All-trans retinoic acid inhibits proliferation of intestinal epithelial cells by inhibiting expression of the gene encoding Krüppel-like factor 5

AbstractRetinoids are known inhibitors of epithelial cell proliferation. Previous studies indicate that Krüppel-like factor 5 (KLF5) is a pro-proliferative transcription factor. Here, we examined the effect of all-trans retinoid acid (ATRA) on proliferation of the intestinal epithelial cell line, IEC6. Treatment of IEC6 cells with ATRA inhibited their proliferation due to G1 cell cycle arrest. This inhibition was correlated with a decrease in the levels of KLF5 mRNA and promoter activity. In contrast, constitutive expression of KLF5 in stably transfected IEC6 cells with a KLF5-expressing plasmid driven by a viral promoter abrogated the growth inhibitory effect of ATRA. Moreover, ATRA inhibited proliferation of several human colon cancer cell lines with high levels of KLF5 expression but not those with low levels of KLF5 expression. Our results indicate that KLF5 is a potential mediator for the inhibitory effect of ATRA on intestinal epithelial cell proliferation

Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined ApcMin and KRASV12 mutations

<p>Abstract</p> <p>Background</p> <p>Both mutational inactivation of the adenomatous polyposis coli (<it>APC</it>) tumor suppressor gene and activation of the <it>KRAS </it>oncogene are implicated in the pathogenesis of colorectal cancer. Mice harboring a germline <it>Apc</it>^{<it>Min </it>}mutation or intestine-specific expression of the <it>KRAS</it>^<it>V</it>12gene have been developed. Both mouse strains develop spontaneous intestinal tumors, including adenoma and carcinoma, though at a different age. The zinc finger transcription factor Krüppel-like factor 5 (KLF5) has previously been shown to promote proliferation of intestinal epithelial cells and modulate intestinal tumorigenesis. Here we investigated the <it>in vivo </it>effect of <it>Klf5 </it>heterozygosity on the propensity of <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12double transgenic mice to develop intestinal tumors.</p> <p>Results</p> <p>At 12 weeks of age, <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12mice had three times as many intestinal tumors as <it>Apc</it>^{<it>Min </it>}mice. This increase in tumor number was reduced by 92% in triple transgenic <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12/<it>Klf5</it>^+/-mice. The reduction in tumor number in <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12/<it>Klf5</it>^+/-mice was also statistically significant compared to <it>Apc</it>^{<it>Min </it>}mice alone, with a 75% decrease. Compared with <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12, tumors from both <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12/<it>Klf5</it>^+/-and <it>Apc</it>^{<it>Min </it>}mice were smaller. In addition, tumors from <it>Apc</it>^{<it>Min </it>}mice were more distally distributed in the intestine as contrasted by the more proximal distribution in <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12and <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12/<it>Klf5</it>^+/-mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both <it>Apc</it>^{<it>Min </it>}and <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12mice but were attenuated in <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12/<it>Klf5</it>^+/-mice. The levels of β-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12/<it>Klf5</it>^+/-mice when compared to <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12mice and modestly reduced in Apc^Min/<it>KRAS</it>^<it>V</it>12/<it>Klf5</it>^+/-mice. Tumor tissues displayed higher levels of both Klf5 and β-catenin, irrespective of the mouse genotype from which tumors were derived.</p> <p>Conclusions</p> <p>Results of the current study confirm the cumulative effect of <it>Apc </it>loss and oncogenic <it>KRAS </it>activation on intestinal tumorigenesis. The drastic reduction in tumor number and size due to <it>Klf5 </it>heterozygosity in <it>Apc</it>^<it>Min</it>/<it>KRAS</it>^<it>V</it>12mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.</p

Krüppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells

Lipopolysaccharide (LPS) is a bacterially-derived endotoxin that elicits a strong proinflammatory response in intestinal epithelial cells. It is well established that LPS activates this response through NF-κB. In addition, LPS signals through the mitogen-activated protein kinase (MAPK) pathway. We previously demonstrated that the Krüppel-like factor 5 [KLF5; also known as intestine-enriched Krüppel-like factor (IKLF)] is activated by the MAPK. In the current study, we examined whether KLF5 mediates the signaling cascade elicited by LPS. Treatment of the intestinal epithelial cell line, IEC6, with LPS resulted in a dose- and time-dependent increase in KLF5 messenger RNA (mRNA) and protein levels. Concurrently, mRNA levels of the p50 and p65 subunits of NF-κB were increased by LPS treatment. Pretreatment with the MAPK inhibitor, U0126, or the LPS antagonist, polymyxin B, resulted in an attenuation of KLF5, p50 and p65 NF-κB subunit mRNA levels from LPS treatment. Importantly, suppression of KLF5 by small interfering RNA (siRNA) resulted in a reduction in p50 and p65 subunit mRNA levels and NF-κB DNA binding activity in response to LPS. LPS treatment also led to an increase in secretion of TNF-α and IL-6 from IEC6, both of which were reduced by siRNA inhibition of KLF5. In addition, intercellular adhesion molecule-1 (ICAM-1) levels were increased in LPS-treated IEC6 cells and this increase was associated with increased adhesion of Jurkat lymphocytes to IEC6. The induction of ICAM-1 expression and T cell adhesion to IEC6 by LPS were both abrogated by siRNA inhibition of KLF5. These results indicate that KLF5 is an important mediator for the proinflammatory response elicited by LPS in intestinal epithelial cells

The role of Krüppel-like factors in the reprogramming of somatic cells to induced pluripotent stem cells

The potential for clinical application of pluripotent embryonic stem cells is immense but hampered by moral and ethical complications. Recent advances in the reprogramming of somatic cells by defined factors to a state that resemble embryonic stem cells have created tremendous excitement in the field. Four factors, Sox2, Oct4, Klf4 and c-Myc, when exogenously introduced into somatic cells, can lead to the formation of induced pluripotent stem (iPS) cells that have the capacity for self-renewal and differentiation into tissues of all three germ layers. In this review, we focus on the role of Krüppel-like factors (KLFs) in regulating somatic cell reprogramming. KLFs are zinc finger-containing transcription factors with diverse biological functions. We first provide an overview of the KLF family of regulatory proteins, paying special attention to the established biological and biochemical functions of KLF4 and KLF5. We then review the role of KLFs in somatic cell reprogramming and delineate the putative mechanism by which KLFs participates the establishment and self-renewal of iPS cells. Further research is likely to provide additional insight into the mechanisms of somatic cell reprogramming and refinement of the technique with which to generate clinically relevant iPS cells

Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells

Krüppel-like factor 5 (KLF5) is a pro-proliferative transcription factor that is expressed in dividing epithelial cells of the intestinal crypt. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) has been identified as a stem cell marker in both small intestinal and colonic epithelial cells. To determine whether KLF5 regulates proliferation of intestinal stem cells, we investigated the effects of Klf5 deletion specifically from the intestinal stem cells in adult mice. Mice with inducible intestinal stem cell-specific deletion of Klf5 (Lgr5-Klf5fl/fl) were injected with tamoxifen for 5 consecutive days to induce Lgr5-driven Cre expression. Intestinal and colonic tissues were examined by immunohistochemistry at various time points up to 112 days following start of tamoxifen treatment. Klf5 is co-localized in the crypt-based columnar (CBC) cells that express Lgr5. By 11 days following the start of tamoxifen treatment, Lgr5-positive crypts from which Klf5 was deleted exhibited a loss of proliferation that was accompanied by an increase in apoptosis. Beginning at 14 days following the start of tamoxifen treatment, both Klf5 expression and proliferation were re-established in the transit-amplifying epithelial cells but not in the Lgr5-positive CBC cells. By 112 days post-treatment, up to 90% of the Lgr5-positive cells from which Klf5 was deleted were lost from the intestinal crypts. These results indicate a critical role for KLF5 in the survival and maintenance of intestinal stem cells