Frédéric Hollande

Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium.

The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active β-catenin–Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis.

Defective Claudin-7 Regulation by Tcf-4 and Sox-9 Disrupts the Polarity and Increases the Tumorigenicity of Colorectal Cancer Cells

Tight junctions have recently emerged as essential signaling regulators of proliferation and differentiation in epithelial tissues. Here, we aimed to identify the factors regulating claudin-7 expression in the colon, and analyzed the consequences of claudin-7 overexpression in colorectal carcinoma (CRC). In healthy human colonic crypts, claudin-7 expression was found to be low in the stem/progenitor cell compartment, where Tcf-4 activity is high, but strong in differentiated and postmitotic cells, where Tcf-4 is inactive. In contrast, claudin-7 was overexpressed in areas with high Tcf-4 target gene levels in CRC samples. In vitro, Tcf-4 was able to repress claudin-7 expression, and the high mobility group-box transcription factor Sox-9 was identified as an essential mediator of this effect. Claudin-7 was strongly expressed in the intestine of Sox-9-deficient mice and in CRC cells with low Sox transcriptional activity. Sox-9 overexpression in these cells reinstated claudin-7 repression, and residual claudin-7 was no longer localized along the basolateral membrane, but was instead restricted to tight junctions. Using HT-29Cl.16E CRC cell spheroids, we found that Sox-9-induced polarization was completely reversed after virus-mediated claudin-7 overexpression. Claudin-7 overexpression in this context increased Tcf-4 target gene expression, proliferation, and tumorigenicity after injection in nude mice. Our results indicate that Tcf-4 maintains low levels of claudin-7 at the bottom of colonic crypts, acting via Sox-9. This negative regulation seems to be defective in CRC, possibly due to decreased Sox-9 activity, and the resulting claudin-7 overexpression promotes a loss of tumor cell polarization and contributes to tumorigenesis.

Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells

Adhesion between neighbouring epithelial cells is a crucial and tightly controlled process. In the gastrointestinal tract, the integrity of cell-cell contacts is essential for the regulation of electrolyte absorption and for the prevention of tumour metastasis. We recently showed that migration of the gastric epithelial cell line IMGE-5 is stimulated by the nonamidated form of the hormone gastrin17. Here, we examine the effect on cell-cell adhesion of the prohormone progastrin, the concentration of which is increased in the plasma of patients with colorectal carcinoma. Progastrin induced the dissociation of both tight junction (TJ) and adherens junction (AJ) complexes in IMGE-5 cells. In progastrin-secreting DLD-1 human colorectal carcinoma cells, expression of an antisense gastrin construct restored membrane localisation of zonula occludens-1 (ZO-1), occludin, β-catenin and E-cadherin. This restoration was reversed by treatment with exogenous progastrin. Endogenous or exogenous progastrin also increased the paracellular flux of mannitol, and induced cell migration of several gastrointestinal cell lines. In addition, progastrin enhanced Src tyrosine kinase activity and induced a spatial delocalisation of protein kinase Cα. Using dominant-negative mutants and pharmacological inhibitors, we showed that the stimulation of Src kinase activity was essential for the regulation of TJs. By contrast, the dissociation of AJs involved phosphatidylinositol 3-kinase, partly through the formation of a complex with protein kinase Cα. We conclude that separate pathways mediate the disruption of AJs and TJs by progastrin. Either pathway may contribute to the co-carcinogenic role of this prohormone in colorectal carcinoma.

Pregnane × Receptor (PXR) expression in colorectal cancer cells restricts irinotecan chemosensitivity through enhanced SN-38 glucuronidation

