improved-swiss-rolling-technique-for-intestinal-tissue-preparation

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

intestinal-epithelial-regeneration-in-response-to-ionizing-irradiation

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

Stony Brook University School of Medicine

Renaissance School of Medicine at Stony Brook University

Krüppel-like factor 5 (KLF5) is a pro-proliferative transcriptional regulator primarily expressed in the intestinal crypt epithelial cells. Constitutive intestine-specific deletion of Klf5 is neonatal lethal suggesting a crucial role for KLF5 in intestinal development and homeostasis. We have previously shown Klf5 to play an active role regulating intestinal tumorigenesis. Here we examine the effect of inducible intestine-specific deletion of Klf5 in adult mice. Klf5 is lost from the intestine beginning at day 3 after the start of a 5-day treatment with the inducer tamoxifen. Although the mice have no significant weight loss or lethality, the colonic tissue shows signs of epithelial distress starting at day 3 following induction. Accompanying the morphological changes is a significant loss of proliferative crypt epithelial cells as revealed by BrdU or Ki67 staining at days 3 and 5 after start of tamoxifen. We also observed a loss of goblet cells from the colon and Paneth cells from the small intestine upon induced deletion of Klf5. In addition, loss of Klf5 from the colonic epithelium is accompanied by a regenerative response that coincides with an expansion in the zone of Sox9 expression along the crypt axis. At day 11, both proliferation and Sox9 expression return to baseline levels. Microarray and quantitative PCR analyses reveal an up-regulation of several regeneration-associated genes (Reg1A, Reg3G and Reg3B) and down-regulation of many Klf5 targets (Ki-67, cyclin B, Cdc2 and cyclin D1). Sox9 and Reg1A protein levels are also increased upon Klf5 loss. Lentiviral-mediated knockdown of KLF5 and exogenous expression of KLF5 in colorectal cancer cell lines confirm that Sox9 expression is negatively regulated by KLF5. Furthermore, ChIP assays reveal a direct association of KLF5 with both the Sox9 and Reg1A promoters. We have shown that disruption of epithelial homeostasis due to Klf5 loss from the adult colon is followed by a regenerative response led by Sox9 and the Reg family of proteins. Our study demonstrates that adult mouse colonic tissue undergoes acute physiological changes to accommodate the loss of Klf5 withstanding epithelial damage further signifying importance of Klf5 in colonic homeostasis.

Inducible intestine-specific deletion of Krüppel-like factor 5 is characterized by a regenerative response in adult mouse colon.

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-Klf5(fl/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 112days following start of tamoxifen treatment. Klf5 is co-localized in the crypt-based columnar (CBC) cells that express Lgr5. By 11days 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 14days 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 112days 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.

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

Colorectal cancer is one of the leading causes of cancer mortality in Western civilization. Studies have shown that colorectal cancer arises as a consequence of the modification of genes that regulate important cellular functions. Deregulation of the WNT and RAS/MAPK/PI3K signaling pathways has been shown to be important in the early stages of colorectal cancer development and progression. Krüppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in the proliferating intestinal crypt epithelial cells. Previously, we showed that KLF5 is a mediator of RAS/MAPK and WNT signaling pathways under homeostatic conditions and that it promotes their tumorigenic functions during the development and progression of intestinal adenomas. Recently, using an ultrahigh-throughput screening approach we identified a number of novel small molecules that have the potential to provide therapeutic benefits for colorectal cancer by targeting KLF5 expression. In the current study, we show that an improved analogue of one of these screening hits, ML264, potently inhibits proliferation of colorectal cancer cells in vitro through modifications of the cell-cycle profile. Moreover, in an established xenograft mouse model of colon cancer, we demonstrate that ML264 efficiently inhibits growth of the tumor within 5 days of treatment. We show that this effect is caused by a significant reduction in proliferation and that ML264 potently inhibits the expression of KLF5 and EGR1, a transcriptional activator of KLF5. These findings demonstrate that ML264, or an analogue, may hold a promise as a novel therapeutic agent to curb the development and progression of colorectal cancer.

