Claudin 7 expression and localization in the normal murine mammary gland and murine mammary tumors

INTRODUCTION: Claudins, membrane-associated tetraspanin proteins, are normally associated with the tight junctions of epithelial cells where they confer a variety of permeability properties to the transepithelial barrier. One member of this family, claudin 7, has been shown to be expressed in the human mammary epithelium and some breast tumors. To set the stage for functional experiments on this molecule, we examined the developmental expression and localization of claudin 7 in the murine mammary epithelium and in a selection of murine mammary tumors. METHOD: We used real-time polymerase chain reaction, in situ mRNA localization, and immunohistochemistry (IHC) to examine the expression and localization of claudin 7. Frozen sections were examined by digital confocal microscopy for colocalization with the tight-junction protein ZO1. RESULTS: Claudin 7 was expressed constitutively in the mammary epithelium at all developmental stages, and the ratio of its mRNA to that of keratin 19 was nearly constant through development. By IHC, claudin 7 was located in the basolateral part of the cell where it seemed to be localized to discrete vesicles. Scant colocalization with the tight-junction scaffolding protein ZO1 was observed. Similar results were obtained from IHC of the airway epithelium and some renal tubules; however, claudin 7 did partly colocalize with ZO1 in EPH4 cells, a normal murine mammary cell line, and in the epididymis. The molecule was localized in the cytoplasm of MMTV-neu and the transplantable murine tumor cell lines TM4, TM10, and TM40A, in which its ratio to cytokeratin was higher than in the normal mammary epithelium. CONCLUSION: Claudin 7 is expressed constitutively in the mammary epithelium at approximately equal levels throughout development as well as in the murine tumors examined. Although it is capable of localizing to tight junctions, in the epithelia of mammary gland, airway, and kidney it is mostly or entirely confined to punctate cytoplasmic structures, often near the basolateral surfaces of the cells and possibly associated with basolateral membranes. These observations suggest that claudin 7 might be involved in vesicle trafficking to the basolateral membrane, possibly stabilizing cytoplasmic vesicles or participating in cell–matrix interactions

Immortalized, premalignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA

Abstract Introduction During selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents, simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy. Methods The glands of 3-week-old immune-competent Balb/C female mice were used intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-Bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells that retain their template DNA. Nulliparous mice were then either injected with [3H]-thymidine (3H-TdR) to distinguish 5-BrdU label-retaining cells that enter the cell cycle and euthanized, or mated, injected with 3H-TdR, and euthanized at various days after coitus. Sections were stained for estrogen receptor-α (ER-α) or progesterone receptor (PR) with immunohistochemistry. Cells labeled with both 5-BrdU and 3H-TdR were indicative of label-retaining epithelial cells (LRECs). Results Cells that retained a 5-BrdU label and cells labeled with [3H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labeled nuclei (LRECs) were found in the intact mammary glands of both pregnant and nulliparous mice, and in mammary glands implanted with premalignant cells. Double-labeled cells (3H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label (3H-TdR) to daughter cells, and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary-implant groups. Conclusions The results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.Peer Reviewe

Evaluation of A2BP1 as an Obesity Gene

OBJECTIVE-A genome-wide association study (GWAS) in Pima Indians (n = 413) identified variation in the ataxin-2 binding protein 1 gene (A2BP1) that was associated with percent body fat. On the basis of this association and the obese phenotype of ataxin-2 knockout mice, A2BP1 was genetically and functionally analyzed to assess its potential role in human obesity. RESEARCH DESIGN AND METHODS-Variants spanning A2BP1 were genotyped in a population-based sample of 3,234 full-heritage Pima Indians, 2,843 of whom were not part of the initial GWAS study and therefore could serve as a sample to assess replication. Published GWAS data across A2BP1 were additionally analyzed in French adult (n = 1,426) and children case/control subjects (n = 1,392) (Meyre et al. Nat Genet 2009;41:157-159). Selected variants were genotyped in two additional samples of Caucasians (Amish, n = 1,149, and German children case/control subjects, n = 998) and one additional Native American (n = 2,531) sample. Small interfering RNA was used to knockdown A2bp1 message levels in mouse embryonic hypothalamus cells. RESULTS-No single variant in A2BP1 was reproducibly associated with obesity across the different populations. However, different variants within intron 1 of A2BP1 were associated with BMI in full-heritage Pima Indians (rs10500331, P = 1.9 x 10(-7)) and obesity in French Caucasian adult (rs4786847, P = 1.9 x 10(-10)) and children (rs8054147, P = 9.2 x 10(-6)) case/control subjects. Reduction of A2bp1 in mouse embryonic hypothalamus cells decreased expression of Atxn2, Insr, and Mc4r. CONCLUSIONS-Association analysis suggests that variation in A2BP1 influences obesity, and functional studies suggest that A2BP1 could potentially affect adiposity via the hypothalamic MC4R pathway. Diabetes 59:2837-2845, 201

