Marya S. Sabir

1,25-Dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D

To investigate vitamin D‐related control of brain‐expressed genes, candidate vitamin D responsive elements (VDREs) at ‐7/‐10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)‐retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25‐dihydroxyvitamin D3 (1,25D). Brain TPH2 mRNA, encoding the rate‐limiting enzyme in serotonin synthesis, was induced 2.2‐fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8‐fold in rat serotonergic RN46A‐B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1‐fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis‐regulatory module; CRM) at ‐28 kb that 1,25D‐dependendy docked VDR, RXR, C/EBPp, and RUNX2. This CRM harbored 3 VDREs and single C/EBPβ and RUNX2 sites. Therefore, the expression of human TPH2 and mouse Lep are governed by 1,25D, potentially via respective VDREs located at ‐7/‐10 kb and ‐28 kb. These results imply that vitamin D affects brain serotonin concentrations, which may be relevant to psychiatric disorders, such as autism, and may control leptin levels and affect eating behavior.—Kaneko, I., Sabir, M. S., Dussik, C. M., Whitfield, G. K., Karrys, A., Hseih, J.‐C., Haussler, M. R., Meyer, M. B., Pike, J. W., Jurutka, P. W. 1,25‐dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D. FASEB J. 29, 4023‐4035 (2015). www.fasebj.org

Resveratrol Potentiates Vitamin D and Nuclear Receptor Signaling

The 1,25‐dihydroxyvitamin D3 (1,25D) hormone is derived from vitamin D generated in skin or obtained from the diet, and binds to and activates the vitamin D receptor (VDR) in target tissues including kidney, colon/small intestine, and bone/muscle. We tested resveratrol for its ability to modulate VDR signaling, using vitamin D responsive element (VDRE) and mammalian 2‐hybrid (M2H) transcriptional system technology. Via VDRE‐based assays in kidney, colon and myoblast cells, VDR‐mediated transcription was activated by resveratrol, and a cooperative effect on transactivation was observed with resveratrol plus 1,25D. The M2H assay revealed a modest, resveratrol‐induced dimerization of VDR with its retinoid X receptor (RXR) heteropartner. Cells treated with both resveratrol and 1,25D displayed synergistic stimulation of VDR–RXR heterodimerization, while resveratrol antagonized rexinoid‐mediated RXR‐RXR homodimerization. Increased transactivation in response to resveratrol was also observed with a subset of other nuclear receptors and their respective cognate responsive elements. Evaluation of wild‐type versus a ligand‐binding domain mutant VDR revealed that hormone‐responsiveness to 1,25D was severely depressed, while the response to resveratrol was only moderately attenuated. Moreover, radiolabeled 1,25D‐displacement assays demonstrated an increase in VDR‐bound 1,25D in the presence of resveratrol. Thus, resveratrol may affect VDR and other nuclear receptors indirectly, likely via the ability of resveratrol to: (1) potentiate 1,25D binding to VDR; (2) activate RXR; and/or (3) stimulate SIRT1, an enzyme known to deacetylate nuclear receptors. The results of this study elucidate a possible pathway for crosstalk between two nutritionally derived lipids, vitamin D and resveratrol, both of which converge on VDR signaling. J. Cell. Biochem. 116: 1130–1143, 2015.

SIRT1 enzymatically potentiates 1,25-dihydroxyvitamin D3 signaling via vitamin D receptor deacetylation

The hormonal metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), binds to the vitamin D receptor (VDR) and promotes heterodimerization of VDR with a retinoid-X-receptor (RXR) to genomically regulate diverse cellular processes. Herein, it is revealed for the first time that VDR is post-translationally acetylated, and that VDR immunoprecipitated from human embryonic kidney (HEK293) cells displays a dramatic decrease in acetylated receptor in the presence of 1,25D-ligand, sirtuin-1 (SIRT1) deacetylase, or the resveratrol activator of SIRT1. To elucidate the functional significance of VDR deacetylation, vitamin-d-responsive-element (VDRE)-based transcriptional assays were performed to determine if deacetylase overexpression affects VDR/VDRE-driven transcription. In HEK293 kidney and TE85 bone cells, co-transfection of low amounts (1-5ng) of a SIRT1-expression vector elicits a reproducible and statistically significant enhancement (1.3- to 2.6-fold) in transcription mediated by VDREs from the CYP3A4 and cyp24a1 genes, where the magnitude of response to 1,25D-ligand is 6- to 30-fold. Inhibition of SIRT1 via EX-527, or utilization of a SIRT1 loss-of-function mutant (H363Y), resulted in abrogation of SIRT1-mediated VDR potentiation. Studies with a novel, non-acetylatable VDR mutant (K413R) showed that the mutant VDR possesses enhanced responsiveness to 1,25D, in conjunction with reduced, but still significant, sensitivity to exogenous SIRT1, indicating that acetylation of lysine 413 is relevant, but that other acetylated residues in VDR contribute to modulation of its activity. We conclude that the acetylation of VDR comprises a negative feedback loop that attenuates 1,25D-VDR signaling. This regulatory loop is reversed by SIRT1-catalyzed deacetylation of VDR to amplify VDR signaling and 1,25D actions.

