Krüppel-like Factor Research Articles

Identification of the EBF1/ETS2/KLF2-miR-126-Gene Feed-Forward Loop in Breast Carcinogenesis and Stemness.

MicroRNA (miR)-126 is frequently downregulated in malignancies, including breast cancer (BC). Despite its tumor-suppressive role, the mechanisms underlying miR-126 deregulation in BC remain elusive. Through silencing experiments, we identified Early B Cell Factor 1 (EBF1), ETS Proto-Oncogene 2 (ETS2), and Krüppel-Like Factor 2 (KLF2) as pivotal regulators of miR-126 expression. These transcription factors were found to be downregulated in BC due to epigenetic silencing or a "poised but not transcribed" promoter state, impairing miR-126 expression. Gene Ontology analysis of differentially expressed miR-126 target genes in the Cancer Genome Atlas: Breast Invasive Carcinoma (TCGA-BRCA) cohort revealed their involvement in cancer-related pathways, primarily signal transduction, chromatin remodeling/transcription, and differentiation/development. Furthermore, we defined interconnections among transcription factors, miR-126, and target genes, identifying a potential feed-forward loop (FFL) crucial in maintaining cellular identity and preventing the acquisition of stemness properties associated with cancer progression. Our findings propose that the dysregulation of the EBF1/ETS2/KLF2/miR-126 axis disrupts this FFL, promoting oncogenic transformation and progression in BC. This study provides new insights into the molecular mechanisms of miR-126 downregulation in BC and highlights potential targets for therapeutic intervention. Further research is warranted to clarify the role of this FFL in BC, and to identify novel therapeutic strategies aimed at modulating this network as a whole, rather than targeting individual signals, for cancer management.

The multifaceted anti-atherosclerotic properties of herbal flavonoids: A comprehensive review.

Atherosclerosis (AS) is a major etiological factor underpinning a spectrum of cardiovascular diseases, leading to cerebral infarction, coronary artery disease, and peripheral vascular disease. The chronic progression of AS, spanning from initial plaque formation to the occurrence of acute cardiovascular events, underscores the complexity of AS and the challenges it presents in terms of treatment. Currently, the clinical management of AS relies predominantly on statins and proprotein convertase subtilisin/kexin type 9 inhibitors, which primarily aim to reduce low-density lipoprotein levels and have demonstrated some therapeutic efficacy. Nevertheless, due to their potential side effects, there is a pressing need to actively investigate alternative treatment approaches. Researches on natural compounds derived from herbal medicines, such as flavonoids, hold significant promise in combating AS by regulating lipid metabolism, reducing oxidative stress and inflammation, inhibiting the proliferation of vascular smooth muscle cells, modulating autophagy and additional pathways. Various targets participate in these physiological processes, encompassing acyl-CoA: cholesterol acyltransferase (ACAT), ATP citrate lyase (ACLY), nuclear factor erythroid 2-related factor 2 (Nrf2), krüppel-like factor 2 (KLF2), NOD-like receptor protein 3 (NLRP3), transcription factor EB (TFEB) and so on. This comprehensive review endeavors to synthesize and analyse the most recent findings on herbal flavonoids, shedding light on their anti-atherosclerotic potential and the underlying protective mechanisms and related-targets, which might pave the way for the development of novel drug candidates or the optimization of flavonoid-based therapies.

The matrix protease ADAMTS1 is transcriptionally activated by KLF6 and contributes to cardiac fibrosis in non-ischemic cardiomyopathy

AimsAberrant cardiac fibrosis, defined as excessive production and deposition of extracellular matrix (ECM), is mediated by myofibroblasts. ECM-producing myofibroblasts are primarily derived from resident fibroblasts during cardiac fibrosis. The mechanism underlying fibroblast-myofibroblast transition is not fully understood. MethodsCardiac fibrosis was induced by transverse aortic constriction (TAC) or by angiotensin II (Ang II) infusion in C57B6/j mice. Cellular transcriptome was evaluated by RNA-seq and CUT&Tag-seq. ResultsIntegrated transcriptomic screening revealed that a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was a novel transcriptional target for Kruppel-like factor 6 (KLF6) in cardiac fibroblasts. Treatment with either TGF-β or Ang II up-regulated ADAMTS1 expression. KLF6 knockdown attenuated whereas KLF6 over-expression enhanced ADAMTS1 induction. ChIP assay and reporter assay showed that KLF6 was recruited to the ADAMTS1 promoter to activate its transcription. Consistently, ADAMTS1 knockdown suppressed fibroblast-myofibroblast transition in vitro. Importantly, myofibroblast-specific ADAMTS1 depletion attenuated cardiac fibrosis and normalized heart function in mice. SignificanceIn conclusion, our data demonstrate that ADAMTS1, as a downstream target of KLF6, contributes to cardiac fibrosis by regulating fibroblast-myofibroblast transition.

