Krüppel-like Factor 5 Knockdown Research Articles

Kruppel‐like Factor 4 is Critical to the Maintenance of Glomerular Endothelial Integrity

BackgroundThrombotic microangiopathy (TMA) represents the most severe manifestation of glomerular endothelial injury consisting of endothelial cell (EC) swelling, subendothelial expansion, inflammation and microthrombi. TMA occurs subsequent to various etiologies and is associated with dysregulation of thrombotic and inflammatory transcripts and complement activation. Expression of membrane bound (CD55, CD59, CD46) complement regulators, is altered in many subtypes of TMA and it is unclear if this is a driver or consequence of EC injury. To investigate transcriptional mediators involved in TMA, we reviewed expression arrays of both global and single cell RNA‐seq from human kidney biopsies with complement mediated renal microvascular injury and observed that Krüppel‐Like Factor 4 (KLF4), a zinc finger transcription factor, is both differentially expressed in ECs and predicted to regulate other differentially expressed genes. Previous studies demonstrate that KLF4 is a critical mediator of anti‐thrombotic and anti‐inflammatory phenotype in systemic vascular beds. We aimed to investigate its role in modulating renal microvascular injury and complement activation in TMA.MethodsEndothelial‐specific Klf4 knockout mice (Klf4ΔEC) were generated by crossing Klf4fl/fl mice withCdh5‐Cre mice. As two separate models of endothelial dysfunction, Klf4ΔEC mice were both aged to 1 year and crossed with Nos3−/− mice to generate double knockout mice (DKO). Periodic acid‐Schiff, electron microscopy, immunofluorescence, ELISA and RT‐PCR were performed to investigate the effects of endothelial Klf4 on the renal microvasculature. Renal histology and ultrastructure were evaluated, blinded, by renal pathology (MPR).ResultsAt 12 weeks of age, while no overt histologic changes were observed, Klf4ΔEC mice exhibited increased glomerular Vcam‐1 and Pai‐1 expression and complement activation (increased C3 and C5b‐9 deposition) as compared to Klf4fl/fl mice. To investigate the basis for this increased complement activation, we measured the expression of the membrane‐bound complement regulators and noted reduced glomerular Cd55 and Cd59 expression in Klf4ΔEC as compared with Klf4fl/fl mice. Interestingly, 1‐year‐old Klf4ΔEC mice exhibited pathology consistent with subacute TMA (glomerular capillary congestion, EC swelling, subendothelial expansion and mesangiolysis as well as increased intraglomerular von Willebrand Factor and albuminuria) as compared to age‐matched Klf4fl/fl mice. In addition, Klf4ΔEC mice crossed with nitric oxide synthase knockout (Nos3−/−) mice also exhibited increased glomerular complement activation and histological and ultrastructural features consistent with subacute TMA (loss of fenestrations, glomerular capillary congestion, tuft collapse with basement membrane wrinkling) as compared to all other groups (12 weeks). Finally, knockdown of KLF4 in cultured human microvascular endothelial cells reduced the expression of CD55, whereas induction of KLF4 increased CD55 expression in HMEC‐1s.ConclusionsLoss of endothelial Klf4 in both aged mice and in Nos3−/− mice recapitulates many pathologic features of renal‐TMA, including complement activation and is potentially mediated through its regulation of Cd55.Support or Funding InformationNIDDK

Pioglitazone protects blood vessels through inhibition of the apelin signaling pathway by promoting KLF4 expression in rat models of T2DM.

Apelin, identified as the endogenous ligand of APJ, exerts various cardiovascular effects. However, the molecular mechanism underlying the regulation of apelin expression in vascular cells is poorly described. Pioglitazone (PIO) and Krüppel-like factor 4 (KLF4) exhibit specific biological functions on vascular physiology and pathophysiology by regulating differentiation- and proliferation-related genes. The present study aimed to investigate the roles of PIO and KLF4 in the transcriptional regulation of apelin in a high-fat diet/streptozotocin rat model of diabetes and in PIO-stimulated vascular smooth muscle cells (VSMCs). Immunohistochemistry, qRT-PCR, and Western blotting assays revealed that the aorta of the Type 2 diabetes mellitus (T2DM) rat models had a high expression of apelin, PIO could decrease the expression of apelin in the PIO-treated rats. In vitro, Western blotting assays and immunofluorescent staining results showed that the basal expression of apelin was decreased but that of KLF4 was increased when VSMCs were stimulated by PIO treatment. Luciferase and chromatin immunoprecipitation assay results suggested that KLF4 bound to the GKLF-binding site of the apelin promoter and negatively regulated the transcription activity of apelin in VSMCs under PIO stimulation. Furthermore, qRT-PCR and Western blotting assay results showed that the overexpression of KLF4 markedly decreased the basal expression of apelin, but the knockdown of KLF4 restored the PIO-induced expression of apelin. In conclusion, PIO inhibited the expression of apelin in T2DM rat models to prevent diabetic macroangiopathy, and negatively regulated the gene transcription of apelin by promoting transcription of KLF4 in the apelin promoter.

