Krüpple-like factor 5 is required for proper maintenance of adult intestinal crypt cellular proliferation.

Abstract

Krüpple-like factor 5 (KLF5) is a transcription factor that is highly expressed in the proliferative compartment of the intestinal crypt. There, it is thought to regulate epithelial turnover and homeostasis. In this study, we sought to determine the role for Klf5 in the maintenance of cellular proliferation, cytodifferentiation, and morphology of the crypt-villus axis. Tamoxifen-induced recombination directed by the epithelial-specific Villin promoter (in Villin-CreERT2 transgenic mice) was used to delete Klf5 (in Klf5 (loxP/loxP) mice) from the adult mouse intestine and analyzed by immunostaining and RT-qPCR. Control mice were tamoxifen-treated Klf5 (loxP/loxP) mice lacking Villin-CreERT2. Three days after tamoxifen-induced recombination, the mitosis marker phospho-histone H3 was significantly reduced within the Klf5-mutant crypt epithelium, coincident with increased expression of the apoptosis marker cleaved-caspase 3 within the crypt where cell death rarely occurs normally. We also observed a reduction in Chromagranin A expressing enteroendocrine cells, though no significant change was seen in other secretory or absorptive cell types. To examine the long-term repercussions of Klf5 loss, we killed mice 5, 14, and 28 days post recombination and found reemerging expression of KLF5. Furthermore, we observed restoration of cellular proliferation, though not to levels seen wildtype intestinal crypts. Reduction of apoptosis to levels comparable to the wildtype intestinal crypt was also observed at later time points. Analysis of cell cycle machinery indicated no significant perturbation upon deletion of Klf5; however, a reduction of stem cell markers Ascl2, Lgr5, and Olfm4 was observed at all time points following Klf5 deletion. These results indicate that Klf5 is necessary to maintain adult intestinal crypt proliferation and proper cellular differentiation. Rapid replacement of Klf5-mutant crypts with wildtype cells and reduction of stem cell markers suggests further that Klf5 is required for self renewal of intestinal stem cells.