(+)4-Cholesten-3-one promotes differentiation of neural stem cells into dopaminergic neurons through TET1 and FoxA2.

In this paper, we report the results of treating cells with an effective small molecule, (+)4-cholesten-3-one (PubChem CID: 91477), which can promote neural stem cell(NSC) differentiation into dopaminergic neurons. This study used rat neural stem cells stimulated with two different concentrations (7.8 µM and 78 µM) of (+)4-cholesten-3-one. Cell phenotypic analysis showed that (+)4-cholesten-3-one induced NSC differentiation into dopaminergic neurons, and the level of tyrosine hydroxylase(TH), which is specific for dopaminergic cells, was significantly increased compared with that of the drug-free control group. Furthermore, in this study, we found that this effect may be related to the transcription factor fork-head box a2 (FoxA2) and ten-eleven translocation 1 (TET1). The expression of TET1 and FoxA2 was upregulated after treatment with (+)4-cholesten-3-one. To verify the relationship between (+)4-cholesten-3-one and these genes, we found that the binding rate of TET1 and FoxA2 increased after the application of (+)4-cholesten-3-one, as confirmed by a coimmunoprecipitation (Co-IP) assay. With a small interfering RNA (siRNA) experiment, we found that only when Tet1 and Foxa2 were not silenced was the mRNA level of Th increased after (+)4-cholesten-3-one treatment. Taken together, these data show that (+)4-cholesten-3-one can promote the differentiation of NSCs into dopaminergic neurons by upregulating the expression of TET1 and FoxA2 and by increasing their binding. Thus, (+)4-cholesten-3-one may help address the application of neural stem cell replacement therapy in neurodegenerative diseases.

Direct and selective lineage conversion of human fibroblasts to dopaminergic precursors.

Transplantation of dopaminergic precursors (DPs) is a promising therapeutic strategy of Parkinson's disease (PD). However, limited cell source for dopaminergic precursors has become a major obstacle for transplantation therapy. Our group demonstrated previously that mouse fibroblasts can be reprogrammed into induced dopaminergic precursors (iDPs) with high differentiation efficiency. In the current study, we hypothesized that a similar strategy can be applied to generate human iDPs for future cell therapy of PD. We overexpressed transcription factors Brn2, Sox2, and Foxa2 in human fibroblasts and observed formation of neurospheres. Subsequent characterization of the precursor colonies confirmed the generation of human induced dopaminergic precursors (hiDPs). These hiDPs were capable of self-renewal, proliferation, and differentiation. The hiDPs demonstrated high immunoreactivity for neural progenitor markers and high levels of gene expression for ventral mesencephalon-related neural progenitor markers such as Lmx1a, NIKX6.1, Corin, Otx2 and Mash1. Furthermore, the hiDPs could be differentiated into dopaminergic neurons with ˜80% efficiency, which significantly increased major functionally relevant proteins such as TH, DAT, AADC, Lmx1B, and VMAT2 compared to hiDPs. Additionally, hiDPs are more dopaminergic progenitor-restricted compare to those hiDP-like cells reprogrammed only by Brn2 and Sox2. Together, these results suggest that hiDPs with high differentiation efficiency can be generated by direct lineage reprogramming of fibroblasts with transcription factors Brn2, Sox2, and Foxa2. These hiDPs may serve as a safe and effective cell source for transplantation treatment of PD.

Reduced Immunohistochemical Expression of Hnf1ß and FoxA2 in Liver Tissue Can Discriminate Between Biliary Atresia and Other Causes of Neonatal Cholestasis.