High Shear Stress Reduces ERG Causing Endothelial-Mesenchymal Transition and Pulmonary Arterial Hypertension.

Computational modeling indicated that pathological high shear stress (HSS; 100 dyn/cm2) is generated in pulmonary arteries (PAs; 100-500 µm) in congenital heart defects causing PA hypertension (PAH) and in idiopathic PAH with occlusive vascular remodeling. Endothelial-to-mesenchymal transition (EndMT) is a feature of PAH. We hypothesize that HSS induces EndMT, contributing to the initiation and progression of PAH. We used Ibidi perfusion system to determine whether HSS applied to human PA endothelial cells (ECs) induces EndMT when compared with physiological laminar shear stress (15 dyn/cm2). The mechanism was investigated and targeted to prevent PAH in a mouse with HSS induced by an aortocaval shunt. EndMT, a feature of PAH not previously attributed to HSS, was observed. HSS did not alter the induction of transcription KLF (Krüppel-like factor) 2/4, but an ERG (ETS-family transcription factor) was reduced, as were histone H3 lysine 27 acetylation enhancer-promoter peaks containing ERG motifs. Consequently, there was reduced interaction between ERG and KLF2/4, a feature important in tethering KLF and the chromatin remodeling complex to DNA. In PA ECs under laminar shear stress, reducing ERG by siRNA caused EndMT associated with decreased BMPR2 (bone morphogenetic protein receptor 2), CDH5 (cadherin 5), and PECAM1 (platelet and EC adhesion molecule 1) and increased SNAI1/2 (Snail/Slug) and ACTA2 (smooth muscle α2 actin). In PA ECs under HSS, transfection of ERG prevented EndMT. HSS was then induced in mice by an aortocaval shunt, causing progressive PAH over 8 weeks. An adeno-associated viral vector (AAV2-ESGHGYF) was used to replenish ERG selectively in PA ECs. Elevated PA pressure, EndMT, and vascular remodeling (muscularization of peripheral arteries) in the aortocaval shunt mice were markedly reduced by ERG delivery. Pathological HSS reduced lung EC ERG, resulting in EndMT and PAH. Agents that upregulate ERG could reverse HSS-mediated PAH and occlusive vascular remodeling resulting from high flow or narrowed PAs.

KLF10 Induced by Hypothermic Machine Perfusion Alleviates Renal Inflammation Through BIRC2/Noncanonical NF-κB Pathway

Background. Hypothermic machine perfusion (HMP) is becoming the main preservation method for donation after circulatory death (DCD) kidneys. It can provide continuous flow and form shear stress (SS) upon endothelial cells (ECs), thereby regulating EC injury. Krüppel-like factor 10 (KLF10) has been shown to lessen vascular damage. However, how SS and KLF10 impact HMP-regulated injury is unclear. Methods. In this study, we investigated the influences of KLF10 on HMP in animal models and human renal biopsy and explored how SS affected KLF10 expression in a parallel-plate flow chamber system. Chromatin Immunoprecipitation sequencing and luciferase assay were performed to seek the target genes of KLF10. The influences of KLF10 on HMP-regulated injury were investigated by transfecting si-KLF10 adeno-associated virus serotype 9 into rat kidneys. The molecular expression was examined using immunofluorescence staining, Western blotting, and quantitative polymerase chain reaction. Results. Our results show KLF10 expression was augmented in human, rabbit, and rat DCD kidneys after HMP. HMP improved ECs and tubule injury and attenuated inflammation; however, the knockdown of KLF10 reversed this effect. SS regulated KLF10 expression in ECs by affecting F-actin, and KLF10 could maintain ECs homeostasis. Chromatin Immunoprecipitation sequencing and luciferase assay revealed that baculoviral inhibitor of apoptosis protein repeat-containing 2 (BIRC2 ) is a target gene of KLF10. Furthermore, BIRC2 linked to nuclear factor kappa B (NF-κB)-inducing kinase, induced NF-κB)-inducing kinase ubiquitination, and resulted in inhibiting the noncanonical NF-κB pathway. Conclusions. SS can mediate KLF10 expression, whereas HMP can protect against warm ischemic injury by reducing inflammation via KLF10/BIRC2/noncanonical NF-κB pathway. Therefore, KLF10 might be a novel target for improving DCD kidney quality.

