Christine Jones

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 β‐catenin protein levels, nor is it sufficient to promote tumorigenesis initiation. However, it severely enhances intestinal tumor load in ApcMin/+ 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.—Langlois, M.‐J., Roy, S. A. B., Auclair, B. A., Jones, C., Boudreau, F., Carrier, J. C., Rivard, N., Perreault, N. Epithelial phosphatase and tensin homolog regulates intestinal architecture and secretory cell commitment and acts as a modifier gene in neoplasia. FASEB J. 23, 1835–1844 (2009)

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.

Loss of Sonic Hedgehog Leads to Alterations in Intestinal Secretory Cell Maturation and Autophagy

Background Intestinal epithelial cells express the Sonic and Indian hedgehog ligands. Despite the strong interest in gut hedgehog signaling in GI diseases, no studies have specifically addressed the singular role of intestinal epithelial cell Sonic hedgehog signaling. The aim of this study was to investigate the specific role of Sonic hedgehog in adult ileal epithelial homeostasis. Methodology/Principal Findings A Sonic hedgehog intestinal epithelial conditional knockout mouse model was generated. Assessment of ileal histological abnormalities, crypt epithelial cell proliferation, epithelial cell fate, junctional proteins, signaling pathways, as well as ultrastructural analysis of intracellular organelles were performed in control and mutant mice. Mice lacking intestinal epithelial Sonic Hedgehog displayed decreased ileal crypt/villus length, decreased crypt proliferation as well as a decrease in the number of ileal mucin-secreting goblet cells and antimicrobial peptide-secreting Paneth cells during adult life. These secretory cells also exhibited disruption of their secretory products in mutant mice. Ultrastructural microscopy analysis revealed a dilated ER lumen in secretory cells. This phenotype was also associated with a decrease in autophagy. Conclusions/Significance Altogether, these findings indicate that the loss of Sonic hedgehog can lead to ileal secretory cell modifications indicative of endoplasmic reticulum stress, accompanied by a significant reduction in autophagy.

Identification of a novel promyelocytic leukemia zinc‐finger isoform required for colorectal cancer cell growth and survival

Promyelocytic leukemia zinc‐finger (PLZF) is a transcriptional repressor that regulates proliferation, differentiation and apoptosis among various cellular origins. PLZF expression is upregulated in colorectal cancer cell lines but its putative functional role in this context is unknown. Here, we report the identification of a novel p65 PLZF isoform that results from the usage of an evolutionarily conserved alternative translational initiation site. This isoform is devoid of the classical BTB/POZ domain required for nuclear localization and transcriptional repression. Depletion of p65 PLZF expression in colorectal cancer cell lines results in reduction of cell growth, loss of cell anchorage and increase in cell apoptosis. Overall, these results indicate that p65 PLZF is crucial to maintain colorectal cancer cell adhesion as well as survival and must occur independently of the traditionally viewed transcriptional role of PLZF in the course of these biological processes.

A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids

Organoids have the potential to bridge 3D cell culture to tissue physiology by providing a model resembling in vivo organs. Long-term growing organoids were first isolated from intestinal crypt cells and recreated the renewing intestinal epithelial niche. Since then, this technical breakthrough was applied to many other organs, including prostate, liver, kidney and pancreas. We describe here how to apply a SILAC-based quantitative proteomic approach to measure protein expression changes in intestinal organoids under different experimental conditions. We generated SILAC organoid media that allow organoids to grow and differentiate normally, and confirmed the incorporation of isotopically labelled amino acids. Furthermore, we used a treatment reported to affect organoid differentiation to demonstrate the reproducibility of the quantification using this approach and to validate the identification of proteins that correlate with the inhibition of cellular growth and development. With the combined use of quantitative mass spectrometry, SILAC and organoid culture, we validated this approach and showed that large-scale proteome variations can be measured in an “organ-like” system.

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.

Subcellular proteomics analysis of different stages of colorectal cancer cell lines.

Studying cell differentiation and transformation allows a better understanding of the mechanisms involved in the initiation and the evolution of cancer. The role of proteins which participate in these processes is dependent on their location within the cell. Determining the subcellular localization of proteins or the changes in localization is, therefore, paramount in elucidating their role. Using quantitative mass spectrometry, we characterized the protein expression and subcellular localization of nearly 5000 proteins from seven different colorectal cancer (CRC) cell lines, as well as normal colon fibroblasts and intestinal epithelial cells. This cellular characterization allowed the identification of colon cancer‐associated proteins with differential expression patterns as well as deregulated protein networks and pathways. Indeed, our results demonstrate differential expression of proteins involved in cell adhesion, cytoskeleton, and transcription in colon cancer cells compared to normal colon‐derived cells. Pathway analyses identified different cellular functions, including endocytosis and eIF2 signaling, whose deregulation correlates with mutations found in the different CRC phenotypes. Our results provide an unbiased, quantitative and high‐throughput approach to measure changes in protein expression and subcellular protein locations in different CRC cell lines.

Identification of GATA-4 as a novel transcriptional regulatory component of regenerating islet-derived family members.

Intestinal epithelial cells are exposed to luminal bacterial threat and require adequate defense mechanisms to ensure host protection and epithelium regeneration against possible deleterious damage. Differentiated intestinal epithelial cells produce antimicrobial and regenerative components that protect against such challenges. Few intestinal specific transcription factors have been identified to control the switching from repression to activation of this class of gene. Herein, we show that gene transcription of some regenerating islet-derived (REG) family members is dependent on the transcription factor GATA-4. Silencing of GATA-4 expression in cultured intestinal epithelial cells identified Reg3β as a target gene using an unbiased approach of gene expression profiling. Co-transfection and RNA interference assays identified complex GATA-4-interactive transcriptional components required for the activation or repression of Reg3β gene activity. Conditional deletion of Gata4 in the mouse intestinal epithelium supported its regulatory role for Reg1, Reg3α, Reg3β and Reg3γ genes. Reg1 dramatic down-modulation of expression in Gata4 conditional null mice was associated with a significant decrease in intestinal epithelial cell migration. Altogether, these results identify a novel and complex role for GATA-4 in the regulation of REG family members gene expression.

P1 promoter-driven HNF4α isoforms are specifically repressed by β-catenin signaling in colorectal cancer cells

ABSTRACT HNF4α is a key nuclear receptor for regulating gene expression in the gut. Although both P1 and P2 isoform classes of HNF4α are expressed in colonic epithelium, specific inhibition of P1 isoforms is commonly found in colorectal cancer. Previous studies have suggested that P1 and P2 isoforms might regulate different cellular functions. Despite these advances, it remains unclear whether these isoform classes are functionally divergent in the context of human biology. Here, the consequences of specific inhibition of P1 or P2 isoform expression was measured in a human colorectal cancer cell transcriptome. Results indicate that P1 isoforms were specifically associated with the control of cell metabolism, whereas P2 isoforms globally supported aberrant oncogenic signalization, promoting cancer cell survival and progression. P1 promoter-driven isoform expression was found to be repressed by β-catenin, one of the earliest oncogenic pathways to be activated during colon tumorigenesis. These findings identify a novel cascade by which the expression of P1 isoforms is rapidly shut down in the early stages of colon tumorigenesis, allowing a change in HNF4α-dependent transcriptome, thereby promoting colorectal cancer progression. This article has an associated First Person interview with the first author of the paper. Summary: This study demonstrates that P1 and P2 promoter-driven HNF4α isoforms regulate different biologically relevant transcriptomic signatures in colorectal cancer cells, and are differently regulated from oncogenic β-catenin activation.

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.