Publications by authors named "Agnieszka B Bialkowska"

48 Publications

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

Circ Res 2021 Feb 2;128(3):335-357. Epub 2020 Dec 2.

Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, PA (I.D.K., M.H., A.M.L., E.M., D.P., W.J.K., K.D.).

Rationale: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism.

Objective: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation.

Methods And Results: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase ()4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice.

Conclusions: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.316738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870005PMC
February 2021

Cardiomyocyte Krüppel-Like Factor 5 Promotes De Novo Ceramide Biosynthesis and Contributes to Eccentric Remodeling in Ischemic Cardiomyopathy.

Circulation 2021 Mar 12;143(11):1139-1156. Epub 2021 Jan 12.

Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, PA (M.H., D.P., I.D.K., C.M., S.R., E.G., R.K., K.D.).

Background: We previously showed that cardiomyocyte Krϋppel-like factor (KLF) 5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure.

Methods: Using real-time polymerase chain reaction and Western blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte-specific KLF5 deletion (αMHC [α-myosin heavy chain]-KLF5). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid chromatography-tandem mass spectrometry, and Western blot and real-time polymerase chain reaction analysis of ceramide metabolism-related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA [reverse tetracycline-controlled transactivator]-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis.

Results: KLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models at 24 hours, 2 weeks, and 4 weeks post-permanent left coronary artery ligation. αMHC-KLF5 mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5 mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI, although basal ceramide content of αMHC-KLF5 mice was not different in control mice. KLF5 ablation suppressed the expression of SPTLC1 and SPTLC2 (serine palmitoyltransferase [SPT] long-chain base subunit ()1 2, respectively), which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2 weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction.

Conclusions: KLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.047420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7965352PMC
March 2021

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

Sci Rep 2020 10 27;10(1):18300. Epub 2020 Oct 27.

Department of Medicine, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, USA.

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.
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http://dx.doi.org/10.1038/s41598-020-75171-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591575PMC
October 2020

IL-22 receptor signaling in Paneth cells is critical for their maturation, microbiota colonization, Th17-related immune responses, and anti-Salmonella immunity.

Mucosal Immunol 2021 03 15;14(2):389-401. Epub 2020 Oct 15.

Department of Microbiology and Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, USA.

Interleukin-22 (IL-22) signaling in the intestines is critical for promoting tissue-protective functions. However, since a diverse array of cell types (absorptive and secretory epithelium as well as stem cells) express IL-22Ra1, a receptor for IL-22, it has been difficult to determine what cell type(s) specifically respond to IL-22 to mediate intestinal mucosal host defense. Here, we report that IL-22 signaling in the small intestine is positively correlated with Paneth cell differentiation programs. Our Il22Ra1;Lgr5-EGFP-cre-specific knockout mice and, independently, our lineage-tracing findings rule out the involvement of Lgr5 intestinal stem cell (ISC)-dependent IL-22Ra1 signaling in regulating the lineage commitment of epithelial cells, including Paneth cells. Using novel Paneth cell-specific IL-22Ra1 knockout mice (Il22Ra1;Defa6-cre), we show that IL-22 signaling in Paneth cells is required for small intestinal host defense. We show that Paneth cell maturation, antimicrobial effector function, expression of specific WNTs, and organoid morphogenesis are dependent on cell-intrinsic IL-22Ra1 signaling. Furthermore, IL-22 signaling in Paneth cells regulates the intestinal commensal bacteria and microbiota-dependent IL-17A immune responses. Finally, we show ISC and, independently, Paneth cell-specific IL-22Ra1 signaling are critical for providing immunity against Salmonella enterica serovar Typhimurium. Collectively, our findings illustrate a previously unknown role of IL-22 in Paneth cell-mediated small intestinal host defense.
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http://dx.doi.org/10.1038/s41385-020-00348-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946635PMC
March 2021

KLF4 Regulates Goblet Cell Differentiation in BMI1 Reserve Intestinal Stem Cell Lineage during Homeostasis.

Int J Stem Cells 2020 Nov;13(3):424-431

Department of Medicine, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, USA.

