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Andrea Hanel, Henna-Riikka Malmberg, and Carsten Carlberg

Molecular endocrinology of vitamin D is based on the activation of the transcription factor vitamin D receptor (VDR) by the vitamin D metabolite 1α,25-dihydroxyvitamin D3. This nuclear vitamin D-sensing process causes epigenome-wide effects, such as changes in chromatin accessibility as well as in the contact of VDR and its supporting pioneer factors with thousands of genomic binding sites, referred to as vitamin D response elements. VDR binding enhancer regions loop to transcription start sites of hundreds of vitamin D target genes resulting in changes of their expression. Thus, vitamin D signaling is based on epigenome- and transcriptome-wide shifts in VDR-expressing tissues. Monocytes are the most responsive cell type of the immune system and serve as a paradigm for uncovering the chromatin model of vitamin D signaling. In this review, an alternative approach for selecting vitamin D target genes is presented, which are most relevant for understanding the impact of vitamin D endocrinology on innate immunity. Different scenarios of the regulation of primary upregulated vitamin D target genes are presented, in which vitamin D-driven super-enhancers comprise a cluster of persistent (constant) and/or inducible (transient) VDR-binding sites. In conclusion, the spatio-temporal VDR binding in the context of chromatin is most critical for the regulation of vitamin D target genes.

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Yawen Xu, Jinhua Lu, Jinxiang Wu, Ruiwei Jiang, Chuanhui Guo, Yedong Tang, Haibin Wang, Shuangbo Kong, and Suqing Wang

Decidualization is a critical process for embryo implantation and pregnancy maintenance in humans. The homeobox gene HOXA10 has been widely studied in endometrial receptivity establishment and decidualization. MEIS1, a three-amino-acid loop extension (TALE) family homeobox gene, has been proven to be a co-factor for HOXA10 in mouse uterus. However, the interaction between MEIS1 and HOXA10 in the human decidual cells remains to be elucidated. siRNA and CRISPR-Cas9 were employed to knockdown and knockout MEIS1 in the cultured human endometrial stromal cells, and it was found that MEIS1 deficiency leads to impaired decidualization. The physical interaction between the MEIS1 and HOXA10 in human endometrial stromal cell was confirmed by immunoprecipitation. Moreover, KAT2B and ETA were proved to be downregulated in the absence of MEIS1, and luciferase reporter and ChIP assays demonstrated that MEIS1-HOXA10 complex binds to the promoters of KAT2B and ETA and regulates their activity. Overexpression of KAT2B and ETA can partially rescue the decidualization defects in MEIS1-knockout HESCs. Taken together, these data suggest that MEIS1 plays an indispensable role in decidualization in human endometrial stromal cells, and MEIS1 interacts with HOXA10 to regulate the downstream genes, such as KAT2B and ETA. These findings will contribute to our understanding about the regulatory network in the process of decidualization in humans.

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Jingyi Luo, Tingting Liu, and Weiping Teng

Hashimoto’s thyroiditis (HT) is a common organ-specific autoimmune disease, which develops in 0.3–1.5/1000 subjects annually. The aims of this study were to determine the lncRNA profile in peripheral blood CD4+ T cells from HT patients and then to characterize the potential function of NONHSAT079547.2. A total of 37 HT patients and 50 sex- and age-matched healthy controls were enrolled for high-throughput sequencing. Another 43 HT patients and 50 sex- and age-matched controls were enrolled for validation via real-time PCR. Flow cytometry and CCK8 assays were used to measure cell apoptosis and growth levels. Western blotting was used for measuring the expression of growth- and apoptosis-associated proteins. IL-17 serum concentration and transcriptional level in CD4+ T cells of participants were detected by ELISA and real-time PCR, respectively. The mechanism of competitive endogenous RNA was determined using real-time PCR, ELISA, RNA immunoprecipitation, and dual-luciferase assays in Jurkat cells. A total of 7564 significantly differentially expressed lncRNAs were found, of which 3913 lncRNAs were upregulated and 3651 lncRNAs were downregulated in HT group when compared to control group. NONHSAT079547.2 was significantly upregulated in HT patients and was positively correlated with serum thyroid peroxidase antibody level. Further studies confirmed that NONHSAT079547.2 could promote cell growth and control IL-17 expression and secretion via the NONHSAT079547.2/miR-4716-5p/IL-17 axis.This is the first study to describe the lncRNA profile in CD4+ T cells of HT patients. The studies on the function of NONHSAT079547.2 might elucidate the underlying molecular mechanisms and represent potential biomarkers for HT.

