KLF4 Inhibits the Activation of Human Hepatic Stellate Cell In Vitro.

OBJECTIVE: Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs. Kruppel-like factor 4 (KLF4) plays a pivotal role in a wide array of physiological and pathological processes. This study aimed to investigate the effect of KLF4 on the proliferation, apoptosis and phenotype of quiescent HSCs METHODS: We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector, to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection. Cell proliferation was assessed using the CCK-8 assay. Flow cytometry was used to detect the cell cycle distribution and apoptosis rate. Western blotting was used to determine the levels of some quiescence and activation markers of HSCs RESULTS: Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1, which are quiescent HSC markers, while significantly decreased the levels of N-cadherin and a-SMA, known activated HSC markers. In contrast, cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced CONCLUSION: KLF4 inhibits the proliferation and activation of human LX-2 HSCs. It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.

Temporal and regional expression changes and co-staining patterns of metabolic and stemness-related markers during glioblastoma progression.

Glioblastomas (GBMs) are characterized by high heterogeneity, involving diverse cell types, including those with stem-like features contributing to GBM's malignancy. Moreover, metabolic alterations promote growth and therapeutic resistance of GBM. Depending on the metabolic state, antimetabolic treatments could be an effective strategy. Against this background, we investigated temporal and regional expression changes and co-staining patterns of selected metabolic markers [pyruvate kinase muscle isozyme 1/2 (PKM1/2), glucose transporter 1 (GLUT1), monocarboxylate transporter 1/4 (MCT1/4)] in a rodent model and patient-derived samples of GBM. To understand the cellular sources of marker expression, we also examined the connection of metabolic markers to markers related to stemness [Nestin, Krüppel-like factor 4 (KLF4)] in a regional and temporal context. Rat tumour biopsies revealed a temporally increasing expression of GLUT1, higher expression of MCT1/4, Nestin and KLF4, and lower expression of PKM1 compared to the contralateral hemisphere. Patient-derived tumours showed a higher expression of PKM2 and Nestin in the tumour centre vs. edge. Whereas rare co-staining of GLUT1/Nestin was found in tumour biopsies, PKM1/2 and MCT1/4 showed a more distinct co-staining with Nestin in rats and humans. KLF4 was mainly co-stained with GLUT1, MCT1 and PKM1/2 in rat and human tumours. All metabolic markers yielded individual co-staining patterns among themselves. Co-staining mainly occurred later in tumour progression and was more pronounced in tumour centres. Also, positive correlations were found amongst markers that showed co-staining. Our results highlight a link between metabolic alterations and stemness in GBM progression, with complex distinctions depending on studied markers, time points and regions.

Hypoxia-inducible factor-1α attenuates renal podocyte injury in male rats in a simulated high-altitude environment by upregulating Krüppel-like factor 4 expression.

Previous studies have shown that podocyte injury is involved in the development of proteinuria in rats under hypobaric hypoxia conditions. Prolyl hydroxylase inhibitors (PHIs) may reduce proteinuria. This study aimed to further investigate whether the protective effects of hypoxia-inducible factor 1α (HIF1α) on podocyte injury induced by hypobaric hypoxia are related to Krüppel-like factor 4 (KLF4). Rats were housed in a low-pressure oxygen chamber to simulate a high-altitude environment (5000 m), and a PHI was intraperitoneally injected. Urinary protein electrophoresis was performed and the morphology of the podocytes was observed by electron microscopy. Rat podocytes were cultured under 1% O2, and siRNA was used to interfere with KLF4 expression. The protein expression levels of HIF1α, KLF4, CD2-associated protein (CD2AP) and nephrin were determined by western blotting. Compared with those in the experimental group, the rats in the intervention group on day 14 had lower urinary protein levels, increased protein expression levels of CD2AP and nephrin, and reduced podocyte injury. The results of in vitro experiments showed that the protein expression levels of KLF4, CD2AP and nephrin were greater in the PHI intervention group and lower in the HIF1α inhibitors group than in the low-oxygen group. The protein expression of CD2AP and nephrin in the siKLF4-transfected podocytes treated with PHI and HIF1α inhibitors did not differ significantly from that in the low-oxygen group. HIF1α may be involved in reducing progressive high-altitude proteinuria by regulating KLF4 expression and contributing to the repair of podocyte injury induced by hypobaric hypoxia.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

MicroRNA-mediated Krüppel-like factor 4 upregulation induces alternatively activated macrophage-associated marker and chemokine transcription in 4,4'-methylene diphenyl diisocyanate exposed macrophages.

1. Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI) is associated with occupational asthma (OA) development. Alveolar macrophage-induced recruitment of immune cells to the lung microenvironment plays an important role during asthma pathogenesis. Previous studies identified that MDI/MDI-glutathione (GSH)-exposure downregulates endogenous hsa-miR-206-3p/hsa-miR-381-3p. Our prior report shows that alternatively activated (M2) macrophage-associated markers/chemokines are induced by MDI/MDI-GSH-mediated Krüppel-Like Factor 4 (KLF4) upregulation in macrophages and stimulates immune cell chemotaxis. However, the underlying molecular mechanism(s) by which MDI/MDI-GSH upregulates KLF4 remain unclear.2. Following MDI-GSH exposure, microRNA(miR)-inhibitors/mimics or plasmid transfection, endogenous hsa-miR-206-3p/hsa-miR-381-3p, KLF4, or M2 macrophage-associated markers (CD206, TGM2), and chemokines (CCL17, CCL22, CCL24) were measured by either RT-qPCR, western blot, or luciferase assay.3. MDI-GSH exposure downregulates hsa-miR-206-3p/hsa-miR-381-3p by 1.46- to 9.75-fold whereas upregulates KLF4 by 1.68- to 1.99-fold, respectively. In silico analysis predicts binding between hsa-miR-206-3p/hsa-miR-381-3p and KLF4. Gain- and loss-of-function, luciferase reporter assays and RNA-induced silencing complex-immunoprecipitation (RISC-IP) studies confirm the posttranscriptional regulatory roles of hsa-miR-206-3p/hsa-miR-381-3p and KLF4 in macrophages. Furthermore, hsa-miR-206-3p/hsa-miR-381-3p regulate the expression of M2 macrophage-associated markers and chemokines via KLF4.4. In conclusion, hsa-miR-206-3p/hsa-miR-381-3p play a major role in regulation of MDI/MDI-GSH-induced M2 macrophage-associated markers and chemokines by targeting the KLF4 transcript, and KLF4-mediated regulation in macrophages.

Intestinal epithelial Krüppel-like factor 4 alleviates endotoxemia and atherosclerosis through improving NF-κB/miR-34a-mediated intestinal permeability.

Maintenance of intestinal barrier function contributes to gastrointestinal homeostasis and therefore cardiovascular diseases. A number of studies show that intestinal permeability is affected by excessive inflammatory responses. Krüppel-like factor (KLF) 4 is one of the critical transcriptional factors, which controls multiple immune responses. In this study we investigated the role of KLF4 in regulating intestinal inflammation and permeability during the atherosclerotic process. Atherosclerotic model was established in ApoE-/- mice by feeding a high fat high cholesterol (HFHC) diet. We showed that colon expression levels of KLF4 and tight junction proteins were significantly decreased whereas inflammatory responses increased in atherosclerotic mice. Overexpression of colon epithelial Klf4 decreased atherosclerotic plaque formation and vascular inflammation in atherosclerotic mice, accompanied by remarkable suppression of intestinal NF-κB activation. We found that overexpression of epithelial Klf4 in atherosclerotic mice significantly increased intestinal tight junction expression and ameliorated endotoxemia, whereas replenishment of LPS abolished these benefits. Overexpression of Klf4 reversed LPS-induced permeability and downregulation of ZO-1 and Occludin in Caco-2 cells in vitro. HFHC diet stimulated the expression of epithelial microRNA-34a, whereas silence of epithelial Klf4 abolished the benefits of microRNA-34a sponge, a specific miR-34a inhibitor, on intestinal permeability and atherosclerotic development. A clinical cohort of 24 atherosclerotic patients supported colon KLF4/NF-κB/tight junction protein axis mediated intestine/cardiovascular interaction in patients with atherosclerosis. Taken together, intestinal epithelial KLF4 protects against intestinal inflammation and barrier dysfunction, ameliorating atherosclerotic plaque formation.