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor, expressed in villus cells of the intestinal epithelium, that promotes cellular differentiation and tissue homeostasis. Previous studies suggest that BMI1 cells represent secretory progenitors with reserve intestinal stem cell (rISC) activity. However, it has not been elucidated how KLF4 contributes to crypt regeneration originated from BMI1 rISC lineage during homeostasis. In this study, -Cre;Rosa26 (Ctrl) and -Cre;Rosa26; () mice were injected with tamoxifen to label BMI1 cells and their lineage and to delete . During homeostasis, MUC2 goblet cells appeared in the BMI1 cell lineage 2, 3 and 7 days after tamoxifen administration. After deletion in BMI1 cells, the number of KLF4 and MUC2 cells in cells decreased in mice compared with Ctrl mice. Thus, KLF4 was positively correlated with goblet cell differentiation in BMI1 cell derived lineage. In analysis, organoids derived from single cells of Ctrl mice contained MUC2-expressing cells that co-expressed KLF4. On the other hand, organoids derived from Klf4-deleted cells from mice showed reduced number of MUC2-expressing cells. In conclusion, these results suggest that KLF4 regulates goblet cell differentiation in BMI1 ISC-derived lineage during homeostasis.
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http://dx.doi.org/10.15283/ijsc20048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691855PMC
November 2020

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

Cell Mol Gastroenterol Hepatol 2020 25;9(4):587-609. Epub 2019 Nov 25.

Department of Medicine, Stony Brook University Renaissance School of Medicine, Stony Brook, New York; Department of Physiology and Biophysics, Stony Brook University Renaissance School of Medicine, Stony Brook, New York. Electronic address:

Background & Aims: 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.

Methods: We performed studies in adult Lgr5;Rosa26 (Lgr5) and Lgr5;Klf5;Rosa26 (Lgr5) mice. Mice were injected with tamoxifen to activate Cre recombinase, which deletes Klf5 from the intestinal epithelium in Lgr5 but not Lgr5 mice. In experiments involving irradiation, mice were subjected to 12 Gy total body irradiation (TBI). Tissues were collected for immunofluorescence (IF) analysis and next generation sequencing. Oganoids were derived from fluoresecence activated cell sorted- (FACS-) single cells from tamoxifen-treated Lgr5 or Lgr5 mice and examined by immunofluorescence stain.

Results: Lgr5 ISCs lacking KLF5 proliferate faster than control ISCs but fail to self-renew, resulting in a depleted ISC compartment. Transcriptome analysis revealed that Klf5-null Lgr5 cells lose ISC identity and prematurely differentiate. Following irradiation injury, which depletes Lgr5 ISCs, reserve Klf5-null progenitor cells fail to dedifferentiate and regenerate the epithelium. Absence of KLF5 inactivates numerous selected enhancer elements and direct transcriptional targets including canonical WNT- and NOTCH-responsive genes. Analysis of human intestinal tissues showed increased levels of KLF5 in the regenerating epithelium as compared to those of healthy controls.

Conclusion: We conclude that ISC self-renewal, lineage specification, and precursor dedifferentiation require KLF5, by its ability to regulate epigenetic and transcriptional activities of ISC-specific gene sets. These findings have the potential for modulating ISC functions by targeting KLF5 in the intestinal epithelium.
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http://dx.doi.org/10.1016/j.jcmgh.2019.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078555PMC
May 2021

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

Mol Cancer Ther 2019 11 29;18(11):1973-1984. Epub 2019 Jul 29.

Department of Medicine, Stony Brook University School of Medicine, Stony Brook, New York.

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.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825545PMC
November 2019

Oncogenic KRAS Reduces Expression of FGF21 in Acinar Cells to Promote Pancreatic Tumorigenesis in Mice on a High-Fat Diet.

Gastroenterology 2019 11 25;157(5):1413-1428.e11. Epub 2019 Jul 25.

School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, Zhejiang, China.

Background & Aims: Obesity is a risk factor for pancreatic cancer. In mice, a high-fat diet (HFD) and expression of oncogenic KRAS lead to development of invasive pancreatic ductal adenocarcinoma (PDAC) by unknown mechanisms. We investigated how oncogenic KRAS regulates the expression of fibroblast growth factor 21, FGF21, a metabolic regulator that prevents obesity, and the effects of recombinant human FGF21 (rhFGF21) on pancreatic tumorigenesis.

Methods: We performed immunohistochemical analyses of FGF21 levels in human pancreatic tissue arrays, comprising 59 PDAC specimens and 45 nontumor tissues. We also studied mice with tamoxifen-inducible expression of oncogenic KRAS in acinar cells (Kras mice) and fElas mice (controls). Kras mice were placed on an HFD or regular chow diet (control) and given injections of rhFGF21 or vehicle; pancreata were collected and analyzed by histology, immunoblots, quantitative polymerase chain reaction, and immunohistochemistry. We measured markers of inflammation in the pancreas, liver, and adipose tissue. Activity of RAS was measured based on the amount of bound guanosine triphosphate.

