Loss of mesenchymal bone morphogenetic protein signaling leads to development of reactive stroma and initiation of the gastric neoplastic cascade.

Bmps are morphogens involved in various gastric cellular functions. Studies in genetically-modified mice have shown that Bmp disruption in gastric epithelial and stromal cell compartments leads to the development of tumorigenesis. Our studies have demonstrated that abrogation of gastric epithelial Bmp signaling alone was not sufficient to recapitulate the neoplastic features associated with total gastric loss of Bmp signaling. Thus, epithelial Bmp signaling does not appear to be a key player in gastric tumorigenesis initiation. These observations suggest a greater role for stromal Bmp signaling in gastric polyposis initiation. In order to identify the specific roles played by mesenchymal Bmp signaling in gastric homeostasis, we generated a mouse model with abrogation of Bmp signaling exclusively in the gastro-intestinal mesenchyme (Bmpr1a(ΔMES)). We were able to expose an unsuspected role for Bmp loss of signaling in leading normal gastric mesenchyme to adapt into reactive mesenchyme. An increase in the population of activated-fibroblasts, suggesting mesenchymal transdifferentiation, was observed in mutant stomach. Bmpr1a(ΔMES) stomachs exhibited spontaneous benign polyps with presence of both intestinal metaplasia and spasmolytic-polypeptide-expressing metaplasia as early as 90 days postnatal. These results support the novel concept that loss of mesenchymal Bmp signaling cascade acts as a trigger in gastric polyposis initiation.

Bmp signaling in colonic mesenchyme regulates stromal microenvironment and protects from polyposis initiation.

In the colon, myofibroblasts are primary contributors in the establishment of the microenvironment involved in tissue homeostasis. Alterations in myofibroblast functions lead to changes resulting in a toxic microenvironment nurturing tumorigenesis. Bone morphogenetic proteins (Bmps) are morphogens known to play key roles in adult gut homeostasis. Studies in genetically-modified mice have shown that Bmp disruption in all cell layers leads to the development of gut polyposis. In contrast, our studies showed that loss of Bmp exclusively in the gastrointestinal epithelium resulted in increased epithelial proliferation without polyposis initiation, thus suggesting a key role for mesenchymal Bmp signaling in polyposis initiation. In order to identify the role of mesenchymal Bmp signaling on the microenvironment and its impact on colonic mucosa, a mouse model was generated with suppression of Bmp signaling exclusively in myofibroblasts (Bmpr1aΔMES). Bmpr1aΔMES mice exhibited increased subepithelial proliferation with changes in cellular composition leading to the development of a primed stroma with modulation of extracellular matrix proteins, immune cells and cytokines as early as 90 days of age. This microenvironmental deregulation was associated with increased polyposis initiation at one year of age. These results are the first to demonstrate that mesenchymal Bmpr1a inactivation alone is sufficient to prompt an expansion of myofibroblasts leading to the development of a reactive mesenchyme that contributes to polyposis initiation in the colon. These findings support the novel concept that inhibition of Bmp signaling in mesenchymal cells surrounding the normal epithelium leads to important changes instructing a toxic microenvironment sufficient to induce colonic polyposis.

Dual regulatory role for phosphatase and tensin homolog in specification of intestinal endocrine cell subtypes.

AIM: To investigate the impact of phosphatase and tensin homolog (Pten) in the specification of intestinal enteroendocrine subpopulations. METHODS: Using the Cre/loxP system, a mouse with conditional intestinal epithelial Pten deficiency was generated. Pten mutant mice and controls were sacrificed and small intestines collected for immunofluorescence and quantitative real-time polymerase chain reaction. Blood was collected on 16 h fasted mice by cardiac puncture. Enzyme-linked immunosorbent assay was used to measure blood circulating ghrelin, somatostatin (SST) and glucose-dependent insulinotropic peptide (GIP) levels. RESULTS: Results show an unexpected dual regulatory role for epithelial Pten signalling in the specification/differentiation of enteroendocrine cell subpopulations in the small intestine. Our data indicate that Pten positively regulates chromogranin A (CgA) expressing subpopulations, including cells expressing secretin, ghrelin, gastrin and cholecystokinin (CCK). In contrast, Pten negatively regulates the enteroendocrine subtype specification of non-expressing CgA cells such as GIP and SST expressing cells. CONCLUSION: The present results demonstrate that Pten signalling favours the enteroendocrine progenitor to specify into cells expressing CgA including those producing CCK, gastrin and ghrelin.