Mechanisms of Epigenomic and Functional Convergence Between Glucocorticoid- and IL4-Driven Macrophage Programming.

Macrophages adopt distinct phenotypes in response to environmental cues, with type-2 cytokine interleukin-4 promoting a tissue-repair homeostatic state (M2IL4). Glucocorticoids, widely used anti-inflammatory therapeutics, reportedly impart a similar phenotype (M2GC), but how such disparate pathways may functionally converge is unknown. We show using integrative functional genomics that M2IL4 and M2GC transcriptomes share a striking overlap mirrored by a shift in chromatin landscape in both common and signal-specific gene subsets. This core homeostatic program is enacted by transcriptional effectors KLF4 and the GC receptor, whose genome-wide occupancy and actions are integrated in a stimulus-specific manner by the nuclear receptor cofactor GRIP1. Indeed, many of the M2IL4:M2GC-shared transcriptomic changes were GRIP1-dependent. Consistently, GRIP1 loss attenuated phagocytic activity of both populations in vitro and macrophage tissue-repair properties in the murine colitis model in vivo. These findings provide a mechanistic framework for homeostatic macrophage programming by distinct signals, to better inform anti-inflammatory drug design.

The miR-25802/KLF4/NF-κB signaling axis regulates microglia-mediated neuroinflammation in Alzheimer's disease.

Microglia-mediated neuroinflammation plays a critical role in the occurrence and progression of Alzheimer's disease (AD). In recent years, studies have increasingly explored microRNAs as biomarkers and treatment interventions for AD. This study identified a novel microRNA termed miR-25802 from our high-throughput sequencing dataset of an AD model and explored its role and the underlying mechanism. The results confirmed the miRNA properties of miR-25802 based on bioinformatics and experimental verification. Expression of miR-25802 was increased in the plasma of AD patients and in the hippocampus of APP/PS1 and 5 × FAD mice carrying two and five familial AD gene mutations. Functional studies suggested that overexpression or inhibition of miR-25802 respectively aggravated or ameliorated AD-related pathology, including cognitive disability, Aß deposition, microglial pro-inflammatory phenotype activation, and neuroinflammation, in 5 × FAD mice and homeostatic or LPS/IFN-γ-stimulated EOC20 microglia. Mechanistically, miR-25802 negatively regulates KLF4 by directly binding to KLF4 mRNA, thus stimulating microglia polarization toward the pro-inflammatory M1 phenotype by promoting the NF-κB-mediated inflammatory response. The results also showed that inhibition of miR-25802 increased microglial anti-inflammatory M2 phenotype activity and suppressed NF-κB-mediated inflammatory reactions in the brains of 5 × FAD mice, while overexpression of miR-25802 exacerbated microglial pro-inflammatory M1 activity by enhancing NF-κB pathways. Of note, AD-associated manifestations induced by inhibition or overexpression of miR-25802 via the NF-κB signaling pathway were reversed by KLF4 silencing or upregulation. Collectively, these results provide the first evidence that miR-25802 is a regulator of microglial activity and establish the role of miR-25802/KLF4/NF-κB signaling in microglia-mediated neuroinflammation, suggesting potential therapeutic targets for AD.

Transcriptional Factors Mediated Reprogramming to Pluripotency.

A unique kind of pluripotent cell, i.e., Induced pluripotent stem cells (iPSCs), now being targeted for iPSC synthesis, are produced by reprogramming animal and human differentiated cells (with no change in genetic makeup for the sake of high efficacy iPSCs formation). The conversion of specific cells to iPSCs has revolutionized stem cell research by making pluripotent cells more controllable for regenerative therapy. For the past 15 years, somatic cell reprogramming to pluripotency with force expression of specified factors has been a fascinating field of biomedical study. For that technological primary viewpoint reprogramming method, a cocktail of four transcription factors (TF) has required: Kruppel-like factor 4 (KLF4), four-octamer binding protein 34 (OCT3/4), MYC and SOX2 (together referred to as OSKM) and host cells. IPS cells have great potential for future tissue replacement treatments because of their ability to self-renew and specialize in all adult cell types, although factor-mediated reprogramming mechanisms are still poorly understood medically. This technique has dramatically improved performance and efficiency, making it more useful in drug discovery, disease remodeling, and regenerative medicine. Moreover, in these four TF cocktails, more than 30 reprogramming combinations were proposed, but for reprogramming effectiveness, only a few numbers have been demonstrated for the somatic cells of humans and mice. Stoichiometry, a combination of reprogramming agents and chromatin remodeling compounds, impacts kinetics, quality, and efficiency in stem cell research.

