Overexpression Of Krüppel-like Factor 4 Research Articles

KLF2 Inhibits Ferroptosis and Improves Mitochondrial Dysfunction in Chondrocyte Through SIRT1/GPX4 Signaling to Improve Osteoarthritis.

Osteoarthritis (OA), a disease of articular joints, is the leading cause of disability in the elderly. Repressing ferroptosis and improving mitochondrial function can delay the progression of OA. Kruppel-like factor 2 (KLF2) exerts a protective effect on OA. However, whether KLF2 affects ferroptosis and mitochondrial function during OA remains unknown. The OA in vivo and in vitro models were constructed in this work. The structural damage of knee joint in OA mice was evaluated through Micro-CT scanning. H&E, SOFG, TB, and TUNEL staining were applied for pathological examination of cartilage tissues. ELISA was employed to examine the contents of inflammatory factors. Additionally, iron deposition in cartilage tissues was measured by Prussian blue staining, and the levels of proteins related to ferroptosis were assessed by immunoblotting. Besides, mitochondrial morphology and function were estimated using a transmission electron microscope and JC-1 staining. In interleukin (IL)-1β-treated C28/I2 cells, the levels of inflammatory factors, intracellular ROS, mitochondrial ROS, lipid ROS, and Fe2+ were measured. Mitochondrial function was evaluated by detecting the levels of mitochondrial membrane potential (MMP), ATP, mPTP, and OCR. KLF2 overexpression ameliorated the structural damage of knee cartilage in OA mice. KLF2 upregulation inhibited ferroptosis and alleviated mitochondrial damage in knee cartilage of OA mice and IL-1β-treated C28/I2 cells. Moreover, KLF2 overexpression activated SIRT1/GPX4 signaling in vivo and in vitro. EX527 addition blocked the influences of KLF2 upregulation on ferroptosis and mitochondrial dysfunction in IL-1β-treated C28/I2 cells. Altogether KLF2 inhibits ferroptosis and improves mitochondrial dysfunction in chondrocytes through SIRT1/GPX4 signaling to improve OA.

Low shear stress protects chondrocytes from IL-1β-induced apoptosis by activating ERK5/KLF4 signaling and negatively regulating miR-143-3p

ObjectiveThis study investigated the protective effects of low fluid shear stress (FSS ≤ 2 dyn/cm²) against interleukin-1β (IL-1β)-induced chondrocyte apoptosis and explored the underlying molecular mechanisms.MethodsChondrocytes were cultured under four conditions: control, IL-1β stimulation, low FSS, and combined low FSS + IL-1β stimulation. Apoptosis was assessed using Hoechst staining and flow cytometry. Western blotting determined the expression of caspase-3 (CASP3), caspase-8 (CASP8), and NF-κB p65. Quantitative real-time PCR measured miR-143-3p expression. The roles of miR-143-3p and the extracellular signal-regulated kinase 5 (ERK5)/Krüppel-like factor 4 (KLF4) signaling pathway were further investigated using miR-143-3p mimics and inhibitors, an ERK5 inhibitor, and a KLF4 overexpression vector.ResultsIL-1β induced significant chondrocyte apoptosis, which was markedly inhibited by low FSS. Mechanistically, low FSS suppressed miR-143-3p expression, thereby enhancing ERK5 signaling. This activated ERK5 subsequently upregulated KLF4 expression, further mitigating IL-1β-induced damage. Importantly, miR-143-3p overexpression under low FSS conditions exacerbated IL-1β-induced apoptosis, while miR-143-3p inhibition attenuated it. Consistent with this, ERK5 inhibition augmented IL-1β-induced apoptosis, whereas KLF4 overexpression suppressed it.ConclusionLow FSS protects chondrocytes from IL-1β-induced apoptosis by suppressing miR-143-3p and activating the ERK5/KLF4 signaling pathway. This study reveals a novel mechanism by which mechanical stimulation protects cartilage.