Integrative Analysis of Chromatin Accessibility and Transcriptional Landscape Identifies Key Genes During Muscle Development in Pigs

Many efforts have been made to reveal the mechanisms underlying skeletal muscle development because of its importance in animals. However, knowledge on chromatin accessibility, a prerequisite for gene expression, remains limited. Here, dynamic changes in chromatin accessibility were analyzed in the skeletal muscles of Min pigs at the ages of 30, 90, and 210 d using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). A total of 16,301 differentially accessible regions (DARs) associated with 7455 genes were identified among three developmental stages. Seven out of eight DARs selected for a functional analysis were found to regulate reporter gene expression significantly (p < 0.05), indicating that DARs are active in gene expression. A total of 2219 differentially expressed genes (DEGs) were identified with RNA sequencing (RNA-seq). Through integrated analyses of ATAC-seq and RNA-seq data, 54 DEG_DAR_genes and 61 transcription factors (TFs) were characterized as critical for muscle development. Among them, Kruppel-like factor 5 (KLF5), targeted to 36 DEG_DAR_genes, was the most important TF. The effects of KLF5 on DEG_DAR_gene expression were then analyzed with molecular biology techniques. KLF5 was found to regulate SLPI (secretory leukocyte proteinase inhibitor) expression by directly binding to the promoter; KLF5 was also involved in APOA1 (apolipoprotein A-I) expression through affecting the regulatory role of DAR located in the intron. These results indicate that the TFs identified were functional. Altogether, the chromatin accessibility region, TFs, and genes important for muscle development in Min pigs were identified. The results provide novel data for further revealing the mechanisms underlying the epigenetic regulation of muscle development.

KLF2 controls the apoptosis of neutrophils and is associated with disease activity of systemic lupus erythematosus

BackgroundNeutropenia is more common in patients with systemic lupus erythematosus (SLE) and is a major cause of life-threatening infections. The increased apoptosis of neutrophils is likely to be an essential cause of neutropenia in SLE. However, the detailed mechanisms of increased neutrophil apoptosis in SLE remain unknown. This study focused on the role of Krüppel-like factor 2 (KLF2) in the regulation of neutrophil apoptosis and its association with SLE disease activity.MethodsThe levels of KLF2 in neutrophils from SLE patients and healthy controls (HCs) were detected by RT-PCR and western blot. The relationship between the levels of KLF2 and the apoptosis levels of neutrophils in SLE patients was analyzed. The KLF2 inhibitor Geranylgeranyl pyrophosphate (GGPP) and the KLF2 inducer geranylgeranyl transferase inhibitor (GGTI-298) were used to incubate with neutrophils to investigate the role of KLF2 in the regulation of neutrophil apoptosis. To clarify whether serum from SLE patients affects neutrophil KLF2 expression and apoptosis, sera from SLE patients were collected and used to incubate with neutrophils from HCs, followed by the detection of KLF2 levels and apoptosis levels of neutrophils. Additionally, the correlation between KLF2 levels and SLE disease activity index (SLEDAI) was analyzed.ResultsThe expression of KLF2 in neutrophils of SLE patients was significantly suppressed, and the decreased KLF2 was associated with the upregulation of neutrophil apoptosis. Moreover, newly diagnosed SLE patients, SLE patients with higher serum IgG and positive anti-Smith antibodies had lower KLF2 expression. Furthermore, we demonstrated that modulating the expression of KLF2 can regulate the apoptosis of neutrophils. The levels of KLF2 in neutrophils were associated with the SLEDAI. In addition, we found that serum from SLE patients could induce apoptosis in neutrophils by down-regulating the expression of KLF2.ConclusionsKLF2 controls the apoptosis of neutrophils and is associated with SLEDAI, which suggests that KLF2 in neutrophils may be involved in the occurrence and development of SLE.