Results: Pancreatic tissues of mice expressed high levels of FGF21 compared with liver tissues. FGF21 and its receptor proteins were expressed by acinar cells. Acinar cells that expressed Kras had significantly lower expression of Fgf21 messenger RNA compared with acinar cells from control mice, partly due to down-regulation of PPARG expression-a transcription factor that activates Fgf21 transcription. Pancreata from Kras mice on a control diet and given injections of rhFGF21 had reduced pancreatic inflammation, infiltration by immune cells, and acinar-to-ductal metaplasia compared with mice given injections of vehicle. HFD-fed Kras mice given injections of vehicle accumulated abdominal fat, developed extensive inflammation, pancreatic cysts, and high-grade pancreatic intraepithelial neoplasias (PanINs); half the mice developed PDAC with liver metastases. HFD-fed Kras mice given injections of rhFGF21 had reduced accumulation of abdominal fat and pancreatic triglycerides, fewer pancreatic cysts, reduced systemic and pancreatic markers of inflammation, fewer PanINs, and longer survival-only approximately 12% of the mice developed PDACs, and none of the mice had metastases. Pancreata from HFD-fed Kras mice given injections of rhFGF21 had lower levels of active RAS than from mice given vehicle.

Conclusions: Normal acinar cells from mice and humans express high levels of FGF21. In mice, acinar expression of oncogenic KRAS significantly reduces FGF21 expression. When these mice are placed on an HFD, they develop extensive inflammation, pancreatic cysts, PanINs, and PDACs, which are reduced by injection of FGF21. FGF21 also reduces the guanosine triphosphate binding capacity of RAS. FGF21 might be used in the prevention or treatment of pancreatic cancer.
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http://dx.doi.org/10.1053/j.gastro.2019.07.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815712PMC
November 2019

Loss of the Krüppel-like factor 4 tumor suppressor is associated with epithelial-mesenchymal transition in colorectal cancer.

J Cancer Metastasis Treat 2019 26;5. Epub 2019 Nov 26.

Department of Medicine, Stony Brook University School of Medicine, Stony Brook, NY 11794, USA.

Aim: Colorectal cancer (CRC) is the third leading cancer-related cause of death due to its propensity to metastasize. Epithelial-mesenchymal transition (EMT) is a multistep process important for invasion and metastasis of CRC. Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor highly expressed in differentiated cells of the intestinal epithelium. KLF4 has been shown to play a tumor suppressor role during CRC tumorigenesis - its loss accelerates development and progression of cancer. The present study examined the relationship between KLF4 and markers of EMT in CRC.

Methods: Immunofluorescence staining for KLF4 and EMT markers was performed on archived patient samples after colorectal cancer resection and on colonic tissues of mice with colitis-associated cancer.

Results: We found that KLF4 expression is lost in tumor sections obtained from CRC patients and in those of mouse colon following azoxymethane and dextran sodium sulfate (AOM/DSS) treatment when compared to their respective normal appearing mucosa. Importantly, in CRC patient tumor sections, we observed a negative correlation between KLF4 levels and mesenchymal markers including TWIST, β-catenin, claudin-1, N-cadherin, and vimentin. Similarly, in tumor tissues from AOM/DSS-treated mice, KLF4 levels were negatively correlated with mesenchymal markers including SNAI2, β-catenin, and vimentin and positively correlated with the epithelial marker E-cadherin.

Conclusion: These findings suggest that the loss of KLF4 expression is a potentially significant indicator of EMT in CRC.
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http://dx.doi.org/10.20517/2394-4722.2019.35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304562PMC
November 2019

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

Mol Cancer Res 2019 01 14;17(1):165-176. Epub 2018 Aug 14.

Department of Medicine, Stony Brook University School of Medicine, Stony Brook, New York.

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.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318049PMC
January 2019

Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation.

JCI Insight 2018 06 21;3(12). Epub 2018 Jun 21.

Division of Nephrology, Department of Medicine, Stony Brook University, Stony Brook, New York, USA.

