KLF4 Inhibits Activation of Human Hepatic Stellate Cells LX-2 in vitro

BackgroundHepatic Stellate Cell (HSC) represents a key factor in liver fibrosis. Early-stage liver fibrosis is still reversible and is intimately associated with the state of HSC. KLF4 has been shown to play a pivotal role in a wide array of physiological and pathological processes. MethodsIn this study, we examined the effect of Kruppel-like factor 4 (KLF4) on the proliferation, apoptosis and phenotype of HSC in resting state in human HSC LX-2 cells, and the role KLF4 plays in the maintenance of the resting state of HSC, with an attempt to provide an experimental basis for the diagnosis, treatment and prognosis evaluation of liver fibrosis. We designed a KLF4 lentiviral vector and a KLF4 shRNA lentiviral vector, to up-regulate and silence KLF4 expression in LX-2 cells by transfection. We examined LX-2 cell proliferation by plate cloning and CCK8 assay, flow cytometrically detected cell cycle distribution and cellular apoptosis rate and determined some quiescence and activation markers of HSC by Western blotting. ResultsOur result showed that E-cadherin and ZO-1, dubbed quiescent HSC markers, were significantly increased. N-cadherin while a-AMA, known as activated HSC marker, was significantly decreased. In contrast, cell proliferation and apoptosis rate were elevated in LX-2 cells whose KLF4 expression had been silenced. ConclusionsOur results indicated that KLF4 inhibited the proliferation and activation of human HSC LX-2 and might be a key regulatory protein in the maintenance of quiescence of HSC and might serve as a target for the inhibition of hepatic fibrosis.

Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4

Perturbed expression of microRNAs (miRs) has been reported in different diseases includingautoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR25-3p and its targets in the central nervous system (CNS) tissue from mice with experimentalautoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets inactivated macrophages and splenocytes.EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyteglycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We nextestablished primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and antiCD3/anti-CD28 antibodies, respectively.MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosisfactor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression ofphosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak ofthe disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significantupregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show anyalterations in miR-25-3p expression compared with vehicle-treated cells.Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators duringautoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.

3,3’-Diindolylmethane Enhances Paclitaxel Sensitivity by Suppressing DNMT1-Mediated KLF4 Methylation in Breast Cancer

Paclitaxel (PTX) is a first-line chemotherapeutic drug for the treatment of breast cancer, but drug resistance seriously limits its clinical use. The aim of the present work was to explore the effect of 3,3’-diindolylmethane (DIM) on PTX sensitivity and its possible mechanism in breast cancer. The expression of Krüppel-like factor 4 (KLF4) and DNA-methyltransferase 1 (DNMT1) in breast cancer tissues were assessed by immunohistochemistry and Western blotting. The methylation of KLF4 was evaluated by the MassARRAY platform. The lentivirus carrying KLF4 and DNMT1 gene or shRNA targeting DNMT1 were used to overexpress KLF4 or knockdown DNMT1 in MCF-7 and T47D breast cancer cells and the role of KLF4 and DNMT1 in regulation of PTX sensitivity was investigated. The effect of PTX on inhibiting the proliferation of MCF-7 and T47D cells was measured by CCK-8 assay. Flow cytometry was used to examine cell apoptosis. The expression of mRNA and protein was evaluated by qRT-PCR and Western blotting analysis, respectively. Our data showed that the expression of DNMT1 was increased, and the methylation level of CpG sites (−148 bp) in the KLF4 promoter was increased while the KLF4 expression was significantly decreased in breast cancer tissues. Overexpression of KLF4 increased the sensitivity of MCF-7 and T47D cells to PTX. DNMT1 increased the methylation of the KLF4 promoter and decrease the expression of KLF4. Knockdown of DNMT1 increased the sensitivity of MCF-7 and T47D cells to PTX. DIM enhanced the PTX sensitivity of MCF-7 and T47D cells, decreased the expression of DNMT1 and the methylation level of KLF4 promoter, thus increasing the level of KLF4. Furthermore, overexpression of DNMT1 attenuated the effect of DIM on the regulation of PTX sensitivity. Collectively, our data indicated that DNMT1-mediated hypermethylation of KLF4 promoter leads to downregulation of KLF4 in breast cancer. The level of KLF4 is correlated with the sensitivity of MCF-7 and T47D cells to PTX. DIM could enhance the antitumor efficacy of PTX on MCF-7 and T47D cells by regulating DNMT1 and KLF4.