Divergent Roles of KLF4 During Primordial Germ Cell Fate Induction from Human Embryonic Stem Cells.

As a core transcriptional factor regulating pluripotency, Krüppel-like factor 4 (KLF4) has gained much attention in the field of stem cells during the past decades. However, few research have focused on the function of KLF4 during human primordial germ cell (PGC) specification. Here, we induced human PGC-like cells (hPGCLCs) from human embryonic stem cells (hESCs) and the derived hPGCLCs upregulated PGC-related genes, like SOX17, BLIMP1, TFAP2C, NANOS3, and the naïve pluripotency gene KLF4. The KLF4-knockout hESCs formed typical multicellular colonies with clear borders, expressed pluripotency genes, such as NANOG, OCT4, and SOX2, and exhibited no differences in proliferation capacity compared with wild type hESCs. Notably, KLF4 deletion in hESCs did not influence the induction of PGCLCs in vitro. In contrast, overexpression of KLF4 during PGC induction process inhibited the efficiency of PGCLC formation from hESCs in vitro. Overexpression of KLF4 may regenerate the naïve ground state in hESCs and results in repression for PGC specification. Thus, KLF4 could be a downstream target of human PGC program and the upregulation of KLF4 is prepared for late stage of germline development.

d-Galactose induces the senescence and phenotype switch of corpus cavernosum smooth muscle cells.

Studies regarding age-related erectile dysfunction (ED) based on naturally aging models are limited by their high costs, especially for the acquisition of primary cells from the corpus cavernosum. Herein, d-galactose ( d-gal) was employed to accelerate cell senescence, and the underlying mechanism was explored. As predominant functional cells involved in the erectile response, corpus cavernosum smooth muscle cells (CCSMCs) were isolated from 2-month-old rats. Following this, d-gal was introduced to induce cell senescence, which was verified via ß-galactosidase staining. The effects of d-gal on CCSMCs were evaluated by terminal deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL), immunofluorescence staining, flow cytometry, western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, RNA interference (RNAi) was carried out for rescue experiments. Subsequently, the influence of senescence on the corpus cavernosum was determined via scanning electron microscopy, qRT-PCR, immunohistochemistry, TUNEL, and Masson stainings. The results revealed that the accelerated senescence of CCSMCs was promoted by d-gal. Simultaneously, smooth muscle alpha-actin (alpha-SMA) expression was inhibited, while that of osteopontin (OPN) and Krüppel-like factor 4 (KLF4), as well as fibrotic and apoptotic levels, were elevated. After knocking down KLF4 expression in d-gal-induced CCSMCs by RNAi, the expression level of cellular alpha-SMA increased. Contrastingly, the OPN expression, apoptotic and fibrotic levels declined. In addition, cellular senescence acquired partial remission. Accordingly, in the aged corpus cavernosum, the fibrotic and apoptotic rates were increased, followed by downregulation in the expression of alpha-SMA and the concurrent upregulation in the expression of OPN and KLF4. Overall, our results signaled that d-gal-induced accelerated senescence of CCSMCs could trigger fibrosis, apoptosis and phenotypic switch to the synthetic state, potentially attributed to the upregulation of KLF4 expression, which may be a multipotential therapeutic target of age-related ED.

KLF4 down-regulation underlies placental angiogenesis impairment induced by maternal glucose intolerance in late pregnancy.