BackgroundClinical efficacy of chemotherapy in colorectal cancer is subjected to broad inter-individual variations leading to the inability to predict outcome and toxicity. The topoisomerase I inhibitor irinotecan (CPT-11) is worldwide approved for the treatment of metastatic colorectal cancer and undergoes extensive peripheral and tumoral metabolism. PXR is a xenoreceptor activated by many drugs and environmental compounds regulating the expression of drug metabolism and transport genes in detoxification organs such as liver and gastrointestinal tract. Considering the metabolic pathway of irinotecan and the tissue distribution of Pregnane × Receptor (PXR), we hypothesized that PXR could play a key role in colon cancer cell response to irinotecan.ResultsPXR mRNA expression was quantified by RT-quantitative PCR in a panel of 14 colon tumor samples and their matched normal tissues. PXR expression was modulated in human colorectal cancer cells LS174T, SW480 and SW620 by transfection and siRNA strategies. Cellular response to irinotecan and its active metabolic SN38 was assessed by cell viability assays, HPLC metabolic profiles and mRNA quantification of PXR target genes. We showed that PXR was strongly expressed in colon tumor samples and displayed a great variability of expression. Expression of hPXR in human colorectal cancer cells led to a marked chemoresistance to the active metabolite SN38 correlated with PXR expression level. Metabolic profiles of SN38 showed a strong enhancement of SN38 glucuronidation to the inactive SN38G metabolite in PXR-expressing cells, correlated with an increase of UDPglucuronosyl transferases UGT1A1, UGT1A9 and UGT1A10 mRNAs. Inhibition of PXR expression by lentivirus-mediated shRNA, led to SN38 chemoresistance reversion concomitantly to a decrease of UGT1A1 expression and SN38 glucuronidation. Similarly, PXR mRNA expression levels correlated to UGT1A subfamily expression in human colon tumor biopsies.ConclusionOur results demonstrate that tumoral metabolism of SN38 is affected by PXR and point to potential therapeutic significance of PXR quantification in the prediction of irinotecan response. Furthermore, our observations are pharmacologically relevant since many patients suffering from cancer diseases are often exposed to co-medications, food additives or herbal supplements able to activate PXR. A substantial part of the variability observed among patients might be caused by such interactions

Biologically active recombinant human progastrin(6-80) contains a tightly bound calcium ion.

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin6–80 and to investigate its structure and biological activities in vitro. Human progastrin6–80 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin6–80 was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin6–80 at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin6–80 did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin6–80suggested that progastrin6–80 stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin6–80 is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

Loss of Parietal Cell Expression of Sonic Hedgehog Induces Hypergastrinemia and Hyperproliferation of Surface Mucous Cells

BACKGROUND & AIMS Sonic Hedgehog (Shh) is expressed in the adult stomach, but its role as a gastric morphogen is unclear. We sought to identify mechanisms by which Shh might regulate gastric epithelial cell function and differentiation. METHODS Mice with a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) were created. Gastric morphology and function were studied in control and HKCre/Shh(KO) mice between 1 and 8 months of age. RESULTS In contrast to control mice, HKCre/Shh(KO) mice developed gastric hypochlorhydria, hypergastrinemia, and a phenotype that resembled foveolar hyperplasia. The fundic mucosa of HKCre/Shh(KO) mice had an expanded surface pit cell lineage that was documented by increased incorporation of bromodeoxyuridine and was attributed to the hypergastrinemia. Compared with controls, numbers of total mucous neck and zymogen cells were significantly decreased in stomachs of HKCre/Shh(KO) mice. In addition, zymogen and neck cell markers were coexpressed in the same cell populations, indicating disrupted differentiation of the zymogen cell lineage from the mucous neck cells in the stomachs of HKCre/Shh(KO) mice. Laser capture microdissection of the surface epithelium, followed by quantitative reverse-transcription polymerase chain reaction, revealed a significant increase in expression of Indian Hedgehog, glioma-associated oncogene homolog 1, Wnt, and cyclin D1. Laser capture microdissection analysis also showed a significant increase in Snail with a concomitant decrease in E-cadherin. CONCLUSIONS In the stomachs of adult mice, loss of Shh from parietal cells results in hypochlorhydria and hypergastrinemia. Hypergastrinemia might subsequently induce increased Hedgehog and Wnt signaling in the surface pit epithelium, resulting in hyperproliferation.

cAMP Response Element Binding Protein Is Required for Mouse Neural Progenitor Cell Survival and Expansion

Development of the mammalian brain relies on the coordinated expansion of neural cells in a relatively short time, spanning for a period of only a few days in mice. The molecular networks regulating neural cell birth and expansion, termed neurogenesis, are still unresolved, although many studies using genetically modified mice have revealed a growing number of genes that are involved in regulating these processes. The cAMP response element binding protein (CREB) lies at the hub of a diverse array of intracellular signaling pathways and is a major transcriptional regulator of numerous functions in adult neural cells, including learning and memory and neuronal survival. Recent studies have shown that activated CREB is highly expressed in immature dividing cells in adult mouse and zebrafish brains and that CREB regulates neural stem/progenitor cells (NSPCs) proliferation in embryonic zebrafish brain. Using genetically modified mice, we show that deletion of CREB, without the concomitant loss of the related compensating factor cAMP response element modifier, leads to defects in neural progenitor cell expansion and survival. Cultured primary CREB−/− NSPCs exhibited decreased expression of several target genes important for neuronal survival and growth, including brain‐derived neurotrophic factor and neural growth factor and showed that the survival and growth defect can be rescued by the addition of wild‐type NSPC‐conditioned medium. This is the first study showing a specific role for CREB in mammalian embryonic neurogenesis. This role appears to be mediated via the expression of factors important for NSPC survival and growth and suggests that CREB is an important signaling regulator within the developing neurogenic niche. STEM CELLS 2009;27:1347–1357