MACC1 ablation suppresses the dedifferentiation process of non-CSCs in lung cancer through stabilizing KLF4

Metastasis-associated in colon cancer-1 (MACC1) was identified as a new player in lung cancer development, and some stemness-related genes can be novel transcriptional targets of MACC1. Cancer stem cells (CSCs) are responsible for sustaining tumorigenesis and plasticity. Both CSCs and non-CSCs are plastic and capable of undergoing phenotypic transition, especially the dedifferentiation of non-CSCs switch to CSC-like cells. However, the precise role of MACC1 during this process is largely unknown. Here, we showed that MACC1 promoted the transition from non-CSC to CSC in lung cancer. We found MACC1 was overexpressed in stemness enriched cells, enhancing the transition from no-CSCs to CSCs, while short-hairpin RNA-mediated Knockdown of MACC1 impaired this process. High-throughput sequencing and tumor specimen analysis revealed that MACC1 was negative correlated with Krüppel-like factor 4 (KLF4) expression level, which acts as a negative stemness regulator in lung cancer. Mechanistically, MACC1 delays the degradation of KLF4 mRNA by repressing the expression of microRNA-25, thereby promoting the KLF4 mRNA stabilization at the post-transcriptional level. Collectively, our findings may facilitate efforts to promote the development of precision targeted therapy for cancer stem cells in lung cancer.

Suppression of KLF5 targets RREB1 to restrain the proliferation of ovarian cancer cells through ERK/MAPK signaling pathway.

The overexpression of Kruppel-like factor 5 (KLF5) appears in several types of cancer. KLF5 may be an effective therapeutic target for treating OC, but its function in ovarian cancer (OC) remains unknown. The KLF5 mRNA expression levels in several OC cell lines were analyzed using RT-qPCR. Then, NC-siRNA or KLF5-siRNA was transfected into SK-OV-3 and OVCAR-3 cells. RT-qPCR and WB were used to detect the efficiency of KLF5 silence, CCK-8, colony formation assay, IHC staining, flow cytometry, and WB were performed to investigate the KLF5 function on OC cell proliferation and the activation of the extracellular signal-regulated Kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway. Next, a dual-luciferase and IF assay were used to determine the relationship between KLF5 and the Ras response element-binding protein (RREB1). SK-OV-3 and OVCAR-3 cells were treated with KLF5-siRNA and C16-PAF + EGF (MAPK agonist), separately or in combination. Proteins including KLF5, RREB1, p-p38, p-ERK1/2, ERK5, p-ERK5, CyclinD1, CDK4, and CDK6 were quantified by WB. Finally, CCK-8, colony formation assay, and flow cytometry were employed again. KLF5 is highly expressed in OC cells compared with normal cells. When KLF5 knockdowns in SK-OV-3 and OVCAR-3 cells, the cell proliferation restrains, andthe G1 phase prolongs. In addition, KLF5 silence caused a decrease of CyclinD1, CDK4, CDK6, p-p38, p-ERK1/2, and p-ERK5/ERK5 expression levels. However, these statuses could be revised by C16-PAF + EGF. Results also found that when the ERK/MAPK signaling is activating, RREB1 is expressed low. The KLF5 silence could up-regulate the RREB1 expression. The KLF5 silence could restrain the OC cell proliferation and cell cycle. KLF5-siRNA may target upregulating RREB1 expression, thereby inhibiting the activation of the ERK/MAPK signaling pathway in OC cells.

KLF2-dependent transcriptional regulation safeguards the heart against pathological hypertrophy.