Hyperhomocysteinemia induces vascular calcification by activating the transcription factor RUNX2 via Krüppel-like factor 4 up-regulation in mice

One of the main characteristics of atherosclerosis is vascular calcification, which is linked to adverse cardiovascular events. Increased homocysteine (Hcy), a feature of hyperhomocysteinemia, is correlated with advanced vascular calcification and phenotypic switching of vascular smooth muscle cells (VSMCs). Oxidative stress and high phosphate levels also induce VSMC calcification, suggesting that the Krüppel-like factor 4 (KLF4) signaling pathway may also contribute to vascular calcification. In this study, we investigated this possibility and the role and mechanisms of Hcy in vascular calcification. We found that in atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice, Hcy significantly increases vascular calcification in vivo, as well as VSMC calcification in vitro Of note, the Hcy-induced VSMC calcification was correlated with elevated KLF4 levels. Hcy promoted KLF4 expression in calcified atherosclerotic lesions in vivo and in calcified VSMCs in vitro shRNA-mediated KLF4 knockdown blocked the Hcy-induced up-regulation of runt-related transcription factor 2 (RUNX2) and VSMC calcification. RUNX2 inhibition abolished Hcy-induced VSMC calcification. Using ChIP analysis, we demonstrate that KLF4 interacts with RUNX2, an interaction promoted by Hcy stimulation. Our experiments also revealed that the KLF4 knockdown attenuates Hcy-induced RUNX2 transactivity, indicating that KLF4 is important in modulating RUNX2 transactivity. These findings support a role for Hcy in regulating vascular calcification through a KLF4-RUNX2 interaction and indicate that Hcy-induced, enhanced RUNX2 transactivity increases VSMC calcification. These insights reveal possible opportunities for developing interventions that prevent or manage vascular calcification.

The long non-coding RNA TUG1-miR-9a-5p axis contributes to ischemic injuries by promoting cardiomyocyte apoptosis via targeting KLF5

Non-coding RNAs participate in many cardiac pathophysiological processes, including myocardial infarction (MI). Here we showed the interplay between long non-coding RNA taurine-upregulated gene 1 (lncR-TUG1), miR-9a-5p (miR-9) and Krüppel-like factor 5 (KLF5). LncR-TUG1 was upregulated in ischemic heart and in cultured cardiomyocytes exposed to H2O2. Knockdown of lncR-TUG1 markedly ameliorated impaired cardiac function of MI mice. Further study showed that lncR-TUG1 acted as a competitive endogenous RNA of miR-9, and silencing of lncR-TUG1 inhibited cardiomyocyte apoptosis by upregulating miR-9 expression. Furthermore, the miR-9 overexpression obviously prevented ischemia injury and significantly inhibited H2O2-induced cardiomyocyte apoptosis via inhibition of mitochondrial apoptotic pathway. KLF5, as a target gene of miR-9 by dual-luciferase reporter assay, was involved in the process of miR-9 in regulating cardiomyocyte apoptosis. Our data identified the KLF5 was downregulated by miR-9 overexpression and knockdown of KLF5 inhibited cardiomyocyte apoptosis induced by H2O2. MiR-9 exerts anti-cardiomyocyte apoptotic affects by targeting KLF5. Collectively, our data identify a novel function of lncR-TUG1/miR-9/KLF5 axis in regulating cardiomyocyte apoptosis that affects myocardial infarction progression.

Mechanism of KLF4 Protection against Acute Liver Injury via Inhibition of Apelin Signaling.