Loss of Smad5 leads to the disassembly of the apical junctional complex and increased susceptibility to experimental colitis.

The regulation of intestinal epithelial cell adhesion and migratory properties is often compromised in inflammatory bowel disease (IBD). Despite an increasing interest in bone morphogenetic protein (Bmp) signaling in gut pathologies, little is known of the specific roles played by individual Smads in intestinal epithelial functions. In the present study, we generated a mouse model with deletion of Smad5 transcriptional effector of the Bmp signaling pathway exclusively in the intestinal epithelium. Proliferation, migration, and apical junctional complex (AJC) protein expression were analyzed by immunofluorescence and Western blot. Human intestinal biopsies from control and IBD patients were analyzed for SMAD5 gene transcript expression by quantitative PCR (qPCR). Smad5(ΔIEC) and control mice were subjected to dextran sulfate sodium (DSS)-induced experimental colitis, and their clinical and histological symptoms were assessed. Loss of Smad5 led to intestinal epithelial hypermigration and deregulation of the expression of claudin-1 and claudin-2. E-cadherin was found to be equally expressed but displaced from the AJC to the cytoplasm in Smad5(ΔIEC) mice. Analysis of SMAD5 gene expression in human IBD patient samples revealed a significant downregulation of the gene transcript in Crohn's disease and ulcerative colitis samples. Smad5(ΔIEC) mice exposed to experimental DSS colitis were significantly more susceptible to the disease and had impaired wound healing during the recovery phase. Our results support that Smad5 is partly responsible for mediating Bmp signals in intestinal epithelial cells. In addition, deficiency in epithelial Smad5 leads to the deregulation of cell migration by disassembling the AJC with increasing susceptibility to experimental colitis and impairment in wound healing.

Loss of hepatocyte-nuclear-factor-1alpha impacts on adult mouse intestinal epithelial cell growth and cell lineages differentiation.

BACKGROUND AND AIMS: Although Hnf1alpha is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1alpha on the maintenance of adult small intestinal epithelial functions. METHODOLOGY/PRINCIPAL FINDINGS: An Hnf1alpha knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1alpha displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1alpha mutant mice. The intestinal epithelium of Hnf1alpha null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway. CONCLUSIONS/SIGNIFICANCE: Together, these results unravel a functional role for Hnf1alpha in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.

Epithelial phosphatase and tensin homolog regulates intestinal architecture and secretory cell commitment and acts as a modifier gene in neoplasia.

Phosphatase and tensin homolog (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/Akt pathway, is one of the most frequently mutated/deleted tumor suppressor genes in human cancers. The aim of this study was to gain insight into the role played by PTEN in intestinal homeostasis and epithelial cell function. Using the Cre/loxP system, we have generated a mouse with a conditional intestinal epithelial Pten deficiency. Pten mutant mice and controls were sacrificed for histology, immunofluorescence, Western blot, and quantitative polymerase chain reaction analysis. Our results show that loss of epithelial Pten leads to an intestinalomegaly associated with an increase in epithelial cell proliferation. Histological analysis demonstrated significant perturbation of the crypt-villus architecture, a marked increase in goblet cells and a decrease in enteroendocrine cells, suggesting a role for Pten in the commitment of the multipotential-secretory precursor cell. Loss of epithelial Pten does not result in induction of nuclear beta-catenin protein levels, nor is it sufficient to promote tumorigenesis initiation. However, it severely enhances intestinal tumor load in Apc(Min/+) mice, in which c-Myc is already deregulated. These results reveal an unknown function for Pten signaling in the commitment of multipotential-secretory progenitor cells and suggest that epithelial Pten functions as a modifier gene in intestinal neoplasia.