iPSC-Derived Pancreatic Progenitors Lacking FOXA2 Reveal Alterations in miRNA Expression Targeting Key Pancreatic Genes.

Recently, we reported that forkhead box A2 (FOXA2) is required for the development of human pancreatic α- and ß-cells. However, whether miRNAs play a role in regulating pancreatic genes during pancreatic development in the absence of FOXA2 expression is largely unknown. Here, we aimed to capture the dysregulated miRNAs and to identify their pancreatic-specific gene targets in pancreatic progenitors (PPs) derived from wild-type induced pluripotent stem cells (WT-iPSCs) and from iPSCs lacking FOXA2 (FOXA2-/-iPSCs). To identify differentially expressed miRNAs (DEmiRs), and genes (DEGs), two different FOXA2-/-iPSC lines were differentiated into PPs. FOXA2-/- PPs showed a significant reduction in the expression of the main PP transcription factors (TFs) in comparison to WT-PPs. RNA sequencing analysis demonstrated significant reduction in the mRNA expression of genes involved in the development and function of exocrine and endocrine pancreas. Furthermore, miRNA profiling identified 107 downregulated and 111 upregulated DEmiRs in FOXA2-/- PPs compared to WT-PPs. Target prediction analysis between DEmiRs and DEGs identified 92 upregulated miRNAs, predicted to target 1498 downregulated genes in FOXA2-/- PPs. Several important pancreatic TFs essential for pancreatic development were targeted by multiple DEmiRs. Selected DEmiRs and DEGs were further validated using RT-qPCR. Our findings revealed that FOXA2 expression is crucial for pancreatic development through regulating the expression of pancreatic endocrine and exocrine genes targeted by a set of miRNAs at the pancreatic progenitor stage. These data provide novel insights of the effect of FOXA2 deficiency on miRNA-mRNA regulatory networks controlling pancreatic development and differentiation.

FoxA-dependent demethylation of DNA initiates epigenetic memory of cellular identity.

Tissue-specific DNA methylation patterns are created by transcription factors that recruit methylation and demethylation enzymes to cis-regulatory elements. To date, it is not known whether transcription factors are needed to continuously maintain methylation profiles in development and mature tissues or whether they only establish these marks during organ development. We queried the role of the pioneer factor FoxA in generating hypomethylated DNA at liver enhancers. We discovered a set of FoxA-binding sites that undergo regional, FoxA-dependent demethylation during organ development. Conditional ablation of FoxA genes in the adult liver demonstrated that continued FoxA presence was not required to maintain the hypomethylated state, even when massive cell proliferation was induced. This study provides strong evidence for the stable, epigenetic nature of tissue-specific DNA methylation patterns directed by lineage-determining transcription factors during organ development.

Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis.

Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine significantly reduced KrasG12D/+; Pdx-1 Cre (KC) (P < 0.01) and KrasG12D/+; p53R172H/+; Pdx-1 Cre (KPC) (P < 0.05) derived mouse tumoroids and KPC-derived murine syngeneic cell line growth compared to gemcitabine/afatinib alone treatment. The drug combination also reduced PC xenograft tumor burden (P < 0.05) and the incidence of metastasis by affecting key stemness markers, as confirmed by co-localization studies. Moreover, the drug combination significantly decreases the growth of various PC patient-derived organoids (P < 0.001). We found that SOX9 is significantly overexpressed in high-grade PC tumors (P < 0.05) and in chemotherapy-treated patients compared to chemo-naïve patients (P < 0.05). These results were further validated using publicly available datasets. Moreover, afatinib alone or in combination with gemcitabine decreased stemness and tumorspheres by reducing phosphorylation of EGFR family proteins, ERK, FAK, and CSC markers. Mechanistically, afatinib treatment decreased CSC markers by downregulating SOX9 via FOXA2. Indeed, EGFR and FOXA2 depletion reduced SOX9 expression in PCSCs. Taken together, pan-EGFR inhibition by afatinib impedes PCSCs growth and metastasis via the EGFR/ERK/FOXA2/SOX9 axis. This novel mechanism of pan-EGFR inhibitor and its ability to eradicate CSC may serve as a tailor-made approach to enhance chemotherapeutic benefits in other cancer types.