LN-439A, a novel BAP1 inhibitor, suppresses the growth of basal-like breast cancer by degrading KLF5.

Basal-like breast cancer (BLBC) is the most malignant subtype of breast cancer because of its aggressive clinical behaviour and lack of effective targeted agents. Krüppel-like factor 5 (KLF5) is an oncogenic transcription factor that is highly expressed in BLBC. The deubiquitinase (DUB) BRCA1-associated protein 1 (BAP1) stabilizes KLF5 and promotes BLBC growth and metastasis. Therefore, pharmacological inhibition of the BAP1‒KLF5 axis is an effective therapeutic strategy for BLBC. Here, through screening, we identified a series of tetrahydro-β-carboline derivatives that effectively reduced the protein expression of KLF5 and exhibited strong antitumour activity. Among the investigated compounds, the lead compound LN-439A presented the strongest antitumour activity and inhibitory effect on KLF5 expression. LN-439A suppressed the proliferation and migration of BLBC cells, induced G2/M arrest, and induced apoptosis. Mechanistically, LN-439A functions as a small molecule catalytic inhibitor of BAP1 by binding to the catalytic pocket of BAP1, leading to the ubiquitination and degradation of KLF5. Consistent with this finding, the overexpression of KLF5 suppressed the antitumour effects of LN-439A. In summary, LN-439A is a promising therapeutic agent for BLBC that functions by targeting the BAP1‒KLF5 axis.

Mechanism of KLF2 in young mice with pneumonia induced by Streptococcus pneumoniae

BackgroundStreptococcus pneumoniae (Spn) is a major causative agent of pneumonia, which can disseminate to the bloodstream and brain. Pneumonia remains a leading cause of death among children aged 1–59 months worldwide. This study aims to investigate the role of Kruppel-like factor 2 (KLF2) in lung injury caused by Spn in young mice.MethodsYoung mice were infected with Spn to induce pneumonia, and the bacterial load in the bronchoalveolar lavage fluid was quantified. KLF2 expression in lung tissues was analyzed using real-time quantitative polymerase chain reaction and Western blotting assays. Following KLF2 overexpression, lung tissues were assessed for lung wet-to-dry weight ratio and Myeloperoxidase activity. The effects of KLF2 on lung injury and inflammation were evaluated through hematoxylin and eosin staining and enzyme-linked immunosorbent assay. Chromatin immunoprecipitation and dual-luciferase assay were conducted to examine the binding of KLF2 to the promoter of microRNA (miR)-222-3p and cyclin-dependent kinase inhibitor 1B (CDKN1B), as well as the binding of miR-222-3p to CDKN1B. Levels of miR-222-3p and CDKN1B in lung tissues were also determined.ResultsIn young mice with pneumonia, KLF2 and CDKN1B were downregulated, while miR-222-3p was upregulated in lung tissues. Overexpression of KLF2 reduced lung injury and inflammation, evidenced by decreased bacterial load, reduced lung injury, and lower levels of proinflammatory factors. Co-transfection of miR-222-3p-WT and oe-KLF2 significantly reduced luciferase activity, suggesting that KLF2 binds to the promoter of miR-222-3p and suppresses its expression. Transfection of CDKN1B-WT with miR-222-3p mimics significantly reduced luciferase activity, indicating that miR-222-3p binds to CDKN1B and downregulates its expression. Overexpression of miR-222-3p or downregulation of CDKN1B increased bacterial load in BALF, lung wet/dry weight ratio, MPO activity, and inflammation, thereby reversing the protective effect of KLF2 overexpression on lung injury in young mice with pneumonia.ConclusionsKLF2 alleviates lung injury in young mice with Spn-induced pneumonia by transcriptional regulation of the miR-222-3p/CDKN1B axis.