Biliary atresia (BA) is a necroinflammatory occlusive cholangiopathy that affects infants. Genetic and environmental factors has been proposed for its occurrence. The objectives of this study was to investigate the protein expression of 2 important genes regulating ductal plate remodeling, hepatocyte nuclear factor 1-beta (Hnf1ß) and the fork head box protein A2 (FoxA2) in liver tissue from patients with BA and to compare their expression with other causes of neonatal cholestasis (NC). This retrospective study included 60 pediatric patients, 30 with BA and 30 with NC. Immunohistochemistry of Hnf1ß and FoxA2 was performed on liver tissues from studied patients as well as 20 healthy subjects. Statistical analysis between immunohistochemistry results and other parameters was performed. Liver tissue from patients with BA revealed reduced Hnf1ß and FoxA2 immunoexpression. A strong significant statistical difference between BA and NC group (P<0.0001) with regard to Hnf1ß and FoxA2 immunoexpression was evident. Moreover, Hnf1ß was significantly correlated with FoxA2 immunoexpression, stage of fibrosis, bile ductular proliferation, and bile plugs in bile ductules. Hnf1ß immunoreaction in BA cases showed 76.7% sensitivity, 90% specificity, 88.5% positive predictive value, 79.4% negative predictive value, and 83.4% accuracy. FoxA2 expression in BA cases revealed 70.0% sensitivity, 80.0% specificity, 77.8% positive predictive value, 72.7% negative predictive value, 75.0% accuracy. Hnf1ß and FoxA2 immunoexpression could differentiate between BA from other cause of NC.

Improvement of hepatogenic differentiation of iPS cells on an aligned polyethersulfone compared to random nanofibers.

The application of stem cells holds great promises in cell and tissue transplants. This study was designed to compare the hepatogenic differentiation of iPSCs on aligned PES/COL versus random. Aligned and random PES/COL nanofibrus scaffolds were fabricated by electrospining and their surface modified through plasma treatment and collagen coating. The scaffolds were characterized using scanning electron microscopy (SEM) and ATR-FTIR. Morphology and biochemical activities of the differentiated hepatocyte-like cells (HLCs) were examined after 5 and 20 days of differentiation. Real-Time RT-PCR and ICC showed no significant difference in the mRNA and protein levels of two important definitive endoderm specific markers, including Sox17 and Foxa2 between two scaffolds. However, Real-Time RT-PCR analysis indicated an increase in the expression of Cyp7A1 gene over the period of the differentiation procedure on the aligned nanofibers but there was no difference in other genes such as Albumin and CK19. Moreover, comparison of hepatogenic differentiation evaluated by Albumin production in conditioned media of HLCs differentiated on aligned PES/COL, showed increase expression of these markers after 20 days compared to that of the random nanofibers. Taken together, the results of this study may indicate that aligned PES/COL nanofibrous scaffolds can improve terminal differentiation of HLCs from iPSCs.

Preferential Generation of 15-HETE-PE Induced by IL-13 Regulates Goblet Cell Differentiation in Human Airway Epithelial Cells.

Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 µM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.

Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.

AIM: To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system. MAIN METHODS: Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test. KEY FINDINGS: Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition. SIGNIFICANCE: This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation.

Foxa2, a novel protein partner of the tumour suppressor menin, is deregulated in mouse and human MEN1 glucagonomas.

Foxa2, known as one of the pioneer factors, plays a crucial role in islet development and endocrine functions. Its expression and biological functions are regulated by various factors, including, in particular, insulin and glucagon. However, its expression and biological role in adult pancreatic α-cells remain elusive. In the current study, we showed that Foxa2 was overexpressed in islets from α-cell-specific Men1 mutant mice, at both the transcriptional level and the protein level. More importantly, immunostaining analyses showed its prominent nuclear accumulation, specifically in α-cells, at a very early stage after Men1 disruption. Similar nuclear FOXA2 expression was also detected in a substantial proportion (12/19) of human multiple endocrine neoplasia type 1 (MEN1) glucagonomas. Interestingly, our data revealed an interaction between Foxa2 and menin encoded by the Men1 gene. Furthermore, using several approaches, we demonstrated the relevance of this interaction in the regulation of two tested Foxa2 target genes, including the autoregulation of the Foxa2 promoter by Foxa2 itself. The current study establishes menin, a novel protein partner of Foxa2, as a regulator of Foxa2, the biological functions of which extend beyond the pancreatic endocrine cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Zic2 mutation causes holoprosencephaly via disruption of NODAL signalling.

