E-CADHERIN mRNA IS DIFFERENTIALLY EXPRESSED DURING CACO-2 CELL DIFFERENTIATION: IDENTIFICATION BY DIFFERENTIAL DISPLAY AND ANALYSIS OF PROMOTER FUNCTION

Abstract

Caco-2 cells differentiate spontaneously during culture, taking on morphologic and biochemical properties of mature small bowel enterocytes. To identify putative differentiation-regulating mRNAs, we used a differential display of mRNA technique (RAP PCR) to analyze multiple transcripts expressed during epithelial differentiation of Caco-2 cells. METHODS: RAP-PCR was performed on poly-A+ RNA purified from Caco-2 cells at days 3,5,7,9, and 12 of culture. A random primer was used under low stringency conditions for first-strand cDNA synthesis. Subsequent amplification of the resulting wide range of cDNAs was performed using the same primer. Individual bands of interest were cut from the gel and cloned into pCDNA. To verify the expression pattern during differentiation, Northern blot analysis was next performed, using clones of interest as the probe. Electrophoretic mobility shift assay (EMSA) was performed for one such clone, E-cadherin, using nuclear proteins isolated during Caco-2 cell differentiation and oligonucleotide probes derived from an E-cadherin promoter SP-1 site. Activity of this promoter in Caco-2 cells was measured using transient transfection of an E-cadherin-green fluorescent protein plasmid construct (pEGFP-Ecad). RESULTS: One band identified by RAP-PCR was prominently expressed at day 3 and again at days 9 and 12, suggesting a possible regulatory role. Sequence analysis of this clone identified it as being human E-cadherin, a cell adhesion molecule known to be important in regulation of the epithelial phenotype. EMSA analysis demonstrated multiple shifted bands which varied during differentiation. Two such bands were present when E-cadherin expression was low, and may represent inhibitory transcription factors. Another was present in a pattern mirroring cadherin mRNA expression, likely representing a transcription-enhancing factor. Caco-2 cells were next shown to activate the E-cadherin promoter when transfected with pEGFP-Ecad. CONCLUSIONS: E-cadherin mRNA is expressed by Caco-2 cells in a differentiation-dependent manner. E-cadherin promoter activity is regulated in a parallel fashion, suggesting direct transcriptional control of E-cadherin expression. E-cadherin is likely an important regulator of Caco-2 cell differentiation.