Maternal glucose intolerance in late pregnancy can easily impair pregnancy outcomes and placental development. The impairment of placental angiogenesis is closely related to the occurrence of glucose intolerance during pregnancy, but the mechanism remains largely unknown. In this study, the pregnant mouse model of maternal high-fat diet and endothelial injury model of porcine vascular endothelial cells (PVECs) was used to investigate the effect of glucose intolerance on pregnancy outcomes and placental development. Feeding pregnant mice, a high-fat diet was shown to induce glucose intolerance in late pregnancy, and significantly increase the incidence of resorbed fetuses. Moreover, a decrease was observed in the proportion of blood sinusoids area and the expression level of CD31 in placenta, indicating that placental vascular development was impaired by high-fat diet. Considering that hyperglycemia is an important symptom of glucose intolerance, we exposed PVECs to high glucose (50 mM), which verified the negative effects of high glucose on endothelial function. Bioinformatics analysis further emphasized that high glucose exposure could significantly affect the angiogenesis-related functions of PVECs and predicted that Krüppel-like factor 4 (KLF4) may be a key mediator of these functional changes. The subsequent regulation of KLF4 expression confirmed that the inhibition of KLF4 expression was an important reason why high glucose impaired the endothelial function and angiogenesis of PVECs. These results indicate that high-fat diet can aggravate maternal glucose intolerance and damage pregnancy outcome and placental angiogenesis, and that regulating the expression of KLF4 may be a potential therapeutic strategy for maintaining normal placental angiogenesis.

Impairment of Autophagy Mediates the Uric-Acid-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells.

INTRODUCTION: Hyperuricemia may be involved in the phenotypic transformation of vascular smooth muscle cells, thus promoting the occurrence of atherosclerosis, and autophagy may be one of the important links, but little is known about the specific molecular mechanism. METHODS: We established a mouse model of hyperuricemia and studied the relationship between changes in autophagy levels and the phenotypic transformation of muscle cells. RESULTS: Our study found that high uric acid levels promote the phenotypic transformation of muscle cells by inhibiting autophagy, thus enhancing their proliferation and migration abilities. If autophagy is restored, phenotypic transformation can be reversed by reducing the levels of the transcription factor Kruppel-like factor 4. CONCLUSION: Uric acid may induce the phenotypic transformation of muscle cells and promote the occurrence of atherosclerosis by disrupting normal autophagy.

Effect of menthol on hypobaric hypoxia-induced pulmonary arterial hypertension in mice and its mechanism / 中国药理学通报

Aim To study the effect of menthol on hypobaric hypoxia-induced pulmonary arterial hypertension and explore the underlying mechanism in mice. Methods 10 to 12 weeks old wild type (WT) mice and TRPM8 gene knockout (TRPM8

Krüppel-like factor 4 in transcriptional control of the three unique isoforms of Agouti-related peptide in mice.

Agouti-related peptide (AgRP/Agrp) within the hypothalamic arcuate nucleus (ARC) contributes to the control of energy balance, and dysregulated Agrp may contribute to metabolic adaptation during prolonged obesity. In mice, three isoforms of Agrp are encoded via distinct first exons. Agrp-A (ENSMUST00000005849.11) contributed 95% of total Agrp in mouse ARC, whereas Agrp-B (ENSMUST00000194654.2) dominated in placenta (73%). Conditional deletion of Klf4 from Agrp-expressing cells (Klf4Agrp-KO mice) reduced Agrp mRNA and increased energy expenditure but had no effects on food intake or the relative abundance of Agrp isoforms in the ARC. Chronic high-fat diet feeding masked these effects of Klf4 deletion, highlighting the context-dependent contribution of KLF4 to Agrp control. In the GT1-7 mouse hypothalamic cell culture model, which expresses all three isoforms of Agrp (including Agrp-C, ENSMUST00000194091.6), inhibition of extracellular signal-regulated kinase (ERK) simultaneously increased KLF4 binding to the Agrp promoter and stimulated Agrp expression. In addition, siRNA-mediated knockdown of Klf4 reduced expression of Agrp. We conclude that the expression of individual isoforms of Agrp in the mouse is dependent upon cell type and that KLF4 directly promotes the transcription of Agrp via a mechanism that is superseded during obesity.NEW & NOTEWORTHY In mice, three distinct isoforms of Agouti-related peptide are encoded via distinct first exons. In the arcuate nucleus of the hypothalamus, Krüppel-like factor 4 stimulates transcription of the dominant isoform in lean mice, but this mechanism is altered during diet-induced obesity.