The ZIC2 transcription factor is one of the genes most commonly mutated in Holoprosencephaly (HPE) probands. Studies in cultured cell lines and mice have shown a loss of ZIC2 function is the pathogenic mechanism but the molecular details of this ZIC2 requirement remain elusive. HPE arises when signals that direct morphological and fate changes in the developing brain and facial primordia are not sent or received. One critical signal is sent from the prechordal plate (PrCP) which develops beneath the ventral forebrain. An intact NODAL signal transduction pathway and functional ZIC2 are both required for PrCP establishment. We now show that ZIC2 acts downstream of the NODAL signal during PrCP development. ZIC2 physically interacts with SMAD2 and SMAD3, the receptor activated proteins that control transcription in a NODAL dependent manner. Together SMAD3 and ZIC2 regulate FOXA2 transcription in cultured cells and Zic2 also controls the foxA2 expression during Xenopus development. Variant forms of the ZIC2 protein, associated with HPE in man or mouse, are deficient in their ability to influence SMAD-dependent transcription. These findings reveal a new mechanism of NODAL signal transduction in the mammalian node and provide the first molecular explanation of how ZIC2 loss-of-function precipitates HPE.

Conditional deletion of Sox17 reveals complex effects on uterine adenogenesis and function.

The importance of canonical Wnt signaling to murine uterine development is well established. Mouse models in which uterine-specific Wnt ligands, ß-catenin, or Lef1 are disrupted result in failure of postnatal endometrial gland development. Sox17 is a transcription factor characterized in numerous tissues as an antagonist of Wnt signaling. Thus, we hypothesized that conditional ablation of Sox17 would lead to hyperproliferation of endometrial glands in mice. Contrary to our prediction, disruption of Sox17 in epithelial and stromal compartments led to inhibition of endometrial adenogenesis and a loss of reproductive capacity. Epithelium-specific Sox17 disruption resulted in normal adenogenesis although reproductive capacity remained impaired. These findings suggest that non-epithelial, Sox17-positive cells are necessary for adenogenesis and that glands require Sox17 to properly function. To our knowledge, these findings are the first to implicate Sox17 in endometrial gland formation and reproductive success. The data presented herein underscore the importance of studying Sox17 in uterine homeostasis and function.

Hepatocyte nuclear factor 1 coordinates multiple processes in a model of intestinal epithelial cell function.

Mutations in hepatocyte nuclear factor 1 transcription factors (HNF1α/ß) are associated with diabetes. These factors are well studied in the liver, pancreas and kidney, where they direct tissue-specific gene regulation. However, they also have an important role in the biology of many other tissues, including the intestine. We investigated the transcriptional network governed by HNF1 in an intestinal epithelial cell line (Caco2). We used chromatin immunoprecipitation followed by direct sequencing (ChIP-seq) to identify HNF1 binding sites genome-wide. Direct targets of HNF1 were validated using conventional ChIP assays and confirmed by siRNA-mediated depletion of HNF1, followed by RT-qPCR. Gene ontology process enrichment analysis of the HNF1 targets identified multiple processes with a role in intestinal epithelial cell function, including properties of the cell membrane, cellular response to hormones, and regulation of biosynthetic processes. Approximately 50% of HNF1 binding sites were also occupied by other members of the intestinal transcriptional network, including hepatocyte nuclear factor 4A (HNF4A), caudal type homeobox 2 (CDX2), and forkhead box A2 (FOXA2). Depletion of HNF1 in Caco2 cells increases FOXA2 abundance and decreases levels of CDX2, illustrating the coordinated activities of the network. These data suggest that HNF1 plays an important role in regulating intestinal epithelial cell function, both directly and through interactions with other intestinal transcription factors.

The lncRNA DEANR1 facilitates human endoderm differentiation by activating FOXA2 expression.

Long non-coding RNAs (lncRNAs) regulate diverse biological processes, including cell lineage specification. Here, we report transcriptome profiling of human endoderm and pancreatic cell lineages using purified cell populations. Analysis of the data sets allows us to identify hundreds of lncRNAs that exhibit differentiation-stage-specific expression patterns. As a first step in characterizing these lncRNAs, we focus on an endoderm-specific lncRNA, definitive endoderm-associated lncRNA1 (DEANR1), and demonstrate that it plays an important role in human endoderm differentiation. DEANR1 contributes to endoderm differentiation by positively regulating expression of the endoderm factor FOXA2. Importantly, overexpression of FOXA2 is able to rescue endoderm differentiation defects caused by DEANR1 depletion. Mechanistically, DEANR1 facilitates FOXA2 activation by facilitating SMAD2/3 recruitment to the FOXA2 promoter. Thus, our study not only reveals a large set of differentiation-stage-specific lncRNAs but also characterizes a functional lncRNA that is important for endoderm differentiation.

