Glucuronidation is an important pathway in the metabolism of nicotine, with previous studies suggesting that ∼22% of urinary nicotine metabolites are in the form of glucuronidated compounds. Recent in vitro studies have suggested that the UDP-glucuronosyltransferases (UGT) 2B10 and 2B17 play major roles in nicotine glucuronidation with polymorphisms in both enzymes shown to significantly alter the levels of nicotineglucuronide, cotinine-glucuronide, and trans-3′-hydroxycotinine (3HC)-glucuronide in human liver microsomes in vitro. In the present study, the relationship between the levels of urinary nicotine metabolites and functional polymorphisms in UGTs 2B10 and 2B17 was analyzed in urine specimens from 104 Caucasian smokers. Based on their percentage of total urinary nicotine metabolites, the levels of nicotine-glucuronide and cotinine-glucuronide were 42% (P < 0.0005) and 48% (P < 0.0001), respectively, lower in the urine from smokers exhibiting the UGT2B10 (*1/*2) genotype and 95% (P < 0.05) and 98% (P < 0.05), respectively, lower in the urine from smokers with the UGT2B10 (*2/*2) genotype compared with the urinary levels in smokers having the wild-type UGT2B10 (*1/*1) genotype. The level of 3HC-glucuronide was 42% (P < 0.001) lower in the urine from smokers exhibiting the homozygous UGT2B17 (*2/*2) deletion genotype compared with the levels in urine from wild-type UGT2B17 subjects. These data suggest that UGTs 2B10 and 2B17 play important roles in the glucuronidation of nicotine, cotinine, and 3HC and suggest that the UGT2B10 codon 67 SNP and the UGT2B17 gene deletion significantly reduce overall glucuronidation rates of nicotine and its major metabolites in smokers.

SummaryMetformin, an oral hypoglycemic agent, has been used for decades to treat type 2 diabetes mellitus. Recent studies indicate that mice treated with metformin live longer and have fewer manifestations of age‐related chronic disease. However, the molecular mechanisms underlying this phenotype are unknown. Here, we show that metformin treatment increases the levels of the microRNA‐processing protein DICER1 in mice and in humans with diabetes mellitus. Our results indicate that metformin upregulates DICER1 through a post‐transcriptional mechanism involving the RNA‐binding protein AUF1. Treatment with metformin altered the subcellular localization of AUF1, disrupting its interaction with DICER1 mRNA and rendering DICER1 mRNA stable, allowing DICER1 to accumulate. Consistent with the role of DICER1 in the biogenesis of microRNAs, we found differential patterns of microRNA expression in mice treated with metformin or caloric restriction, two proven life‐extending interventions. Interestingly, several microRNAs previously associated with senescence and aging, including miR‐20a, miR‐34a, miR‐130a, miR‐106b, miR‐125, and let‐7c, were found elevated. In agreement with these findings, treatment with metformin decreased cellular senescence in several senescence models in a DICER1‐dependent manner. Metformin lowered p16 and p21 protein levels and the abundance of inflammatory cytokines and oncogenes that are hallmarks of the senescence‐associated secretory phenotype (SASP). These data lead us to hypothesize that changes in DICER1 levels may be important for organismal aging and to propose that interventions that upregulate DICER1 expression (e.g., metformin) may offer new pharmacotherapeutic approaches for age‐related disease.

ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.ATF5 (activating transcription factor 5; also known as ATFx) is a member of the ATF/cAMP response element-binding protein (CREB) 2 family of bZIP (basic zipper) proteins (1). It can bind to several transcription regulatory DNA elements that include cAMP response elements (CRE), an amino acid response element, and an ATF5-specific response element (ARE) and regulate gene expression in different types of cells (1-4). ATF5 has been identified as a factor whose down-regulation is essential for differentiation of rat neural progenitor cells and PC12 pheochromocytoma cells (5-7). ATF5 is expressed in a number of cancer cells and down-regulated in those cells following growth factor deprivation; exogenous expression of ATF5 suppresses apoptosis in HeLa cells induced by serum withdrawal and in FL5.12 cells, an IL-3-dependent cell line, by IL-3 deprivation (8). Conversely, dominant-negative ATF5 induces apoptosis of HeLa and FL5.12 cells and a number of glioma and breast cancer cell lines cultured in the presence of growth factors (8 -10). Similar interference of ATF5 function in non-neoplastic breast cells or in non-tumor brain cells, such as mature neurons and glial cells, does not affect their survival (9, 10). The mechanism of such selective survival requirement for ATF5 in different cell types is not understood.The BCL-2 family of proteins includes both anti-apoptotic (BCL-2, BCL-X L , and MCL-1) and apoptotic (BAK, BAX, BID, and BIM) proteins. The regulation and balance of this BCL-2 family of proteins in a particular cell result in inhibition or induction of the apoptotic signaling pathways (11,12). Notably, the expression pattern ...