Senescent endothelial cells promote liver metastasis of uveal melanoma in single-cell resolution

BackgroundUveal melanoma (UM), the most common adult intraocular tumor, is characterized by high malignancy and poor prognosis in advanced stages. Angiogenesis is critical for UM development, however, not only the role of vascular endothelial dysfunction in UM remains unknown, but also their analysis at the single-cell level has been lacking. A comprehensive analysis is essential to clarify the role of the endothelium in the development of UM.MethodsBy using single-cell RNA transcriptomics data of 11 cases of primary and liver metastasis UM, we analyzed the endothelial cell status. In addition, we analyzed and validated ECs in the in vitro model and collected clinical specimens. Subsequently, we explored the impact of endothelial dysfunction on UM cell migration and explored the mechanisms responsible for the endothelial cell abnormalities and the reasons for their peripheral effects.ResultsUM metastasis has a significantly higher percentage of vascular endothelial cells compared to in situ tumors, and endothelial cells in metastasis show significant senescence. Senescent endothelial cells in metastatic tumors showed significant Krüppel-like factor 4 (KLF4) upregulation, overexpression of KLF4 in normal endothelial cells induced senescence, and knockdown of KLF4 in senescent endothelium inhibited senescence, suggesting that KLF4 is a driver gene for endothelial senescence. KLF4-induced endothelial senescence drove tumor cell migration through a senescence-associated secretory phenotype (SASP), of which the most important component of the effector was CXCL12 (C-X-C motif chemokine ligand 12), and participated in the composition of the immunosuppressive microenvironment.ConclusionThis study provides an undesirable insight of senescent endothelial cells in promoting UM metastasis.

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

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.

Influences and mechanism of extracellular vesicles from dermal papilla cells of mice on human hypertrophic scar fibroblasts

Objective: To investigate the influences and mechanism of extracellular vesicles from dermal papilla cells (DPC-EVs) of mice on human hypertrophic scar fibroblasts (HSFs). Methods: The study was an experimental research. The primary dermal papilla cells (DPCs) of whiskers were extracted from 10 6-week-old male C57BL/6J mice and identified successfully. The DPC-EVs were extracted from the 3rd to 5th passage DPCs by ultracentrifugation, and the morphology was observed through transmission electron microscope and the particle diameter was detected by nanoparticle tracking analyzer (n=3) at 24 h after culture. The 3rd passage of HSFs were divided into DPC-EV group and phosphate buffer solution (PBS) group, which were cultured with DPC-EVs and PBS, respectively. The cell scratch test was performed and cell migration rate at 24 h after scratching was calculated (n=5). The cell proliferation levels at 0 (after 12 h of starvation treatment and before adding DPC-EVs or PBS), 24, 48, 72, and 96 h after culture were detected by using cell counting kit 8 (n=4). The protein expressions of α-smooth muscle actin (α-SMA) and collagen typeⅠ (ColⅠ) in cells at 24 h after culture were detected by immunofluorescence method and Western blotting, and the protein expression of Krüppel-like factor 4 (KLF4) in cells at 24 h after culture was detected by Western blotting. After the 3rd passage of HSFs were cultured with DPC-EVs for 24 h, the cells were divided into blank control group, KLF4 knockdown group, and KLF4 overexpression group according to the random number table. The cells in blank control group were only routinely cultured for 48 h. The cells in KLF4 knockdown group and KLF4 overexpression group were incubated with KLF4 knockdown virus for 24 h, then the cells in KLF4 knockdown group were routinely cultured for 24 h while the cells in KLF4 overexpression group were incubated with KLF4 overexpression virus for 24 h. The protein expressions of KLF4, α-SMA, and ColⅠ in cells were detected by Western blotting at 48 h after culture. Results: At 24 h after culture, the extracted DPC-EVs showed vesicular structure with an average particle diameter of 108.8 nm. At 24 h after scratching, the migration rate of HSFs in PBS group was (54±10)%, which was significantly higher than (29±8)% in DPC-EV group (t=4.37, P<0.05). At 48, 72, and 96 h after culture, the proliferation levels of HSFs in DPC-EV group were significantly lower than those in PBS group (with t values of 4.06, 5.76, and 6.41, respectively, P<0.05). At 24 h after culture, the protein expressions of α-SMA and ColⅠ of HSFs in DPC-EV group were significantly lower than those in PBS group, while the protein expression of KLF4 was significantly higher than that in PBS group. At 48 h after culture, compared with those in blank control group, the protein expression of KLF4 of HSFs in KLF4 knockdown group was down-regulated, while the protein expressions of α-SMA and ColⅠ were both up-regulated; compared with those in KLF4 knockdown group, the protein expression of KLF4 of HSFs in KLF4 overexpression group was up-regulated, while the protein expressions of ColⅠ and α-SMA were down-regulated. Conclusions: The DPC-EVs of mice can inhibit the proliferation and migration of human HSFs and significantly inhibit the expressions of fibrosis markers α-SMA and ColⅠ in human HSFs by activating KLF4.