Resetting the transcription factor network reverses terminal chronic hepatic failure.

The cause of organ failure is enigmatic for many degenerative diseases, including end-stage liver disease. Here, using a CCl4-induced rat model of irreversible and fatal hepatic failure, which also exhibits terminal changes in the extracellular matrix, we demonstrated that chronic injury stably reprograms the critical balance of transcription factors and that diseased and dedifferentiated cells can be returned to normal function by re-expression of critical transcription factors, a process similar to the type of reprogramming that induces somatic cells to become pluripotent or to change their cell lineage. Forced re-expression of the transcription factor HNF4α induced expression of the other hepatocyte-expressed transcription factors; restored functionality in terminally diseased hepatocytes isolated from CCl4-treated rats; and rapidly reversed fatal liver failure in CCl4-treated animals by restoring diseased hepatocytes rather than replacing them with new hepatocytes or stem cells. Together, the results of our study indicate that disruption of the transcription factor network and cellular dedifferentiation likely mediate terminal liver failure and suggest reinstatement of this network has therapeutic potential for correcting organ failure without cell replacement.

Propofol Attenuates Lipopolysaccharide-Induced Monocyte Chemoattractant Protein-1 Production Through Enhancing apoM and foxa2 Expression in HepG2 Cells.

Monocyte chemoattractant protein-1 (MCP-1) is a cytokine that mediates the influx of cells to sites of inflammation. Our group recently reported that propofol exerted an anti-inflammatory effect and could inhibit lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines. However, the effect and possible mechanisms of propofol on MCP-1 expression remain unclear. LPS-stimulated HepG2 cells were treated with 50 µM propofol for 0, 6, 12, and 24 h, respectively. The transcript and protein levels were measured by real-time quantitative PCR and Western blot analyses, respectively. We found that propofol markedly decreased both MCP-1 messenger RNA (mRNA) and protein levels in LPS-stimulated HepG2 cells in a time-dependent manner. Expression of apolipoprotein M (apoM) and forkhead box protein A2 (foxa2) was increased by propofol treatment in HepG2 cells. In addition, the inhibitory effect of propofol on MCP-1 expression was significantly abolished by small interfering RNA against apoM and foxa2 in LPS-stimulated HepG2 cells. Propofol attenuates LPS-induced MCP-1 production through enhancing apoM and foxa2 expression in HepG2 cells.

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.

FOXA2 suppresses the metastasis of hepatocellular carcinoma partially through matrix metalloproteinase-9 inhibition.

The forkhead box transcription factor A2 (FOXA2) is a member of the hepatocyte nuclear factor family and plays an important role in liver development and metabolic homeostasis, but its role in the metastasis of hepatocellular carcinoma (HCC) has not been evaluated. In this study, we found that the expression of FOXA2 was decreased in 68.1% (49/72) of human HCC tissues compared with their paired non-cancerous adjacent tissues. Clinicopathological analysis revealed that reduced FOXA2 expression was correlated with aggressive characteristics (venous invasion, poor differentiation, high tumor node metastasis grade). FOXA2 level was even lower in portal vein tumor thrombus compared with primary tumor tissues and correlated with epithelial-mesenchymal transition in HCC cells. Overexpression of FOXA2 inhibited migration and invasion of Focus cells, whereas knockdown of FOXA2 in HepG2 showed the opposite effect. Moreover, upregulation of FOXA2 suppressed HCC metastasis to bone, brain and lung in two distinct mouse models. Finally, we proved that FOXA2 repressed the transcription of matrix metalloproteinase (MMP)-9 and exerted its antimetastasis effect partially through downregulation of MMP-9. In conclusion, our findings indicate that FOXA2 plays a critical role in HCC metastasis and may serve as a novel therapeutic target for HCC.

