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The mammalian intestine is a rapidly self-renewing organ with epithelial cell turn over occurring every 4-6 days. Along the length of the small and large intestine are crypt invaginations that contain rapidly proliferating progenitors and stem cells that continuously divide and give rise to all cell types. Regulation of this activity requires an intricate network of signaling and gene regulation to maintain homeostasis. KLF5 is a zinc-finger transcription factor that is found to be highly expressed in the proliferative crypts. It has been shown to mediate the onset and progression of diseases in the intestine; however its function in normal intestinal homeostasis is not well defined. To examine this role, we first utilized inducible knockout mouse models to delete KLF5 from the adult intestine, and found it to be required for normal proliferation. We discovered that loss of KLF5 correlated with decreased expression of active stem cell transcripts, Lgr5, Ascl2, and Olfm4. Restoration of protein expression in the crypt occurred within 14 days; however this had no impact on the loss of stem cell expression and did not restore progenitor proliferative to wildtype levels. We found a transient decrease in the enteroendocrine cells, but no other differentiated cell types were affected. These studies indicated that KLF5 is required for stem cell renewal and proliferation.We then specifically overexpressed KLF5 in the intestine discern its effects on proliferation, cytodifferentiation, and stem cell dynamics. After 2.5 days of overexpression villus blunting and crypt hyperplasia were associated with decreased villus cytodifferentiation and increased proliferative progenitor cell numbers in mice overexpressing KLF5 in the intestinal epithelium. While no effect was seen on stem cells, we were only able to examine these mice 2.5 days after induction as ectopic KLF5 expression in the intestine proved to be fatal by day 4. Results from these experiments indicated that this transcription factor could maintain and extend the transient amplifying zone and prevent intestinal cell differentiation.To elucidate a mechanism for KLF5’s effects on proliferation and stem cell maintenance we performed RNA-sequencing on isolated crypts obtained from both wildtype and knockout intestines. Differentially expressed genes were then broadly classified into gene ontologies. Results from this analysis indicated that loss of KLF5 could impact MAPK signaling. Utilizing jejunal tissue from both our knockout and transgenic mice, we discovered that KLF5 expression levels in the intestine correlated with the expression of activated ERK1/2 and MEK. We further investigated this finding by performing rescue experiments in vivo. We found that constitutively active KRAS could rescue proliferation in the absence of KLF5, but had no effect stem cell marker expression. These results were confirmed in vitro when we saw that activation of KRAS could rescue the loss of crypt formation seen in enteroids lacking KLF5. However, the mean survival of enteroids from both groups was significantly decreased when compared to mean survival of wildtype enteroids. This suggests that KLF5’s regulation MEK-ERK signaling is only relevant in the progenitor cell types indicating that KLF5 may differentially regulate stem and progenitor cell populations. |