Nobiletin Alleviates Non-alcoholic Steatohepatitis in MCD-Induced Mice by Regulating Macrophage Polarization

Non-alcoholic steatohepatitis (NASH) is an inflammatory disorder that is characterized by chronic activation of the hepatic inflammatory response and subsequent liver damage. The regulation of macrophage polarization in liver is closely related to the progression of NASH. The orphan nuclear receptor retinoic-acid-related orphan receptor α (RORα) and Krüppel-like factor 4 (KLF4) are key regulators which promote hepatic macrophages toward M2 phenotype and protect against NASH in mice. Nobiletin (NOB), a natural polymethoxylated flavone, is previously reported as a RORα regulator in diet-induced obese mice. However, it is still unclear whether NOB has the protective effect on NASH. In this study, we investigated the role of NOB in NASH using a methionine and choline deficient (MCD)-induced NASH mouse model. Our results showed that NOB ameliorated hepatic damage and fibrosis in MCD fed mice. NOB treatment reduced the infiltration of macrophages and neutrophils in the liver in MCD-fed mice. Of importance, NOB significantly increased the proportion of M2 macrophages and the expression of anti-inflammatory factors in vivo and in vitro. Meanwhile, NOB also decreased the population of M1 macrophages and the expression of proinflammatory cytokines. Mechanistically, NOB elevated KLF4 expression in macrophages. Inhibition of KLF4 abolished NOB regulated macrophage polarization. Furthermore, the regulation of NOB in KLF4 expression was dependent on RORα.

5-Aza-2′-Deoxycytidine and Valproic Acid in Combination with CHIR99021 and A83-01 Induce Pluripotency Genes Expression in Human Adult Somatic Cells

A generation of induced pluripotent stem cells (iPSC) by ectopic expression of OCT4, SOX2, KLF4, and c-MYC has established promising opportunities for stem cell research, drug discovery, and disease modeling. While this forced genetic expression represents an advantage, there will always be an issue with genomic instability and transient pluripotency genes reactivation that might preclude their clinical application. During the reprogramming process, a somatic cell must undergo several epigenetic modifications to induce groups of genes capable of reactivating the endogenous pluripotency core. Here, looking to increase the reprograming efficiency in somatic cells, we evaluated the effect of epigenetic molecules 5-aza-2′-deoxycytidine (5AZ) and valproic acid (VPA) and two small molecules reported as reprogramming enhancers, CHIR99021 and A83-01, on the expression of pluripotency genes and the methylation profile of the OCT4 promoter in a human dermal fibroblasts cell strain. The addition of this cocktail to culture medium increased the expression of OCT4, SOX2, and KLF4 expression by 2.1-fold, 8.5-fold, and 2-fold, respectively, with respect to controls; concomitantly, a reduction in methylated CpG sites in OCT4 promoter region was observed. The epigenetic cocktail also induced the expression of the metastasis-associated gene S100A4. However, the epigenetic cocktail did not induce the morphological changes characteristic of the reprogramming process. In summary, 5AZ, VPA, CHIR99021, and A83-01 induced the expression of OCT4 and SOX2, two critical genes for iPSC. Future studies will allow us to precise the mechanisms by which these compounds exert their reprogramming effects.

LncRNA MEG3 Protects Chondrocytes From IL-1β-Induced Inflammation via Regulating miR-9-5p/KLF4 Axis

BackgroundOsteoarthritis (OA) is a chronic degenerative disease of the joints characterized by articular cartilage damage, subchondral bone remodeling, osteophyte formation, and inflammatory changes. This work aims to investigate the protective role of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) against the apoptosis of chondrocytes.MethodsChondrocyte cell lines, CHON-001, and ATDC5 were treated with different doses of interleukin-1β (IL-1β) to mimic the inflammatory response during OA pathogenesis. Quantitative real-time polymerase chain reaction was performed to measure MEG3, miR-9-5p, and Krüppel-like factor 4 (KLF4) mRNA expression levels. MEG3 and KLF4 overexpression plasmids, MEG3 shRNA, miR-9-5p mimics, and miR-9-5p inhibitors were transfected into the cells. Cell counting kit-8, wound healing assay, and flow cytometry were conducted to determine cell viability, migration, and apoptotic rate. Dual-luciferase reporter assay was adopted to verify the targeting relationships among MEG3, miR-9-5p, and KLF4. Western blot was used to detect KLF4 protein expression. Enzyme-linked immunosorbent assay was employed to measure the levels of inflammatory factors.ResultsMEG3 expression in chondrocytes was down-regulated by the stimulation of IL-1β, and MEG3 negatively regulated miR-9-5p expression but positively regulated KLF4 expression. MEG3 overexpression strengthened the viability and migration of CHON-001 and ATDC5 cells but restrained the apoptosis and inflammatory response, while MEG3 knockdown had opposite effects. miR-9-5p inhibition or KLF4 overexpression could counteract the effects of MEG3 knockdown on chondrocytes. Besides that, MEG3 was proved to be a molecular sponge for miR-9-5p, and KLF4 was verified as the target of miR-9-5p.ConclusionMEG3 can promote chondrocyte proliferation and migration and inhibit apoptosis and inflammation by sponging miR-9-5p to induce KLF4 expression, which provides a promising therapy target for OA treatment.

