Pancreatic ductal deletion of Hnf1b disrupts exocrine homeostasis, leads to pancreatitis and facilitates tumorigenesis

BACKGROUND AND AIMS: The exocrine pancreas consists of acinar cells that produce digestive enzymes transported to the intestine through a branched ductal epithelium. Chronic pancreatitis is characterized by progressive inflammation, fibrosis and loss of acinar tissue. These changes of the exocrine tissue are risk factors for pancreatic cancer. The cause of chronic pancreatitis cannot be identified in one-quarter of patients. Here, we investigated how duct dysfunction could contribute to pancreatitis development. METHODS: The transcription factor Hnf1b, first expressed in pancreatic progenitors, is strictly restricted to ductal cells from late embryogenesis. We have previously shown that Hnf1b is crucial for pancreas morphogenesis but its postnatal role still remains unelucidated. To investigate the role of pancreatic ducts in exocrine homeostasis, we inactivated Hnf1b gene in vivo in mouse ductal cells. RESULTS: We uncovered that postnatal Hnf1b inactivation in pancreatic ducts leads to chronic pancreatitis in adults. Hnf1bΔduct mutants display dilatation of ducts, loss of acinar cells, acinar-to-ductal metaplasia (ADM) and lipomatosis. We deciphered the early events involved, with downregulation of cystic disease-associated genes, loss of primary cilia, upregulation of signaling pathways, especially Yap pathway involved in ADM. Remarkably, Hnf1bΔduct mutants developed pancreatic intraepithelial neoplasia and promote PanIN progression in concert with KRAS. We further showed that adult Hnf1b inactivation in pancreatic ducts is associated with impaired regeneration after injury, with persistent metaplasia and initiation of neoplasia. CONCLUSION: Loss of Hnf1b in ductal cells leads to chronic pancreatitis and neoplasia. This reveals that Hnf1b deficiency may contribute to diseases of the exocrine pancreas and could gain further insight into the etiology of pancreatitis and tumorigenesis.Support to CH was received from theCentre National de la Recherche Scientifique (CNRS), the Universite Pierre et Marie Curie (UPMC)- Sorbonne Université , the GEFLUC - Les entreprises contre le Cancer, the Societe Francophone du Diabete (SFD)-Ypsomed, the programme Emergence UPMC. EQ was supported by a PhD fellowship from the French Ministère de la Recherche et de la Technologie. MF is an assistant engineer of the CNRS. TD and AS were supported by Sorbonne Université. MDV was supported by a PhD student fellowship from the European Marie Curie Initial Training Network (ITN)-Biology of Liver and Pancreatic Development and Disease (BOLD). O. O. was supported by a Master1 fellowship. RCP was supported by a postdoctoral fellowship from the American Heart Association (14POST20380262). MG was supported by the National Institutes of Health (U01 DK089540) and the Juvenile Diabetes Research Foundation (1-2011-592). CH is a permanent senior researcher of the Institut National de la Sante et de la Recherche Medicale (INSERM).S

Special Issue: “Digestive Inflammation and New Therapeutical Targets”

Inflammatory diseases commonly associated with humans are chronic inflammatory gastrointestinal diseases (CIGDs) [...

Rôle du facteur de transcription vHNF1/HNF1b/TCF2 au cours du développement chez la souris et l' homme

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Histone Deacetylase Inhibitors Modify Pancreatic Cell Fate Determination and Amplify Endocrine Progenitors

International audienceABSTRACT During pancreas development, transcription factors play critical roles in exocrine and endocrine differentiation. Transcriptional regulation in eukaryotes occurs within chromatin and is influenced by posttranslational histone modifications (e.g., acetylation) involving histone deacetylases (HDACs). Here, we show that HDAC expression and activity are developmentally regulated in the embryonic rat pancreas. We discovered that pancreatic treatment with different HDAC inhibitors (HDACi) modified the timing and determination of pancreatic cell fate. HDACi modified the exocrine lineage via abolition and enhancement of acinar and ductal differentiation, respectively. Importantly, HDACi treatment promoted the NGN3 proendocrine lineage, leading to an increased pool of endocrine progenitors and modified endocrine subtype lineage choices. Interestingly, treatments with trichostatin A and sodium butyrate, two inhibitors of both class I and class II HDACs, enhanced the pool of β cells. These results highlight the roles of HDACs at key points in exocrine and endocrine differentiation. They show the powerful use of HDACi to switch pancreatic cell determination and amplify specific cellular subtypes, with potential applications in cell replacement therapies in diabetes

Rôle du facteur de transcription HNF1B et réseaux de régulation impliqués dans le développement du pancréas chez la souris

