Beth B. McConnell

Mammalian Krüppel-Like Factors in Health and Diseases

The Krüppel-like factor (KLF) family of transcription factors regulates diverse biological processes that include proliferation, differentiation, growth, development, survival, and responses to external stress. Seventeen mammalian KLFs have been identified, and numerous studies have been published that describe their basic biology and contribution to human diseases. KLF proteins have received much attention because of their involvement in the development and homeostasis of numerous organ systems. KLFs are critical regulators of physiological systems that include the cardiovascular, digestive, respiratory, hematological, and immune systems and are involved in disorders such as obesity, cardiovascular disease, cancer, and inflammatory conditions. Furthermore, KLFs play an important role in reprogramming somatic cells into induced pluripotent stem (iPS) cells and maintaining the pluripotent state of embryonic stem cells. As research on KLF proteins progresses, additional KLF functions and associations with disease are likely to be discovered. Here, we review the current knowledge of KLF proteins and describe common attributes of their biochemical and physiological functions and their pathophysiological roles.

Haploinsufficiency of Krüppel-Like Factor 4 Promotes Adenomatous Polyposis Coli–Dependent Intestinal Tumorigenesis

The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently down-regulated in colorectal cancer. Previous studies showed that the expression of KLF4 was activated by the colorectal cancer tumor suppressor adenomatous polyposis coli (APC) and that KLF4 repressed the Wnt/beta-catenin pathway. Here, we examined whether KLF4 plays a role in modulating intestinal tumorigenesis by comparing the tumor burdens in mice heterozygous for the Apc(Min) allele (Apc(Min/+)) and those heterozygous for both the Apc(Min) and Klf4 alleles (Klf4(+/-)/Apc(Min/+)). Between 10 and 20 weeks of age, Klf4(+/-)/Apc(Min/+) mice developed, on average, 59% more intestinal adenomas than Apc(Min/+) mice (P < 0.0001). Immunohistochemical staining showed that Klf4 protein levels were lower in the normal-appearing intestinal tissues of Klf4(+/-)/Apc(Min/+) mice compared with wild-type, Klf4(+/-), or Apc(Min/+) mice. In contrast, the levels of beta-catenin and cyclin D1 were higher in the normal-appearing intestinal tissues of Klf4(+/-)/Apc(Min/+) mice compared with the other three genotypes. Klf4 levels were further decreased in adenomas from both Apc(Min/+) and Klf4(+/-)/Apc(Min/+) mice compared with their corresponding normal-appearing tissues. Reverse transcription-PCR showed an inverse correlation between adenoma size and Klf4 mRNA levels in both Klf4(+/-)/Apc(Min/+) and Apc(Min/+) mice. There was also a progressive loss of heterozygosity of the wild-type Apc allele in adenomas with increasing size from Klf4(+/-)/Apc(Min/+) and Apc(Min/+) mice. Results from this study show that KLF4 plays an important role in promoting the development of intestinal adenomas in the presence of Apc(Min) mutation.

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.

SUMOylation regulates nuclear localization of Kruppel-like factor 5.

SUMOylation is a form of post-translational modification shown to control nuclear transport. Krüppel-like factor 5 (KLF5) is an important mediator of cell proliferation and is primarily localized to the nucleus. Here we show that mouse KLF5 is SUMOylated at lysine residues 151 and 202. Mutation of these two lysines or two conserved nearby glutamates results in the loss of SUMOylation and increased cytoplasmic distribution of KLF5, suggesting that SUMOylation enhances nuclear localization of KLF5. Lysine 151 is adjacent to a nuclear export signal (NES) that resembles a consensus NES. The NES in KLF5 directs a fused green fluorescence protein to the cytoplasm, binds the nuclear export receptor CRM1, and is inhibited by leptomycin and site-directed mutagenesis. SUMOylation facilitates nuclear localization of KLF5 by inhibiting this NES activity, and enhances the ability of KLF5 to stimulate anchorage-independent growth of HCT116 colon cancer cells. A survey of proteins whose nuclear localization is regulated by SUMOylation reveals that SUMOylation sites are frequently located in close proximity to NESs. A relatively common mechanism for SUMOylation to regulate nucleocytoplasmic transport may lie in the interplay between neighboring NES and SUMOylation motifs.

