID: PMC3378787  PMID: 22386952
breast cancer; depurinating estrogen-DNA adducts; biomarker; breast cancer prevention
25.  Discovery of novel vitamin D receptor interacting proteins that modulate 1,25-dihydroxyvitamin D3 signaling 
The nuclear vitamin D receptor (VDR) modulates gene transcription in 1,25-dihydroxyvitamin D3 (1,25D) target tissues such as kidney, intestine, and bone. VDR is also expressed in heart, and 1,25D deficiency may play a role in the acceleration of cardiovascular disease. Employing a yeast two-hybrid system and a human heart library, using both a 1,25D-independent and 1,25D-dependent screen, we discovered six candidate VDR interacting proteins (VIPs). These novel VIPs include CXXC5, FASTK, NR4A1, TPM2, MYL3 and XIRP1. Mammalian two-hybrid assays as well as GST pull-downs were used to confirm VIP-VDR interaction, and the combination of these two assays reveals that CXXC5, XIRP1, FASTK and NR4A1 interactions with VDR may be modulated by 1,25D. The functional effects of these VIPs on 1,25D-mediated gene expression were explored in transcriptional assays employing three separate and distinct 1,25D-responsive element (VDRE)-driven luciferase reporter genes in transfected Caco-2 and HEK-293 cells, and in a C2C12 myoblast line. FASTK and TPM2 activated expression in all cell line and promoter contexts, while CXXC5 and XIRP1 exhibited differing effects depending on the cell line and promoter employed, suggesting promoter and cell-specific effects of these unique VIPs on VDR signaling. Further evaluation of the interaction between CXXC5 and VDR revealed that CXXC5 acts in a dose-dependent manner to stimulate VDR-mediated transcription on select VDREs. Identification of novel heart VIPs and their influence on VDR activity may increase our understanding of how vitamin D impacts cardiac physiology and may facilitate development of VDR/VIP drug analogs to combat heart disease.
PMCID: PMC3408799  PMID: 22626544
vitamin D; vitamin D receptor; vitamin D responsive elements; transcription; co-modulators; CXXC5

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