Alanna Roff

A Novel SNP in a Vitamin D Response Element of the CYP24A1 Promoter Reduces Protein Binding, Transactivation, and Gene Expression

The active form of vitamin D (1alpha,25(OH)(2)D(3)) is known to have antiproliferative effects and has been implicated in cancers of the colon, breast, and prostate. These cancers occur more frequently among African Americans than Caucasians, and individuals with African ancestry are known to have approximately twofold lower levels of serum vitamin D (25(OH)D) compared with individuals of European ancestry. However, epidemiological studies of the vitamin D receptor (VDR) have shown inconsistent associations with cancer risk, suggesting that differences in other genes in the pathway may be important. We sought to identify functionally significant polymorphic variants in CYP24A1, a gene that is highly inducible by 1alpha,25(OH)(2)D(3) and that encodes the primary catabolic enzyme in the pathway. Here we report the identification of six novel SNPs in the human CYP24A1 promoter, including one at nucleotide -279 occurring within the distal vitamin D response element (VDRE2). Our experiments demonstrate that the VDRE2 variant results in decreased protein binding and transactivation in vitro, and reduced expression of CYP24A1 in cultured primary human lymphocytes provides evidence for an effect in vivo. This variant was only observed in our African American population, and represents a first step toward understanding differences in disease risk among racial/ethnic groups.

Genetic Ancestry, Skin Reflectance and Pigmentation Genotypes in Association with Serum Vitamin D Metabolite Balance.

Abstract Background: Lower serum vitamin D (25(OH)D) among individuals with African ancestry is primarily attributed to skin pigmentation. However, the influence of genetic polymorphisms controlling for skin melanin content has not been investigated. Therefore, we investigated differences in non-summer serum vitamin D metabolites according to self-reported race, genetic ancestry, skin reflectance and key pigmentation genes (SLC45A2 and SLC24A5). Materials and methods: Healthy individuals reporting at least half African American or half European American heritage were frequency matched to one another on age (±2 years) and sex. One hundred and seventy-six autosomal ancestry informative markers were used to estimate genetic ancestry. Melanin index was measured by reflectance spectrometry. Serum vitamin D metabolites [25(OH)D3, 25(OH)D2 and 24,25(OH)2D3] were determined by high performance liquid chromatography (HPLC) tandem mass spectrometry. Percent 24,25(OH)2D3 was calculated as a percent of the parent metabolite [25(OH)D3]. Stepwise and backward selection regression models were used to identify leading covariates. Results: Fifty African Americans and 50 European Americans participated in the study. Compared with SLC24A5 111Thr homozygotes, individuals with the SLC24A5 111Thr/Ala and 111Ala/Ala genotypes had, respectively, lower levels of 25(OH)D3 (23.0 and 23.8 nmol/L lower, p-dominant=0.007), and percent 24,25(OH)2D3 (4.1% and 5.2% lower, p-dominant=0.003), controlling for tanning bed use, vitamin D/fish oil supplement intake, race/ethnicity and genetic ancestry. Results were similar with melanin index adjustment, and were not confounded by glucocorticoid, oral contraceptive, or statin use. Conclusions: The SLC24A5 111Ala allele was associated with lower serum vitamin 25(OH)D3 and lower percent 24,25(OH)2D3, independently from melanin index and West African genetic ancestry.

Corrigendum to “A novel SNP in a vitamin D response element of the CYP24A1 promoter reduces protein binding, transactivation, and gene expression” [J. Steroid Biochem. Mol. Biol. 112 (2008) 47–54]

The authors realized after the publication of the above article that the numbering used to indicate the positions of the variants and response elements relative to the transcription start site were not accurate. The correct positions for the eight polymorphisms described in Table 1 are one nucleotide further from the start site, i.e. −104, −128, −227, etc. The novel variants have since been submitted to NCBI with the correct annotation. In addition, the positions of the promoter elements shown in Figure 1 should be −290 to −276 (VDRE2) and −170 to−147 (VDRE1). We apologize for any inconvenience or confusion this may have caused the readers. Dr. Wilson regrets that during publication, Dr. Carastro was not included as an author. The corrected author list is published above.

Post-transcriptional Regulation of Meprin α by the RNA-binding Proteins Hu Antigen R (HuR) and Tristetraprolin (TTP)

Background: Meprin α is a metalloproteinase implicated in the pathogenesis of inflammatory bowel disease (IBD). Results: RNA-binding proteins post-transcriptionally regulate meprin α, and RNA stability decreases after induction of an inflammatory response. Conclusion: Inflammatory stimuli downregulate meprin α by inducing TTP expression and binding to the transcript. Significance: Determining how inflammation alters meprin α regulation is crucial to understanding its role in IBD. Meprins are multimeric proteases that are implicated in inflammatory bowel disease by both genetic association studies and functional studies in knock-out mice. Patients with inflammatory bowel disease show decreased colonic expression of meprin α, although regulation of expression, particularly under inflammatory stimuli, has not been studied. The studies herein demonstrate that the human meprin α transcript is bound and stabilized by Hu antigen R at baseline, and that treatment with the inflammatory stimulus phorbol 12-myristate 13-acetate downregulates meprin α expression by inducing tristetraprolin. The enhanced binding of tristetraprolin to the MEP1A 3′-UTR results in destabilization of the transcript and occurs at a discrete site from Hu antigen R. This is the first report to describe a mechanism for post-transcriptional regulation of meprin α and will help clarify the role of meprins in the inflammatory response and disease.

