Key Role of the Cdx2 Homeobox Gene in Extracellular Matrix–mediated Intestinal Cell Differentiation

Abstract

To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell–cell and cell–substratum interactions and in transduction process: indeed, E-cadherin, integrin-β4 subunit, laminin-γ2 chain, hemidesmosomal protein, APC, and α-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures.Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-α1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-α1 chain mRNA and that of sucrase-isomaltase expression in these cell lines.Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix–mediated intestinal cell differentiation.

The intestine-specific homeobox gene Cdx2 decreases mobility and antagonizes dissemination of colon cancer cells

“…Cdx2 plays a key role in intestinal differentiation by upregulating intestinal-specific genes and inhibiting cell proliferation, which may contribute to antagonize cancer cell dissemination. In addition, the inhibitory effect of Cdx2 on cell migration and spreading is in line with the fact that it controls the expression of extracellular matrix molecules and stimulates LI-cadherin and E-cadherin levels (Lorentz et al, 1997;Hinoi et al, 2002;Keller et al, 2004). Moreover, we showed here that the decline of Cdx2 correlates with increased amounts of integrin-b1 and the metalloproteinase MMP2, two molecules involved in tumor cell invasion.…”

Hedgehog signalling in colorectal tumour cells: Induction of apoptosis with cyclopamine treatment

“…Single-stranded cDNA was prepared from 10 g RNA in a 50 l volume containing 100 pmoles oligo-dT, 15 units AMV reverse transcriptase (Promega, Southampton, UK) and 0.4 mM each dNTP, in appropriate buffer. Primers were designed using Lasergene primer design software (DNAStar, Madison, WI) with the exception of positive control primers to the ribosomal protein 36B4, which were described by Lorentz et al 18 cDNA (5 l) was amplified in 100 l PCRs containing 50 pmoles of each primer, 200 M dNTPs and 0.5 units of Taq DNA polymerase (Roche, Lewes, UK) in the buffer supplied. PCR was carried out for 35 cycles.…”

Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras

“…Semi-quantitative RT ± PCR was performed as described previously Lorentz et al, 1997) for increasing number of cycles (24 ± 40 cycles). The speci®c primers used for the Cdx-1, Cdx-2, c-jun and c-fos mRNAs were respectively cdx1 a/b: dGCGCCAAGGCG-GACG CCCTACGAAT / d TGTTTACTTTGCGCTCCTT-GGCCCG , cdx2 b/c: dCCCA-GCGGCCAGCGGCGAAACCTGT / dTATTTGTCTTTT-GTCCTGGTTTTCA , c-jun a/b: dGAA TTGTC AAAGAGAAGATTCCAAA / dGAGTTG-AAAGAGTTAAGAATGCTCG and c-fos a/b: dAAACA-CATCTTCCCTAGAGGGTTCC / dACACA CTATTGCC-AGGAACACAGTA.…”

“…Cdx-1 is mainly expressed in the crypt compartment although not restricted to proliferative cells (Silberg et al, 1997), and its inhibition by antisense RNA reduces cell proliferation in vitro . The CDX-2 homeoprotein occurs mainly in the differentiating enterocytes (James et al, 1994) and it triggers growth retardation and cell di erentiation in several intestinal lines in vitro (Suh and Traber, 1996;Lorentz et al, 1997). In addition, Cdx-1 and Cdx-2 are controlled by epithelial-mesenchymal cell interactions , via extracellular matrix components .…”