The Transcription Factor Rfx3 Regulates β-Cell Differentiation, Function, and Glucokinase Expression

OBJECTIVE: Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing beta-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency. RESEARCH DESIGN AND METHODS: Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of beta-cells from endocrine progenitors, and the expression of mature beta-cell markers during development in Rfx3(-/-) and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 beta-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes. RESULTS: Reduced development of insulin-positive cells in Rfx3(-/-) mice was not due to deficiencies in endocrine progenitors or beta-lineage specification, but reflected the accumulation of insulin-positive beta-cell precursors and defective beta-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated beta-cells developed in pancreas-specific Rfx3-deficient embryos. Defective beta-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference-mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene. CONCLUSIONS: Our results show that Rfx3 is required for the differentiation and function of mature beta-cells and regulates the beta-cell promoter of the glucokinase gene

ESRRA-C11orf20 Is a Recurrent Gene Fusion in Serous Ovarian Carcinoma

Many ovarian cancers have a chromosomal rearrangement that fuses two neighboring genes, ESRRA and c11orf20. Similar rearrangements may be common, important features of cancer genomes that have largely escaped detection

Clusters of Conserved Beta Cell Marker Genes for Assessment of Beta Cell Phenotype

The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Functional Characterization of MODY2 Mutations Highlights the Importance of the Fine-Tuning of Glucokinase and Its Role in Glucose Sensing

Glucokinase (GK) acts as a glucose sensor in the pancreatic beta-cell and regulates insulin secretion. Heterozygous mutations in the human GK-encoding GCK gene that reduce the activity index increase the glucose-stimulated insulin secretion threshold and cause familial, mild fasting hyperglycaemia, also known as Maturity Onset Diabetes of the Young type 2 (MODY2). Here we describe the biochemical characterization of five missense GK mutations: p.Ile130Thr, p.Asp205His, p.Gly223Ser, p.His416Arg and p.Ala449Thr. The enzymatic analysis of the corresponding bacterially expressed GST-GK mutant proteins show that all of them impair the kinetic characteristics of the enzyme. In keeping with their position within the protein, mutations p.Ile130Thr, p.Asp205His, p.Gly223Ser, and p.His416Arg strongly decrease the activity index of GK, affecting to one or more kinetic parameters. In contrast, the p.Ala449Thr mutation, which is located in the allosteric activator site, does not affect significantly the activity index of GK, but dramatically modifies the main kinetic parameters responsible for the function of this enzyme as a glucose sensor. The reduced Kcat of the mutant (3.21±0.28 s−1 vs 47.86±2.78 s−1) is balanced by an increased glucose affinity (S0.5 = 1.33±0.08 mM vs 7.86±0.09 mM) and loss of cooperativity for this substrate. We further studied the mechanism by which this mutation impaired GK kinetics by measuring the differential effects of several competitive inhibitors and one allosteric activator on the mutant protein. Our results suggest that this mutation alters the equilibrium between the conformational states of glucokinase and highlights the importance of the fine-tuning of GK and its role in glucose sensing

Expression of nuclear retinoid receptors in normal, premalignant and malignant gastric tissues determined by in situ hybridization

[[abstract]]Retinoids exhibit multiple functions through interaction with nuclear retinoid receptors and have growth-suppressive activity on gastric cancer cells. To better understand the roles of nuclear retinoid receptors during gastric carcinogenesis, we have used in situ hybridization to investigate expression of retinoic acid receptors (RARs) and retinoid x receptors (RXRs) in premalignant and malignant formalin-fixed paraffin-embedded gastric tissues. Histological sections of eight normal, 17 distal normal and nine gastric cancer tissues were hybridized with non-radioactive RNA probes for subtypes of RAR and RXR. Expression of RARα, RARβ, RARγ, RXRα and RXRβ was found in most cell types in gastric mucosa tissues from normal individuals as well as in distal normal tissues from cancer patients. Expression of RARα and RARβ were found in three and seven cancer tissues, respectively, and levels of RXRα mRNA were significantly decreased in poorly differentiated cancer tissues. Among the five investigated nuclear retinoid receptors, only expression of RARα mRNA was significantly decreased in intestinal metaplasia, dysplasia and cancer tissues when compared to adjacent normal tissues. In conclusion, normal gastric mucosa expressed both RARs and RXRs, which supports the physiological role of retinoic acid on normal gastric mucosa. The decrease in RARα expression in premalignant and malignant gastric tissues suggests a significant role of RARα during gastric carcinogenesis.[[notice]]補正完畢[[incitationindex]]SC

