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Immune parameters show rhythmic changes with a 24-h periodicity driven by an internal circadian timing system that relies on clock genes (CGs). CGs form interlocked transcription-translation feedback loops to generate and maintain 24-h mRNA and protein oscillations. In this study we evaluate and compare the profiles and the dynamics of variation of CG expression in peripheral blood, and two lymphoid tissues of mice. Expression levels of seven recognized key CGs (mBmal1, mClock, mPer1, mPer2, mCry1, mCry2, and Rev-erbalpha) were evaluated by quantitative RT- PCR in spleen, thymus and peripheral blood of C57BL/6 male mice housed on a 12-h light (L)-dark (D) cycle and sacrificed every 4 h for 24 h (3-4 mice/time point). We found a statistically significant time-effect in spleen (S), thymus (T) and blood (B) for the original values of expression level of mBmal1 (S), mClock (T, B), mPer1 (S, B), mPer2 (S), mCry1 (S), mCry2 (B) and mRev-Erbalpha (S, T, B) and for the fractional variation calculated between single time-point expression value of mBmal1 (B), mPer2 (T), mCry2 (B) and mRev-Erbalpha (S). A significant 24-h rhythm was validated for five CGs in blood (mClock, mPer1, mPer2, mCry2, mRev-Erbalpha), for four CGs in the spleen (mBmal1, mPer1, mPer2, mRev-Erbalpha), and for three CGs in the thymus (mClock, mPer2, mRev-Erbalpha). The original values of acrophases for mBmal1, mClock, mPer1, mPer2, mCry1 and mCry2 were very similar for spleen and thymus and advanced by several hours for peripheral blood compared to the lymphoid tissues, whereas the phases of mRev-Erbalpha were coincident for all three tissues. In conclusion, central and peripheral lymphoid tissues in the mouse show different sequences of activation of clock gene expression compared to peripheral blood. These differences may underlie the compartmental pattern of web functioning in the immune system.

The collecting duct is the site of final Na reabsorption according to Na balance requirements. Using isolated rat cortical collecting ducts (CCD) and mpkCCD(cl4) cells, a mouse cortical collecting duct cell line, we have studied the physiological control of Na,K-ATPase, the key enzyme that energizes Na reabsorption. Aldosterone, a major regulator of Na transport by the collecting duct, stimulates Na,K-ATPase activity through both recruitment of intracellular pumps and increased total amounts of Na pump subunits. This effect is observed after a lag time of 1 hour and is independent of Na entry through ENaC, but requires de novo transcription and translation. Vasopressin and cAMP, its second messenger, stimulate Na,K-ATPase activity within minutes through translocation of Na pumps from a brefeldin A-sensitive intracellular pool to the plasma membrane. Dysregulation of collecting duct Na,K-ATPase activity is at least in part responsible of the Na retention observed in nephritic syndrome. In this setting, Na,K-ATPase activity and subunit synthesis are specifically increased in CCD. In conclusion, aldosterone, vasopressin, and intracellular Na control the cell surface expression of Na,K-ATPase and translocation from intracellular stores is a major mechanism of regulation of Na,K-ATPase activity in collecting duct principal cells.

Background Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far. Methods We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines. Results CRY1 (p = 0.01) and CRY2 (p

Hepatocellular carcinoma (HCC) is a deadly malignancy characterized at the epigenetic level by global DNA hypomethylation and focal hypermethylation on the promoter of tumor suppressor genes. In most cases it develops on a background of liver steatohepatitis, fibrosis, and cirrhosis. Guadecitabine (SGI-110) is a second-generation hypomethylating agent, which inhibits DNA methyltransferases. Guadecitabine is formulated as a dinucleotide of decitabine and deoxyguanosine that is resistant to cytidine deaminase (CDA) degradation and results in prolonged in vivo exposure to decitabine following small volume subcutaneous administration of guadecitabine. Here we found that guadecitabine is an effective demethylating agent and is able to prevent HCC progression in pre-clinical models. In a xenograft HCC HepG2 model, guadecitabine impeded tumor growth and inhibited angiogenesis, while it could not prevent liver fibrosis and inflammation in a mouse model of steatohepatitis. Demethylating efficacy of guadecitabine on LINE-1 elements was found to be the highest 8 d post-infusion in blood samples of mice. Analysis of a panel of human HCC vs. normal tissue revealed a signature of hypermethylated tumor suppressor genes (CDKN1A, CDKN2A, DLEC1, E2F1, GSTP1, OPCML, E2F1, RASSF1, RUNX3, and SOCS1) as detected by methylation-specific PCR. A pronounced demethylating effect of guadecitabine was obtained also in the promoters of a subset of tumor suppressors genes (CDKN2A, DLEC1, and RUNX3) in HepG2 and Huh-7 HCC cells. Finally, we analyzed the role of macroH2A1, a variant of histone H2A, an oncogene upregulated in human cirrhosis/HCC that synergizes with DNA methylation in suppressing tumor suppressor genes, and it prevents the inhibition of cell growth triggered by decitabine in HCC cells. Guadecitabine, in contrast to decitabine, blocked growth in HCC cells overexpressing macroH2A1 histones and with high CDA levels, despite being unable to fully demethylate CDKN2A, RUNX3, and DLEC1 promoters altered by macroH2A1. Collectively, our findings in human and mice models reveal novel epigenetic anti-HCC effects of guadecitabine, which might be effective specifically in advanced states of the disease.