Krüppel-like factor 4 (KLF4) is a key transcription factor that regulates genes involved in the proliferation or differentiation in different tissues. Apelin plays roles in cardiovascular functions, metabolic disease, and homeostatic disorder. However, the biological function of apelin in liver disease is still ongoing. In this study, we investigated the mechanism of KLF4-mediated protection against acute liver injury via the inhibition of the apelin signaling pathway. Mice were intraperitoneally injected with carbon tetrachloride (CCl4; 0.2 mL dissolved in 100 mL olive oil, 10 mL/kg) to establish an acute liver injury model. A KLF4 expression plasmid was injected through the tail vein 48 h before CCl4 treatment. In cultured LX-2 cells, pAd-KLF4 or siRNA KLF4 was overexpressed or knockdown, and the mRNA and protein levels of apelin were determined. The results showed that the apelin serum level in the CCl4-injected group was higher than that of control group, and the expression of apelin in the liver tissues was elevated while KLF4 expression was decreased in the CCl4-injected group compared to the KLF4-plasmid-injected group. HE staining revealed serious hepatocellular steatosis in the CCl4-injected mice, and KLF4 alleviated this steatosis in the mice injected with KLF4 plasmid. In vitro experiments showed that tumor necrosis factor-alpha (TNF-α) could downregulate the transcription and translation levels of apelin in LX-2 cells and also upregulate KLF4 mRNA and protein expression. RT-PCR and Western blotting showed that the overexpression of KLF4 markedly decreased basal apelin expression, but knockdown of KLF4 restored apelin expression in TNF-α-treated LX-2 cells. These in vivo and in vitro experiments suggest that KLF4 plays a key role in inhibiting hepatocellular steatosis in acute liver injury, and that its mechanism might be the inhibition of the apelin signaling pathway.

The profibrotic effects of MK‐8617 on tubulointerstitial fibrosis mediated by the KLF5 regulating pathway

The discovery of hypoxia-inducible factor (HIF)-prolyl hydroxylase inhibitor (PHI) has revolutionized the treatment strategy for renal anemia. However, the presence of multiple transcription targets of HIF raises safety concerns regarding HIF-PHI. Here, we explored the dose-dependent effect of MK-8617 (MK), a kind of HIF-PHI, on renal fibrosis. MK was administered by oral gavage to mice for 12 wk at doses of 1.5, 5, and 12.5 mg/kg. In vitro, the human proximal tubule epithelial cell line HK-2 was treated with increasing doses of MK administration. Transcriptome profiling was performed, and fibrogenesis was evaluated. The dose-dependent biphasic effects of MK on tubulointerstitial fibrosis (TIF) were observed in chronic kidney disease mice. Accordingly, high-dose MK treatment could significantly enhance TIF. Using RNA-sequencing, combined with in vivo and in vitro experiments, we found that Krüppel-like factor 5 (KLF5) expression level was significantly increased in the proximal tubular cells, which could be transcriptionally regulated by HIF-1α with high-dose MK treatment but not low-dose MK. Furthermore, our study clarified that HIF-1α-KLF5-TGF-β1 signaling activation is the potential mechanism of high-dose MK-induced TIF, as knockdown of KLF5 reduced TIF in vivo. Collectively, our study demonstrates that high-dose MK treatment initiates TIF by activating HIF-1α-KLF5-TGF-β1 signaling. These findings provide novel insights into TIF induction by high-dose MK (HIF-PHI), suggesting that the safety dosage window needs to be emphasized in future clinical applications.-Li, Z.-L., Lv, L.-L., Wang, B., Tang, T.-T., Feng, Y., Cao, J.-Y., Jiang, L.-Q., Sun, Y.-B., Liu, H., Zhang, X.-L., Ma, K.-L., Tang, R.-N., Liu, B.-C. The profibrotic effects of MK-8617 on tubulointerstitial fibrosis mediated by the KLF5 regulating pathway.

Krüppel-like Factor 5 Promotes Sonic Hedgehog Signaling and Neoplasia in Barrett's Esophagus and Esophageal Adenocarcinoma

Krüppel-like Factor 5 (KLF5) is a zinc-finger transcription factor associated with cell cycle progression and cell survival. KLF5 plays a key role in mammalian intestinal epithelium development and maintenance, expressed at high levels in basal proliferating cells and low levels in terminally differentiated cells. Considering Barrett's esophagus (BE) and esophageal adenocarcinoma's (EAC) histopathological similarities to intestinal epithelium, we sought to determine KLF5’s role in BE and EAC, as well as KLF5’s possible connection to the sonic hedgehog (SHH) pathway which is highly active in BE and EAC development. Low levels of KLF5 mRNA were found in BE cell lines and tissue– similar to what has been reported in differentiated intestinal epithelium. In contrast, higher KLF5 levels were observed in EAC cells and tissues. KLF5 knockdown in EAC cells caused significant decreases in cell migration, proliferation, and EAC-associated gene expression. Moreover, KLF5 knockdown led to decreased SHH signaling. These results suggest that KLF5 is connected to the SHH pathway in BE and EAC and may represent a potential drug target in EAC; further studies are now indicated to verify these findings and elucidate underlying mechanisms involved.