MicroRNA-29a-3p Accelerates Inflammatory Damage in Neonatal Pneumonia Via Targeting Krüppel-like Factor 4.

Neonatal pneumonia (NP) is a frequently occurring illness during the neonatal phase. The study investigated the molecular process and the role of microRNA (miR)-29a-3p in NP. Peripheral blood was collected from NP patients and healthy newborns. Human lung fibroblasts cell line (WI-38) were treated with lipopolysaccharide (LPS)) to establish a cellular model for NP. Then, miR-29a-3p and Krüppel-like Factor 4 (KLF4) levels were detected by RT-qPCR or Western blot. The relationship between miR-29a-3p and KLF4 was confirmed by dual luciferase reporter gene assay. Cell survival was assessed using the CCK-8 assay, whereas the levels of interleukin-6, tumor necrosis factor-α, and IL-1ß were quantified using ELISA. Additionally, apoptosis was evaluated through flow cytometry. Meanwhile, Bax and Bcl-2 were detected by RT-qPCR. Neonatal rats were administered LPS intraperitoneally (3 mg/kg) to induce NP, and pathological injury and inflammatory reaction were analyzed. MiR-29a-3p was elevated but KLF4 was silenced in NP patient's serum, LPS-treated WI-38 cell line, and LPS-treated newborn rats. Silence of miR-29a-3p or elevation of KLF4 constrained cell proliferation with inflammation of LPS-treated WI-38 cell line. MiR-29a-3p immediately targeted KLF4. Additionally, silence of miR-29a-3p alleviated LPS-stimulated lung injury and inflammation in neonatal rats. The protective action of silenced miR-29a-3p in LPS-treated WI-38 cell line and newborn rats was turned around by silencing KLF4. This study demonstrates originally that miR-29a-3p boosts inflammatory damage in NP via targeting KLF4, offering a basis for clinically diagnosing and treating NP.

KLF4 suppresses the proliferation of perihilar cholangiocarcinoma by negatively regulating GDF15 and phosphorylating AKT.

Krüppel­like factor 4 (KLF4) is a transcription factor which functions as a tumor suppressor or an oncogene in numerous types of solid tumors. However, its expression levels and function in perihilar cholangiocarcinoma (pCCA) have yet to be elucidated. In the present study, in order to investigate its roles in pCCA, reverse transcription­quantitative PCR (RT­qPCR), western blot analysis and immunohistochemistry were used to detect KLF4 expression in pCCA. The Chi­squared test was used to analyze the associations between KLF4 and the clinicopathological features of patients with pCCA. Univariate and multivariate analyses were subsequently used to analyze the prognostic significance of KLF4. The tumor suppression of KLF4 was investigated for the purposes of illustrating its biological function both in vitro and in vivo. Furthermore, the association between KLF4 and growth/differentiation factor 15 (GDF15) was determined using pCCA tissue microarray (TMA) analysis and RT­qPCR. The underlying molecular mechanisms between KLF4 and GDF15 were subsequently investigated in vitro. In pCCA tissues, KLF4 was found to be downregulated, and this was negatively associated with the histological grade and tumor size. The knockdown of KLF4 was also found to be a prognostic indicator of the poorer survival of patients with pCCA. Based on in vitro and in vivo analyses, KLF4 was found to suppress tumor progression and induce cell apoptosis. Furthermore, it was found that KLF4 executed its tumor suppressive effects via the regulation of the GDF15/AKT signaling pathway. Taken together, the findings of the present study demonstrate that KLF4 may be considered as an independent biomarker of a favorable prognosis of patients with pCCA, and the KLF4/GDF15/AKT signaling pathway may potentially be a novel molecular therapeutic target for patients with pCCA.

4,4'-Methylene diphenyl diisocyanate exposure induces expression of alternatively activated macrophage-associated markers and chemokines partially through Krüppel-like factor 4 mediated signaling in macrophages.

