The Wnt/ß-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body ß-hydroxybutyrate (ßHB), contributes to the regulation of intestinal cell differentiation. Here, we have shown that HMGCS2 is a novel target of Wnt/ß-catenin/PPAR¿ signaling in intestinal epithelial cancer cell lines and normal intestinal organoids. Inhibition of the Wnt/ß-catenin pathway resulted in increased protein and mRNA expression of HMGCS2 and ßHB production in human colon cancer cell lines LS174T and Caco2. In addition, Wnt inhibition increased expression of PPAR¿ and its target genes, FABP2 and PLIN2, in these cells. Conversely, activation of Wnt/ß-catenin signaling decreased protein and mRNA levels of HMGCS2, ßHB production, and expression of PPAR¿ and its target genes in LS174T and Caco2 cells and mouse intestinal organoids. Moreover, inhibition of PPAR¿ reduced HMGCS2 expression and ßHB production, while activation of PPAR¿ increased HMGCS2 expression and ßHB synthesis. Furthermore, PPAR¿ bound the promoter of HMGCS2 and this binding was enhanced by ß-catenin knockdown. Finally, we showed that HMGCS2 inhibited, while Wnt/ß-catenin stimulated, glycolysis, which contributed to regulation of intestinal cell differentiation. Our results identified HMGCS2 as a downstream target of Wnt/ß-catenin/PPAR¿ signaling in intestinal epithelial cells. Moreover, our findings suggest that Wnt/ß-catenin/PPAR¿ signaling regulates intestinal cell differentiation, at least in part, through regulation of ketogenesis.

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