Mathieu Darsigny

Hepatocyte nuclear factor 4α contributes to an intestinal epithelial phenotype in vitro and plays a partial role in mouse intestinal epithelium differentiation

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a regulator of hepatocyte and pancreatic transcription. Hnf4alpha deletion in the mouse is embryonically lethal with severe defects in visceral endoderm formation. It has been concluded in the past that the role of Hnf4alpha in the developing colon was much less important than in the liver. However, the precise role of Hnf4alpha in the homeostasis of the small intestinal epithelium remains unclear. Our aim was to evaluate the potential of Hnf4alpha to support an intestinal epithelial phenotype. First, Hnf4alpha potential to dictate this phenotype was assessed in nonintestinal cell lines in vitro. Forced expression of Hnf4alpha in fibroblasts showed an induction of features normally restricted to epithelial cells. Combinatory expression of Hnf4alpha with specific transcriptional regulators of the intestine resulted in the induction of intestinal epithelial genes in this context. Second, the importance of Hnf4alpha in maintaining the homeostasis of the intestinal epithelium was investigated in mice. Mice conditionally deficient for intestinal Hnf4alpha developed normally throughout adulthood with an epithelium displaying normal morphological and functional structures with minor alterations. Subtle but statistical differences were observed at the proliferation and the cytodifferentiation levels. Hnf4alpha mutant mice displayed an increase in the number of goblet and enteroendocrine cells compared with controls. Given the fundamental role of this transcription factor in other tissues, these findings dispute the crucial role for this regulator in the maintenance of intestinal epithelial cell function at a period of time that follows cytodifferentiation but may suggest a functional role in instructing cells to become specific to the intestinal epithelium.

Loss of cathepsin L activity promotes claudin-1 overexpression and intestinal neoplasia

Intestinal epithelial integrity and polarity are maintained by cohesive interactions between cells via the formation of tight junctions. Irregularities in tight junctions have only recently been found to be associated with the initiation and progression of intestinal neoplasia. The claudin family of proteins is integral to the structure and function of the tight junction but little is known of the molecular events that regulate the expression of these components. The present report identifies cathepsin L, classically a lysosomal cysteine protease, as being induced during intestinal epithelial cell polarization and differentiation. Inhibition of intracellular cathepsin L activity results in the accumulation of disorganized cell layers and a decline in the expression of differentiation markers in cultured intestinal epithelial cells. This coincides with a rapid up‐ regulation of claudin‐1 protein accumulation. Mutant mice defective in cathepsin L activity (furless) display an elevated level of intestinal claudin‐1 and claudin‐2 expression. Loss of cathepsin L activity leads to a marked increase in tumor multiplicity in the intestine of ApcMin mice. Given the traditionally viewed biological role of cathepsin L in the processing of lysosomal content as well as in pathological extracellular matrix remodeling, the results here demonstrate an as yet unsuspected intracellular role for this protease in normal intestinal epithelial polarization and initiation of neoplasia.— Boudreau, F., Lussier, C. R., Mongrain, S., Darsigny, M., Drouin, J. L., Doyon, G., Suh, E. R., Beaulieu, J.‐F., Rivard, N., Perreault, N. Loss of cathepsin L activity promotes claudin‐1 overexpression and intestinal neoplasia. FASEB J. 21, 3853–3865 (2007)

Hepatocyte Nuclear Factor-4α Promotes Gut Neoplasia in Mice and Protects against the Production of Reactive Oxygen Species

Hepatocyte nuclear factor-4α (Hnf4α) is a transcription factor that controls epithelial cell polarity and morphogenesis. Hnf4α conditional deletion during postnatal development has minor effects on intestinal epithelium integrity but promotes activation of the Wnt/β-catenin pathway without causing tumorigenesis. Here, we show that Hnf4α does not act as a tumor-suppressor gene but is crucial in promoting gut tumorigenesis in mice. Polyp multiplicity in ApcMin mice lacking Hnf4α is suppressed compared with littermate ApcMin controls. Analysis of microarray gene expression profiles from mice lacking Hnf4α in the intestinal epithelium identifies novel functions of this transcription factor in targeting oxidoreductase-related genes involved in the regulation of reactive oxygen species (ROS) levels. This role is supported with the demonstration that HNF4α is functionally involved in the protection against spontaneous and 5-fluorouracil chemotherapy-induced production of ROS in colorectal cancer cell lines. Analysis of a colorectal cancer patient cohort establishes that HNF4α is significantly upregulated compared with adjacent normal epithelial resections. Several genes involved in ROS neutralization are also induced in correlation with HNF4A expression. Altogether, the findings point to the nuclear receptor HNF4α as a potential therapeutic target to eradicate aberrant epithelial cell resistance to ROS production during intestinal tumorigenesis.