Smooth muscle-specific Gsα deletion exaggerates angiotensin II-induced abdominal aortic aneurysm formation in mice in vivo

ObjectiveAbdominal aortic aneurysm (AAA) is a life-threatening vascular disease without an effective pharmaceutical treatment. Genetic studies have proved the involvement of smooth muscle phenotype switch in the development of AAA. The alpha subunit of the heterotrimeric G stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of smooth muscle Gsα in AAA formation remains unknown. Approach and resultsIn this study, mice with knockout of smooth muscle-specific Gsα (GsαSMKO) were generated by cross-breeding Gsαflox/flox mice with SM22-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. Gsα deficiency induced a smooth muscle phenotype switch from a contractile to a synthetic state. Mechanically, Gsα deletion reduced cAMP level and increased the level of human antigen R (HuR), which binds with the adenylate uridylate–rich elements of the 3′ untranslated region of Krüppel-like factor 4 (KLF4) mRNA, thereby increasing the stability of KLF4. Moreover, genetic knockdown of HuR or KLF4 rescued the phenotype switch in Gsα-deficient smooth muscle cells. Furthermore, with acute infusion of angiotensin II, the incidence of AAA was markedly higher in ApoE−/−/GsαSMKO than ApoE−/−/Gsαflox/flox mice and induced increased elastic lamina degradation and aortic expansion. Finally, the levels of Gsα and SM α-actin were significantly lower while those of HuR and KLF4 were higher in human AAA samples than adjacent nonaneurysmal aortic sections. ConclusionsGsα may play a protective role in AAA formation by regulating the smooth muscle phenotype switch and could be a potential therapeutic target for AAA disease.

Podocyte‐parietal epithelial cell cross‐talk: a role for paracrine KLF4‐STAT3 signaling in parietal epithelial cell proliferation in proliferative glomerulopathies

Podocyte injury contributing to parietal epithelial cell (PEC) proliferation is a dominant histologic feature in both rapidly progressive glomerulonephritis (RPGN) and subtypes of focal segmental glomerulosclerosis (FSGS). Activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling is critical to the progression of both. We recently showed that podocyte‐specific loss of Krüppel‐Like Factor 4 (KLF4), a zinc‐finger transcription factor, activates STAT3 signaling leading to mitotic catastrophe in podocytes and eventual FSGS, while triggering PEC proliferation.To determine the mechanism by which podocyte‐specific knockdown of KLF4 leads to podocyte loss and triggers PEC proliferation with dysregulated STAT3 signaling, we generated cultured human podocytes with KLF4 knockdown (KLF4‐shRNA) and screened several pharmacological STAT3‐specific inhibitors to determine that S3I‐201 prevented podocyte loss to a higher degree than other STAT3 inhibitors or DMSO‐treated KLF4‐shRNA podocytes. Furthermore, we demonstrated that S3I‐201‐treated PodCre‐KLF4fl/fl mice exhibited less proteinuria and improved renal function as compared to DMSO‐treated PodCre‐KLF4fl/fl mice. We further validated the reno‐protective effects of S3I‐201 in HIV‐1 transgenic mice, a known model of podocyte‐specific Klf4 loss and STAT3 activation, by demonstrating reduced albuminuria, podocyte loss, FSGS lesions, and parietal epithelial cell activation, with improved renal function.We previously demonstrated that supernatant harvested from KLF4‐shRNA podocytes induces PEC proliferation. To elucidate the mechanism by which loss of KLF4 specifically in podocytes triggers PEC activation and proliferation, we performed unbiased quantitative proteomics on the supernatant of KLF4‐shRNA podocytes as compared to EV‐shRNA podocytes. We subsequently cross‐matched proteins upregulated in supernatant of KLF4‐shRNA podocytes with differentially expressed transcripts from RNA‐sequencing of isolated glomeruli from PodCre‐KLF4fl/fl mice as compared to wildtype KLF4fl/fl mice. Enrichment analysis of these differentially expressed targets revealed activation of STAT‐dependent and independent pathways involved in inflammation, extracellular matrix organization, and proliferation. Interestingly, the highest expressed STAT3 downstream targets were Plasminogen Activator Inhibitor 1 (PAI‐1) and Galectin‐1 (LGALS1), which are key mediators of epithelial cell migration and proliferation.Collectively, these findings demonstrate the role of dysregulated KLF4‐STAT3 signaling in proliferative glomerulopathies and identify potential paracrine mediators of podocyte‐PEC cross‐talk.Support or Funding InformationNIH/NIDDK, SBU URECAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Knockdown of KLF5 promotes cisplatin-induced cell apoptosis via regulating DNA damage checkpoint proteins in non-small cell lung cancer.