Functional interaction between the ZO-1-interacting transcription factor ZONAB/DbpA and the RNA processing factor symplekin

Epithelial tight junctions participate in the regulation of gene expression by controlling the activity of transcription factors that can interact with junctional components. One such protein is the Y-box transcription factor ZONAB/DbpA that binds to ZO-1, a component of the junctional plaque. Symplekin, another nuclear protein that can associate with tight junctions, functions in the regulation of polyadenylation and thereby promotes gene expression. Here, we addressed the question of whether these two proteins interact and whether this is of functional relevance. We demonstrate that ZONAB/DbpA and symplekin form a complex in kidney and intestinal epithelial cells that can be immunoprecipitated and that exists in the nucleus. The interaction between ZONAB/DbpA and symplekin can be reconstituted with recombinant proteins. In reporter gene assays in which ZONAB/DbpA functions as a repressor, symplekin functionally interacts with ZONAB/DbpA, indicating that symplekin can also promote transcriptional repression. RNAi experiments indicate that symplekin depletion reduces the nuclear accumulation and the transcriptional activity of ZONAB/DbpA in colon adenocarcinoma cells, resulting in inhibition of proliferation and reduced expression of the ZONAB/DbpA-target gene cyclin D1. Our data thus indicate that symplekin and ZONAB/DbpA cooperate in the regulation of transcription, and that they promote epithelial proliferation and cyclin D1 expression.

Gastric Sonic Hedgehog acts as a macrophage chemoattractant during the immune response to Helicobacter pylori.

BACKGROUND & AIMS Macrophages mediate the epithelial response to Helicobacter pylori and are involved in the development of gastritis. Sonic Hedgehog (Shh) regulates gastric epithelial differentiation and function, but little is known about its immunoregulatory role in the stomach. We investigated whether gastric Shh acts as a macrophage chemoattractant during the innate immune response to H pylori infection. METHODS Mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and control mice were infected with H pylori. Levels of gastric Shh, cytokines, and chemokines were assayed by quantitative reverse-transcriptase polymerase chain reaction or by a Luminex-based multiplex assay 2, 7, or 180 days after infection. Circulating concentrations of Shh were measured by enzyme-linked immunosorbent assay. Bone marrow chimera experiments were performed with mice that have myeloid cell-specific deletion of the Hedgehog signal transduction protein Smoothened (LysMCre/Smo(KO)). Macrophage recruitment was measured in gastric tissue and peripheral blood by fluorescence-activated cell sorting analysis. RESULTS Control mice infected with H pylori for 6 months developed an inflammatory response characterized by infiltration of CD4(+) T cells and increased levels of interferon gamma and interleukin 1β in the stomach. PC-Shh(KO) mice did not develop gastritis, even after 6 months of infection with H pylori. Control mice had increased concentrations of Shh, accompanied by the recruitment of CD11b(+)F4/80(+)Ly6C(high) macrophages 2 days after infection. Control mice that received bone marrow transplants from control mice had an influx of macrophages to the gastric mucosa in response to H pylori infection; this was not observed in H pylori-infected control mice that received bone marrow transplants from LysMCre/Smo(KO) mice. CONCLUSIONS H pylori induces release of Shh from the stomach; Shh acts as a macrophage chemoattractant during initiation of gastritis.

The Wnt Target Jagged-1 Mediates the Activation of Notch Signaling by Progastrin in Human Colorectal Cancer Cells

The Wnt and Notch signaling pathways are both abnormally activated in colorectal cancer (CRC). We recently showed that progastrin depletion inhibited Wnt signaling and increased goblet cell differentiation of CRC cells. Here, we show that progastrin down-regulation restores the expression by CRC cells of the early secretory lineage marker Math-1/Hath-1 due to an inhibition of Notch signaling. This effect is mediated by a decreased transcription of the Notch ligand Jagged-1, downstream of beta-catenin/Tcf-4. Accordingly, recombinant progastrin sequentially activated the transcription of Wnt and Notch target genes in progastrin-depleted cells. In addition, restoration of Jagged-1 levels in these cells is sufficient to activate Tcf-4 activity, demonstrating the occurrence of a feedback regulation from Notch toward Wnt signaling. These results suggest that progastrin could be instrumental in maintaining the concomitant activation of Wnt and Notch pathways in CRC cells, further highlighting the interest of progastrin targeting for the clinical management of CRC.