Philippe Blache

SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes

TCF and SOX proteins belong to the high mobility group box transcription factor family. Whereas TCFs, the transcriptional effectors of the Wnt pathway, have been widely implicated in the development, homeostasis and disease of the intestine epithelium, little is known about the function of the SOX proteins in this tissue. Here, we identified SOX9 in a SOX expression screening in the mouse fetal intestine. We report that the SOX9 protein is expressed in the intestinal epithelium in a pattern characteristic of Wnt targets. We provide in vitro and in vivo evidence that a bipartite β-catenin/TCF4 transcription factor, the effector of the Wnt signaling pathway, is required for SOX9 expression in epithelial cells. Finally, in colon epithelium-derived cells, SOX9 transcriptionally represses the CDX2 and MUC2 genes, normally expressed in the mature villus cells of the intestinal epithelium, and may therefore contribute to the Wnt-dependent maintenance of a progenitor cell phenotype.

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.

Expression of the Carcinoembryonic Antigen Gene Is Inhibited by SOX9 in Human Colon Carcinoma Cells

The human carcinoembryonic antigen (CEA) is overexpressed in many types of human cancers and is commonly used as a clinical marker. In colon cancer, this overexpression protects cells against apoptosis and contributes to carcinogenesis. Therefore, CEA-expressing cells as well as CEA expression itself constitute potential therapeutic targets. In this report, we show that the transcription factor SOX9 down-regulates CEA gene expression and, as a probable consequence, induces apoptosis in the human colon carcinoma cell line HT29Cl.16E.

Metabolic clearance rates of oxyntomodulin and glucagon in the rat : contribution of the kidney

The half-life (t1/2) and metabolic clearance rate (MCR) of exogenous natural porcine oxyntomodulin (porcine OXM) and the synthetic analog of rat oxyntomodulin, [Nle27]-OXM (rat OXM), were compared with that of glucagon in control, sham-operated and acutely nephrectomized rats using the primed-continuous infusion technique. The half-disappearance times for porcine OXM (8.2 +/- 0.5 min) and rat OXM (6.4 +/- 0.5 min) were 3-fold slower than that of glucagon (1.9 +/- 0.1 min). Acute bilateral nephrectomy significantly prolonged the half-disappearance time of rat OXM (8.2 +/- 0.7 min) and glucagon (3.6 +/- 0.4 min) compared with that of sham-operated animals (6.5 +/- 0.8 min and 2.5 +/- 0.2 min, respectively). The mean MCRs were similar for porcine and rat OXM (11.3 +/- 0.7 and 11.9 +/- 0.5 ml.kg-1.min-1) but were 3 times lower than that measured with glucagon (36 +/- 5 ml.kg-1.min-1). Bilateral nephrectomy reduced the MCR of OXM and glucagon by 38% and 34%, respectively. No significant increase in C-terminal glucagon immunoreactivity was noticed during infusion of either porcine or rat OXM, measured directly in plasma, with a specific C-terminal glucagon antiserum or after HPLC. In the course of the glucagon infusion, blood glucose was increased 2-fold, while the same dose of porcine OXM or of rat OXM induced only a small increase over the values in phosphate buffer-infused rats. 10 times higher doses of rat OXM were necessary to obtain a similar hyperglycemic effect. These results indicate that: (1) the metabolism of OXM is 3-fold slower than that of glucagon, (2) renal clearance contributed close to 35% of the overall metabolic plasma extraction for OXM and glucagon and (3) OXM, although effective at a higher dose, when compared with glucagon, displays a hyperglycemic effect probably through the glucagon receptors.

Creating and exploiting multimodal annotated corpora: the ToMA project

The paper presents a project aiming at collecting, annotating and exploiting a dialogue corpus from a multimodal perspective. The goal of the project is the description of the different parameters involved in a natural interaction process. Describing such complex mechanism requires corpora annotated in different domains. This paper first presents the corpus and the scheme used in order to annotate the different domains that have to be taken into consideration, namely phonetics, morphology, syntax, prosody, discourse and gestures. Several examples illustrating the interest of such a resource are then proposed.

CEACAM1 , a SOX9 direct transcriptional target identified in the colon epithelium

A deletion of the transcription factor SOX9 gene in the mice intestine affects the morphology of the colon epithelium and leads to hyperplasia. Nevertheless, direct transcriptional targets of SOX9 in this tissue are still unknown. A microarray analysis identified the tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as a possible SOX9 target gene and we demonstrate here that SOX9 upregulates CEACAM1 in human colonic cells. Moreover, CEACAM1 expression is reduced in colon of SOX9-deficient mouse, suggesting an important function for SOX9 in the transcriptional activation of the CEACAM1 gene. We further identified SOX9-binding sequences in the human and rat CEACAM1 promoters, and an electrophoretic mobility shift together with a chromatin immunoprecipitation provided an additional evidence of the SOX9 binding to the human promoter. In addition, we established that histone acyl-transferase p300 behaves as an SOX9 co-activator of the rat and human CEACAM1promoters. These results highlight CEACAM1 as the first direct target of SOX9 identified in the colon epithelium.

Immunological detection of prohormone convertases in two different proglucagon processing cell lines

The distribution of the prohormone convertases, PCI/3, PC2 and PC5/6, was determined by immunoblotting in two cell lines. In αTC1–6 cells, the proglucagon processing occured according to the pancreatic A‐cell type. In STC‐1 cells, proglucagon was processed in a manner reminiscent of the intestinal L‐cell type. PC1/3 was undetectable in both proglucagon processing cell lines whereas PC2 displayed a strong immunostaining in the αTC1–6 cells and was barely detectable in the STC‐1 cells. PC5/6 was detected as a 70 kDa protein in both cell lines. These results suggest a possible role of PC2 in the processing of proglucagon into glucagon in the A‐cells, whereas in L‐cells it would require still undetermined endoproteases.

α-Endosulfine, a new entity in the control of insulin secretion

Abstract. ATP-dependent potassium (KATP) channels occupy a key position in the control of insulin release from the pancreatic β cell since they couple cell polarity to metabolism. These channels close when more ATP is produced via glucose metabolism. They are also controlled by sulfonylureas, a class of drugs used in type 2 diabetic patients for triggering insulin secretion from β cells that have lost part of their sensitivity to glucose. We have demonstrated the existence of endogenous counterparts to sulfonylureas which we have called ‘endosulfines.’ In this review, we describe the discovery, isolation, cloning, and biological features of the high-molecular-mass form, α-endosulfine, and discuss its possible role in the physiology of the β cell as well as in pathology.

A new mechanism of SOX9 action to regulate PKCα expression in the intestine epithelium

Variations of protein kinase C (PKC) expression greatly influence the proliferation-to-differentiation transition (PDT) of intestinal epithelial cells and might have an important impact on intestinal tumorigenesis. We demonstrate here that the expression of PKCα in proliferating intestinal epithelial cells is repressed both in vitro and in vivo by the SOX9 transcription factor. This repression does not require DNA binding of the SOX9 high-mobility group (HMG) domain but is mediated through a new mechanism of SOX9 action requiring the central and highly conserved region of SOXE members. Because SOX9 expression is itself upregulated by Wnt-APC signaling in intestinal epithelial cells, the present study points out this transcription factor as a molecular link between the Wnt-APC pathway and PKCα. These results provide a potential explanation for the decrease of PKCα expression in colorectal cancers with constitutive activation of the Wnt-APC pathway.