BackgroundPrevious research has revealed that Krüppel‐like factor 5 (KLF5) may affect DNA damage repair pathways; however, the associated molecular mechanisms are unclear.MethodsThe expression of KLF5 was studied by immunohistochemical staining in paired tumour and normal tissues from 90 patients with ESCC. We studied the effects of KLF5 knockdown on cell proliferation and apoptosis with or without cisplatin treatment in A549 and H1299 cell lines. Moreover, we examined the effect of KLF5 on the DNA damage response.Results KLF5 was significantly overexpressed in non‐small cell lung cancer (NSCLC) tissues, and high KLF5 expression predicted poor prognosis for NSCLC patients. The inhibition of KLF5 markedly augmented cisplatin‐induced cell apoptosis. In addition, we observed that KLF5 knockdown could decrease DNA repair potential by inhibiting H2AX S139 phosphorylation in response to cisplatin. Moreover, silencing of KLF5 in NSCLC cell lines inhibited the phosphorylation of checkpoint kinases Chk1 S345 and Chk2 T68. KLF5 knockdown permits cells with broken or damaged DNA strands to enter mitosis by inhibiting the activation of H2AX, Chk1 and Chk2, resulting in mitotic catastrophe.Conclusion KLF5 plays a significant role in the DNA damage response by regulating DNA damage checkpoint proteins. Inhibition of KLF5 may be a potential therapeutic target for NSCLC patients with cisplatin resistance.

Role of Krüppel-like Factor 4-p21CIP1 Axis in Breast Cancer Stem-like Cell Inhibition by Benzyl Isothiocyanate.

Cancer chemoprevention by benzyl isothiocyanate (BITC), which is derived from cruciferous vegetables like garden cress, in a transgenic mouse model of breast cancer is associated with inhibition of breast cancer stem-like cells (bCSC), but the molecular regulators of this effect remain elusive. This study demonstrates a protective effect of Krüppel-like factor 4 (KLF4)-p21CIP1 axis in bCSC inhibition by BITC. Exposure of human breast cancer cells (MCF-7, MDA-MB-231, and SUM159) to plasma-achievable concentrations of BITC resulted in a robust induction of KLF4 mRNA and its protein expression as determined by qRT-PCR and Western blotting or confocal microscopy. BITC-mediated suppression of bCSC markers, including aldehyde dehydrogenase 1 activity and mammosphere frequency, was significantly augmented by transient or stable knockdown of KLF4. Western blotting and IHC revealed relatively higher levels of KLF4 protein in mammary tumor sections from BITC-treated mice in comparison with controls, but the difference was insignificant. Analysis of the breast cancer RNA-Seq data from The Cancer Genome Atlas indicated significant positive correlation between expression of KLF4 and that of p21CIP1 (CDKN1A) but not β-Catenin (CTNNB1). Knockdown of p21CIP1 protein also amplified BITC-mediated suppression of bCSC. Finally, KLF4 was recruited to the promoter of p21CIP1 as indicated by chromatin immunoprecipitation assay. These results indicate that induction of KLF4-p21CIP1 axis attenuates inhibitory effect of BITC on bCSC self-renewal. Translational implication of these findings is that breast cancer chemoprevention by BITC may be augmented with a combination regimen involving BITC and an inhibitor of KLF4.

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-β Signaling and Upregulating CDK Inhibitors P16 and P27.

PurposeKrüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) cell fate while suppressing mesenchymal properties. TGF-β plays a crucial role in cell differentiation and development, and if dysregulated, it induces epithelial-mesenchymal transition (EMT). As KLF4 and TGF-β regulate each other in a context-dependent manner, we evaluated the role of the crosstalk between KLF4 and TGF-β-signaling in CE homeostasis.MethodsWe used spatiotemporally regulated ablation of Klf4 within the adult mouse CE in ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/ Krt12rtTA/rtTA/ Tet-O-Cre) mice and short hairpin RNA (shRNA)-mediated knockdown or lentiviral vector-mediated overexpression of KLF4 in human corneal limbal epithelial (HCLE) cells to evaluate the crosstalk between KLF4 and TGF-β-signaling components. Expression of TGF-β signaling components and cyclin-dependent kinase (CDK) inhibitors was quantified by quantitative PCR, immunoblots, and/or immunofluorescent staining.ResultsCE-specific ablation of Klf4 resulted in (1) upregulation of TGF-β1, -β2, -βR1, and -βR2; (2) downregulation of inhibitory Smad7; (3) hyperphosphorylation of Smad2/3; (4) elevated nuclear localization of phospho-Smad2/3 and Smad4; and (5) downregulation of CDK inhibitors p16 and p27. Consistently, shRNA-mediated knockdown of KLF4 in HCLE cells resulted in upregulation of TGF-β1 and -β2, hyperphosphorylation and nuclear localization of SMAD2/3, downregulation of SMAD7, and elevated SMAD4 nuclear localization. Furthermore, overexpression of KLF4 in HCLE cells resulted in downregulation of TGF-β1, -βR1, and -βR2 and upregulation of SMAD7, p16, and p27.ConclusionsCollectively, these results demonstrate that KLF4 regulates CE cell cycle progression by suppressing canonical TGF-β signaling and overcomes the undesirable concomitant decrease in TGF-β–dependent CDK inhibitors p16 and p27 expression by directly upregulating them.