Background Obesity has tremendous impact on the health systems. Its epigenetic bases are unclear. MacroH2A1 is a variant of histone H2A, present in two alternatively exon-spliced isoforms macroH2A1.1 and macroH2A1.2, regulating cell plasticity and proliferation, during pluripotency and tumorigenesis. Their role in adipose tissue plasticity is unknown. Results Here, we show evidence that macroH2A1.1 protein levels in the visceral adipose tissue of obese humans positively correlate with BMI, while macroH2A1.2 is nearly absent. We thus introduced a constitutive GFP-tagged transgene for macroH2A1.2 in mice, and we characterized their metabolic health upon being fed a standard chow diet or a high fat diet. Despite unchanged food intake, these mice exhibit lower adipose mass and improved glucose metabolism both under a chow and an obesogenic diet. In the latter regimen, transgenic mice display smaller pancreatic islets and significantly less inflammation. MacroH2A1.2 overexpression in the mouse adipose tissue induced dramatic changes in the transcript levels of key adipogenic genes; genomic analyses comparing pre-adipocytes to mature adipocytes uncovered only minor changes in macroH2A1.2 genomic distribution upon adipogenic differentiation and suggested differential cooperation with transcription factors. MacroH2A1.2 overexpression markedly inhibited adipogenesis, while overexpression of macroH2A1.1 had opposite effects. Conclusions MacroH2A1.2 is an unprecedented chromatin component powerfully promoting metabolic health by modulating anti-adipogenic transcriptional networks in the differentiating adipose tissue. Strategies aiming at enhancing macroH2A1.2 expression might counteract excessive adiposity in humans. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0098-9) contains supplementary material, which is available to authorized users.

Background: Hunter syndrome (HS) is a lysosomal storage disease caused by iduronate-2-sulfatase (IDS) deficiency and loss of ability to break down and recycle the glycosaminoglycans, heparan and dermatan sulfate, leading to impairment of cellular processes and cell death. Cell activities and functioning of intracellular organelles are controlled by the clock genes (CGs), driving the rhythmic expression of clock controlled genes (CCGs). We aimed to evaluate the expression of CGs and downstream CCGs in HS, before and after enzyme replacement treatment with IDS. Methods: The expression levels of CGs and CCGs were evaluated by a whole transcriptome analysis through Next Generation Sequencing in normal primary human fibroblasts and fibroblasts of patients affected by HS before and 24 h/144 h after IDS treatment. The time related expression of CGs after synchronization by serum shock was also evaluated by qRT-PCR before and after 24 hours of IDS treatment. Results: In HS fibroblasts we found altered expression of several CGs and CCGs, with dynamic changes 24 h and 144 h after IDS treatment. A semantic hypergraph-based analysis highlighted five gene clusters significantly associated to important biological processes or pathways, and five genes, AHR, HIF1A, CRY1, ITGA5 and EIF2B3, proven to be central players in these pathways. After synchronization by serum shock and 24 h treatment with IDS the expression of ARNTL2 at 10 h (p = 0.036), PER1 at 4 h (p = 0.019), PER2 at 10 h (p = 0.041) and 16 h (p = 0.043) changed in HS fibroblasts. Conclusion: CG and CCG expression is altered in HS fibroblasts and IDS treatment determines dynamic modifications, suggesting a direct involvement of the CG machinery in the physiopathology of cellular derangements that characterize HS.

Sorafenib is a small molecular inhibitor of intracellular tyrosine and serine/threonine protein kinases (VEGFR, PDGFR, CRAF and BRAF), and is thought also to induce autophagy, a chief mechanism influencing tumor growth. Sorafenib shows efficacy in the management of non-resectable hepatocellular carcinoma (HCC), which is refractory to other chemotherapeutic drugs. HCC represents a major end point of chronic liver diseases and the third leading cause of cancer-related death. In HCC patients Sorafenib increases overall survival compared to placebo. The most common chronic liver disease affecting up to 30% of the population in Western countries is non-alcoholic fatty liver disease (NAFLD), an intra-hepatic amassing of triglycerides deemed as the hepatic manifestation of insulin resistance and obesity. NAFLD encompasses a range of disorders with grades of liver damage varying from steatosis to non-alcoholic steatohepatitis (NASH), hallmarked by hepatocellular injury/inflammation in the presence or not of fibrosis. NAFLD patients progress to NASH in 10% of cases, which may progress to cirrhosis and HCC. Recent exciting studies uncovered a potential therapeutic role for Sorafenib that goes beyond HCC, and extends to cirrhotic portal hypertensive syndrome during cirrhosis, and to selective anti-fibrotic effects mediated through direct inhibition of activated hepatic stellate cells (HSC), the cellular mediators of intra-hepatic matrix deposition. The aim of this review is to concisely summarize our current knowledge of the biology, epidemiology and clinical aspects of HCC, as well as the previously under-appreciated therapeutic efficacy of Sorafenib beyond HCC. The review therefore utilizes data along the spectrum of liver diseases, including from experimental via pre-clinical to clinical.