P-169 Claudin 23 Regulation by TCF7l2 and HNF4-Alpha Promotes Epithelial Barrier Function

Colonic enterocytes form a rapidly renewing epithelium and barrier to luminal antigens. During colonocyte differentiation, coordinated claudin gene expression is vital to epithelia barrier maintenance and disruption of this process is believed to play a role in inflammatory diseases. However, little is known about the regulation of this critical aspect of epithelial cell differentiation. We therefore sought to determine factors that regulate claudin gene expression along the colonic crypt-base-to-surface cell axis. Using high-throughput transcriptome surveys of laser-capture micro-dissected cells, we identified nearly 3 thousand differentially expressed genes between the crypt-base and surface cell populations. Real-time PCR confirmed differential claudin expression gradients and detected 11 claudin genes throughout the tissue. Quantitative ddPCR revealed stable total claudin transcript levels along the crypt axis with heterogeneous expression within the family. Expression correlation studies then identified the transcription factors Hnf4-α and Tcf7l2 as potential claudin regulators. Using combined in vitro and in vivo methods we identified Tcf7l2 as a global activator of claudin family genes, with coincident enhancement of Hnf4-α expression. In contrast, Hnf4-α specifically upregulated claudin 23. Importantly, Hnf4-α conditional knockout mice failed to induce the claudin 23 gene in surface cells populations. From these findings we conclude that the enterocyte claudin differentiation cascade includes Tcf7l2 activation of Hnf4-α, and subsequent claudin 23 expression, thereby strengthening the colonic barrier.