Role of Kruppel-Like Factor 5 in Deoxycholic Acid-Mediated Intestinal Transdifferentiation of Esophageal Squamous Epithelium.

Barrett's esophagus (BE) is an acquired condition in which normal squamous epithelium is replaced with metaplastic columnar epithelium as a consequence of gastroesophageal reflux disease. BE is known as a precursor of esophageal adenocarcinoma. Currently, the molecular mechanism underlying epithelial metaplasia in BE patients remains unknown. Therefore, we investigated the role of Krüppel-like factor 5 (KLF5) signaling in the initiation of BE-associated metaplasia. Sprague-Dawley (SD) rats were used to create a surgical model of bile reflux injury. Immunohistochemistry was performed to analyze human and mouse esophageal specimens. Human esophageal squamous epithelial (HET-1A) cells were treated with bile acid and used in transfection experiments. Quantitative real-time PCR and western blot analysis were performed to detect the expression of KLF5, CDX2, MUC2 and villin.Epithelial tissue from both the rat BE model and human BE patients strongly expressed KLF5, CDX2, MUC2, and villin. Bile acid treatment also increased the expression of KLF5, CDX2, MUC2 and villin in esophageal epithelial cells in a time-dependent manner. Moreover, siRNA-mediated knockdown of KLF5 blocked the expression of CDX2, MUC2 and villin, but transfection of a KLF5 expression vector into esophageal epithelial cells promoted their transdifferentiation into columnar-like cells, as demonstrated by increased expression of the intestinal markers CDX2, MUC2 and villin. Thus, in addition to its function as a transcription factor, KLF5 may be linked to an increased risk of BE development.

LncRNA SNHG5 enhances astrocytes and microglia viability via upregulating KLF4 in spinal cord injury

This study aims to explore the role and mechanism of lncRNA SNHG5 in spinal cord injury (SCI). The interaction between SNHG5 and Krüppel-like factor 4 (KLF4) was verified by RNA pull-down and RNA immunoprecipitation (RIP) assay. Rat neural function was evaluated by BBB and BMS scores. Results showed that GFAP and Iba-1 (specific proteins for astrocytes and microglia respectively) were upregulated in spinal cord of SCI rats. Simultaneously, spinal cord also expressed substantially higher levels of SNHG5, KLF4 and eNOS (endothelial Nitric Oxide Synthase) than sham group. In traumatically injured astrocytes and microglia, SNHG5 overexpression increased cells viability, which was significantly inhibited by SNHG5 knockdown. KLF4 is a directly target for SNHG5 and is positively regulated by SNHG5. The knockdown of KLF4 effectively decreased astrocytes and microglia viability induced by SHNG5 overexpression and attenuated the pcDNA-SNHG5-mediated repression of the apoptosis. In SCI rats, the injection of Lenti-SNHG5 reduced BBB and BMS scores and also enhanced the protein expression of KLF4, eNOS, GFAP and Iba-1. In summary, our data suggested that SNHG5 promotes SCI via increasing the viability of astrocytes and microglia. The mechanism by which SNHG5 works is its directive interaction to KLF4 and contribution to eNOS upregulation.

Krüppel-like factor 4 promotes c-Met amplification-mediated gefitinib resistance in non-small-cell lung cancer.