Induction of Krüppel-Like Factor 4 Mediates Polymorphonuclear Neutrophil Activation in Streptococcus pneumoniae Infection

The recruitment and activation of polymorphonuclear neutrophils (PMNs) are of central importance for the elimination of pathogens in bacterial infections. We investigated the Streptococcus pneumoniae-dependent induction of the transcription factor Krüppel-like factor (KLF) 4 in PMNs as a potential regulator of PMN activation. We found that KLF4 expression is induced in human blood-derived PMNs in a time- and dose-dependent manner by wild-type S. pneumoniae and capsule knockout mutants. Unencapsulated knockout mutants induced stronger KLF4 expression than encapsulated wild types. The presence of autolysin LytA-competent (thus viable) pneumococci and LytA-mediated bacterial autolysis were required for KLF4 induction in human and murine PMNs. LyzMcre-mediated knockdown of KLF4 in murine blood-derived PMNs revealed that KLF4 influences pneumococci killing and increases the release of the proinflammatory cytokines tumor necrosis factor α and keratinocyte chemoattractant and decreases the release of the anti-inflammatory cytokine interleukin-10. Thus, S. pneumoniae induces KLF4 expression in PMNs, which contributes to PMN activation in S. pneumoniae infection.

Overexpression of Krüppel-Like Factor 4 (KLF4) Inhibits the Proliferation of Pulmonary Arterial Smooth Muscle Cells Through ERK1/2 Signaling Under Hypoxia Environment

Background This study is designed to examine the role of Krüppel-like factor 4 in mediating the proliferation of pulmonary artery smooth muscle cells (PASMCs) in hypoxic pulmonary hypertension and the underlying mechanisms. Methods Experiments were conducted using PASMCs isolated from male Sprague-Dawley rats. The cells were divided into 24 h group, 48 h group and 72 h group according to different hypoxia treatment time. For specific inhibition of the ERK1/2 and p38 signaling pathways, PASMCs were treated with the ERK1/2-specific inhibitor. KLF4 was cloned from plasmid into the eukaryotic expression vector and transfected into PASMCs for KLF4 overexpression and was transfected into PASMCs for KLF4 knockdown. Results PASMCs dedifferentiation and proliferation phenotypes under hypoxia conditioned culture were not synchronous. Overexpression of KLF4 attenuated extracellular signal-regulated kinase (ERK)1/2-mediated proliferative signals in PASMCs. Knockdown of KLF4 expression resulted in increased ERK1/2 phosphorylation and significantly increased hypoxia-induced PASMCs proliferation, while p38 did not change and showed no influence on proliferation during the above process. Conclusions Our results indicate for the first time that KLF4 plays inhibitor role through activating ERK1/2 in PASMCs proliferation under hypoxia condition, expanding existing reports on the participation of KLF4 in the development of HPH. Our findings may provide pharmacological targets for therapy of HPH.

MYOD1 Inhibits Avian Adipocyte Differentiation via miRNA-206/KLF4 Axis

Background: A considerable number of muscle development-related genes were differentially expressed in the early stage of avian adipocyte differentiation. However, the functions of them in adipocyte differentiation remain largely known. In this study, the myoblast determination protein 1 (MYOD1) was selected as a representative of muscle development and we investigated its expression, function and regulation in avian adipocyte differentiation.Results: The expression of MYOD1 decreased significantly in the early stage of avian adipocyte differentiation. CRISPR/CAS9-mediated deletion of MYOD1 induced adipocyte differentiation, whereas over-expression of MYOD1 inhibited adipogenesis. mRNA-seq showed that MYOD1 could perturb the lipid biosynthetic process during differentiation. Mechanistically, MYOD1 directly up-regulates the miR-206 expression by binding upstream 1200 bp region, and over-expression of miR-206 also inhibits adipogenesis. Furthermore, MYOD1 affected the expression of endogenous miR-206 and its target gene Kruppel Like Factor 4 (KLF4), which is an important activator of adipogenesis. Accordingly, the inhibition of miR-206 or over-expression of KLF4 could counteract the inhibitory effect of MYOD1 on adipocyte differentiation. Conclusions: These findings suggest that MYOD1 inhibited adipocyte differentiation by up-regulating miR-206 to suppress the KLF4 expression. Collectively, these findings identify a novel function of MYOD1 in adipocyte differentiation, suggesting a potential role in body-fat distribution regulation.

Invasion by Fusobacterium nucleatum Causes Upregulation of dll4 and klf4 Expression in Caco2 Cells

Fusobacterium nucleatum is an emerging microbe of importance in the pathogenesis of colorectal cancer. Strains of this enigmatic bacterial species vary in their capacity to invade human epithelial cells, a virulence determinant which has important implications in disease. Here, we infected human colorectal epithelial (Caco-2) cells in vitro with a known, highly invasive strain of F. nucleatum isolated from a Crohn’s Disease patient, as well as a further invasive isolate of F. nucleatum derived from a colorectal cancer tumour. We used transcriptional profiling to determine the human genes upregulated during the invasion process compared to exposure to a non-invasive E.coli control strain. Infection with F. nucleatum strains resulted in the upregulation of several host genes, including two associated with tumorigenesis: dll4 and klf4.