Angiopoietin-1 accelerates Alzheimer's disease via FOXA2/PEN2/APP pathway in APP/PS1 mice.

Angiopoietin-1 (Ang-1), a regulatory angiogenesis protein and it has been found to be involved in the occurrence and progression of Alzheimer's disease. However, it was still to be addressed the distinctly role and the molecular mechanisms of Ang-1 affects Alzheimer's disease. Our data suggest that Ang-1 aggravated the accumulation of Aß42 and cognitive decline in APP/PS1 mice. The upregulation of APPß is essential for Aß42 production in N2a cells overexpressing the mutational human APP gene (N2a/APP695 cells), while downregulation of PEN2 could reduce APP expression. Silencing of FOXA2 lead to inhibition of APP expression, as well as decrease of Aß42 contents. In conclusion, Ang-1 has an accelerative effect on Alzheimer's disease by increasing the secretion of Aß42 via FOXA2/PEN2/APP pathway.

Tissue-specific transcription reprogramming promotes liver metastasis of colorectal cancer.

Metastasis, the development of secondary malignant growths at a distance from a primary tumor, is the cause of death for 90% of cancer patients, but little is known about how metastatic cancer cells adapt to and colonize new tissue environments. Here, using clinical samples, patient-derived xenograft (PDX) samples, PDX cells, and primary/metastatic cell lines, we discovered that liver metastatic colorectal cancer (CRC) cells lose their colon-specific gene transcription program yet gain a liver-specific gene transcription program. We showed that this transcription reprogramming is driven by a reshaped epigenetic landscape of both typical enhancers and super-enhancers. Further, we identified that the liver-specific transcription factors FOXA2 and HNF1A can bind to the gained enhancers and activate the liver-specific gene transcription, thereby driving CRC liver metastasis. Importantly, similar transcription reprogramming can be observed in multiple cancer types. Our data suggest that reprogrammed tissue-specific transcription promotes metastasis and should be targeted therapeutically.

FOXA2 Is Required for Enhancer Priming during Pancreatic Differentiation.

Transcriptional regulatory mechanisms of lineage priming in embryonic development are largely uncharacterized because of the difficulty of isolating transient progenitor populations. Directed differentiation of human pluripotent stem cells (hPSCs) combined with gene editing provides a powerful system to define precise temporal gene requirements for progressive chromatin changes during cell fate transitions. Here, we map the dynamic chromatin landscape associated with sequential stages of pancreatic differentiation from hPSCs. Our analysis of chromatin accessibility dynamics led us to uncover a requirement for FOXA2, known as a pioneer factor, in human pancreas specification not previously shown from mouse knockout studies. FOXA2 knockout hPSCs formed reduced numbers of pancreatic progenitors accompanied by impaired recruitment of GATA6 to pancreatic enhancers. Furthermore, FOXA2 is required for proper chromatin remodeling and H3K4me1 deposition during enhancer priming. This work highlights the power of combining hPSC differentiation, genome editing, and computational genomics for discovering transcriptional mechanisms during development.

The nuclear bile acid receptor FXR is a PKA- and FOXA2-sensitive activator of fasting hepatic gluconeogenesis.