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Karin J Bosma, Mohsin Rahim, Kritika Singh, Slavina B Goleva, Martha L Wall, Jing Xia, Kristen E Syring, James K Oeser, Greg Poffenberger, Owen P McGuinness, Anna L Means, Alvin C Powers, Wen-hong Li, Lea K Davis, Jamey D Young, and Richard M O’Brien

The G6PC1, G6PC2 and G6PC3 genes encode distinct glucose-6-phosphatase catalytic subunit (G6PC) isoforms. In mice, germline deletion of G6pc2 lowers fasting blood glucose (FBG) without affecting fasting plasma insulin (FPI) while, in isolated islets, glucose-6-phosphatase activity and glucose cycling are abolished and glucose-stimulated insulin secretion (GSIS) is enhanced at submaximal but not high glucose. These observations are all consistent with a model in which G6PC2 regulates the sensitivity of GSIS to glucose by opposing the action of glucokinase. G6PC2 is highly expressed in human and mouse islet beta cells however, various studies have shown trace G6PC2 expression in multiple tissues raising the possibility that G6PC2 also affects FBG through non-islet cell actions. Using real-time PCR we show here that expression of G6pc1 and/or G6pc3 are much greater than G6pc2 in peripheral tissues, whereas G6pc2 expression is much higher than G6pc3 in both pancreas and islets with G6pc1 expression not detected. In adult mice, beta cell-specific deletion of G6pc2 was sufficient to reduce FBG without changing FPI. In addition, electronic health record-derived phenotype analyses showed no association between G6PC2 expression and phenotypes clearly unrelated to islet function in humans. Finally, we show that germline G6pc2 deletion enhances glycolysis in mouse islets and that glucose cycling can also be detected in human islets. These observations are all consistent with a mechanism by which G6PC2 action in islets is sufficient to regulate the sensitivity of GSIS to glucose and hence influence FBG without affecting FPI.

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R Alhamdan, W Maalouf, B K Campbell, J H Hernandez-Medrano, and P Marsters

Natriuretic peptides (NPs) have been reported to have critical roles in follicular development and oocyte maturation in rodents. This study aimed to extend our current understanding of NP-mediated signalling pathways and mechanisms of action in the follicles of a monovulatory species. Ovine granulosa cells (GCs) and theca cells (TCs) were cultured under conditions designed to allow gonadotrophin-stimulated cell differentiation. Gene expression analysis was performed by qualitative (q)PCR for NPs and NPRs (between 16 and 96 h of culture) and VEGF 120 and VEGF 164 (between 16 and 144 h of culture). A qualitative analysis of the production of NP/NPR family members and NP ligand/receptor associations was carried out utilising a highly sensitive immunological approach known as ‘proximity ligation assay’ (PLA). All NPRs were observed in GCs, while NPRA was absent in TCs. In GCs, gene expression of NPRA, NPRB and NPRC was apparent but only active BNP and CNP and not ANP, were detected. Also in GCs, ANP but not CNP was able to significantly (P < 0.05) reduce oestradiol and increase (P < 0.05) progesterone. Inhibition of VEGF164 by ANP and CNP (P < 0.01) after 48 h of culture preceded up-regulation of VEGF120 by ANP (P < 0.01) after 144 h, but not CNP. Taken together, these findings appear to demonstrate that NP responsiveness in the GC compartment of sheep follicles is multi-facilitated, utilising both autocrine and paracrine stimulation pathways.