Pathologic glomerular epithelial cell (GEC) hyperplasia is characteristic of both rapidly progressive glomerulonephritis (RPGN) and subtypes of focal segmental glomerulosclerosis (FSGS). Although initial podocyte injury resulting in activation of STAT3 signals GEC proliferation in both diseases, mechanisms regulating this are unknown. Here, we show that the loss of Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, enhances GEC proliferation in both RPGN and FSGS due to dysregulated STAT3 signaling. We observed that podocyte-specific knockdown of Klf4 (C57BL/6J) increased STAT3 signaling and exacerbated crescent formation after nephrotoxic serum treatment. Interestingly, podocyte-specific knockdown of Klf4 in the FVB/N background alone was sufficient to activate STAT3 signaling, resulting in FSGS with extracapillary proliferation, as well as renal failure and reduced survival. In cultured podocytes, loss of KLF4 resulted in STAT3 activation and cell-cycle reentry, leading to mitotic catastrophe. This triggered IL-6 release into the supernatant, which activated STAT3 signaling in parietal epithelial cells. Conversely, either restoration of KLF4 expression or inhibition of STAT3 signaling improved survival in KLF4-knockdown podocytes. Finally, human kidney biopsy specimens with RPGN exhibited reduced KLF4 expression with a concomitant increase in phospho-STAT3 expression as compared with controls. Collectively, these results suggest the essential role of KLF4/STAT3 signaling in podocyte injury and its regulation of aberrant GEC proliferation.
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http://dx.doi.org/10.1172/jci.insight.98214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124441PMC
June 2018

KLF5 mediates the hyper-proliferative phenotype of the intestinal epithelium in mice with intestine-specific endogenous K-Ras expression.

Am J Cancer Res 2018 1;8(4):723-731. Epub 2018 Apr 1.

Department of Medicine, School of Medicine, Stony Brook University, Stony Brook NY 11794, USA.

Oncogenic activation is a common mutational event in colorectal cancer. We previously showed that transcription factor, Krüppel-like factor 5 (KLF5), contributes to intestinal polyposis in mice with activation. At 14 months of age, mice developed small intestinal and colonic hyperplastic polyps while had none. The intestinal crypts of mice contained a higher number of mitotic figures and increased crypt heights compared to controls. The intestinal epithelium of mice showed prolific KLF5 expression throughout and above the elongated crypts. In contrast, KLF5 expression was limited to the upper crypt region in the controls. The levels of K-Ras effectors were significantly increased in as compared to controls. The mice showed decreased survival upon treatment with azoxymethane (AOM) as compared to controls. Furthermore, loss of one of alleles reduced levels of K-Ras effector proteins and prevented mortality of mice upon AOM treatment. The mice spontaneously develop hyperplastic intestinal polyps and display a hyper-proliferative intestinal phenotype with elongated crypts, increased numbers of mitotic figures, elevated expression of KLF5, and other pro-proliferative targets. Induction of colonic tumorigenesis with AOM is detrimental to mice that is in part dependent of KLF5.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934562PMC
April 2018

Correction to: The Role of Intestinal Stem Cells in Epithelial Regeneration Following Radiation-Induced Gut Injury.

Curr Stem Cell Rep 2018;4(1):95. Epub 2018 Feb 19.

1Department of Medicine, Stony Brook University School of Medicine, HSC T-17, Rm. 090, Stony Brook, NY 11794 USA.

[This corrects the article DOI: 10.1007/s40778-017-0103-7.].
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http://dx.doi.org/10.1007/s40778-018-0121-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866267PMC
February 2018

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

Gastroenterology 2018 04 15;154(5):1494-1508.e13. Epub 2017 Dec 15.

Department of Medicine, Stony Brook University School of Medicine, Stony Brook, New York. Electronic address:

Background & Aims: 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.

Methods: We performed studies in adult mice with conditional disruption of Klf5 (Klf5) and/or expression of Kras (LSL-Kras) via Cre recombinase regulated by an acinar cell-specific promoter (Ptf1a). Activation of Kras and loss of KLF5 was achieved by administration of tamoxifen. Pancreatitis was induced in mice by administration of cerulein; pancreatic tissues were collected, analyzed by histology and immunohistochemistry, and transcriptomes were compared between mice that did or did not express KLF5. We performed immunohistochemical analyses of human tissue microarrays, comparing levels of KLF5 among 96 human samples of PDAC. UN-KC-6141 cells (pancreatic cancer cells derived from Pdx1-Cre;LSL-Kras mice) were incubated with inhibitors of different kinases and analyzed in proliferation assays and by immunoblots. Expression of KLF5 was knocked down with small hairpin RNAs or CRISPR/Cas9 strategies; cells were analyzed in proliferation and gene expression assays, and compared with cells expressing control vectors. Cells were subcutaneously injected into flanks of syngeneic mice and tumor growth was assessed.