BACKGROUND & AIMS: Embedded into a complex signaling network that coordinates glucose uptake, usage and production, the nuclear bile acid receptor FXR is expressed in several glucose-processing organs including the liver. Hepatic gluconeogenesis is controlled through allosteric regulation of gluconeogenic enzymes and by glucagon/cAMP-dependent transcriptional regulatory pathways. We aimed to elucidate the role of FXR in the regulation of fasting hepatic gluconeogenesis. METHODS: The role of FXR in hepatic gluconeogenesis was assessed in vivo and in mouse primary hepatocytes. Gene expression patterns in response to glucagon and FXR agonists were characterized by quantitative reverse transcription PCR and microarray analysis. FXR phosphorylation by protein kinase A was determined by mass spectrometry. The interaction of FOXA2 with FXR was identified by cistromic approaches and in vitro protein-protein interaction assays. The functional impact of the crosstalk between FXR, the PKA and FOXA2 signaling pathways was assessed by site-directed mutagenesis, transactivation assays and restoration of FXR expression in FXR-deficient hepatocytes in which gene expression and glucose production were assessed. RESULTS: FXR positively regulates hepatic glucose production through two regulatory arms, the first one involving protein kinase A-mediated phosphorylation of FXR, which allowed for the synergistic activation of gluconeogenic genes by glucagon, agonist-activated FXR and CREB. The second arm involves the inhibition of FXR's ability to induce the anti-gluconeogenic nuclear receptor SHP by the glucagon-activated FOXA2 transcription factor, which physically interacts with FXR. Additionally, knockdown of Foxa2 did not alter glucagon-induced and FXR agonist enhanced expression of gluconeogenic genes, suggesting that the PKA and FOXA2 pathways regulate distinct subsets of FXR responsive genes. CONCLUSIONS: Thus, hepatic glucose production is regulated during physiological fasting by FXR, which integrates the glucagon/cAMP signal and the FOXA2 signal, by being post-translationally modified, and by engaging in protein-protein interactions, respectively. LAY SUMMARY: Activation of the nuclear bile acid receptor FXR regulates gene expression networks, controlling lipid, cholesterol and glucose metabolism, which are mostly effective after eating. Whether FXR exerts critical functions during fasting is unknown. The results of this study show that FXR transcriptional activity is regulated by the glucagon/protein kinase A and the FOXA2 signaling pathways, which act on FXR through phosphorylation and protein-protein interactions, respectively, to increase hepatic glucose synthesis.

Pioneer Factors FOXA1 and FOXA2 Assist Selective Glucocorticoid Receptor Signaling in Human Endometrial Cells.

Successful pregnancy relies on dynamic control of cell signaling to achieve uterine receptivity and the necessary biological changes required for endometrial decidualization, embryo implantation, and fetal development. Glucocorticoids are master regulators of intracellular signaling and can directly regulate embryo implantation and endometrial remodeling during murine pregnancy. In immortalized human uterine cells, we have shown that glucocorticoids and estradiol (E2) coregulate thousands of genes. Recently, glucocorticoids and E2 were shown to coregulate the expression of Left-right determination factor 1 (LEFTY1), previously implicated in the regulation of decidualization. To elucidate the molecular mechanism by which glucocorticoids and E2 regulate the expression of LEFTY1, immortalized and primary human endometrial cells were evaluated for gene expression and receptor recruitment to regulatory regions of the LEFTY1 gene. Glucocorticoid administration induced expression of LEFTY1 messenger RNA and protein and recruitment of the glucocorticoid receptor (GR) and activated polymerase 2 to the promoter of LEFTY1. Glucocorticoid-mediated recruitment of GR was dependent on pioneer factors FOXA1 and FOXA2. E2 was found to antagonize glucocorticoid-mediated induction of LEFTY1 by reducing recruitment of GR, FOXA1, FOXA2, and activated polymerase 2 to the LEFTY1 promoter. Gene expression analysis identified several genes whose glucocorticoid-dependent induction required FOXA1 and FOXA2 in endometrial cells. These results suggest a molecular mechanism by which E2 antagonizes GR-dependent induction of specific genes by preventing the recruitment of the pioneer factors FOXA1 and FOXA2 in a physiologically relevant model.

Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3ß in Human Liver Cells.

CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3ß (HNF3ß) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3ß in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3ß binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3ß expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3ß located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3ß-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3ß to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3ß represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population.

The global gene expression profile of the secondary transition during pancreatic development.