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Mark C Turner, Neil R W Martin, Darren J Player, Richard A Ferguson, Patrick Wheeler, Charlotte J Green, Elizabeth C Akam, and Mark P Lewis

Hyperinsulinaemia potentially contributes to insulin resistance in metabolic tissues, such as skeletal muscle. The purpose of these experiments was to characterise glucose uptake, insulin signalling and relevant gene expression in primary human skeletal muscle-derived cells (HMDCs), in response to prolonged insulin exposure (PIE) as a model of hyperinsulinaemia-induced insulin resistance. Differentiated HMDCs from healthy human donors were cultured with or without insulin (100 nM) for 3 days followed by an acute insulin stimulation. HMDCs exposed to PIE were characterised by impaired insulin-stimulated glucose uptake, blunted IRS-1 phosphorylation (Tyr612) and Akt (Ser473) phosphorylation in response to an acute insulin stimulation. Glucose transporter 1 (GLUT1), but not GLUT4, mRNA and protein increased following PIE. The mRNA expression of metabolic (PDK4) and inflammatory markers (TNF-α) was reduced by PIE but did not change lipid (SREBP1 and CD36) or mitochondrial (UCP3) markers. These experiments provide further characterisation of the effects of PIE as a model of hyperinsulinaemia-induced insulin resistance in HMDCs.

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Maria K Tsoumpra, Shun Sawatsubashi, Michihiro Imamura, Seiji Fukumoto, Shin’ichi Takeda, Toshio Matsumoto, and Yoshitsugu Aoki

The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (VD3), exerts its tissue-specific actions through binding to its intracellular vitamin D receptor (VDR) which functions as a heterodimer with retinoid X receptor (RXR) to recognize vitamin D response elements (VDRE) and activate target genes. Upregulation of VDR in murine skeletal muscle cells occurs concomitantly with transcriptional regulation of key myogenic factors upon VD3 administration, reinforcing the notion that VD3 exerts beneficial effects on muscle. Herein we elucidated the regulatory role of VD3/VDR axis on the expression of dystrobrevin alpha (DTNA), a member of dystrophin-associated protein complex (DAPC). In C2C12 cells, Dtna and VDR gene and protein expression were upregulated by 1–50 nM of VD3 during all stages of myogenic differentiation. In the dystrophic-derived H2K-mdx52 cells, upregulation of DTNA by VD3 occurred upon co-transfection of VDR and RXR expression vectors. Silencing of MyoD1, an E-box binding myogenic transcription factor, did not alter the VD3-mediated Dtna induction, but Vdr silencing abolished this effect. We also demonstrated that VD3 administration enhanced the muscle-specific Dtna promoter activity in presence of VDR/RXR only. Through site-directed mutagenesis and chromatin immunoprecipitation assays, we have validated a VDRE site in Dtna promoter in myogenic cells. We have thus proved that the positive regulation of Dtna by VD3 observed during in vitro murine myogenic differentiation is VDR mediated and specific. The current study reveals a novel mechanism of VDR-mediated regulation for Dtna, which may be positively explored in treatments aiming to stabilize the DAPC in musculoskeletal diseases.

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Belen Brie, Ana Ornstein, Maria Cecilia Ramirez, Isabel Lacau-Mengido, and Damasia Becu-Villalobos

Many sex differences in liver gene expression originate in the brain, depend on GH secretion and may underlie sex disparities in hepatic disease. Because epigenetic mechanisms may contribute, we studied promoter methylation and microRNA abundance in the liver, associated with expression of sexual dimorphic genes in mice with selective disruption of the dopamine D2 receptor in neurons (neuroDrd2KO), which decreases hypothalamic Ghrh, pituitary GH, and serum IGFI and in neonatally androgenized female mice which have increased pituitary GH content and serum IGFI. We evaluated mRNA levels of the female predominant genes prolactin receptor (Prlr), alcohol dehydrogenase 1 (Adh1), Cyp2a4, and hepatocyte nuclear transcription factor 6 (Hnf6) and the male predominant gene, Cyp7b1. Female predominant genes had higher mRNA levels compared to males, but lower methylation was only detected in the Prlr and Cyp2a4 female promoters. In neuroDrd2KO mice, sexual dimorphism was lost for all genes; the upregulation (feminization) of Prlr and Cyp2a4 in males correlated with decreased methylation of their promoters, and the downregulation (masculinization) of Hnf-6 mRNA in females correlated inversely with its promoter methylation. Neonatal androgenization of females evoked a loss of sexual dimorphism only for the female predominant Hnf6 and Adh1 genes, but no differences in promoter methylation were found. Finally, mmu-miR-155-5p, predicted to target Cyp7b1 expression, was lower in males in association with higher Cyp7b1 mRNA levels compared to females and was not modified in neuroDrd2KO or TP mice. Our results suggest specific regulation of gene sexually dimorphic expression in the liver by methylation or miRNAs.