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 Crohns 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.

Cux1 transcription factor is induced in inflammatory bowel disease and protects against experimental colitis.

Background: Cux1 is a ubiquitous transcriptional factor that has been associated with cell proliferation, migration, invasion, and differentiation. Cux1 is an effector of the transforming growth factor beta (TGF&bgr;) pathway, PAR2 receptor signaling, and cellular migration, mechanisms intimately related to inflammatory bowel diseases (IBD). Methods: CD1 mice treated with dextran sulfate sodium (DSS) in drinking water and cultured intestinal epithelial cells were used to determine Cux1 expression under inflammatory conditions. A commercial cDNA library was used to monitor CUX1 expression in IBD patients. The Cux1&Dgr;HD/&Dgr;HD hypomorphic mouse model (Cux1&Dgr;HD) treated with DSS in drinking water was used and the disease severity assessed. Results: Cux1 expression increased in cultured intestinal epithelial cells stimulated with tumor necrosis factor alpha (TNF&agr;), in the mouse intestinal epithelium during experimental colitis and in human IBD patient samples. DSS‐induced colitis in Cux1&Dgr;HD mice was more severe according to clinical observations such as weight loss, colon length, and rectal bleeding. Histological observations confirmed an increase of IBD‐related morphological changes including ulceration and mucosal infiltration of leukocytes in Cux1&Dgr;HD mice. An increased number of pSer276‐RelA‐positive cells and higher expression levels of proinflammatory cytokines were also measured in the colon of Cux1&Dgr;HD diseased animals. Elevated levels of Cxcl1 were measured before and after DSS‐treatment and a greater neutrophilic infiltration was quantified in DSS‐treated Cux1&Dgr;HD mice. Finally, mucosal healing was significantly impaired in Cux1&Dgr;HD mice during recovery from DSS treatment. Conclusions: CUX1 is increased in response to inflammatory stress and its nuclear expression is crucial to protect against DSS‐induced colitis and subsequent mucosal healing. (Inflamm Bowel Dis 2010)

The Promyelocytic Leukemia Zinc Finger (PLZF ) gene is a novel transcriptional target of the CCAAT‐Displacement‐Protein (CUX1) repressor

The CCAAT‐Displacement‐Protein (CUX1) can transcriptionally repress sucrase–isomaltase gene expression, a specific product of enterocytes that becomes re‐expressed during human colonic polyposis. Little is known of the gene repertoire that is directly affected by CUX1 in the intestinal epithelial context. This article identifies the Promyelocytic Leukemia Zinc Finger (PLZF) gene as a transcriptional target for the CUX1 repressor. CUX1 interacts in vivo with multiple DNA‐binding sites in the 5′‐UTR and promoter of the PLZF gene in colorectal cancer cells, a region that is functionally targeted by CUX1 in cotransfection assays. PLZF was found to be induced in colorectal cancer cell lines, correlating with a low detectable level of CUX1, a pattern that was reversed in normal human colonocytes. Reduction of p200CUX1 expression by RNAi in the Caco‐2/15 cell line increased PLZF gene transcript expression. Because of the implication of Plzf in the regulation of stem cell maintenance, as well as Wnt and Ras signaling, in other systems, our observations suggest that the novel genetic relationship between CUX1 and PLZF could be of relevance to human diseases, such as leukemia, and open up a new field of investigation for the implication of these regulators during intestinal polyposis and cancer.

Transcription factor CUX1 is required for intestinal epithelial wound healing and targets the VAV2-RAC1 Signalling complex

Intestinal epithelial cells form a protective barrier in limiting gut luminal content potentially harmful to the host. Upon gut epithelium injury, several signals instruct epithelial cells to undergo a rapid healing process. Defects in this process induce inflammatory responses and can further evolve into chronic gut inflammatory diseases. We previously identified the transcription factor CUX1 as crucial for protecting against experimental colitis in mice. However, the precise molecular mechanisms by which CUX1 intervenes during this biological process are unknown. Our aim was to evaluate CUX1 biological and functional roles during intestinal epithelial cell wound healing. RNAi knockdown of CUX1 in intestinal epithelial cells revealed a crucial role for this regulator in migratory response following wounding assays. Gene expression profiling identified several gene transcripts modulated in absence of CUX1 during wound healing for which a significant number was associated with cell motility and cytoskeleton function. Chromatin immunoprecipitation assays identified the guanine nucleotide exchange factor Vav2 gene as a direct target for CUX1. Coincidently, reduction of VAV2 in absence of CUX1 was associated with a significant decrease of RAC1 activity in response to epithelial wounding. Our results identify a novel pathway by which CUX1 regulates normal intestinal epithelial cell restitution.