Impact of vitamin D metabolism on clinical epigenetics

The bioactive vitamin D (VD) metabolite, 1,25-dihydroxyvitamin D3 regulates essential pathways of cellular metabolism and differentiation via its nuclear receptor (VDR). Molecular mechanisms which are known to play key roles in aging and cancer are mediated by complex processes involving epigenetic mechanisms contributing to efficiency of VD-activating CYP27A1 and CYP27B1 or inactivating CYP24 enzymes as well as VDR which binds to specific genomic sequences (VD response elements or VDREs). Activity of VDR can be modulated epigenetically by histone acetylation. It co-operates with other nuclear receptors which are influenced by histone acetyl transferases (HATs) as well as several types of histone deacetylases (HDACs). HDAC inhibitors (HDACi) and/or demethylating drugs may contribute to normalization of VD metabolism. Studies link VD signaling through the VDR directly to distinct molecular mechanisms of both HAT activity and the sirtuin class of HDACs (SIRT1) as well as the forkhead transcription factors thus contributing to elucidate complex epigenetic mechanisms for cancer preventive actions of VD

Disruption of Retinoic Acid Receptor Alpha Reveals the Growth Promoter Face of Retinoic Acid

Retinoic acid (RA), the bioactive derivative of Vitamin A, by epigenetically controlling transcription through the RA-receptors (RARs), exerts a potent antiproliferative effect on human cells. However, a number of studies show that RA can also promote cell survival and growth. In the course of one of our studies we observed that disruption of RA-receptor alpha, RARalpha, abrogates the RA-mediated growth-inhibitory effects and unmasks the growth-promoting face of RA (Ren et al., Mol. Cell. Biol., 2005, 25:10591). The objective of this study was to investigate whether RA can differentially govern cell growth, in the presence and absence of RARalpha, through differential regulation of the "rheostat" comprising ceramide (CER), the sphingolipid with growth-inhibitory activity, and sphingosine-1-phosphate (S1P), the sphingolipid with prosurvival activity.We found that functional inhibition of endogenous RARalpha in breast cancer cells by using either RARalpha specific antagonists or a dominant negative RARalpha mutant hampers on one hand the RA-induced upregulation of neutral sphingomyelinase (nSMase)-mediated CER synthesis, and on the other hand the RA-induced downregulation of sphingosine kinase 1, SK1, pivotal for S1P synthesis. In association with RA inability to regulate the sphingolipid rheostat, cells not only survive, but also grow more in response to RA both in vitro and in vivo. By combining genetic, pharmacological and biochemical approaches, we mechanistically demonstrated that RA-induced growth is, at least in part, due to non-RAR-mediated activation of the SK1-S1P signaling.In the presence of functional RARalpha, RA inhibits cell growth by concertedly, and inversely, modulating the CER and S1P synthetic pathways. In the absence of a functional RARalpha, RA-in a non-RAR-mediated fashion-promotes cell growth by activating the prosurvival S1P signaling. These two distinct, yet integrated processes apparently concur to the growth-promoter effects of RA

The DAC system and associations with acute leukemias and myelodysplastic syndromes

Imbalances of histone acetyltransferase (HAT) and deacetylase activity (DAC) that result in deregulated gene expression are commonly observed in leukemias. These alterations provide the basis for novel therapeutic approaches that target the epigenetic mechanisms implicated in leukemogenesis. As the acetylation status of histones has been linked to transcriptional regulation of genes involved particularly in differentiation and apoptosis, DAC inhibitors (DACi) have attracted considerable attention for treatment of hematologic malignancies. DACi encompass a structurally diverse family of compounds that are being explored as single agents as well as in combination with chemotherapeutic drugs, small molecule inhibitors of signaling pathways and hypomethylating agents. While DACi have shown clear evidence of activity in acute myeloid leukemia, myelodysplastic syndromes and lymphoid malignancies, their precise role in treatment of these different entities remain to be elucidated. Successful development of these compounds as elements of novel targeted treatment strategies for leukemia will require that clinical studies be performed in conjunction with translational research including efforts to identify predictive biomarkers