Arx together with FoxA2, regulates Shh floor plate expression.

Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.

Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix.

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated "RoGel," which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC-HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.

Topical interferon-gamma neutralization prevents conjunctival goblet cell loss in experimental murine dry eye.

Evidence suggests that the cytokine interferon (IFN)-γ released by natural killer and CD4(+) T cells contributes to the conjunctival goblet cell (GC) loss in dry eye. The purpose of this study was to investigate if topical neutralization of IFN-γ prevents or alleviates GC loss in an experimental desiccating stress (DS) model of dry eye. In this study, we found that topical IFN-γ neutralization significantly decreased DS-induced conjunctival GC loss. This was accompanied by decreased epithelial apoptosis, and increased IL-13 and decreased FoxA2 expression in the forniceal conjunctiva. To establish that IFN-γ produced by pathogenic CD4(+) T cells contributes to DS-induced GC loss, adoptive transfer of CD4(+) T cells isolated from DS exposed donors to naïve RAG-1(-/-) recipient mice was performed. Similar to the donor mice, topical IFN-γ neutralization decreased conjunctival GC loss, suppressed apoptosis and increased IL-13 expression in adoptive transfer recipients. In summary, this study demonstrated that topical neutralization of IFN-γ prevents GC loss via modulating apoptosis and maintaining IL-13 signaling.

Neurogenin3 cooperates with Foxa2 to autoactivate its own expression.

The transcription factor Neurogenin3 functions as a master regulator of endocrine pancreas formation, and its deficiency leads to the development of diabetes in humans and mice. In the embryonic pancreas, Neurogenin3 is transiently expressed at high levels for a narrow time window to initiate endocrine differentiation in scattered progenitor cells. The mechanisms controlling these rapid and robust changes in Neurogenin3 expression are poorly understood. In this study, we characterize a Neurogenin3 positive autoregulatory loop whereby this factor may rapidly induce its own levels. We show that Neurogenin3 binds to a conserved upstream fragment of its own gene, inducing deposition of active chromatin marks and the activation of Neurog3 transcription. Additionally, we show that the broadly expressed endodermal forkhead factors Foxa1 and Foxa2 can cooperate synergistically to amplify Neurogenin3 autoregulation in vitro. However, only Foxa2 colocalizes with Neurogenin3 in pancreatic progenitors, thus indicating a primary role for this factor in regulating Neurogenin3 expression in vivo. Furthermore, in addition to decreasing Neurog3 autoregulation, inhibition of Foxa2 by RNA interference attenuates Neurogenin3-dependent activation of the endocrine developmental program in cultured duct mPAC cells. Hence, these data uncover the potential functional cooperation between the endocrine lineage-determining factor Neurogenin3 and the widespread endoderm progenitor factor Foxa2 in the implementation of the endocrine developmental program in the pancreas.

ß-Catenin signaling regulates Foxa2 expression during endometrial hyperplasia formation.

The Wnt/ß-catenin signaling is essential for various organogenesis and is often implicated during tumorigenesis. Dysregulated ß-catenin signaling is associated with the formation of endometrial adenocarcinomas (EACs), which is considered as the common form of endometrial cancer in women. In the current study, we investigate the downstream target of Wnt/ß-catenin signaling in the uterine epithelia and the mechanism leading to the formation of endometrial hyperplasia. We report that conditional ablation and activation of ß-catenin in the uterine epithelia lead to aberrant epithelial structures and endometrial hyperplasia formation, respectively. We demonstrate that ß-catenin regulates Foxa2 with its candidate upstream region for the uterine epithelia. Furthermore, knockdown of Foxa2 leads to defects in cell cycle regulation, suggesting a possible function of Foxa2 in the control of cell proliferation. We also observe that ß-catenin and Foxa2 expression levels are augmented in the human specimens of complex atypical endometrial hyperplasia, which is considered to have a greater risk of progression to EACs. Thus, our study indicates that ß-catenin regulates Foxa2 expression, and this interaction is possibly essential to control cell cycle progression during endometrial hyperplasia formation. Altogether, the augmented expression levels of ß-catenin and Foxa2 are essential features during the formation of endometrial hyperplasia.