Occupational exposure to the most widely used monomeric diisocyanate (dNCO), 4,4'-methylene diphenyl diisocyanate (MDI), may lead to the development of occupational asthma (OA). Alveolar macrophages with alternatively activated (M2) phenotype have been implicated in allergic airway responses and the pathogenesis of asthma. Recent in vivo studies demonstrate that M2 macrophage-associated markers and chemokines are induced by MDI-exposure, however, the underlying molecular mechanism(s) by which this proceeds is unclear.Following MDI exposure (in vivo and in vitro) M2 macrophage-associated transcription factors (TFs), markers, and chemokines were determined by RT-qPCR, western blots, and ELISA.Expression of M2 macrophage-associated TFs and markers including Klf4/KLF4, Cd206/CD206, Tgm2/TGM2, Ccl17/CCL17, Ccl22/CCL22, and CCL24 were induced by MDI/MDI-GSH exposure in bronchoalveolar lavage cells (BALCs)/THP-1 macrophages. The expression of CD206, TGM2, CCL17, CCL22, and CCL24 are upregulated by 3.83-, 7.69-, 6.22-, 6.08-, and 1.90-fold in KLF4-overexpressed macrophages, respectively. Endogenous CD206 and TGM2 were downregulated by 1.65-5.17-fold, and 1.15-1.78-fold, whereas CCL17, CCL22, and CCL24 remain unchanged in KLF4-knockdown macrophages. Finally, MDI-glutathione (GSH) conjugate-treated macrophages show increased chemotactic ability to T-cells and eosinophils, which may be attenuated by KLF4 knockdown.Our data suggest that MDI exposure may induce M2 macrophage-associated markers partially through induction of KLF4.

Overexpression of KLF4 Suppresses Pulmonary Fibrosis through the HIF-1α/Endoplasmic Reticulum Stress Signaling Pathway.

The hypoxia-inducible factor-1α/endoplasmic reticulum stress signaling pathway (HIF-1α/ERS) has a crucial role in the pathogenetic mechanism of pulmonary fibrosis (PF). However, the upstream regulatory mediators of this pathway remain unclear. In the present study, by conducting bioinformatics analysis, we found that Krüppel-like factor 4 (KLF4) expression was decreased in the lung tissues of patients with idiopathic pulmonary fibrosis (IPF) as compared to that in patients with non-IPF. Furthermore, KLF4 expression was significantly reduced (p = 0.0331) in bleomycin-induced fibrotic HFL-1 cells. Moreover, in mice with bleomycin-induced PF, the degree of fibrosis was significantly reduced in mice overexpressing KLF4 as compared to that in wild-type mice. In mice and HFL-1 cells, KLF4 overexpression significantly reduced bleomycin-induced protein expression of HIF-1α (p = 0.0027) and ERS markers, particularly p-IRE1α (p = 0.0255) and ATF6 (p = 0.0002). By using the JASPAR database, we predicted that KLF4 has five binding sites for the HIF-1α promoter. The results of in vitro and in vivo studies suggest that KLF4 may inhibit PF through the HIF-1α/ERS pathway. This finding could guide the development of future therapies for PF and facilitate the identification of appropriate biomarkers for routine clinical diagnosis of PF.

EGR1 Regulation of Vasculogenic Mimicry in the MDA-MB-231 Triple-Negative Breast Cancer Cell Line through the Upregulation of KLF4 Expression.

Vasculogenic mimicry (VM) is an intriguing phenomenon observed in tumor masses, in which cancer cells organize themselves into capillary-like channels that closely resemble the structure and function of blood vessels. Although VM is believed to contribute to alternative tumor vascularization, the detailed regulatory mechanisms controlling these cellular processes remain poorly understood. Our study aimed to investigate the role of Early Growth Response 1 (EGR1) in regulating VM in aggressive cancer cells, specifically MDA-MB-231 triple-negative breast cancer cells. Our study revealed that EGR1 promotes the formation of capillary-like tubes by MDA-MB-231 cells in a 3-dimensional Matrigel matrix. EGR1 was observed to upregulate Kruppel-like factor 4 (KLF4) expression, which regulates the formation of the capillary-like tube structure. Additionally, our findings highlight the involvement of the ERK1/2 and p38 mitogen-activated protein kinase pathways in mediating the expression of EGR1 and KLF4, underscoring their crucial role in VM in MDA-MB-231 cells. Understanding these regulatory mechanisms will provide valuable insights into potential therapeutic targets for preventing VM during the treatment of triple-negative breast cancer.