Bioactive Dietary VDR Ligands Regulate Genes Encoding Biomarkers of Skin Repair That Are Associated with Risk for Psoriasis

Treatment with 1,25-dihydroxyvitamin D3 (1,25D) improves psoriasis symptoms, possibly by inducing the expression of late cornified envelope (LCE)3 genes involved in skin repair. In psoriasis patients, the majority of whom harbor genomic deletion of LCE3B and LCE3C (LCE3C_LCE3B-del), we propose that certain dietary analogues of 1,25D activate the expression of residual LCE3A/LCE3D/LCE3E genes to compensate for the loss of LCE3B/LCE3C in the deletant genotype. Herein, human keratinocytes (HEKn) homozygous for LCE3C_LCE3B-del were treated with docosahexaenoic acid (DHA) and curcumin, two low-affinity, nutrient ligands for the vitamin D receptor (VDR). DHA and curcumin induce the expression of LCE3A/LCE3D/LCE3E mRNAs at concentrations corresponding to their affinity for VDR. Moreover, immunohistochemical quantitation revealed that the treatment of keratinocytes with DHA or curcumin stimulates LCE3 protein expression, while simultaneously opposing the tumor necrosis factor-alpha (TNFα)-signaled phosphorylation of mitogen activated protein (MAP) kinases, p38 and Jun amino-terminal kinase (JNK), thereby overcoming inflammation biomarkers elicited by TNFα challenge. Finally, DHA and curcumin modulate two transcription factors relevant to psoriatic inflammation, the activator protein-1 factor Jun B and the nuclear receptor NR4A2/NURR1, that is implicated as a mediator of VDR ligand-triggered gene control. These findings provide insights into the mechanism(s) whereby dietary VDR ligands alter inflammatory and barrier functions relevant to skin repair, and may provide a molecular basis for improved treatments for mild/moderate psoriasis.

Gene Expression Profiling and Assessment of Vitamin D and Serotonin Pathway Variations in Patients With Irritable Bowel Syndrome

Background/Aims Irritable bowel syndrome (IBS) is a multifaceted disorder that afflicts millions of individuals worldwide. IBS is currently diagnosed based on the presence/duration of symptoms and systematic exclusion of other conditions. A more direct manner to identify IBS is needed to reduce healthcare costs and the time required for accurate diagnosis. The overarching objective of this work is to identify gene expression-based biological signatures and biomarkers of IBS. Methods Gene transcripts from 24 tissue biopsy samples were hybridized to microarrays for gene expression profiling. A combination of multiple statistical analyses was utilized to narrow the raw microarray data to the top 200 differentially expressed genes between IBS versus control subjects. In addition, quantitative polymerase chain reaction was employed for validation of the DNA microarray data. Gene ontology/pathway enrichment analysis was performed to investigate gene expression patterns in biochemical pathways. Finally, since vitamin D has been shown to modulate serotonin production in some models, the relationship between serum vitamin D and IBS was investigated via 25-hydroxyvitamin D (25[OH]D) chemiluminescence immunoassay. Results A total of 858 genetic features were identified with differential expression levels between IBS and asymptomatic populations. Gene ontology enrichment analysis revealed the serotonergic pathway as most prevalent among the differentially expressed genes. Further analysis via real-time polymerase chain reaction suggested that IBS patient-derived RNA exhibited lower levels of tryptophan hydroxylase-1 expression, the enzyme that catalyzes the rate-limiting step in serotonin biosynthesis. Finally, mean values for 25(OH)D were lower in IBS patients relative to non-IBS controls. Conclusions Values for serum 25(OH)D concentrations exhibited a trend towards lower vitamin D levels within the IBS cohort. In addition, the expression of select IBS genetic biomarkers, including tryptophan hydroxylase 1, was modulated by vitamin D. Strikingly, the direction of gene regulation elicited by vitamin D in colonic cells is “opposite” to the gene expression profile observed in IBS patients, suggesting that vitamin D may help “reverse” the pathological direction of biomarker gene expression in IBS. Thus, our results intimate that IBS pathogenesis and pathophysiology may involve dysregulated serotonin production and/or vitamin D insufficiency.