Gefitinib has been widely used in the first‐line treatment of advanced EGFR‐mutated non‐small‐cell lung cancer (NSCLC). However, many NSCLC patients will acquire resistance to gefitinib after 9‐14 months of treatment. This study revealed that Krüppel‐like factor 4 (KLF4) contributes to the formation of gefitinib resistance in c‐Met‐overexpressing NSCLC cells. We observed that KLF4 was overexpressed in c‐Met‐overexpressing NSCLC cells and tissues. Knockdown of KLF4 increased tumorigenic properties in gefitinib‐resistant NSCLC cell lines without c‐Met overexpression, but it reduced tumorigenic properties and increased gefitinib sensitivity in gefitinib‐resistant NSCLC cells with c‐Met overexpression, whereas overexpression of KLF4 reduced gefitinib sensitivity in gefitinib‐sensitive NSCLC cells. Furthermore, Western blot analysis revealed that KLF4 contributed to the formation of gefitinib resistance in c‐Met‐overexpressing NSCLC cells by inhibiting the expression of apoptosis‐related proteins under gefitinib treatment and activating the c‐Met/Akt signaling pathway by decreasing the inhibition of β‐catenin on phosphorylation of c‐Met to prevent blockade by gefitinib. In summary, this study's results suggest that KLF4 is a promising candidate molecular target for both prevention and therapy of NSCLC with c‐Met overexpression.

Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 in cerebral edema treatment

BackgroundHypertonic saline (HS) has been used clinically for treatment of cerebral edema for decades. Previously we have demonstrated that HS alleviates cerebral edema via regulating water/ion channel protein and attenuating neuroinflammation. However, whether HS treatment triggers microglia polarization and its regulatory mechanism during this process is unclear. Methods and resultsThe Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of neuroinflammation and microglia functions. Treatment of 10% HS not only significantly reduced infarct size and ipsilateral ischemic hemispheric brain water content (BWC) via attenuating ischemia-induction of TNF-α, IL-1β, microglia M1 markers (iNOS, CD86) and miR-200b, but also increased neurotrophic factors such as IL-10 and IL-4, microglia M2 markers (Arg1, CD206) and Krüppel-like factor 4 (KLF4). Similar changes were confirmed in primary microglial cells subjected to hypoxia with/without HS in vitro. Importantly, overexpression of miR-200b was able to induce microglia M1 polarization via directly targeting KLF4. Restoring KLF4 expression abolished this effect. On the contrary, miR-200b inhibitor or KLF4 overexpression led to microglia M2 polarization. Mechanistically, KLF4 directly binds to promoter region of Agr1, thus inducing its transcription. Similar to treatment of HS, experimental overexpression of KLF4 in vivo exerted significant beneficial effects on ischemia-induced cerebral edema. However, knockdown of KLF4 abrogated the benefits of HS. ConclusionsHypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 during its treatment of cerebral edema. This study may provide new insights of HS-related therapy for cerebral edema.

MiR-25 enhances cell migration and invasion in non-small-cell lung cancer cells via ERK signaling pathway by inhibiting KLF4.

In recent years, microRNAs (miRNAs/miRs) have gained increasing interest in cancer research. Increasing evidences demonstrated that miRNAs are important for tumor early detection and prognosis. The present study aimed to explore the function of miR-25 in non-small-cell lung cancer (NSCLC) and its underlying mechanisms. The expression levels of miR-25 and Krüppel-like factor 4 (KLF4) were assessed in 31 pairs of tissue from patients with NSCLC. In addition, the biological roles of miR-25 in NSCLC were analyzed via a cell wound healing assay, Transwell invasion and migration assays. Target genes of miR-25 were predicted using TargetScan and verified via a dual luciferase activity assay, western blotting and reverse transcription-quantitative polymerase chain reaction. The downstream signaling pathway was confirmed by western blot analysis. In the present study, miR-25 was overexpressed in 31 NSCLC samples compared with in corresponding normal tissues. Overexpression of miR-25 using miR-25 mimics markedly promoted NSCLC cell migration and invasion, while inhibition of miR-25 exerted the opposite effect. KLF4 was suggested to be a novel target gene of miR-25 in NSCLC cells. Knockdown of KLF4 promoted the migration and invasion of NSCLC cells, whereas rescue of KLF4 expression reduced cell motion ability in miR-25-overexpressing NSCLC cells. Furthermore, it was demonstrated that miR-25 activated the extracellular signal-regulated kinase (ERK) signaling pathway, which eventually led to increased vimentin, matrix metalloproteinase 11 and N-cadherin levels, and the downregulation of E-cadherin expression by inhibiting the expression of KLF4. In conclusion, miR-25 was demonstrated to activate the ERK signaling pathway by directly targeting KLF4, promoting cell migration and invasion. The findings of the present study indicated that miR-25 or KLF4 may serve as a therapeutic target for the treatment of NSCLC.

Ascl2 activation by YAP1/KLF5 ensures the self-renewability of colon cancer progenitor cells.