Our previous single-cell RNA sequencing study in the adult human heart revealed that cardiomyocytes from both the atrium and ventricle display high activities of Krüppel-like factor 2 (KLF2) regulons. However, the role of the transcription factor KLF2 in cardiomyocyte biology remains largely unexplored. We employed transverse aortic constriction surgery in male C57BL/6 J mice to develop an in vivo model of cardiac hypertrophy, and generated different in vitro cardiac hypertrophy models in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes. Our results demonstrated a significant reduction in KLF2 expression during the progression of cardiac hypertrophy. In vitro, Klf2 deficiency exacerbates cardiac hypertrophy and enhances hypertrophic reprogramming, while KLF2 overexpression attenuates cardiac hypertrophy and reverses hypertrophic transcriptome reprogramming. Mechanistically, combined RNA-seq and cleavage under targets & tagmentation (CUT&Tag) analysis revealed that KLF2 exerts its protective effects by directly regulating a set of genes associated with cardiac hypertrophy. In vivo, KLF2 overexpression specifically in cardiomyocytes effectively prevents TAC-induced cardiac hypertrophy in mice. Additionally, we found that simvastatin elevates KLF2 expression in cardiomyocytes, which subsequently alleviates cardiomyocyte hypertrophy. This study provides the first evidence that transcription factor KLF2 serves as a negative regulator of cardiac hypertrophy. Our findings highlight the therapeutic potential of enhancing KLF2 expression, particularly through simvastatin administration, as a promising strategy in the treatment of cardiac hypertrophy.

MicroRNA-34a-5p regulates agouti-related peptide via krüppel-like factor 4 and is disrupted by bisphenol A in hypothalamic neurons

Obesity is a complex disease marked by increased adiposity and impaired metabolic function. While diet and lifestyle are primary causes, endocrine-disrupting chemicals (EDCs), such as bisphenol A (BPA), significantly contribute to obesity. BPA, found in plastic consumer products, accumulates in the hypothalamus and dysregulates energy homeostasis by disrupting the neuropeptide Y (NPY)/agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) neurons.However, the precise molecular mechanisms of how BPA disrupts neuropeptide expression remains unclear. We hypothesized that microRNAs (miRNAs), which regulate approximately 60% of the human protein-coding genome and are crucial for hypothalamic energy regulation, may mediate the effects of BPA on Agrp. Using the TargetScanMouse 8.0 and DIANA microT bioinformatics tools, we identified miR-501-5p as a potential miRNA that directly regulates Agrp and the miR-34 family as miRNAs that indirectly regulate Agrp through its transcription factor krüppel-like factor 4 (KLF4). We found that in an immortalized NPY/AgRP-expressing cell line, mHypoE-41, miR-501-5p unexpectedly upregulated Agrp, while miR-34a-5p reduced Klf4 and Agrp mRNA levels. Serum starvation reduced miR-34a-5p levels and elevated Agrp mRNA levels, suggesting a potential role in AgRP regulation. Inhibiting the miR-34a-5p interaction with the Klf4 3′UTR using a specific target site blocker prevented the downregulation of both Klf4 and Agrp, suggesting miR-34a-5p alters Agrp mRNA levels via regulation of KLF4. BPA treatment increased Agrp and Klf4 expression while simultaneously decreasing miR-34a-5p levels, indicating miR-34a-5p may play a role in BPA-mediated dysregulation of Agrp. Overall, this study highlights indirect miRNA-based regulation of Agrp, which can also be dysregulated by obesogens, such as BPA.

KLF14 inhibits tumor progression via FOSL1 in glioma

BackgroundGlioma, the most frequent central nervous system malignancy, is often promoted by the overexpression of Fos-like antigen 1 (FOSL1). However, the regulation of FOSL1 remains unexplored. The present study aimed to investigate the regulatory mechanism of FOSL1 to identify potential therapeutic targets for glioblastoma. MethodsThis study's initial investigation utilized dual-luciferase reporter gene assays and quantitative polymerase chain reaction (qPCR) assays to establish that Kruppel-like factor 14 (KLF14) inhibits the transcription of FOSL1. Subsequent immunohistochemistry and western blotting (WB) assays on glioma tissues confirmed a negative association between FOSL1 and KLF14. This study generated KLF14 knockdown cells and double knockdown cells of KLF14 and FOSL1 and further assessed cell growth through various experimental methods. The impact of KLF14 on tumor cell migration via FOSL1 was determined using qPCR and WB assays. A xenograft tumor model was utilized to verify tumor growth suppression by KLF14. ResultsThe present study demonstrated that KLF14 restrains FOSL1 transcription and is inversely correlated with FOSL1 in glioma tissues. KLF14 overexpression was found to counteract FOSL1's effect on cell migration and epithelial-to-mesenchymal transition in glioma cells, which coincided with decreased Snail2 and cluster of differentiation 44 (CD44) expressions. Further, KLF14 overexpression was shown to hinder tumor progression in vivo. ConclusionThis study highlights that FOSL1 is negatively regulated by KLF14 in glioblastoma and suggests that KLF14 overexpression can mitigate tumor growth by inhibiting FOSL1, thus identifying KLF14 as a novel molecular target for treating glioblastoma. Further research into the interplay and regulatory dynamics between KLF14 and FOSL1 under varying stress conditions can enhance the precision of glioblastoma treatment.