Implications of language barrier on the diagnostic yield of computed tomography in pulmonary embolism

Objectives: To determine if a physician‐patient language barrier impacts the diagnostic accuracy of pulmonary embolism (PE) evaluation. Methods: A retrospective chart review, conducted between June 2015 and December 2016, of a consecutive sample of diagnostic computed tomography pulmonary angiogram (CTPA) studies performed on adult patients. Positive and negative CTPA scans were further categorized by patient language and the positive diagnostic yield was determined for each language group. A post collection sub‐analysis was performed to determine the yield when interpreter services were identified as necessary. Results: The yield for English speaking patients was 10.24% (92/898, 95% CI 8.39% to 12.36%), similar to the yield in Spanish speaking patients of 9.40% (25/266, 95% CI 6.31% to 13.37%, P = 0.69). This contrasted with the yield in patients who identified as bilingual, which was significantly lower at 1.41% (1/71, 95% CI 0.07% to 6.75%) compared to both English‐(P < 0.02) and Spanish‐only speakers (P < 0.03). The yield for non‐English speaking patients who requested an interpreter was 7.37% (14/190, 95% CI 4.26% to 11.77%) versus 3.23% (2/62, 95% CI 0.54% to 10.25%, P = 0.25) in those who did not. Conclusions: The diagnostic yield in English‐ and Spanish‐only speaking patients was similar, however, the yield in those that self‐identified as bilingual was significantly lower. In patient groups in which a language barrier existed and an interpreter was not utilized, there was a trend toward a lower diagnostic yield. This suggests an increased propensity to order diagnostic imaging when potential communication barriers exist.

Vitamin D Nutrient-Gene Interactions and Healthful Aging

Abstract 1,25-Dihydroxyvitamin D 3 (1,25D) is the endocrine metabolite of vitamin D that signals through binding to the vitamin D receptor (VDR). The ligand–receptor complex transcriptionally regulates genes that encode factors promoting intestinal calcium and phosphate absorption plus bone remodeling, maintaining a skeleton with reduced risk of age-related osteoporotic fractures. 1,25D/VDR signaling further exerts feedback control of mineral ions via regulation of FGF23, klotho, and CYP24A1 to prevent age-related, ectopic calcification, and associated pathologies. Vitamin D also elicits xenobiotic detoxification, oxidative stress reduction, antimicrobial defense, immunoregulation, anti-inflammatory/anticancer actions, and cardiovascular benefits. 1,25D exerts neuroprotective actions against excitotoxicity, and induces serotonin mood elevator to support cognitive function and prosocial behavior. Nutrient, low-affinity VDR ligands including curcumin, polyunsaturated fatty acids, and delphinidin/anthocyanidins initiate VDR signaling, whereas longevity agents such as resveratrol and SIRT1 potentiate VDR signaling. Therefore, liganded VDR modulates the expression of a network of genes that facilitates health span by delaying the chronic diseases of aging.

Abstract B24: Targeting promoter regions of oncogenic drivers in pediatric AML cells using G-quadruplex Interacting Drugs (GQIDs)

Cancer is primarily a disease characterized by aberrant gene expression that is manifested by the overexpression of key genes that support tumor development and maintenance. In many instances, oncogenic drivers are frequently ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. Our laboratory investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific genes used by tumor cells for growth and survival. We tested the activity of two GQIDs GQC-05 and GSA-1103 on cell growth of a panel of eight pediatric and eight adult AML cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 µM for GQC-05 and from 40 nM to 2 µM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-myc and/or bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of cells with the GQC-05 decreased expression of c-myc and bcl-2. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures. Validation of such an approach for AML will have important implications for testing this therapeutic approach to other childhood cancers in order to improve the length and quality of survival. Citation Format: Apurvi Patel, Justin J. Montoya, Megan Turnidge, Marya Sabir, Daniel H. Wai, David W. Lee, Vijay Gokhale, Eiman Aleem, Laurence J. Hurley, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of oncogenic drivers in pediatric AML cells using G-quadruplex Interacting Drugs (GQIDs). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B24.