Achaete scute-like 2 (Ascl2) is the Wnt signaling target, its regulation by other signaling is undefined. Now we demonstrated that CD133+/CD44+ cell population from HT-29 or Caco-2 cells exhibited cancer stem cell (CSC) properties with highly expressed Ascl2, which is related to the Hippo signaling pathway. YAP1 interference in CD133+/CD44+ HT-29 or Caco-2 cells reduced their proliferation, colony-forming ability and tumorsphere formation in vitro and inhibited the ‘stemness’-associated genes and Ascl2 expression. Enforcing YAP1 expression in HT-29 or Caco-2 cells triggered the opposite changes. Ascl2 interference reversed the phenotype of YAP1-enforced expressed HT-29 or Caco-2 cells. Krüppel-like factor 5 (KLF5) protein, not KLF5 mRNA levels, were increased due to YAP1 overexpression which is reported to prevent KLF5 degradation. Co-immunoprecipitation (Co-IP) assays demonstrated that YAP1 bound with KLF5 in HT-29 and Caco-2 cells. Luciferase and chromatin immunoprecipitation (ChIP) assays indicated that both YAP1 and KLF5 bound to the first two loci with GC-boxes in Ascl2 promoter and induced Ascl2 transcription. The decreased Ascl2 transcription by YAP1 interference required an intact KLF5 binding site (GC-box) within Ascl2 promoter, KLF5 knockdown reduced YAP1 binding and Ascl2 luciferase reporter activity upon YAP1 overexpression. Positive correlation among YAP1 and Ascl2 mRNA levels was observed in colorectal cancer (CRC) samples. Thus, our study demonstrated that Ascl2, a fate decider of CRC progenitor cells can be activated by the Hippo signaling pathway in CRC progenitor cells, and ensured their self-renewability.

Role of Krüppel-Like Factor 4 in the Maintenance of Chemoresistance of Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) has a very poor prognosis due to its aggressive nature and resistance to conventional treatment. Radiotherapy and chemotherapy are not fully effective because of the undifferentiated phenotype and enhanced drug resistance of ATC. The objective of this study was to evaluate the involvement of Krüppel-like factor 4 (KLF4), a stemness-associated transcription factor, in the undifferentiated phenotype and drug resistance of ATC. ATC cells were compared to papillary thyroid cancer cells in drug resistance and gene expression. The effects of KLF4 knockdown in ATC cells on in vitro and in vivo drug resistance were measured. The effects of KLF4 overexpression and knockdown on ABC transporter activity were determined. ATC cells, such as HTH83, 8505C, and SW1736, exhibited higher resistance to the anticancer drug paclitaxel and higher expression of KLF4 than TPC-1 papillary thyroid cancer cells. Knockdown of KLF4 expression in ATC cells increased the expression of the thyroid-specific differentiation genes, such as thyrotropin receptor, thyroid peroxidase, thyroglobulin, and sodium-iodide symporter. Knockdown of KLF4 expression in ATC cells decreased the resistance to doxorubicin and paclitaxel, and reduced ABC transporter expression. Luciferase reporter assay results showed that KLF4 overexpression increased ABCG2 promoter activity, which was abolished by KLF4 knockdown. A tumorigenicity assay showed that the combination of paclitaxel treatment and KLF4 knockdown significantly decreased tumor mass originated from HTH83 cells in mice. ATC cells show high expression of KLF4, and KLF4 expression is necessary for maintaining the undifferentiated phenotype and drug resistance in vitro and in vivo. The present study identifies KLF4 as a potential therapeutic target for eliminating ATC cells.

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

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 Klf5 knockout mouse models (Villin-CreERT2;Klf5fl/fl designated as Klf5ΔIND and Villin-Cre;Klf5fl/fl as Klf5ΔIS ) and studied an inducible KLF5 knockdown in Caco-2 BBe cells using a lentiviral Tet-on system (Caco-2 BBe KLF5ΔIND). Specific knockout of Klf5 in colonic tissues, either inducible or constitutive, resulted in increased intestinal permeability. The phenotype was accompanied by a significant reduction in Dsg2, which encodes desmoglein-2, a desmosomal cadherin, at both mRNA and protein levels. Transmission electron microscopy showed alterations of desmosomal morphology in both KLF5 knockdown Caco-2 BBe cells and Klf5 knockout mouse colonic tissues. Inducible knockdown of KLF5 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 KLF5 knockdown cells, and DSG2 overexpression partially rescued the impaired barrier function caused by KLF5 knockdown. Electron microscopy studies demonstrated altered desmosomal morphology after KLF5 knockdown. In combination with chromatin immunoprecipitation analysis and promoter study, our data show that KLF5 regulates intestinal barrier function by mediating the transcription of DSG2, a gene encoding a major component of desmosome structures.NEW & NOTEWORTHY 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.