Results: Of the 96 PDAC samples analyzed, 73% were positive for KLF5 (defined as nuclear staining in more than 5% of tumor cells). Pancreata from Ptf1a-Cre;LSL-Kras mice contained ADM and PanIN lesions, which contained high levels of nuclear KLF5 within these structures. In contrast, Ptf1a-Cre;LSL-Kras;Klf5 mice formed fewer PanINs. After cerulein administration, Ptf1a-Cre;LSL-Kras mice formed more extensive ADM than Ptf1a-Cre;LSL-Kras;Klf5 mice. Pancreata from Ptf1a-Cre;LSL-Kras;Klf5 mice had increased expression of the tumor suppressor NDRG2 and reduced phosphorylation (activation) of STAT3, compared with Ptf1a-Cre;LSL-Kras mice. In UN-KC-6141 cells, PI3K and MEK signaling increased expression of KLF5; a high level of KLF5 increased proliferation. Cells with knockdown of Klf5 had reduced proliferation, compared with control cells, had reduced expression of ductal markers, and formed smaller tumors (71.61 ± 30.79 mm vs 121.44 ± 34.90 mm from control cells) in flanks of mice.

Conclusion: Levels of KLF5 are increased in human PDAC samples and in PanINs of Ptf1a-Cre;LSL-Kras mice, compared with controls. KLF5 disruption increases expression of NDRG2 and reduces activation of STAT3 and reduces ADM and PanINs formation in mice. Strategies to reduce KLF5 activity might reduce progression of acinar cells from ADM to PanIN and pancreatic tumorigenesis.
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http://dx.doi.org/10.1053/j.gastro.2017.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880723PMC
April 2018

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

Am J Physiol Gastrointest Liver Physiol 2017 Nov 1;313(5):G478-G491. Epub 2017 Sep 1.

Department of Medicine, Stony Brook University School of Medicine, Stony Brook, New York;

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.
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http://dx.doi.org/10.1152/ajpgi.00172.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792213PMC
November 2017

The loss of Krüppel-like factor 15 in Foxd1 stromal cells exacerbates kidney fibrosis.

Kidney Int 2017 11 24;92(5):1178-1193. Epub 2017 Jun 24.

Kidney Institute of PLA, Department of Medicine, Changzheng Hospital, Second Military Medical University, Shanghai 200003, People's Republic of China. Electronic address:

Large epidemiological studies clearly demonstrate that multiple episodes of acute kidney injury contribute to the development and progression of kidney fibrosis. Although our understanding of kidney fibrosis has improved in the past two decades, we have limited therapeutic strategies to halt its progression. Myofibroblast differentiation and proliferation remain critical to the progression of kidney fibrosis. Although canonical Wnt signaling can trigger the activation of myofibroblasts in the kidney, mediators of Wnt inhibition in the resident progenitor cells are unclear. Recent studies demonstrate that the loss of a Krüppel-like factor 15 (KLF15), a kidney-enriched zinc-finger transcription factor, exacerbates kidney fibrosis in murine models. Here, we tested whether Klf15 mRNA and protein expression are reduced in late stages of fibrosis in mice that underwent unilateral ureteric obstruction, a model of progressive renal fibrosis. Knockdown of Klf15 in Foxd1-expressing cells (Foxd1-Cre Klf15fl/fl) increased extracellular matrix deposition and myofibroblast proliferation as compared to wildtype (Foxd1-Cre Klf15+/+) mice after three and seven days of ureteral obstruction. This was validated in mice receiving angiotensin II treatment for six weeks. In both these murine models, the increase in renal fibrosis was found in Foxd1-Cre Klf15 mice and accompanied by the activation of Wnt/β-catenin signaling. Furthermore, knockdown of Klf15 in cultured mouse embryonic fibroblasts activated canonical Wnt/β-catenin signaling, increased profibrotic transcripts, and increased proliferation after treatment with a Wnt1 ligand. Conversely, the overexpression of KLF15 inhibited phospho-β-catenin (Ser552) expression in Wnt1-treated cells. Thus, KLF15 has a critical role in attenuating kidney fibrosis by inhibiting the canonical Wnt/β-catenin pathway.
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http://dx.doi.org/10.1016/j.kint.2017.03.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651204PMC
November 2017

Novel sphingosine kinase-1 inhibitor, LCL351, reduces immune responses in murine DSS-induced colitis.

Prostaglandins Other Lipid Mediat 2017 05 2;130:47-56. Epub 2017 Apr 2.