Overexpression of tuftelin and KLF-5 and its clinicopathological features in hepatitis B virus-related hepatocellular carcinoma

Objective: To analyze and evaluate the expressions and clinical value of tuftelin (TUFT1) and Krüppel-like factor 5 (KLF5) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) tissues. Method: KLF5 mRNA and TUFT1 mRNA transcriptional status in cancer and non-cancer groups were compared according to the Cancer Genome Atlas (TCGA) database. The differences and prognostic value between the groups were analyzed. Postoperative liver cancer and its paired pericancerous tissues, with the approval of the ethics committee, were collected to build tissue chips. The expression of KLF5 and TUFT1 and their intracellular localization were verified by immunohistochemistry. Tissue expression and clinicopathological characteristics were analyzed by immunoblotting. SPSS software was used to analyze the relationship between SPSS and patient prognosis. Results: The transcription level of TUFT1 or KLF5 mRNA was significantly higher in the HCC group than the non-cancer group (P < 0.001), according to TCGA data. Immunohistochemistry and Western blotting examination confirmed the overexpression of TUFT1 and KLF5 in human HCC tissues, which were mainly localized in the cytoplasm and cell membrane. The positivity rates of TUFT1 and KLF5 were 87.1% ( χ(2) = 18.563, P < 0.001) and 95.2% ( χ(2) = 96.435, P < 0.001) in HCC tissues, and both were significantly higher than those in the adjacent group. The expression intensity was higher in stage III-IV than stage I-II of the International Union Against Cancer standard (P < 0.01). The clinicopathological features showed that the abnormalities of the two were significantly related to HBV infection, tumor size, extrahepatic metastasis, TNM stage, and ascites. Univariate analysis was related to tumor size, HBV infection, and survival. Multivariate analysis was an independent prognostic factor for patients with HCC. Conclusion: TUFT1 and KLF5 may both be novel markers possessing clinical value in the diagnosis and prognosis of HBV-related HCC.

Krüppel-like factor 4 modulates the miR-101/COL10A1 axis to inhibit renal fibrosis after AKI by regulating epithelial–mesenchymal transition