Neurologic Factors in Female Sexual Function and Dysfunction

Sexual dysfunction affects both men and women, involving organic disorders, psychological problems, or both. Overall, the state of our knowledge is less advanced regarding female sexual physiology in comparison with male sexual function. Female sexual dysfunction has received little clinical and basic research attention and remains a largely untapped field in medicine. The epidemiology of female sexual dysfunction is poorly understood because relatively few studies have been done in community settings. In the United States, female sexual dysfunction has been estimated to affect 40% of women in the general population. Among the elderly, however, it has been reported that up to 87% of women complain of sexual dissatisfaction. Several studies have shown that the prevalence of female sexual arousal disorders correlates significantly with increasing age. These studies have shown that sexual arousal and frequency of coitus in the female decreases with increasing age. The pathophysiology of female sexual dysfunction appears more complex than that of males, involving multidimensional hormonal, neurological, vascular, psychological, and interpersonal aspects. Organic female sexual disorders may include a wide variety of vascular, neural, or neurovascular factors that lead to problems with libido, lubrication, and orgasm. However, the precise etiology and mechanistic pathways of age-related female sexual arousal disorders are yet to be determined. In the past two decades, some advances have been made in exploring the basic hemodynamics and neuroregulation of female sexual function and dysfunction in both animal models and in human studies. In this review, we summarize neural regulation of sexual function and neurological causes of sexual dysfunction in women

Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-alpha

AIMS/HYPOTHESIS: MicroRNAs (miRNAs) are short endogenous RNAs that regulate multiple biological processes including adipogenesis and fat metabolism. We sought to identify miRNAs that correlate with BMI and to elucidate their upstream regulation and downstream targets. METHODS: Microarray-based expression profiling of 233 miRNAs was performed on subcutaneous abdominal adipose tissue biopsies from 29 non-diabetic Pima Indian participants. Correlation of the expression levels of eight miRNAs with BMI was assessed by quantitative reverse transcription (QRT) PCR in adipose samples from 80 non-diabetic Pima Indians with a BMI of 21.6–54.0 kg/m(2). The upstream regulation of one of these miRNAs, miR-221, was tested by treating cultured human pre-adipocytes with leptin, TNF-α and insulin. Predicted targets of miR-221 were validated using QRT-PCR, immunoblots and luciferase assays. The downstream effects of miR-221 overexpression were assayed by proteomic analysis. RESULTS: Expression levels of miR-221 were positively correlated with BMI (particularly in women) and fasting insulin concentrations, while the levels of miR-193a-3p and miR-193b-5p were negatively correlated with BMI; other miRNAs did not show significant associations in the 80 samples. miR-221 was downregulated by leptin and TNF-α treatment in cultured human pre-adipocytes. Conversely, miR-221 overexpression upregulated several proteins involved in fat metabolism, mimicking peroxisome proliferator-activated receptor (PPAR) activation. Furthermore, miR-221 directly downregulated the adiponectin receptor 1 (ADIPOR1) and the transcription factor v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1). Adiponectin signalling is known to promote insulin sensitivity, and ETS1 is crucial for angiogenesis. CONCLUSIONS/INTERPRETATION: Our data suggest that miR-221 may contribute to the development of the insulin resistance that typically accompanies obesity, by affecting PPAR signalling pathways and by directly downregulating ADIPOR1 and ETS1. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00125-013-2950-9) contains peer-reviewed but unedited supplementary material, which is available to authorised users

Differential methylation of genes in individuals exposed to maternal diabetes in utero

Aims/hypothesis Individuals exposed to maternal diabetes in utero are more likely to develop metabolic and cardiovascular diseases later in life. This may be partially attributable to epigenetic regulation of gene expression. We performed an epigenome-wide association study to examine whether differential DNA methylation, a major source of epigenetic regulation, can be observed in offspring of mothers with type 2 diabetes during the pregnancy (OMD) compared with offspring of mothers with no diabetes during the pregnancy (OMND). Methods DNA methylation was measured in peripheral blood using the Illumina HumanMethylation450K BeadChip. A total of 423,311 CpG sites were analysed in 388 Pima Indian individuals, mean age at examination was 13.0 years, 187 of whom were OMD and 201 were OMND. Differences in methylation between OMD and OMND were assessed. Results Forty-eight differentially methylated CpG sites (with an empirical false discovery rate ≤0.05), mapping to 29 genes and ten intergenic regions, were identified. The gene with the strongest evidence was LHX3, in which six CpG sites were hypermethylated in OMD compared with OMND (p ≤ 1.1 × 10−5). Similarly, a CpG near PRDM16 was hypermethylated in OMD (1.1% higher, p = 5.6 × 10−7), where hypermethylation also predicted future diabetes risk (HR 2.12 per SD methylation increase, p = 9.7 × 10−5). Hypermethylation near AK3 and hypomethylation at PCDHGA4 and STC1 were associated with exposure to diabetes in utero (AK3: 2.5% higher, p = 7.8 × 10−6; PCDHGA4: 2.8% lower, p = 3.0 × 10−5; STC1: 2.9% lower, p = 1.6 × 10−5) and decreased insulin secretory function among offspring with normal glucose tolerance (AK3: 0.088 SD lower per SD of methylation increase, p = 0.02; PCDHGA4: 0.08 lower SD per SD of methylation decrease, p = 0.03; STC1: 0.072 SD lower per SD of methylation decrease, p = 0.05). Seventeen CpG sites were also associated with BMI (p ≤ 0.05). Pathway analysis of the genes with at least one differentially methylated CpG (p < 0.005) showed enrichment for three relevant biological pathways. Conclusions/interpretation Intrauterine exposure to diabetes can affect methylation at multiple genomic sites. Methylation status at some of these sites can impair insulin secretion, increase body weight and increase risk of type 2 diabetes