MicroRNA-146a is induced by inflammatory stimuli in airway epithelial cells and augments the anti-inflammatory effects of glucocorticoids

Background MicroRNAs (miRNAs) are emerging as central regulators of inflammation, but their role in asthma and airway epithelial cells is not well studied. Glucocorticoids are the cornerstone of therapy in asthma and other inflammatory disease, yet their mechanisms of action are not completely elucidated, and it is not clear whether miRNAs modulate their effects. Objective We aimed to identify miRNAs that regulate cytokine and chemokine expression in airway epithelial cells and whether these miRNAs are subject to the effects of glucocorticoids. Methods and results MicroRNAomic analyses of immortalized, normal human bronchial epithelial cells identified 7 miRNAs that were altered by inflammatory cytokine treatment and 22 that were regulated by glucocorticoids (n = 3 for each treatment condition). MiR-146a emerged as a central candidate, whose expression was induced by TNF-α and repressed by glucocorticoids. Its role as a candidate in asthmatic inflammation was supported by expression profiling in human asthmatics, which showed that plasma miR-146a expression was elevated in asthma and associated with measures related to worse asthma outcomes, including elevated blood eosinophil counts, higher asthma control questionnaire scores, and need for higher doses of inhaled glucocorticoids. However, transfection of miR-146a in A549 cells treated with TNF-α +/- glucocorticoids produced an anti-inflammatory effect and increased efficacy of glucocorticoids. Conclusions We propose a model whereby miR-146a is induced by inflammatory conditions as a feedback mechanism to limit inflammation. Exogenous administration of miR-146a augmented the effects of glucocorticoids and could be a novel therapeutic strategy to enhance efficacy of these medications.

Abstract 670: Cigarette smoking, race, and genetic ancestry in association with plasma vitamin D-binding protein (VDBP) in healthy African Americans (AA) and Caucasian Americans (CA)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Vitamin D and VDBP affect several pathways involved in inflammation, tumor growth, and immune surveillance relevant to carcinogenesis. In a previous pilot study (Bortner et al., 2010), using a proteomics approach, VDBP was down-regulated in the plasma of chronic cigarette smokers by as much as 3-fold. Therefore, using an existing study of healthy AA and CA participants, we conducted an investigation of the differences in VDBP between 39 current smokers and 82 never smokers, frequency matched on age, sex and race/ethnicity. VDBP plasma concentrations were determined in duplicate using the Quantikine® Human VDBP ELISA (CV%, 5.6%). Genotyping was conducted for a set of 112 previously validated Ancestry Informative Markers for the percent West African and European genetic ancestry, and VDBP coding region changes via rs7041 (Asp416Glu, T/G) and rs4588 (Thr420Lys, C/A). Vitamin D metabolites (25OHD3 and 24,25(OH)2D3) were determined using LC-MS/MS (CV% 2.7 and 6.3, respectively). T-test, Chi-Square, Fishers Exact, and Spearmans correlation coefficients were used to determine statistically significant bivariate differences. Multiple logistic regression was used to adjust for multiple factors using the log-normal transformation of VDBP concentration with the Cochran-Armitage test for trend for VDBP genotype. Multiplicative interaction terms were tested along with their main-effects, adjusted for co-variates. There were no significant differences between current smokers and never smokers by age, VDBP genotype, body mass index, vitamin D metabolites, or genetic ancestry. VDBP concentrations were significantly negatively correlated with body mass index (BMI, r=−0.19, p=0.033), West African ancestry (r=−0.66, p<0.001), vitamin D metabolites (p-values<0.001), VDBP rs4588-A and rs7041-T alleles (VDBP genotypes were 100% correlated). VDBP genotypes coding Asp416Asp and Lys420Lys were at least 3.3-fold lower, compared with Glu416Glu and Thr420Thr, respectively, and this was regardless of smoking status (p-trend<0.001). There was no association between VDBP concentration and age or smoking status. In multiple regression models, serum vitamin D metabolites, self-reported race/ethnicity, West African ancestry and GC genotypes remained significantly associated with VDBP concentration. The magnitude of difference in these variables was similar among current and never smokers, although BMI remained significantly negatively associated with VDBP concentrations among never smokers (p-interaction =0.028). Smoking status was not highly correlated with VDBP plasma concentration, although may have some interactive effect with BMI. Future studies will need to account for the strong correlations with VDBP genotype and genetic ancestry. Support: 1K22CA120092-01A2 and 3K22CA120092-02S1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 670. doi:1538-7445.AM2012-670