Acute kidney injury (AKI) can progress to renal fibrosis and chronic kidney disease (CKD), which reduces quality of life and increases the economic burden on patients. However, the molecular mechanisms underlying renal fibrosis following AKI remain unclear. This study tested the hypothesis that the Krüppel-like factor 4 (KLF4)/miR-101/Collagen alpha-1X (COL10A1) axis could inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis after AKI in a mouse model of ischemia-reperfusion (I/R)-induced renal fibrosis and HK-2 cells by gene silencing, overexpression, immunofluorescence, immunohistochemistry, real-time quantitative PCR, Western blotting, dual-luciferase reporter assay, fluorescence in situ hybridization (FISH) and ELISA. Compared with the Sham group, I/R induced renal tubular and glomerular injury and fibrosis, and increased the levels of BUN, serum Scr and neutrophil gelatinase-associated lipocalin (NGAL), Col10a1 and Vimentin expression, but decreased E-cadherin expression in the kidney tissues of mice at 42 days post-I/R. Similarly, hypoxia promoted fibroblastic morphological changes in HK-2 cells and enhanced NGAL, COL10A1, Vimentin, and α-SMA expression, but reduced E-cadherin expression in HK-2 cells. These pathological changes were significantly mitigated in COL10A1-silenced renal tissues and HK-2 cells. KLF4 induces miR-101 transcription. More importantly, hypoxia upregulated Vimentin and COL10A1 expression, but decreased miR-101, KLF4, and E-cadherin expression in HK-2 cells. These hypoxic effects were significantly mitigated or abrogated by KLF4 over-expression in the HK-2 cells. Our data indicate that KLF4 up-regulates miR-101 expression, leading to the downregulation of COL10A1 expression, inhibition of EMT and renal fibrosis during the pathogenic process of I/R-related renal fibrosis.

P53 exerts anticancer effects by regulating enhancer formation and activity.

The abnormality of the p53 tumor suppressor is crucial in lung cancer development, because p53 regulates target gene promoters to combat cancer. Recent studies have shown extensive p53 binding to enhancer elements. However, whether p53 exerts a tumor suppressor role by shaping the enhancer landscape remains poorly understood. In the current study, we employed several functional genomics approaches to assess the enhancer activity at p53 binding sites throughout the genome based on our established TP53 knockout (KO) human bronchial epithelial cells (BEAS-2B). A total of 943 active regular enhancers and 370 super-enhancers (SEs) disappeared upon the deletion of p53, indicating that p53 modulates the activity of hundreds of enhancer elements. We found that one p53-dependent SE, located on chromosome 9 and designated as KLF4-SE, regulated the expression of the Krüppel-like factor 4 ( KLF4) gene. Furthermore, the deletion of p53 significantly decreased the KLF4-SE enhancer activity and the KLF4 expression, but increased colony formation ability in the nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced cell transformation model. Subsequently, in TP53 KO cells, the overexpression of KLF4 partially reversed the increased clonogenic capacity caused by p53 deficiency. Consistently, KLF4 expression also decreased in lung cancer tissues and cell lines. It appeared that overexpression of KLF4 significantly suppressed the proliferation and migration of lung cancer cells. Collectively, our results suggest that the regulation of enhancer formation and activity by p53 is an integral component of the p53 tumor suppressor function. Therefore, our findings offer some novel insights into the regulation mechanism of p53 in lung oncogenesis and introduce a new strategy for screening therapeutic targets.

Kruppel‐like factor 5 (KLF5) as a genetic factor associated with the etiopathogenesis of Keratoconus

Aims: To analyse expression and genotype of Krüppel‐Like Factor 5 (KLF5) gene in the corneal epithelium of eyes with Keratoconus (KC) compared to normal eyes (controls); as well as, to determine the effect of several pro‐inflammatory cytokines on the KLF5 expression by an in vitro study.Methods: 64 corneal epithelium samples from 53 KC and 11 controls were included in this study. KC samples were collected from patients who received crosslinking epi‐off or penetrating keratoplasty for their mild to advanced KC. Control samples were collected from dead donors with healthy corneas. Molecular determinations to measure KLF5 expression and genotype on these samples were performed respectively by qPCR and TaqMan targeting the SNP of interest (rs17285550). In addition, using a cell line of healthy corneal epithelial cells, incubations with increasing concentrations of interleukin 6 (IL6), interleukin 1 beta (IL1β), and tumour necrosis factor alpha (TNFα) were performed to determine the KLF5 response to a pro‐inflammatory environment. KLF5 expression in the stimulated cell cultures was assessed by qPCR analysis.Results: KC samples evidenced significantly higher levels of KLF5 gene expression than the controls (p &lt; 0.0001). The GG genotypes showed an upward tendency for higher expression of KLF5 in the KC group. Higher IL6, IL1β, and TNFα concentrations contribute to a significatively KLF5 overexpression in corneal epithelial cell cultures.Conclusions: Overexpression of KLF5 in the corneal epithelium of KC patients and its upregulation against a proinflammatory microenvironment, could lead to an inadequate epithelial barrier function that destabilizes the surrounding corneal layers and contributes to the KC pathology. KLF5 represents a promising target for gene therapy in KC.