Pancreas organogenesis is a highly dynamic process where neighboring tissue interactions lead to dynamic changes in gene regulatory networks that orchestrate endocrine, exocrine, and ductal lineage formation. To understand the spatio-temporal regulatory logic we have used the Forkhead transcription factor Foxa2-Venus fusion (FVF) knock-in reporter mouse to separate the FVF(+) pancreatic epithelium from the FVF(−) surrounding tissue (mesenchyme, neurons, blood, and blood vessels) to perform a genome-wide mRNA expression profiling at embryonic days (E) 12.5-15.5. Annotating genes and molecular processes suggest that FVF marks endoderm-derived multipotent epithelial progenitors at several lineage restriction steps, when the bulk of endocrine, exocrine and ductal cells are formed during the secondary transition. In the pancreatic epithelial compartment, we identified most known endocrine and exocrine lineage determining factors and diabetes-associated genes, but also unknown genes with spatio-temporal regulated pancreatic expression. In the non-endoderm-derived compartment, we identified many well-described regulatory genes that are not yet functionally annotated in pancreas development, emphasizing that neighboring tissue interactions are still ill defined. Pancreatic expression of over 635 genes was analyzed with them RNA in situ hybridization Genepaint public database. This validated the quality of the profiling data set and identified hundreds of genes with spatially restricted expression patterns in the pancreas. Some of these genes are also targeted by pancreatic transcription factors and show active chromatin marks in human islets of Langerhans. Thus, with the highest spatio-temporal resolution of a global gene expression profile during the secondary transition, our study enables to shed light on neighboring tissue interactions, developmental timing and diabetes gene regulation.

Transcription factors FOXA1 and FOXA2 maintain dopaminergic neuronal properties and control feeding behavior in adult mice.

Midbrain dopaminergic (mDA) neurons are implicated in cognitive functions, neuropsychiatric disorders, and pathological conditions; hence understanding genes regulating their homeostasis has medical relevance. Transcription factors FOXA1 and FOXA2 (FOXA1/2) are key determinants of mDA neuronal identity during development, but their roles in adult mDA neurons are unknown. We used a conditional knockout strategy to specifically ablate FOXA1/2 in mDA neurons of adult mice. We show that deletion of Foxa1/2 results in down-regulation of tyrosine hydroxylase, the rate-limiting enzyme of dopamine (DA) biosynthesis, specifically in dopaminergic neurons of the substantia nigra pars compacta (SNc). In addition, DA synthesis and striatal DA transmission were reduced after Foxa1/2 deletion. Furthermore, the burst-firing activity characteristic of SNc mDA neurons was drastically reduced in the absence of FOXA1/2. These molecular and functional alterations lead to a severe feeding deficit in adult Foxa1/2 mutant mice, independently of motor control, which could be rescued by L-DOPA treatment. FOXA1/2 therefore control the maintenance of molecular and physiological properties of SNc mDA neurons and impact on feeding behavior in adult mice.

Transcription factor FOXA2-centered transcriptional regulation network in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-mediated death. Although various therapeutic approaches are used for lung cancer treatment, these mainly target the tumor suppressor p53 transcription factor, which is involved in apoptosis and cell cycle arrest. However, p53-targeted therapies have limited application in lung cancer, since p53 is found to be mutated in more than half of lung cancers. In this study, we propose tumor suppressor FOXA2 as an alternative target protein for therapies against lung cancer and reveal a possible FOXA2-centered transcriptional regulation network by identifying new target genes and binding partners of FOXA2 by using various screening techniques. The genes encoding Glu/Asp-rich carboxy-terminal domain 2 (CITED2), nuclear receptor subfamily 0, group B, member 2 (NR0B2), cell adhesion molecule 1 (CADM1) and BCL2-associated X protein (BAX) were identified as putative target genes of FOXA2. Additionally, the proteins including highly similar to heat shock protein HSP 90-beta (HSP90A), heat shock 70 kDa protein 1A variant (HSPA1A), histone deacetylase 1 (HDAC1) and HDAC3 were identified as novel interacting partners of FOXA2. Moreover, we showed that FOXA2-dependent promoter activation of BAX and p21 genes is significantly reduced via physical interactions between the identified binding partners and FOXA2. These results provide opportunities to understand the FOXA2-centered transcriptional regulation network and novel therapeutic targets to modulate this network in p53-deficient lung cancer.