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Kelly L Short, A Daniel Bird, Bennet K L Seow, Judy Ng, Annie R A McDougall, Megan J Wallace, Stuart B Hooper, and Timothy J Cole

Glucocorticoid (GC) signaling via the glucocorticoid receptor (GR) is essential for lung maturation in mammals. Previous studies using global or conditional mouse model knockouts of the GR gene have established that GR-mediated signaling in the interstitial mesenchyme of the fetal lung is critical for normal lung development. Screens for downstream GC-targets in conditional mesenchymal GR deficient mouse lung (GRmesKO) identified Versican (Vcan), an important extracellular matrix component and cell proliferation regulator, as a potential GR-regulated target. We show that, of the five major VCAN isoforms, the VCAN-V1 isoform containing the GAGβ domain is the predominant VCAN isoform in the fetal mouse lung distal mesenchyme at both E16.5 and E18.5, whereas the GAGα-specific VCAN-V2 isoform was only localized to the smooth muscle surrounding proximal airways. Both Vcan-V1 mRNA and protein levels were strongly overexpressed in the GRmesKO lung at E18.5. Finally, we investigated the GC regulation of the ECM protease ADAMTS 12 and showed that Adamts 12 mRNA levels were markedly reduced at E18.5 in GRmesKO fetal mouse lung and were strongly induced by both cortisol and betamethasone in cultures of primary rat fetal lung fibroblasts. ADAMTS12 protein immunoreactivity was also strongly increased in the distal lung at E18.5, after dexamethasone treatment in utero. In summary, glucocorticoid signaling via GR represses GAGβ domain-containing VCAN isoforms in distal lung mesenchyme in vivo by repressing Vcan gene expression and, in part, by inducing the ECM protease ADAMTS12, thereby contributing to the control of ECM remodelling and lung cell proliferation prior to birth.

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Takumi Nakamura, Kazuki Harada, Taichi Kamiya, Mai Takizawa, Jim Küppers, Kazuo Nakajima, Michael Gütschow, Tetsuya Kitaguchi, Kunihiro Ohta, Tadafumi Kato, and Takashi Tsuboi

Glucagon-like peptide-1 (GLP-1), secreted by gastrointestinal enteroendocrine L cells, induces insulin secretion and is important for glucose homeostasis. GLP-1 secretion is induced by various luminal nutrients, including amino acids. Intracellular Ca2+ and cAMP dynamics play an important role in GLP-1 secretion regulation; however, several aspects of the underlying mechanism of amino acid-induced GLP-1 secretion are not well characterized. We investigated the mechanisms underlying the L-glutamine-induced increase in Ca2+ and cAMP intracellular concentrations ([Ca2+]i and [cAMP]i, respectively) in murine enteroendocrine L cell line GLUTag cells. Application of L-glutamine to cells under low extracellular [Na+] conditions, which inhibited the function of the sodium-coupled L-glutamine transporter, did not induce an increase in [Ca2+]i. Application of G protein-coupled receptor family C group 6 member A and calcium-sensing receptor antagonist showed little effect on [Ca2+]i and [cAMP]i; however, taste receptor type 1 member 3 (TAS1R3) antagonist suppressed the increase in [cAMP]i. To elucidate the function of TAS1R3, which forms a heterodimeric umami receptor with taste receptor type 1 member 1 (TAS1R1), we generated TAS1R1 and TAS1R3 mutant GLUTag cells using the CRISPR/Cas9 system. TAS1R1 mutant GLUTag cells exhibited L-glutamine-induced increase in [cAMP]i, whereas some TAS1R3 mutant GLUTag cells did not exhibit L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. These findings suggest that TAS1R3 is important for L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. Thus, TAS1R3 may be coupled with Gs and related to cAMP regulation.