Systemic arterial hypertension is an important cause of cardiovascular disease morbidity and mortality. African Americans are disproportionately affected by hypertension, in fact the incidence, prevalence, and severity of hypertension is highest among African American (AA) women. Previous data suggests that differential gene expression influences individual susceptibility to selected diseases and we hypothesized that this phenomena may affect health disparities in hypertension. Transcriptional profiling of peripheral blood mononuclear cells from AA or white, normotensive or hypertensive females identified thousands of mRNAs differentially-expressed by race and/or hypertension. Predominant gene expression differences were observed in AA hypertensive females compared to AA normotensives or white hypertensives. Since microRNAs play important roles in regulating gene expression, we profiled global microRNA expression and observed differentially-expressed microRNAs by race and/or hypertension. We identified novel mRNA-microRNA pairs potentially involved in hypertension-related pathways and differently-expressed, including MCL1/miR-20a-5p, APOL3/miR-4763-5p, PLD1/miR-4717-3p, and PLD1/miR-4709-3p. We validated gene expression levels via RT-qPCR and microRNA target validation was performed in primary endothelial cells. Altogether, we identified significant gene expression differences between AA and white female hypertensives and pinpointed novel mRNA-microRNA pairs differentially-expressed by hypertension and race. These differences may contribute to the known disparities in hypertension and may be potential targets for intervention.

The UDP-glucuronosyltransferase (UGT) 1A enzymes are involved in the phase II metabolism of many important endogenous and exogenous compounds. The nine UGT1A isoforms exhibit high interindividual differences in expression, but their epigenetic regulation is not well understood. The purpose of the present study was to examine microRNA (miRNA) regulation of hepatic UGT1A enzymes and determine whether or not that regulation impacts enzymatic activity. In silico analysis identified miRNA 491-3p (miR-491-3p) as a potential regulator of the UGT1A gene family via binding to the shared UGT1A 39-untranslated region common to all UGT1A enzymes. Transfection of miR-491-3p mimic into HuH-7 cells significantly repressed UGT1A1 (P , 0.001), UGT1A3 (P , 0.05), and UGT1A6 (P , 0.05) mRNA levels. For UGT1A1, this repression correlated with significantly reduced metabolism of raloxifene into raloxifene-6-glucuronide (ral-6-gluc; P , 0.01) and raloxifene-49-glucuronide (ral-49-gluc; P , 0.01). In HuH-7 cells with repressed miR-491-3p expression, there was a significant increase (∼80%; P , 0.01) in UGT1A1 mRNA and a corresponding increase in glucuronidation of raloxifene into ral-6-gluc (50%; P , 0.05) and ral-49-gluc (22%; P , 0.01). Knockdown of endogenous miR-491-3p in HepG2 cells did not significantly alter UGT1A1 mRNA levels but did increase the formation of ral-6-gluc (50%; P , 0.05) and ral-49-gluc (34%; P , 0.001). A significant inverse correlation between miR-491-3p expression and both UGT1A3 (P , 0.05) and UGT1A6 (P , 0.01) mRNA levels was observed in a panel of normal human liver specimens, with a significant (P , 0.05) increase in UGT1A3 and UGT1A6 mRNA levels observed in miR-491-3p nonexpressing versus expressing liver specimens. These results suggest that miR-491-3p is an important factor in regulating the expression of UGT1A enzymes in vivo.