The transcription factor FOXA2 suppresses gastric tumorigenesis in vitro and in vivo.

BACKGROUND AND AIMS: The transcription factor forkhead box A2 (FOXA2) plays a central role in the development of endoderm-derived organs. It has been reported that FOXA2 acts as a suppressor in many kinds of tumor. However, little is known about the role of FOXA2 in gastric cancer. METHODS: The expression of FOXA2 in gastric cancer tissue samples from 89 patients was assessed by immunohistochemistry, and the clinicopathological characteristics of the samples were analyzed. The human gastric cancer cell line, BGC-823, was used to investigate the effects of FOXA2 in gastric cancer in vitro and in vivo and the potential mechanism involved was explored. RESULTS: FOXA2 expression in human gastric cancer cell lines and human gastric cancer tissues was lower compared with the normal gastric epithelium cell line GES1 and normal adult gastric tissues, respectively. Patients with high FOXA2 expression level had longer 5-year overall survival than those with low FOXA2 expression level. FOXA2 markedly inhibited growth of BGC-823 cells accompanied with the cell cycle arrest and apoptosis. Infection of BGC-823 cells by FOXA2 lentivirus resulted in reduced cell tumorigenesis in vitro and in vivo. Moreover, expression of Mucin 5AC was up-regulated along with increased expression of exogenous FOXA2 in BGC-823 cells; in contrast, dedifferentiation markers, BMI, CD54 and CD24, were down-regulated. CONCLUSIONS: These results suggest that FOXA2 induces the differentiation of gastric cancer and highlight FOXA2 as a novel therapeutic target and prognostic marker for human gastric cancer.

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett's metaplasia.

Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett's esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett's metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett's pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett's metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett's esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett's metaplasia.

Foxa1 and Foxa2 regulate α-cell differentiation, glucagon biosynthesis, and secretion.

The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse ß-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes.

Developmental lung expression and transcriptional regulation of claudin-6 by TTF-1, Gata-6, and FoxA2.

BACKGROUND: Claudins are transmembrane proteins expressed in tight junctions that prevent paracellular transport of extracellular fluid and a variety of other substances. However, the expression profile of Claudin-6 (Cldn6) in the developing lung has not been characterized. METHODS AND RESULTS: Cldn6 expression was determined during important periods of lung organogenesis by microarray analysis, qPCR and immunofluorescence. Expression patterns were confirmed to peak at E12.5 and diminish as lung development progressed. Immunofluorescence revealed that Cldn6 was detected in cells that also express TTF-1 and FoxA2, two critical transcriptional regulators of pulmonary branching morphogenesis. Cldn6 was also observed in cells that express Sox2 and Sox9, factors that influence cell differentiation in the proximal and distal lung, respectively. In order to assess transcriptional control of Cldn6, 0.5, 1.0, and 2.0-kb of the proximal murine Cldn6 promoter was ligated into a luciferase reporter and co-transfected with expression vectors for TTF-1 or two of its important transcriptional co-regulators, FoxA2 and Gata-6. In almost every instance, TTF-1, FoxA2, and Gata-6 activated gene transcription in cell lines characteristic of proximal airway epithelium (Beas2B) and distal alveolar epithelium (A-549). CONCLUSIONS: These data revealed for the first time that Cldn6 might be an important tight junctional component expressed by pulmonary epithelium during lung organogenesis. Furthermore, Cldn6-mediated aspects of cell differentiation may describe mechanisms of lung perturbation coincident with impaired cell junctions and abnormal membrane permeability.

