MElal, a 16-base-pair nuclear factor binding site residing between the c-MYC P1 and P2 transcription initiation sites, is required for P2 activity.

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Constitutive activation of the MAP kinase kinase MEK1 induces oncogenic transformation in intestinal epithelial cells. Loss of cellcell adhesion followed by the dissociation of epithelial structures is a prerequisite for increased cell motility and tumor invasion. This phenotypic switch is designated epithelial-to-mesenchymal transition (EMT). EMT also plays an important role in determining the dissemination of tumors. However, the role of MEK1 in intestinal EMT, tumor invasion and metastasis has not been elucidated. To determine the functions of activated MEK1 in intestinal tumorigenesis, we established intestinal epithelial cell lines that overexpress wild-type MEK1 (wtMEK) or activated MEK1 (caMEK). Our results indicate that expression of caMEK is sufficient to induce EMT as confirmed with the induction of N-cadherin, vimentin, Snail1 and Snail2, whereas a reduction in E-cadherin, occludin, ZO-1 and cortical F-actin was noted. The Snail1 and Snail2 promoter analyses revealed that Egr-1 and Fra-1, an AP-1 protein, are responsible for MEK1-induced Snail1 and Snail2 expression, respectively. Cells expressing activated MEK1 clearly acquired an invasive capacity when compared to wtMEK-expressing cells. Zymography studies confirmed elevated levels of MMP2 and MMP9 activities in media of caMEK-expressing cells. Importantly, cells expressing activated MEK1 induced tumors with short latency in correlation with their ability to induce experimental metastasis in vivo and to express factors known to promote colorectal cancer cell metastasis. In conclusion, our results demonstrate, for the first time, that constitutive activation of MEK1 in intestinal epithelial cells is sufficient to induce an EMT associated with tumor invasion and metastasis. ' UICCKey words: MEK; ERK; intestinal epithelium; epithelialmesenchymal transition; snail; metastasis Ras proteins act as molecular switches that cycle between active GTP-bound and inactive GDP-bound forms and function as essential components of signal transduction pathways regulating cell growth. Activating mutations of the Ras family members are among the most common genetic events in human tumorigenesis. 1 For example, K-ras is mutated in nearly 50% of colorectal tumors at a relatively early stage of the carcinogenic process 2 and despite extensive research, the primary reason for this high frequency remains unclear.The most studied downstream effector pathway of K-ras is the mitogenic serine/threonine kinase cascade called Raf/MEK/mitogen-activated protein kinase (MAPK). Indeed, upon activation by growth factor-stimulated receptors, activated Ras complexes with and promotes Raf kinases, which in turn activate MAPK kinases (MEK1 and MEK2), resulting in activation of extracellular signal-regulated kinases (ERK1 and ERK2). 3 Activated ERKs then translocate into the nucleus where they phosphorylate and activate nuclear transcription factors, such as Elk-1, ATF-2 and ETS1/2 resulting in immediate-early gene induction. 4 Studies on cultured intestinal epithelial cells and many othe...

Acetylation and deacetylation of histones and other proteins depends on histone acetyltransferases and histone deacetylases (HDACs) activities, leading to either positive or negative gene expression. HDAC inhibitors have uncovered a role for HDACs in proliferation, apoptosis and inflammation. However, little is known of the roles of specific HDACs in intestinal epithelial cells (IEC). We investigated the consequences of ablating both HDAC1 and HDAC2 in murine IECs. Floxed Hdac1 and Hdac2 homozygous mice were crossed with villin-Cre mice. Mice deficient in both IEC HDAC1 and HDAC2 weighed less and survived more than a year. Colon and small intestinal sections were stained with hematoxylin and eosin, or with Alcian blue and Periodic Acid Schiff for goblet cell identification. Tissue sections from mice injected with BrdU for 2 h, 14 h and 48 h were stained with anti-BrdU. To determine intestinal permeability, 4-kDa FITC-labeled dextran was given by gavage for 3 h. Microarray analysis was performed on total colon RNAs. Inflammatory and IEC-specific gene expression was assessed by Western blot or semi-quantitative RT-PCR and qPCR with respectively total colon protein and total colon RNAs. HDAC1 and HDAC2-deficient mice displayed: 1) increased migration and proliferation, with elevated cyclin D1 expression and phosphorylated S6 ribosomal protein, a downstream mTOR target; 2) tissue architecture defects with cell differentiation alterations, correlating with reduction of secretory Paneth and goblet cells in jejunum and goblet cells in colon, increased expression of enterocytic markers such as sucrase-isomaltase in the colon, increased expression of cleaved Notch1 and augmented intestinal permeability; 3) loss of tissue homeostasis, as evidenced by modifications of claudin 3 expression, caspase-3 cleavage and Stat3 phosphorylation; 4) chronic inflammation, as determined by inflammatory molecular expression signatures and altered inflammatory gene expression. Thus, epithelial HDAC1 and HDAC2 restrain the intestinal inflammatory response, by regulating intestinal epithelial cell proliferation and differentiation.