ML264, A Novel Small-Molecule Compound That Potently Inhibits Growth of Colorectal Cancer.

The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently downregulated in colorectal cancer. Previous studies showed that KLF4 is a tumor suppressor in the intestinal tract and plays an important role in DNA damage-repair mechanisms. Here, thein vivoeffects ofKlf4deletion were examined from the mouse intestinal epithelium (Klf4(ΔIS)) in a genetic or pharmacological setting of colonic tumorigenesis:Apc(Min/+)mutation or carcinogen treatment with azoxymethane (AOM), respectively.Klf4(ΔIS)/Apc(Min/+)mice developed significantly more colonic adenomas with 100% penetrance as compared withApc(Min/+)mice with intactKlf4(Klf4(fl/fl)/Apc(Min/+)). The colonic epithelium ofKlf4(ΔIS)/Apc(Min/+)mice showed increased mTOR pathway activity, together with dysregulated epigenetic mechanism as indicated by altered expression of HDAC1 and p300. Colonic adenomas from both genotypes stained positive for γH2AX, indicating DNA double-strand breaks. InKlf4(ΔIS)/Apc(Min/+)mice, this was associated with reduced nonhomologous end joining (NHEJ) repair and homologous recombination repair (HRR) mechanisms as indicated by reduced Ku70 and Rad51 staining, respectively. In a separate model, following treatment with AOM,Klf4(ΔIS)mice developed significantly more colonic tumors thanKlf4(fl/fl)mice, with moreKlf4(ΔIS)mice harboringK-Rasmutations thanKlf4(fl/fl)mice. Compared with AOM-treatedKlf4(fl/fl)mice, adenomas of treatedKlf4(ΔIS)mice had suppressed NHEJ and HRR mechanisms, as indicated by reduced Ku70 and Rad51 staining. This study highlights the important role of KLF4 in suppressing the development of colonic neoplasia under different tumor-promoting conditions.

KLF4 Suppresses Tumor Formation in Genetic and Pharmacological Mouse Models of Colonic Tumorigenesis.

In response to ionizing radiation-induced injury, the normally quiescent intestinal stem cells marked by BMI1 participate in the regenerative response. Previously, we established a protective role for Krüppel-like factor 4 (KLF4) in the intestinal epithelium where it reduces senescence, apoptosis, and crypt atrophy following γ-radiation-induced gut injury. We also described a pro-proliferative function for KLF4 during the regenerative phase post irradiation. In the current study, using a mouse model in which Klf4 is deleted from quiescent BMI1(+) intestinal stem cells, we observed increased proliferation from the BMI1(+) lineage during homeostasis. In contrast, following irradiation, Bmi1-specific Klf4 deletion leads to decreased expansion of the BMI1(+) lineage due to a combination of reduced proliferation and increased apoptosis. Our results support a critical role for KLF4 in modulating BMI1(+) intestinal stem cell fate in both homeostasis and the regenerative response to radiation injury.

Krüppel-like Factor 4 Modulates Development of BMI1(+) Intestinal Stem Cell-Derived Lineage Following γ-Radiation-Induced Gut Injury in Mice.

Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell growth, proliferation, and differentiation. Since its discovery in 1996, KLF4 has been gaining a lot of attention, particularly after it was shown in 2006 as one of four factors involved in the induction of pluripotent stem cells (iPSCs). Here we review the current knowledge about the different functions and roles of KLF4 in various tissue and organ systems.

Krüppel-like factor 4 (KLF4): What we currently know.