MicroRNA-29 fine-tunes the expression of key FOXA2-activated lipid metabolism genes and is dysregulated in animal models of insulin resistance and diabetes.

MicroRNAs (miRNAs) have emerged as biomarkers of metabolic status, etiological factors in complex disease, and promising drug targets. Recent reports suggest that miRNAs are critical regulators of pathways underlying the pathophysiology of type 2 diabetes. In this study, we demonstrate by deep sequencing and real-time quantitative PCR that hepatic levels of Foxa2 mRNA and miR-29 are elevated in a mouse model of diet-induced insulin resistance. We also show that Foxa2 and miR-29 are significantly upregulated in the livers of Zucker diabetic fatty (fa/fa) rats and that the levels of both returned to normal upon treatment with the insulin-sensitizing agent pioglitazone. We present evidence that miR-29 expression in human hepatoma cells is controlled in part by FOXA2, which is known to play a critical role in hepatic energy homeostasis. Moreover, we demonstrate that miR-29 fine-tunes FOXA2-mediated activation of key lipid metabolism genes, including PPARGC1A, HMGCS2, and ABHD5. These results suggest that miR-29 is an important regulatory factor in normal metabolism and may represent a novel therapeutic target in type 2 diabetes and related metabolic syndromes.

Thioredoxin-interacting protein promotes islet amyloid polypeptide expression through miR-124a and FoxA2.

Thioredoxin-interacting protein (TXNIP) is up-regulated by glucose and diabetes and plays a critical role in glucotoxicity, inflammation, and beta-cell apoptosis, whereas we have found that TXNIP deficiency protects against diabetes. Interestingly, human islet amyloid polypeptide (IAPP) is also induced by glucose, aggregates into insoluble amyloid fibrils found in islets of most individuals with type 2 diabetes and promotes inflammation and beta-cell cytotoxicity. However, so far no connection between TXNIP and IAPP signaling had been reported. Using TXNIP gain and loss of function experiments, INS-1 beta-cells and beta-cell-specific Txnip knock-out mice, we now found that TXNIP regulates IAPP expression. Promoter analyses and chromatin-immunoprecipitation assays further demonstrated that TXNIP increases IAPP expression at the transcriptional level, and we discovered that TXNIP-induced FoxA2 (forkhead box A2) transcription factor expression was conferring this effect by promoting FoxA2 enrichment at the proximal FoxA2 site in the IAPP promoter. Moreover, we found that TXNIP down-regulates miR-124a expression, a microRNA known to directly target FoxA2. Indeed, miR-124a overexpression led to decreased FoxA2 expression and IAPP promoter occupancy and to a significant reduction in IAPP mRNA and protein expression and also effectively inhibited TXNIP-induced IAPP expression. Thus, our studies have identified a novel TXNIP/miR-124a/FoxA2/IAPP signaling cascade linking the critical beta-cell signaling pathways of TXNIP and IAPP and thereby provide new mechanistic insight into an important aspect of transcriptional regulation and beta-cell biology.

Wnt signaling regulates left-right axis formation in the node of mouse embryos.

The node triggers formation of the left-right axis in mouse embryos by establishing local asymmetry of Nodal and Cerl2 expression. We found that Wnt3 is expressed in perinodal crown cells preferentially on the left side. The enhancer responsible for Wnt3 expression was identified and found to be regulated by Foxa2 and Rbpj under the control of Notch signaling. Rbpj binding sites suppress enhancer activity in pit cells of the node, thereby ensuring crown cell-specific expression. In addition, we found that the expression of Gdf1 and Cerl2 is also regulated by Notch signaling, suggesting that such signaling may induce the expression of genes related to left-right asymmetry as a set. Furthermore, Cerl2 expression became symmetric in response to inhibition of Wnt-ß-catenin signaling. Our results suggest that Wnt signaling regulates the asymmetry of Cerl2 expression, which likely generates a left-right difference in Nodal activity at the node for further amplification in lateral plate mesoderm.