Department of Medicine and the, Stony Brook Cancer Center, Stony Brook University, Stony Brook, NY, USA; Northport Veterans Affairs Medical Center, Northport, NY, USA. Electronic address:

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite which has been implicated in many diseases including cancer and inflammatory diseases. Recently, sphingosine kinase 1 (SK1), one of the isozymes which generates S1P, has been implicated in the development and progression of inflammatory bowel disease (IBD). Based on our previous work, we set out to determine the efficacy of a novel SK1 selective inhibitor, LCL351, in a murine model of IBD. LCL351 selectively inhibits SK1 both in vitro and in cells. LCL351, which accumulates in relevant tissues such as colon, did not have any adverse side effects in vivo. In mice challenged with dextran sodium sulfate (DSS), a murine model for IBD, LCL351 treatment protected from blood loss and splenomegaly. Additionally, LCL351 treatment reduced the expression of pro-inflammatory markers, and reduced neutrophil infiltration in colon tissue. Our results suggest inflammation associated with IBD can be targeted pharmacologically through the inhibition and degradation of SK1. Furthermore, our data also identifies desirable properties of SK1 inhibitors.
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http://dx.doi.org/10.1016/j.prostaglandins.2017.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509055PMC
May 2017

SP and KLF Transcription Factors in Digestive Physiology and Diseases.

Gastroenterology 2017 06 30;152(8):1845-1875. Epub 2017 Mar 30.

Department of Medicine, Stony Brook University School of Medicine, Stony Brook, New York; Department of Physiology and Biophysics, Stony Brook University School of Medicine, Stony Brook, New York. Electronic address:

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.
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http://dx.doi.org/10.1053/j.gastro.2017.03.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815166PMC
June 2017

Krüppel-like factors in mammalian stem cells and development.

Development 2017 03;144(5):737-754

Division of Nephrology, Department of Medicine, Stony Brook University School of Medicine, Stony Brook, NY 11794-8176, USA

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.
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http://dx.doi.org/10.1242/dev.145441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374354PMC
March 2017

The Role of Intestinal Stem Cells in Epithelial Regeneration Following Radiation-Induced Gut Injury.

Curr Stem Cell Rep 2017 5;3(4):320-332. Epub 2017 Oct 5.

1Department of Medicine, Stony Brook University School of Medicine, HSC T-17, Rm. 090, Stony Brook, NY 11794 USA.

Purpose Of Review: Intestinal epithelial cells show remarkable plasticity in regenerating the epithelium following radiation injury. In this review, we explore the regenerative capacity and mechanisms of various populations of intestinal stem cells (ISCs) in response to ionizing radiation.

Recent Findings: Ionizing radiation targets mitotic cells that include "active" ISCs and progenitor cells. Lineage-tracing experiments showed that several different cell types identified by a single or combination of markers are capable of regenerating the epithelium, confirming that ISCs exhibit a high degree of plasticity. However, the identities of the contributing cells marked by various markers require further validation.

Summary: Following radiation injury, quiescent and/or radioresistant cells become active stem cells to regenerate the epithelium. Looking forward, understanding the mechanisms by which ISCs govern tissue regeneration is crucial to determine therapeutic approaches to promote intestinal epithelial regeneration following injury.
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http://dx.doi.org/10.1007/s40778-017-0103-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818549PMC
October 2017

Epithelial derived-matrix metalloproteinase (MMP9) exhibits a novel defensive role of tumor suppressor in colitis associated cancer by activating MMP9-Notch1-ARF-p53 axis.

Oncotarget 2017 Jan;8(1):364-378

Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.

Colitis associated cancer (CAC) is chronic inflammation driven colon cancer, prevalent among individuals with Inflammatory Bowel Disease. Matrix-metalloproteinase (MMP9) is one of the essential regulators of extra cellular matrix components. We have shown that MMP9 is protective in CAC contrary to its inflammatory role in acute-colitis. Aim of our study is to identify the mechanism of the protective role of epithelial derived-MMP9 in CAC. We used homozygous transgenic mice constitutively-expressing MMP9 in colonic-epithelium (TgM9) and wild-type (WT) littermates for in vivo experiments. Stably-transfected HCT116 with/without MMP9, and mouse embryonic-fibroblasts (WT and MMP9-/-, MEFs) were used for in vitro experiments. TgM9 mice exhibited less tumor burden, increased apoptosis, and increased expressions of active-Notch1, p53, p21WAF1/Cip1, caspase-3 and cyclin E in CAC compared to WTs. These results were supported by MEFs data. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation, S-phase cell-cycle arrest and less DNA damage compared to vector. MMP9-/- mice showed attenuation of MMP9 was directly associated with p19ARF. Our study identifies the tumor suppressor role of epithelial derived-MMP9 in CAC via novel mechanistic pathway "MMP9-Notch1-ARF-p53 axis" regulating apoptosis, cell-cycle arrest and DNA damage implying, that MMP9 expression might be a natural/biological way to suppress colonic ulceration due to chronic inflammation.
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http://dx.doi.org/10.18632/oncotarget.13406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352126PMC
January 2017

Intestinal stem cell resurgence by enterocyte precursors.