Astilbin improves the therapeutic effects of mesenchymal stem cells in AKI-CKD mice by regulating macrophage polarization through PTGS2-mediated pathway

BackgroundAlthough mesenchymal stem cells (MSCs) have been proven to be appropriate candidates for the treatment of AKI-CKD, their efficacy is limited and variable. Astilbin (AST) had a protective effect on MSCs from oxidative stress via ROS-scavenging, however, whether it can improve MSCs’ renoprotection and the underlying mechanism need to be elucidated.MethodsAST-pretreated MSCs were administered intravenously into the ischemia–reperfusion injury mice models and the renal function, pathological changes and inflammation. Were evaluated. In addition, DARTS, molecular docking, surface plasma resonance(SPR), dual-luciferase reporter gene assay and the ChIP-PCR were utilized to explore the potential signaling pathways through which AST exert renal protective effects on MSCs.ResultsAST-pretreated MSCs markedly improved kidney function, reduced kidney pathological injury and inflammation in AKI and AKI-CKD mice. RNA-seq results showed that PTGS2 related pathway was significantly up-regulated in MSCs after AST pretreatment. DARTS assay, molecular docking and SPR assay revealed that AST could bind with the transcriptional factor of Kruppel-Like Factor 4(KLF4) protein. The promoter of PTGS2 had the binding and transcriptional activation by KLF4. Furthermore, AST pretreatment promoted the secretion of PGE2 in MSCs. And then the westren blot results showed that the protein levels of CD163 and CD206 were upregulated after coculture in AST-pretreated MSCs, indicating that the polarization of RAW264.7 cells towards M2-like macrophages was induced. Knockdown of PTGS2 reversed the ability of AST-pretreated MSCs in converting macrophages to M2 phenotype and reducing their therapeutic effects on AKI-CKD mice.ConclusionAST pretreatment enhances the efficacy of MSCs on AKI and AKI-CKD mice by inducing of M2-like phenotype polarization in macrophages through the PTGS2-mediated pathway. This approach not only provides a novel strategy to strengthen the capability of MSCs but also helps elucidate the beneficial effects of the Chinese herbal medicine AST.

Exercise training attenuates cardiac dysfunction induced by excessive sympathetic activation through an AMPK-KLF4-FMO2 axis

Cardiovascular diseases (CVDs) are a leading cause of mortality worldwide and are associated with an overactivated sympathetic system. Although exercise training has shown promise in mitigating sympathetic stress-induced cardiac remodeling, the precise mechanisms remain elusive. Here, we demonstrate that exercise significantly upregulates cardiac flavin-containing monooxygenase 2 (FMO2) expression. Notably, we find that exercise training effectively counteracts sympathetic overactivation-induced cardiac dysfunction and fibrosis by enhancing FMO2 expression via adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) activation. Functional investigations employing FMO2 knockdown with adeno-associated virus 9 (AAV9) underscore the necessity for FMO2 expression to protect the heart during exercise in vivo. Furthermore, we identify the krüppel-like factor 4 (KLF4) as a transcriptional mediator of FMO2 that is crucial for the mechanism through which AMPK activation protects against sympathetic overactivation-induced cardiac dysfunction and fibrosis. Taken together, our study reveals a cardioprotective mechanism for exercise training through an AMPK-KLF4-FMO2 signaling pathway that underscores how exercise alleviates cardiac dysfunction induced by excessive sympathetic activation.

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.

Overexpression of miR-451a Aggravates Renal Ischemia-Reperfusion Injury by Targeting KLF1-ACSL4 to Promote Ferroptosis.