The UDP-glucuronosyltransferase (UGT) 2B enzymes are important in the detoxification of a variety of endogenous and exogenous compounds, including many hormones, drugs, and carcinogens. Identifying novel mechanisms governing their expression is important in understanding patient-specific response to drugs and cancer risk factors. In silico prediction algorithm programs were used to screen for microRNAs (miRNAs) as potential regulators of UGT2B enzymes, with miR-216b-5p identified as a potential candidate. Luciferase data suggested the presence of a functional miR-216b-5p binding motif within the 39 untranslated regions of UGTs 2B7, 2B4, and 2B10. Overexpression of miR-216b-5p mimics significantly repressed UGT2B7 (P , 0.001) and UGT2B10 (P 5 0.0018) mRNA levels in HuH-7 cells and UGT2B4 (P , 0.001) and UGT2B10 (P 5 0.018) mRNA in Hep3B cells. UGT2B7 protein levels were repressed in both HuH-7 and Hep3B cells in the presence of increasing miR-216b-5p concentrations, corresponding with significant (P , 0.001 and P 5 0.011, respectively) decreases in glucuronidation activity against the UGT2B7-specific substrate epirubicin. Inhibition of endogenous miR-216b-5p levels significantly increased UGT2B7 mRNA levels in HuH-7 (P 5 0.021) and Hep3B (P 5 0.0068) cells, and increased epirubicin glucuronidation by 85% (P 5 0.057) and 50% (P 5 0.012) for HuH-7 and Hep3B cells, respectively. UGT2B4 activity against codeine and UGT2B10 activity against nicotine were significantly decreased in both HuH-7 and Hep3B cells (P , 0.001 and P 5 0.0048, and P 5 0.017 and P 5 0.043, respectively) after overexpression of miR-216b-5p mimic. This is the first evidence that miRNAs regulate UGT 2B7, 2B4, and 2B10 expression, and that miR-216b-5p regulation of UGT2B proteins may be important in regulating the metabolism of UGT2B substrates.

UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Realtime polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > larynx > kidney. A novel splice variant of UGT2A2 lacking exon 3 (termed UGT2A2Dexon3) was investigated, with UGT2A2Dexon3 expression determined to be 25-50% that of wild-type UGT2A2 in all tissues examined. UGT2A3 was determined to be well expressed in the liver and colon, followed by pancreas > kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from human embryonic kidney (HEK)293 cells overexpressing wild-type UGT2A2 (termed UGT2A2_i1) exhibited glucuronidation activity, as observed by reverse-phase ultra-pressure liquid chromatography, against 1-hydroxy-(OH)-pyrene, 1-naphthol, and hydroxylated benzo(a) pyrene metabolites, whereas homogenates prepared from HEK293 cells overexpressing UGT2A3 only showed activity against simple PAHs like 1-OH-pyrene and 1-naphthol. Activity assays showed the UGT2A2Dexon3 protein (termed UGT2A2_i2) exhibited no detectable glucuronidation activity against all substrates examined; however, coexpression studies suggested that UGT2A2_i2 negatively modulates UGT2A2_i1 activity. Both UGT2A2 and UGT2A3 exhibited no detectable activity against complex PAH proximate carcinogens, tobacco-specific nitrosamines, or heterocyclic amines. These data suggest that, although UGT2A1 is the only UGT2A enzyme active against PAH proximate carcinogens (including PAH diols), both UGTs 2A1 and 2A2 play an important role in the local detoxification of procarcinogenic monohydroxylated PAH metabolites.

Since the discovery of extracellular RNA (exRNA) in circulation and other bodily fluids, there has been considerable effort to catalog and assess whether exRNAs can be used as markers for health and disease. A variety of exRNA species have been identified including mRNA, and non-coding RNA (ncRNA) such as microRNA (miRNA), small nucleolar RNA (snRNA), transfer RNA (tRNA), and long non-coding RNA (lncRNA). Age-related changes in exRNA abundance have been observed, and it is likely some of these transcripts play a role in aging. In this review, we summarize the current state of exRNA profiling in various body fluids and discuss age-related changes in exRNA abundance that have been identified in humans and other model organisms. MiRNAs, in particular, are a major focus of current research and we will highlight and discuss the potential role specific miRNAs might play in age-related phenotypes and disease. We will also review challenges facing this emerging field and various strategies that can be used for the validation and future use of exRNAs as markers of aging and age-related disease.