Haploinsufficiency of Krüppel-Like Factor 5 Rescues the Tumor-Initiating Effect of the ApcMin Mutation in the Intestine

Inactivation of the tumor suppressor adenomatous polyposis coli, with the resultant activation of beta-catenin, is the initiating event in the development of a majority of colorectal cancers. Krüppel-like factor 5 (KLF5), a proproliferative transcription factor, is highly expressed in the proliferating intestinal crypt epithelial cells. To determine whether KLF5 contributes to intestinal adenoma formation, we examined tumor burdens in Apc(Min/+) mice and Apc(Min/+)/Klf5(+/-) mice. Compared with Apc(Min/+) mice, Apc(Min/+)/Klf5(+/-) mice had a 96% reduction in the number of intestinal adenomas. Reduced tumorigenicity in the Apc(Min/+)/Klf5(+/-) mice correlated with reduced levels and nuclear localization of beta-catenin as well as reduced expression of two beta-catenin targets, cyclin D1 and c-Myc. In vitro studies revealed a physical interaction between KLF5 and beta-catenin that enhanced the nuclear localization and transcriptional activity of beta-catenin. Thus, KLF5 is necessary for the tumor-initiating activity of beta-catenin during intestinal adenoma formation in Apc(Min/+) mice, and reduced expression of KLF5 offsets the tumor-initiating activity of the Apc(Min) mutation by reducing the nuclear localization and activity of beta-catenin.

Altered intestinal epithelial homeostasis in mice with intestine-specific deletion of the Krüppel-like factor 4 gene

The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is expressed in the post-mitotic, differentiated epithelial cells lining the intestinal tract and exhibits a tumor suppressive effect on intestinal tumorigenesis. Here we report a role for KLF4 in maintaining homeostasis of intestinal epithelial cells. Mice with conditional ablation of the Klf4 gene from the intestinal epithelium were viable. However, both the rates of proliferation and migration of epithelial cells were increased in the small intestine of mutant mice. In addition, the brush-border alkaline phosphatase was reduced as was expression of ephrine-B1 in the small intestine, resulting in mispositioning of Paneth cells to the upper crypt region. In the colon of mutant mice, there was a reduction of the differentiation marker, carbonic anhydrase-1, and failure of differentiation of goblet cells. Mechanistically, deletion of Klf4 from the intestine resulted in activation of genes in the Wnt pathway and reduction in expression of genes encoding regulators of differentiation. Taken together, these data provide new insights into the function of KLF4 in regulating postnatal proliferation, migration, differentiation, and positioning of intestinal epithelial cells and demonstrate an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis in vivo.

Krüppel-like Factor 5 is Important for Maintenance of Crypt Architecture and Barrier Function in Mouse Intestine

BACKGROUND & AIMS Krüppel-like factor 5 (KLF5) is transcription factor that is expressed by dividing epithelial cells of the intestinal epithelium. KLF5 promotes proliferation in vitro and in vivo and is induced by mitogens and various stress stimuli. To study the role of KLF5 in intestinal epithelial homeostasis, we examined the phenotype of mice with conditional deletion of Klf5 in the gut. METHODS Mice were generated with intestinal-specific deletion of Klf5 (Vil-Cre;Klf5fl/fl). Morphologic changes in the small intestine and colon were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction. RESULTS Klf5 mutant mice were born at a normal Mendelian ratio but had high mortality compared with controls. Complete deletion of Klf5 from the intestinal mucosa resulted in neonatal lethality that corresponded with an absence of epithelial proliferation. Variegated intestinal-specific deletion of Klf5 in adult mice resulted in morphologic changes that included a regenerative phenotype, impaired barrier function, and inflammation. Adult mutant mice exhibited defects in epithelial differentiation and migration. These changes were associated with reduced expression of Caudal type homeobox (Cdx) 1, Cdx2, and Eph and ephrin signaling proteins. Concomitantly, Wnt signaling to β-catenin was reduced. Proliferation in regenerative crypts was associated with increased expression of the progenitor cell marker Sox9. CONCLUSIONS Deletion of Klf5 in the gut epithelium of mice demonstrated that KLF5 maintains epithelial proliferation, differentiation, and cell positioning along the crypt radial axis. Morphologic changes that occur with deletion of Klf5 are associated with disruption of canonical Wnt signaling and increased expression of Sox9.