Stem Cell Investig 2016 23;3:49. Epub 2016 Sep 23.

Department of Medicine, Stony Brook University School of Medicine, Stony Brook, NY, USA;; Department of Physiology and Biophysics, Stony Brook University School of Medicine, Stony Brook, NY, USA.

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http://dx.doi.org/10.21037/sci.2016.09.01DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067364PMC
September 2016

Role of neutral ceramidase in colon cancer.

FASEB J 2016 12 8;30(12):4159-4171. Epub 2016 Sep 8.

Department of Medicine, Stony Brook University, New York, USA;

Alterations in sphingolipid metabolism, especially ceramide and sphingosine 1-phosphate, have been linked to colon cancer, suggesting that enzymes of sphingolipid metabolism may emerge as novel regulators and targets in colon cancer. Neutral ceramidase (nCDase), a key enzyme in sphingolipid metabolism that hydrolyzes ceramide into sphingosine, is highly expressed in the intestine; however, its role in colon cancer has not been defined. Here we show that molecular and pharmacological inhibition of nCDase in colon cancer cells increases ceramide, and this is accompanied by decreased cell survival and increased apoptosis and autophagy, with minimal effects on noncancerous cells. Inhibition of nCDase resulted in loss of β-catenin and inhibition of ERK, components of pathways relevant for colon cancer development. Furthermore, inhibition of nCDase in a xenograft model delayed tumor growth and increased ceramide while decreasing proliferation. It is noteworthy that mice lacking nCDase treated with azoxymethane were protected from tumor formation. Taken together, these studies show that nCDase is pivotal for regulating initiation and development of colon cancer, and these data suggest that this enzyme is a suitable and novel target for colon cancer therapy.-García-Barros, M., Coant, N., Kawamori, T., Wada, M., Snider, A. J., Truman, J.-P., Wu, B. X., Furuya, H., Clarke, C. J., Bialkowska, A. B., Ghaleb, A., Yang, V. W., Obeid, L. M., Hannun, Y. A. Role of neutral ceramidase in colon cancer.
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http://dx.doi.org/10.1096/fj.201600611RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102116PMC
December 2016

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

J Vis Exp 2016 07 13(113). Epub 2016 Jul 13.

Department of Medicine, Stony Brook University School of Medicine; Department of Physiology & Biophysics, Stony Brook University School of Medicine;

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.
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http://dx.doi.org/10.3791/54161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993444PMC
July 2016

Krüppel-Like Factor 15 Mediates Glucocorticoid-Induced Restoration of Podocyte Differentiation Markers.

J Am Soc Nephrol 2017 Jan 10;28(1):166-184. Epub 2016 Jun 10.

Department of Pharmacology and Systems Therapeutics and.

Podocyte injury is the inciting event in primary glomerulopathies, such as minimal change disease and primary FSGS, and glucocorticoids remain the initial and often, the primary treatment of choice for these glomerulopathies. Because inflammation is not readily apparent in these diseases, understanding the direct effects of glucocorticoids on the podocyte, independent of the immunomodulatory effects, may lead to the identification of targets downstream of glucocorticoids that minimize toxicity without compromising efficacy. Several studies showed that treatment with glucocorticoids restores podocyte differentiation markers and normal ultrastructure and improves cell survival in murine podocytes. We previously determined that Krüppel-like factor 15 (KLF15), a kidney-enriched zinc finger transcription factor, is required for restoring podocyte differentiation markers in mice and human podocytes under cell stress. Here, we show that in vitro treatment with dexamethasone induced a rapid increase of KLF15 expression in human and murine podocytes and enhanced the affinity of glucocorticoid receptor binding to the promoter region of KLF15 In three independent proteinuric murine models, podocyte-specific loss of Klf15 abrogated dexamethasone-induced podocyte recovery. Furthermore, knockdown of KLF15 reduced cell survival and destabilized the actin cytoskeleton in differentiated human podocytes. Conversely, overexpression of KLF15 stabilized the actin cytoskeleton under cell stress in human podocytes. Finally, the level of KLF15 expression in the podocytes and glomeruli from human biopsy specimens correlated with glucocorticoid responsiveness in 35 patients with minimal change disease or primary FSGS. Thus, these studies identify the critical role of KLF15 in mediating the salutary effects of glucocorticoids in the podocyte.
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http://dx.doi.org/10.1681/ASN.2015060672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198263PMC
January 2017

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

Stem Cell Reports 2016 06 26;6(6):815-824. Epub 2016 May 26.

Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA; Stony Brook University Medical Center, HSC T-16, Room 020, Stony Brook, NY 11794-8160, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stemcr.2016.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911500PMC
June 2016

Murine Model for Colitis-Associated Cancer of the Colon.

Methods Mol Biol 2016 ;1438:245-54

Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), significantly increases the risk for development of colorectal cancer. Specifically, dysplasia and cancer associated with IBD (colitis-associated cancer or CAC) develop as a result of repeated cycles of injury and healing in the intestinal epithelium. Animal models are utilized to examine the mechanisms of CAC, the role of epithelial and immune cells in this process, as well as the development of novel therapeutic targets. These models typically begin with the administration of a carcinogenic compound, and inflammation is caused by repeated cycles of colitis-inducing agents. This review describes a common CAC model that utilizes the pro-carcinogenic compound azoxymethane (AOM) followed by dextran sulfate sodium (DSS) which induces the inflammatory insult.
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http://dx.doi.org/10.1007/978-1-4939-3661-8_14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657253PMC
December 2017

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

Mol Cancer Res 2016 Apr 2;14(4):385-96. Epub 2016 Feb 2.

Department of Medicine, Stony Brook University, Stony Brook, New York. Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York.

Unlabelled: 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, the in vivo effects of Klf4 deletion 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 with Apc(Min/⁺) mice with intact Klf4 (Klf4(fl/fl)/Apc (Min/⁺)). The colonic epithelium of Klf4 (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 than Klf4 (fl/fl) mice, with more Klf4 (ΔIS) mice harboring K-Rasmutations than Klf4 (fl/fl)mice. Compared with AOM-treated Klf4 (fl/fl)mice, adenomas of treated Klf4 (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.

Implications: The study demonstrates that KLF4 plays a significant role in the pathogenesis of colorectal neoplasia.
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http://dx.doi.org/10.1158/1541-7786.MCR-15-0410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834227PMC
April 2016

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

Mol Cancer Ther 2016 Jan 30;15(1):72-83. Epub 2015 Nov 30.

Department of Medicine, Stony Brook University School of Medicine, Stony Brook, New York.

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.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707060PMC
January 2016

Role of Krüppel-like factor 5 in the maintenance of the stem cell niche in the intestinal crypt.

Stem Cell Transl Investig 2015;2(2)

Department of Medicine, Stony Brook University School of Medicine, HSC-T16 Room 020, Stony Brook, NY 11794, United States ; Department of Physiology and Biophysics, Stony Brook University School of Medicine, HSC-T16 Room 020, Stony Brook, NY 11794, United States.

The intestinal epithelium is a tissue that undergoes continuous self-renewal initiated at the bottom of the crypts, which harbor the intestinal stem cell (ISC) pool. The ISC pool is sub-divided into crypt base columnar (CBC) cells at the crypt bottom and label retention cells (LRC) at position +4 from the crypt bottom. CBC cells are marked by Leucine-rich repeat-containing G-protein coupled receptor (Lgr5) while LRC cells are identified by several markers including Bmi1, mTert, Hopx, Lrig1, and Sox9. Krüppel-like factors (KLFs) belong to a family of transcription factors that exert important physiological function in various tissues. In the intestine, KLF4 is predominantly expressed in the terminally differentiated, non-proliferating cells lining the villus. Its deletion in the adult mouse intestine results in perturbed homeostasis. In contrast, KLF5 is expressed in actively proliferating cells of the intestinal crypt, including CBC cells and transit amplifying (TA) cells. We recently investigated the effect of deletion specifically from the Lgr5-expressing CBC cells in adult mouse intestine using an inducible Cre recombinase system. Shortly (3-5 days) after Cre induction, proliferation of both CBC and TA cells ceased, which was accompanied by an increase in apoptosis in the crypt. Beginning at two weeks following Cre induction, both Klf5 expression and proliferation re-appeared but without the re-emergence of Lgr5-positive CBC cells, which were eventually depleted by four months following induction. These findings indicate that KLF5 plays an important role in regulating proliferation and survival of CBC stem cells in the intestine.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474380PMC
January 2015