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.

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.

Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors that are found in many species. Recent studies have shown that KLFs play a fundamental role in regulating diverse biological processes such as cell proliferation, differentiation, development and regeneration. Of note, several KLFs are also crucial for maintaining pluripotency and, hence, have been linked to reprogramming and regenerative medicine approaches. Here, we review the crucial functions of KLFs in mammalian embryogenesis, stem cell biology and regeneration, as revealed by studies of animal models. We also highlight how KLFs have been implicated in human diseases and outline potential avenues for future research.

Krüppel-like factors in mammalian stem cells and development.

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 5 (KLF5) is a member of the zinc finger family of transcription factors that regulates homeostasis of the intestinal epithelium. Previous studies suggested an indispensable role of KLF5 in maintaining intestinal barrier function. In the current study, we investigated the mechanisms by which KLF5 regulates colonic barrier function in vivo and in vitro. We used an inducible and a constitutive intestine-specific  knockout mouse models ( designated as  and  as  ) and studied an inducible  knockdown in Caco-2 BBe cells using a lentiviral Tet-on system (Caco-2 BBe ). Specific knockout of  in colonic tissues, either inducible or constitutive, resulted in increased intestinal permeability. The phenotype was accompanied by a significant reduction in , which encodes desmoglein-2, a desmosomal cadherin, at both mRNA and protein levels. Transmission electron microscopy showed alterations of desmosomal morphology in both  knockdown Caco-2 BBe cells and  knockout mouse colonic tissues. Inducible knockdown of  in Caco-2BBe cells grown on Transwell plates led to impaired barrier function as evidenced by decreased transepithelial electrical resistance and increased paracellular permeability to fluorescein isothiocyanate-4 kDa dextran. Furthermore, DSG2 was significantly decreased in  knockdown cells, and DSG2 overexpression partially rescued the impaired barrier function caused by  knockdown. Electron microscopy studies demonstrated altered desmosomal morphology after  knockdown. In combination with chromatin immunoprecipitation analysis and promoter study, our data show that KLF5 regulates intestinal barrier function by mediating the transcription of , a gene encoding a major component of desmosome structures. The study is original research on the direct function of a Krüppel-like factor on intestinal barrier function, which is commonly exerted by cell junctions, including tight junctions, adherens junctions, and desmosomes. Numerous previous studies were focused on tight junctions and adherens junctions. However, this study provided a new perspective on how the intestinal barrier function is regulated by KLF5 through DSG2, a component of desmosome complexes.

Krüppel-like factor 5 is essential for maintenance of barrier function in mouse colon.

Activating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)-a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar-to-ductal metaplasia (ADM). We investigated the role of the transcription factor Krüppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs.

Krüppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice.

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 .

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.

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.

Agnieszka B. Bialkowska

Department of Medicine

Stony Brook University School of Medicine

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

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

Agnieszka B. Bialkowska

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medicine

Stony Brook University School of Medicine

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

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

Department of Medicine