Chez l'homme, des mutations hétérozygotes d HNF1B sont associées au syndrome Maturity-Onset Diabetes of the Young type 5 (MODY5). Nous avons précédemment montré que l absence d HNF1B chez la souris provoque une agénésie du pancréas. Afin d'élucider le rôle spécifique d HNF1B dans le pancréas, indépendamment de son rôle dans la régionalisation de l'intestin primitif, nous avons réalisé une inactivation conditionnelle d Hnf1b dans les progéniteurs pancréatiques. Ceci conduit à une forte hypoplasie pancréatique, un pool réduit de progéniteurs multipotents dû à une diminution de la prolifération et une augmentation de l'apoptose, avec une dérégulation des voies de signalisation FGF et Notch. Le compartiment exocrine est affecté chez ces mutants Hnf1b, avec une baisse drastique des cellules acinaires, et une métaplasie acinaire-vers-canalaire . La morphogenèse canalaire est altérée, révélant des canaux kystiques dépourvus de cils primaires, et une diminution de l'expression de gènes kystiques , dont certains identifiés comme cibles directes d HNF1B. L'inactivation d Hnf1b au cours de l embryogenèse engendre une absence de progéniteurs endocrines. Remarquablement, nous avons identifié Ngn3 comme cible directe d HNF1B. L inactivation plus tardive d Hnf1b dans les canaux pancréatiques conduit à un défaut de morphogenèse des îlots et une pancréatite après la naissance. Nos résultats placent HNF1B au centre des réseaux de régulation contrôlant l'organogenèse du pancréas, la morphogenèse exocrine et les précurseurs endocrines. Ces données devraient permettre une meilleure compréhension de l'étiologie MODY5 et le développement de traitements alternatifs pour le diabète.In humans, heterozygous mutations in the gene HNF1B are associated with the maturity-onset diabetes of the young type 5 (MODY5). We previously showed that lack of this transcription factor causes pancreas agenesis in Hnf1b mutant mouse embryos rescued by tetraploid aggregation. In order to elucidate the role of HNF1B specifically in pancreas development independently of its role in gut regionalization, we performed a conditional gene inactivation specifically in pancreatic progenitors. Hnf1b deletion in the early pancreas leads to a strong hypoplasia, with a reduced pool of pancreatic multipotent progenitor cells due to decreased proliferation and increased apoptosis, and deregulation of FGF and Notch pathways. Exocrine compartment is severely affected in Hnf1b mutants, with a dramatic decrease in acinar cells, associated with an acinar-to-ductal metaplasia. Duct morphogenesis is defective showing cystic ducts devoid of primary cilia, and decreased expression of cystic-related genes, some of which identified as direct targets of HNF1B. Moreover, inactivation of Hnf1b at distinct time points results in an absence of NGN3+ endocrine progenitors throughout embryogenesis. Remarkably, Ngn3 was found to be directly regulated by HNF1B. Duct-specific deletion of Hnf1b leads to impaired islet morphogenesis, and pancreatitis after birth. Our results place HNF1B at a central position in the regulatory networks controlling pancreas organogenesis, exocrine morphogenesis and maintenance, and generation of endocrine progenitors. These findings are expected to provide new insights for understanding the MODY5 etiology and the development of alternative treatments for diabetes.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Acinar-to-Ductal Metaplasia (ADM): On the Road to Pancreatic Intraepithelial Neoplasia (PanIN) and Pancreatic Cancer

Adult pancreatic acinar cells show high plasticity allowing them to change in their differentiation commitment. Pancreatic acinar-to-ductal metaplasia (ADM) is a cellular process in which the differentiated pancreatic acinar cells transform into duct-like cells. This process can occur as a result of cellular injury or inflammation in the pancreas. While ADM is a reversible process allowing pancreatic acinar regeneration, persistent inflammation or injury can lead to the development of pancreatic intraepithelial neoplasia (PanIN), which is a common precancerous lesion that precedes pancreatic ductal adenocarcinoma (PDAC). Several factors can contribute to the development of ADM and PanIN, including environmental factors such as obesity, chronic inflammation and genetic mutations. ADM is driven by extrinsic and intrinsic signaling. Here, we review the current knowledge on the cellular and molecular biology of ADM. Understanding the cellular and molecular mechanisms underlying ADM is critical for the development of new therapeutic strategies for pancreatitis and PDAC. Identifying the intermediate states and key molecules that regulate ADM initiation, maintenance and progression may help the development of novel preventive strategies for PDAC

Hnf1b controls pancreas morphogenesis and the generation of Ngn3(+) endocrine progenitors