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

BackgroundBoth mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene and activation of the KRAS oncogene are implicated in the pathogenesis of colorectal cancer. Mice harboring a germline ApcMinmutation or intestine-specific expression of the KRASV 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 in vivo effect of Klf5 heterozygosity on the propensity of ApcMin/KRASV 12double transgenic mice to develop intestinal tumors.ResultsAt 12 weeks of age, ApcMin/KRASV 12mice had three times as many intestinal tumors as ApcMinmice. This increase in tumor number was reduced by 92% in triple transgenic ApcMin/KRASV 12/Klf5+/- mice. The reduction in tumor number in ApcMin/KRASV 12/Klf5+/- mice was also statistically significant compared to ApcMinmice alone, with a 75% decrease. Compared with ApcMin/KRASV 12, tumors from both ApcMin/KRASV 12/Klf5+/- and ApcMinmice were smaller. In addition, tumors from ApcMinmice were more distally distributed in the intestine as contrasted by the more proximal distribution in ApcMin/KRASV 12and ApcMin/KRASV 12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both ApcMinand ApcMin/KRASV 12mice but were attenuated in ApcMin/KRASV 12/Klf5+/- mice. The levels of β-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of ApcMin/KRASV 12/Klf5+/- mice when compared to ApcMin/KRASV 12mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of ApcMin/KRASV 12mice and modestly reduced in ApcMin/KRASV 12/Klf5+/- mice. Tumor tissues displayed higher levels of both Klf5 and β-catenin, irrespective of the mouse genotype from which tumors were derived.ConclusionsResults of the current study confirm the cumulative effect of Apc loss and oncogenic KRAS activation on intestinal tumorigenesis. The drastic reduction in tumor number and size due to Klf5 heterozygosity in ApcMin/KRASV 12mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.

Krüppel-like factor 5 mediates transmissible murine colonic hyperplasia caused by Citrobacter rodentium infection.

BACKGROUND & AIMS Krüppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in proliferating crypt cells of the intestinal epithelium. KLF5 has a pro-proliferative effect in vitro and is induced by mitogenic and stress stimuli. To determine whether KLF5 is involved in mediating proliferative responses to intestinal stressors in vivo, we examined its function in a mouse model of transmissible murine colonic hyperplasia triggered by colonization of the mouse colon by the bacteria Citrobacter rodentium. METHODS Heterozygous Klf5 knockout (Klf5(+/-)) mice were generated from embryonic stem cells carrying an insertional disruption of the Klf5 gene. Klf5(+/-) mice or wild-type (WT) littermates were infected with C rodentium by oral gavage. At various time points postinfection, mice were killed and distal colons were harvested. Colonic crypt heights were determined morphometrically from sections stained with H&E. Frozen tissues were stained by immunofluorescence using antibodies against Klf5 and the proliferation marker, Ki67, to determine Klf5 expression and numbers of proliferating cells per crypt. RESULTS Infection of WT mice with C rodentium resulted in a 2-fold increase in colonic crypt heights at 14 days postinfection and was accompanied by a 1.7-fold increase in Klf5 expression. Infection of Klf5(+/-) mice showed an attenuated induction of Klf5 expression, and hyperproliferative responses to C rodentium were reduced in the Klf5(+/-) animals as compared with WT littermates. CONCLUSION Our study shows that Klf5 is a key mediator of crypt cell proliferation in the colon in response to pathogenic bacterial infection.

Krüppel-Like Factor 5 Protects Against Dextran Sulfate Sodium−Induced Colonic Injury in Mice by Promoting Epithelial Repair

BACKGROUND & AIMS Krüppel-like factor 5 (KLF5) is a transcription factor that promotes proliferation, is highly expressed in dividing crypt cells of the gastrointestinal epithelium, and is induced by various stress stimuli. We sought to determine the role of KLF5 in colonic inflammation and recovery by studying mice with dextran sulfate sodium (DSS)-induced colitis. METHODS Wild-type (WT) and Klf5(+/-) mice were given DSS in the drinking water to induce colitis. For recovery experiments, mice were given normal drinking water for 5 days after DSS administration. The extent of colitis was determined using established clinical and histological scoring systems. Immunohistochemical and immunoblotting analyses were used to examine proliferation, migration, and expression of the epidermal growth factor receptor. RESULTS Klf5 expression was increased in colonic tissues of WT mice given DSS; induction of Klf5 was downstream of mitogen-activated protein kinase signaling. In DSS-induced colitis, Klf5(+/-) mice exhibited greater sensitivity to DSS than WT mice, with significantly higher clinical and histological colitis scores. In recovery experiments, Klf5(+/-) mice showed poor recovery, with continued weight loss and higher mortality than WT mice. Klf5(+/-) mice from the recovery period had reduced epithelial proliferation and cell migration at sites of ulceration compared to WT mice; these reductions correlated with reduced expression of epidermal growth factor receptor. CONCLUSIONS Epithelial repair is an important aspect of recovery from DSS-induced colitis. The transcription factor KLF5 regulates mucosal healing through its effects on epithelial proliferation and migration.