Suppression of Super-Enhancer-Driven TAL1 Expression By KLF4 in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by differentiation arrest and clonal proliferation of immature thymocytes. Many oncogenes in T-ALL are genes encoding transcription factors. TAL bHLH transcription factor 1 ( TAL1) is one of the most frequently dysregulated transcription factors in T-ALL and is ectopically overexpressed in approximately 50% of T-ALL cases, owing to chromosome translocations, SIL (SCL-interrupting locus) -TAL1 fusion, and 5‘ super-enhancer (SE)-generating mutations. TAL1 overexpression caused by 5‘ TAL1 SE is found in 5% of patients with T-ALL and is associated with unfavorable clinical outcomes. However, no clinically available drugs specifically inhibit TAL1 or TAL1 SE components. Kruppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors that exhibits both oncogenic and tumor-suppressive functions in a context-dependent manner. In T-cell malignancies, KLF4 is regarded as a tumor suppressor because KLF4 expression is silenced by promoter methylation, and the induction of KLF4 promotes apoptosis through the BCL-2/BCL-XL pathway. However, whether KLF4 affects the expression of T-ALL-related transcription factors remains unclear. We first analyzed three independent previously reported microarray datasets to explore the essential downstream targets of KLF4 in T-ALL cells. The following groups of genes were extracted: genes upregulated in patients with T-ALL compared to normal thymocytes (GSE46170), genes upregulated in Klf4-deficient T-ALL mice (GSE75663), and genes downregulated in KLF4-overexpressing human T-ALL cells (Li W et al. Mol. Cancer. 2015). Overlapping genes in these groups were identified as potential downstream targets of KLF4. Among these, we focused on TAL1, a well-known oncogenic transcription factor gene in T-ALL. We performed a functional analysis of KLF4 using TAL1-positive T-ALL cell lines (Jurkat, MOLT-3, CCRF-CEM, and RPMI-8402). Genetic induction of KLF4 showed stronger anti-leukemic effects in Jurkat and MOLT-3 cells, 5‘ TAL1 SE-positive T-ALL cell lines, compared with CCRF-CEM and RPMI-8402 cells, SIL-TAL1-positive T-ALL cell lines. KLF4 overexpression significantly reduced mRNA expression levels of TAL1 in Jurkat and MOLT-3 cells but not in CCRF-CEM and RPMI-8402 cells. Thus, we hypothesized that KLF4 specifically suppressed SE-driven TAL1 expression by impairing mutated TAL1 enhancer activity. The luciferase reporter assay confirmed that the activity of the mutated TAL1 enhancer was significantly reduced by the exogenous expression of KLF4, whereas that of the wild-type reporter was not affected. According to previous studies, mutations in TAL1 enhancers in Jurkat and MOLT-3 cells create MYB primary motifs, and MYB binding at this location is essential for the formation of 5‘ TAL1 SE. Therefore, we examined whether KLF4 represses MYB expression. The chromatin immunoprecipitation assay and qPCR analysis revealed that KLF4 bound to the proximal gene promoter of MYB and directly downregulated its expression. The luciferase reporter assay using the MYB promoter showed a significant reduction of reporter activity upon KLF4 overexpression. Finally, we investigated whether pharmacological induction of KLF4 using APTO-253, a small-molecule inducer ofKLF4, could control leukemia. APTO-253 inhibited the growth of all TAL1-positive T-ALL cell lines at nanomolar concentrations. The GI 50 values of APTO-253 for Jurkat and MOLT-3 cells were lower than those for CCRF-CEM and RPMI-8402 cells, which was consistent with the KLF4-overexpression-induced growth inhibition of T-ALL cell lines. APTO-253 induced KLF4 expression and decreased TAL1 and MYB expression in Jurkat and MOLT-3 cells. Furthermore, exogenous MYB expression rescued growth inhibition by APTO-253. Collectively, these results indicated that APTO-253 suppressed the growth of T-ALL cells by targeting SE-driven TAL1 expression via the induction of KLF4. In summary, we revealed that KLF4 suppressed SE-driven TAL1 expression via the direct inhibition of MYB. Moreover, pharmacological induction of KLF4 demonstrated anti-leukemic activity in T-ALL cells. These findings propose a promising strategy for patients with T-ALL and 5' TAL1 SE.