International audienceHeterozygous mutations in the human HNF1B gene are associated with maturity-onset diabetes of the young type 5 (MODY5) and pancreas hypoplasia. In mouse, Hnf1b heterozygous mutants do not exhibit any phenotype, whereas the homozygous deletion in the entire epiblast leads to pancreas agenesis associated with abnormal gut regionalization. Here, we examine the specific role of Hnf1b during pancreas development, using constitutive and inducible conditional inactivation approaches at key developmental stages. Hnf1b early deletion leads to a reduced pool of pancreatic multipotent progenitor cells (MPCs) due to decreased proliferation and increased apoptosis. Lack of Hnf1b either during the first or the secondary transitions is associated with cystic ducts. Ductal cells exhibit aberrant polarity and decreased expression of several cystic disease genes, some of which we identified as novel Hnf1b targets. Notably, we show that Glis3, a transcription factor involved in duct morphogenesis and endocrine cell development, is downstream Hnf1b. In addition, a loss and abnormal differentiation of acinar cells are observed. Strikingly, inactivation of Hnf1b at different time points results in the absence of Ngn3(+) endocrine precursors throughout embryogenesis. We further show that Hnf1b occupies novel Ngn3 putative regulatory sequences in vivo. Thus, Hnf1b plays a crucial role in the regulatory networks that control pancreatic MPC expansion, acinar cell identity, duct morphogenesis and generation of endocrine precursors. Our results uncover an unappreciated requirement of Hnf1b in endocrine cell specification and suggest a mechanistic explanation of diabetes onset in individuals with MODY5

Tissue sample stability: thawing effect on multi-organ samples

International audienceCorrect handling of samples is essential in metabolomic studies. Improper handling and prolonged storage of samples has unwanted effects on the metabolite levels. The aim of this study was to identify the effects that thawing has on different organ samples. Organ samples from gut, kidney, liver, muscle and pancreas were analyzed for a number of endogenous metabolites in an untargeted metabolomics approach, using gas chromatography time of flight mass spectrometry at the Swedish Metabolomics Centre, Umeao University, Sweden. Multivariate data analysis was performed by means of principal component analysis and orthogonal projection to latent structures discriminant analysis. The results showed that the metabolic changes caused by thawing were almost identical for all organs. As expected, there was a marked increase in overall metabolite levels after thawing, caused by increased protein and cell degradation. Cholesterol was one of the eight metabolites found to be decreased in the thawed samples in all organ groups. The results also indicated that the muscles are less susceptible to oxidation compared to the rest of the organ samples

A vHNF1/TCF2-HNF6 cascade regulates the transcription factor network that controls generation of pancreatic precursor cells

Generation of pancreatic precursor cells in the endoderm is controlled by a network of transcription factors. Hepatocyte nuclear factor-6 (HNF6) is a key player in this network, because it controls the initiation of the expression of pancreatic and duodenal homeobox 1 (Pdx1), the earliest marker of pancreatic precursor cells. To further characterize this network, we have investigated how the expression of HNF6 is controlled in mouse endoderm, by using in vitro and in vivo protein-DNA interaction techniques combined with endoderm electroporation, transgenesis, and gene inactivation in embryos. We delineated Hnf6 regulatory regions that confer expression of a reporter gene in the embryonic endoderm but not in extraembryonic visceral endoderm. HNF6 expression in the embryonic endoderm was found to depend on an intronic enhancer. This enhancer contains functional binding sites for the tissue-specific factors of the forkhead box A and HNF1 families. Among the latter, variant HNF1 (vHNF1)/TCF2, which is expressed before HNF6 in the endoderm, was found to be critical for HNF6 expression. Therefore, the sequential activation of vHNF1, HNF6, and Pdx1 in the endoderm appears to control the generation of pancreatic precursors. This cascade may be used to benchmark in vitro differentiation of pancreatic precursor cells from embryonic stem cells, for cell therapy of diabetes

Insights into the etiology and physiopathology of MODY5/HNF1B pancreatic phenotype with a mouse model of the human disease

Maturity‐onset diabetes of the young type 5 (MODY5) is due to heterozygous mutations or deletion of HNF1B. No mouse models are currently available to recapitulate the human MODY5 disease. Here, we investigate the pancreatic phenotype of a unique MODY5 mouse model generated by heterozygous insertion of a human HNF1B splicing mutation at the intron‐2 splice donor site in the mouse genome. This Hnf1bsp2/+ model generated with targeted mutation of Hnf1b mimicking the c.544+1G>T (<IVS2nt+1G>T) mutation identified in humans, results in alternative transcripts and a 38% decrease of native Hnf1b transcript levels. As a clinical feature of MODY5 patients, the hypomorphic mouse model Hnf1bsp2/+ displays glucose intolerance. Whereas Hnf1bsp2/+ isolated islets showed no altered insulin secretion, we found a 65% decrease in pancreatic insulin content associated with a 30% decrease in total large islet volume and a 20% decrease in total β‐cell volume. These defects were associated with a 30% decrease in expression of the pro‐endocrine gene Neurog3 that we previously identified as a direct target of Hnf1b, showing a developmental etiology. As another clinical feature of MODY5 patients, the Hnf1bsp2/+ pancreases display exocrine dysfunction with hypoplasia. We observed chronic pancreatitis with loss of acinar cells, acinar‐to‐ductal metaplasia, and lipomatosis, with upregulation of signaling pathways and impaired acinar cell regeneration. This was associated with ductal cell deficiency characterized by shortened primary cilia. Importantly, the Hnf1bsp2/+ mouse model reproduces the pancreatic features of the human MODY5/HNF1B disease, providing a unique in vivo tool for molecular studies of the endocrine and exocrine defects and to advance basic and translational research