Specificity proteins (SPs) and Krüppel-like factors (KLFs) belong to the family of transcription factors that contain conserved zinc finger domains involved in binding to target DNA sequences. Many of these proteins are expressed in different tissues and have distinct tissue-specific activities and functions. Studies have shown that SPs and KLFs regulate not only physiological processes such as growth, development, differentiation, proliferation, and embryogenesis, but pathogenesis of many diseases, including cancer and inflammatory disorders. Consistently, these proteins have been shown to regulate normal functions and pathobiology in the digestive system. We review recent findings on the tissue- and organ-specific functions of SPs and KLFs in the digestive system including the oral cavity, esophagus, stomach, small and large intestines, pancreas, and liver. We provide a list of agents under development to target these proteins.

SP and KLF Transcription Factors in Digestive Physiology and Diseases.

Krüppel-like factor 4 (KLF4), a zinc finger transcription factor, regulates homeostasis of the intestinal epithelium. Previously, it was reported that KLF4 functions as a tumor suppressor in colorectal cancer. Here, evidence demonstrates that KLF4 mitigates the development and progression of colitis-associated colorectal cancer (CAC) in a murine model. Mice with intestinal epithelium-specific deletion of  ( ) and control mice ( ) were used to explore the role of KLF4 in the development of azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced CAC. Upon AOM and DSS treatment, KLF4 expression was progressively lost in colonic tissues of  mice during tumor development.  mice treated with AOM/DSS developed significantly more adenomatous polyps and carcinomas  in comparison with treated  mice. Adenomatous polyps, but not normal-appearing mucosa, from colonic tissues of treated  mice contained a significantly increased number of mitotic cells with more than 2 centrosomes relative to treated control mice. KLF4 and p53 colocalize to the centrosomes in mouse embryonic fibroblasts (MEF). Absence of KLF4 in  MEFs inhibits and its overexpression restores p53 localization to the centrosomes in  MEFs. IMPLICATIONS: Taken together, these results indicate that KLF4 plays a protective role against progression of CAC by guarding against genetic instability.

Increased Genetic Instability and Accelerated Progression of Colitis-Associated Colorectal Cancer through Intestinal Epithelium-specific Deletion of .

Krüppel-like factor 5 (KLF5), a member of the SP/KLF family of zinc finger transcription factors, is overexpressed in human colorectal cancer specimens, and this overabundance is associated with aggressive cancer development and progression. We demonstrated that mice haploinsufficient for  had reduced intestinal tumor burden in the background of germline mutation in , a gatekeeper of intestinal tumorigenesis. Based on a high-throughput screening strategy, we developed ML264, a small-molecule compound that inhibits KLF5, and showed that it inhibits growth of colorectal cancer  and  Through optimization efforts based on the structure of ML264, we have now identified a new lead compound, SR18662. We find that treatment with SR18662 significantly reduces growth and proliferation of colorectal cancer cells as compared with treatment with vehicle control, ML264, or SR15006 (a less optimized analogue from SAR efforts leading to SR18662). SR18662 showed improved efficacy in reducing the viability of multiple colorectal cancer cell lines. Flow cytometry analysis following SR18662 treatment showed an increase in cells captured in either S or G-M phases of the cell cycle and a significant increase in the number of apoptotic cells, the latter a unique property compared with ML264 or SR15006. SR18662 treatment also reduces the expression of cyclins and components of the MAPK and WNT signaling pathways. Importantly, we observed a significant dose-dependent inhibition of xenograft growth in mice following SR18662 treatment that exceeded the effect of ML264 at equivalent doses. These findings support further development of SR18662 and its analogues for colorectal cancer therapy.

The Novel Small-Molecule SR18662 Efficiently Inhibits the Growth of Colorectal Cancer  and .

Self-renewal and multipotent differentiation are cardinal properties of intestinal stem cells (ISCs), mediated in part by WNT and NOTCH signaling. Although these pathways are well characterized, the molecular mechanisms that control the 'stemness' of ISCs are still not well defined. Here, we investigated the role of Krüppel-like factor 5 (KLF5) in regulating ISC functions.

Krüppel-like Factor 5 Regulates Stemness, Lineage Specification, and Regeneration of Intestinal Epithelial Stem Cells.

Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-Cre to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21 in Bmi1-Cre cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21 mRNA translation, which promotes exit of the Bmi1-Cre cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1 cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21, MSI1 and KLF4 regulates Bmi1-Cre cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.

Interplay among p21, MUSASHI-1 and Krüppel-like factor 4 in activation of Bmi1-Cre reserve intestinal stem cells after gamma radiation-induced injury.

Aberrant differentiation of intestinal stem cells due to inflammation-induced mitochondrial dysfunction predicts postoperative recurrence of Crohn's disease.

Inflammatory bowel disease (IBD) is a chronic illness characterized by dysregulated immune cascades in the intestines, in which the Th17 immune response plays an important role. We demonstrated that mice with intestinal epithelium-specific deletion of Krüppel-like factor 5 (Klf5) developed Th17-dependent colonic inflammation. In the absence of KLF5, there was aberrant cellular localization of phosphorylated STAT3, an essential mediator of the Th17-associated cytokine, IL-22, which is required for epithelial tissue regeneration. In contrast, mitigation of IL-17A with anti-IL-17A neutralizing antibody attenuated colitis in Klf5-deficient mice. There was also a considerable shift in the colonic microbiota of Klf5-deficient mice that phenocopied human IBD. Notably, the inflammatory response due to Klf5 deletion was alleviated by antibiotic treatment, implicating the role of microbiota in pathogenesis. Finally, human colitic tissues had reduced KLF5 levels when compared with healthy tissues. Together, these findings demonstrated the importance of KLF5 in protecting the intestinal epithelium against Th17-mediated immune and inflammatory responses. The mice described herein may serve as a potential model for human IBD.

ArticleTotal : 2	Year
Publication title Cited by 166	2016
Publication title Cited by 6	2022

Article	Year
Inducible intestine-specific deletion of Krüppel-like factor 5 is characterized by a regenerative response in adult mouse colon. Developmental biology\| PubMed ID: 24440658	2014
Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells. Stem cell research\| PubMed ID: 25460247	2015
ML264, A Novel Small-Molecule Compound That Potently Inhibits Growth of Colorectal Cancer. Molecular cancer therapeutics\| PubMed ID: 26621868	2016
KLF4 Suppresses Tumor Formation in Genetic and Pharmacological Mouse Models of Colonic Tumorigenesis. Molecular cancer research : MCR\| PubMed ID: 26839262	2016
Krüppel-like Factor 4 Modulates Development of BMI1(+) Intestinal Stem Cell-Derived Lineage Following γ-Radiation-Induced Gut Injury in Mice. Stem cell reports\| PubMed ID: 27237377	2016
Krüppel-like factor 4 (KLF4): What we currently know. Gene\| PubMed ID: 28237823	2017
SP and KLF Transcription Factors in Digestive Physiology and Diseases. Gastroenterology\| PubMed ID: 28366734	2017
Increased Genetic Instability and Accelerated Progression of Colitis-Associated Colorectal Cancer through Intestinal Epithelium-specific Deletion of . Molecular cancer research : MCR\| PubMed ID: 30108164	2019
The Novel Small-Molecule SR18662 Efficiently Inhibits the Growth of Colorectal Cancer and . Molecular cancer therapeutics\| PubMed ID: 31358661	2019
Krüppel-like Factor 5 Regulates Stemness, Lineage Specification, and Regeneration of Intestinal Epithelial Stem Cells. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 31778829	2020
Interplay among p21, MUSASHI-1 and Krüppel-like factor 4 in activation of Bmi1-Cre reserve intestinal stem cells after gamma radiation-induced injury. Scientific reports\| PubMed ID: 33110120	2020
Aberrant differentiation of intestinal stem cells due to inflammation-induced mitochondrial dysfunction predicts postoperative recurrence of Crohn's disease. Digestive medicine research\| PubMed ID: 33447825	2020
KLF5 protects the intestinal epithelium against Th17 immune response in a murine colitis model. JCI insight\| PubMed ID: 35393949	2022

Vincent W. Yang

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