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.

Krüppel-like factor 4 promotes autophagy in macrophages under high glucose concentration by inhibiting the AKT/mTOR signaling pathway

Background: Diabetic atherosclerosis (AS) is the main cause of disability and death in diabetes. In the progression of AS, autophagic activity plays an important role. Krüppel-like factor 4 (KLF4) is a member of the zinc finger protein transcription factor family and is believed to play a protective role in the pathogenesis of atherosclerosis. This study aimed to explore the role of KLF4 in diabetic atherosclerosis and the autophagic mechanism. Methods: A diabetic mouse model was established and the expression level of KLF4 protein in the aorta of the mice was detected after a high-fat diet. The effects of KLF4 on cholesterol content, apoptosis, autophagy-related proteins, and the AKT/mTOR signaling pathway of THP-1 macrophages were also evaluated. Results: The expression level of KLF4 protein in the aorta of diabetic mice was decreased. Meanwhile, overexpression of KLF4 in THP-1 macrophages significantly decreased cholesterol accumulation, increased beclin-1 expression, decreased P62 expression, enhanced LC3 fluorescence intensity decreased cell apoptosis and p-mTOR and p-AKT expression were decreased under the condition of high glucose. After the reduction of KLF4 expression, the result is reversed. Conclusion: KLF4 induces autophagy by inhibiting the AKT/mTOR pathway and alleviates cholesterol deposition in THP-1 macrophages under high glucose concentration.

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.

KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy.

Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC. We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer. Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC's commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC's cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage. Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.

KLF2 transcription suppresses endometrial cancer cell proliferation, invasion, and migration through the inhibition of NPM1.

Endometrial cancer (EC) is the most common gynaecologic malignancy. This study was to explore the role of kruppel-like factor 2 (KLF2) in EC cell behaviours. The expression of KLF2 in EC and its correlation with NPM1 were first predicted on the database. Levels of KLF2 and nucleophosmin 1 (NPM1) in EC cell lines were then determined. After transfection of the overexpression vector of KLF2 or NPM1, cell proliferation, invasion, and migration were evaluated. The binding relationship between KLF2 and the NPM1 promoter was analysed. KLF2 was downregulated while NPM1 was upregulated in EC cells. KLF2 overexpression reduced the proliferation potential of EC cells and the number of invaded and migrated cells. KLF2 was enriched in the NPM1 promoter and inhibited NPM1 transcriptional level. NPM1 overexpression neutralised the effects of KLF2 overexpression on suppressing EC cell growth. Collectively, KLF2 was decreased in EC cells and KLF2 overexpression increased the binding to the NPM1 promoter to inhibit NPM1 transcription, thus suppressing EC cell growth.

Ursolic Acid Inhibits the Growth of Gastric Cancer by Targeting KLF4/YAP1

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor which has various mechanisms in different tumors. Ursolic acid (UA), a natural compound that exists in many herbs, is known to prevent tumor progression and has anticancer effects on many human cancers. The present study was to evaluate the antitumor effect of UA on gastric cancer (GC) through KLF4 and the Hippo pathway. Our data showed that UA inhibited the growth of GC in vivo and in vitro. UA treatment significantly increased the expression of KLF4 and decreased the expression of CTGF. The overexpression of KLF4 inhibited the proliferation and cell cycle of GC and decreased the expression of CTGF, whereas the knockdown of KLF4 attenuated the effects of UA. Furthermore, the inhibition of CTGF arrested tumor cells in G2/M which blocked proliferation progress. Confocal laser scanning and molecular simulation software MOE showed that KLF4 combined with YAP1 which may block the formation of the TEADs-YAP1 complex to interrupt the expression of CTGF and the downstream oncogenic process. In conclusion, UA can inhibit GC growth both in vivo and in vitro, and it activated KLF4 which may competitively bind with YAP1 against TEADs and block the oncogenic Hippo pathways.

Effect of overexpression of KLF4 on the growth and development of hair follicles in mice.

Hair follicle growth is cyclical, and hair cycle dysfunction can lead to hair follicle-related disorders, including alopecia and hirsutism. The objective was to investigate the influence and underlying mechanism of Krüppel-like factor 4 (KLF4) overexpression on hair follicle growth and development in C57BL/6 mice. To provide a theoretical basis for the biological functions of KLF4 gene in hair follicle development and hair follicle cycle, mice were assigned to three groups: experimental, overexpressing KLF4 (Ad-KLF4); control, expressing green fluorescent protein (Ad-NC); and blank, no treatment. Fur was removed from the dorsal surface, and the mice were intradermally injected with 25 μL 1 × 1010 PFU/mL adenovirus vector (Ad-KLF4 or Ad-NC) at three points. Samples were collected for molecular biological and histological analysis. It was found that mRNA and protein levels of Wnt pathway-associated factors β-catenin, LEF1, hair follicle cell proliferation-related factor Ki67, and hair follicle inner caledrin marker AE15 were all significantly greater in the Ad-NC and blank groups than in Ad-KLF4 mice (P < 0.01). These findings were confirmed by immunohistochemical analysis. Hair growth was monitored photographically for 14days, showing an absence of growth in the injected region of the KLF4-overexpressing mice in contrast to non-overexpressing areas where hair growth was normal. HE staining showed that hair follicles in the blank and Ad-NC mice were normal, while those in the KLF4-overexpressing areas remained in telogen or early anagen with spherical dermal papillae situated at the edge of the dermis and subcutaneous tissue without an inner heel sheath. In conclusion, it was found that KLF4 downregulated key Wnt/β-catenin-associated factors during follicular regeneration in mice, reducing both follicular development and growth.

Circular RNA circPLOD2 regulates pericyte function by targeting the transcription factor KLF4

Circular RNAs are generated by backsplicing and control cellular signaling and phenotypes. Pericytes stabilize capillary structures and play important roles in the formation and maintenance of blood vessels. Here, we characterize hypoxia-regulated circular RNAs (circRNAs) in human pericytes and show that the circular RNA of procollagen-lysine,2-oxoglutarate 5-dioxygenase-2 (circPLOD2) is induced by hypoxia and regulates pericyte functions. Silencing of circPLOD2 affects pericytes and increases proliferation, migration, and secretion of soluble angiogenic proteins, thereby enhancing endothelial migration and network capability. Transcriptional and epigenomic profiling of circPLOD2-depleted cells reveals widespread changes in gene expression and identifies the transcription factor krüppel-like factor 4 (KLF4) as a key effector of the circPLOD2-mediated changes. KLF4 depletion mimics circPLOD2 silencing, whereas KLF4 overexpression reverses the effects of circPLOD2 depletion on proliferation and endothelial-pericyte interactions. Together, these data reveal an important function of circPLOD2 in controlling pericyte proliferation and capillary formation and show that the circPLOD2-mediated regulation of KLF4 significantly contributes to the transcriptional response to hypoxia.