Ischemia-reperfusion injury (IRI) is a predominant factor leading to delayed graft function (DGF) following kidney transplantation. MicroRNAs (miRNAs) play a pivotal role in the pathogenesis of renal IRI, with ferroptosis being a critical driving force throughout the process. In this study, we utilized bioinformatics methods to construct a network diagram of differentially expressed miRNAs, transcription factors (TFs), and ferroptosis-related genes. An I/R-induced renal injury model in mice and an in vitro H/R-induced HK-2 cell injury model were established. Quantitative real-time PCR (qRT-PCR) and Western blot analysis were used to measure the mRNA and miRNA levels in cells and tissues. The MDA concentration, iron levels, and GSH concentration were measured to evaluate the ferroptosis levels. CCK-8 assays were performed to assess cell viability. Luciferase reporter assays were conducted to validate the downstream targets of miRNA, and chromatin immunoprecipitation assays were performed to verify the interaction between TFs and mRNAs. Both the in vivo and in vitro results demonstrate that miR-451a was significantly enriched in the IRI renal tissues and cells, exacerbating ferroptosis. MiR-451a was found to reduce the expression of Kruppel-like factor 1 (KLF1) by directly binding to the 3'UTR of KLF1 mRNA. Additionally, KLF1 was identified as a negative transcription factor for acyl-CoA synthetase long-chain family member 4 (ACSL4). We demonstrated that IRI induced the upregulation of miR-451a, which reduced KLF1 expression, thereby promoting ferroptosis by upregulating ACSL4 expression, ultimately aggravating IRI-induced renal damage.

Integrated mRNA-seq and miRNA-seq analysis reveals key transcription factors of HNF4α and KLF4 in ADPKD

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most prevalent genetic disorder affecting the kidneys. Understanding epigenetic regulatory mechanisms and the role of microRNAs (miRNAs) is crucial for developing therapeutic interventions. Two mRNA datasets (GSE7869 and GSE35831) and miRNA expression data (GSE133530) from ADPKD patients were used to find differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs), with a focus on genes regulated by hub transcription factors (TFs) and their target genes. The expression of hub TFs was validated in human kidneys and animal models through Western Blot (WB) and RT-PCR analysis. The location of the hub TF proteins in kidney cells was observed by a laser confocal microscope. A total of 2037 DEGs were identified. DEM analysis resulted in 59 up-regulated and 107 down-regulated miRNAs. Predicted target DEGs of DEMs indicated two top dysregulated TFs: hepatocyte nuclear factor 4 alpha (HNF4α) and Kruppel-like factor 4 (KLF4). RT-PCR, WB, and immunochemistry results showed that mRNA and protein levels of HNF4α were significantly decreased while KLF4 levels were significantly up-regulated in human ADPKD kidneys and Pkd1 conditional knockout mice compared with normal controls. Laser confocal microscopy revealed that KLF4 was mainly located in the cytoplasm while HNF4α was in the nucleus. Functional enrichment analysis indicated that genes regulated by HNF4α were mainly associated with metabolic pathways, while KLF4-regulated genes were linked to kidney development. Drug response prediction analysis revealed potential drug candidates for ADPKD treatment, including BI-2536, Sepantronium, and AZD5582. This integrated analysis provides new epigenetic insights into the complex miRNA-TF-mRNA network in ADPKD and identifies HNF4α and KLF4 as key TFs. These findings offer valuable resources for further research and potential drug development for ADPKD.

Application prospects of urine-derived stem cells in neurological and musculoskeletal diseases.

Urine-derived stem cells (USCs) are derived from urine and harbor the potential of proliferation and multidirectional differentiation. Moreover, USCs could be reprogrammed into pluripotent stem cells [namely urine-derived induced pluripotent stem cells (UiPSCs)] through transcription factors, such as octamer binding transcription factor 4, sex determining region Y-box 2, kruppel-like factor 4, myelocytomatosis oncogene, and Nanog homeobox and protein lin-28, in which the first four are known as Yamanaka factors. Mounting evidence supports that USCs and UiPSCs possess high potential of neurogenic, myogenic, and osteogenic differentiation, indicating that they may play a crucial role in the treatment of neurological and musculoskeletal diseases. Therefore, we summarized the origin and physiological characteristics of USCs and UiPSCs and their therapeutic application in neurological and musculoskeletal disorders in this review, which not only contributes to deepen our understanding of hallmarks of USCs and UiPSCs but also provides the theoretical basis for the treatment of neurological and musculoskeletal disorders with USCs and UiPSCs.

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.

Pterosin B improves cognitive dysfunction by promoting microglia M1/M2 polarization through inhibiting Klf5/Parp14 pathway

BackgroundPterosin B (PB) exhibits strong neuroprotective effects in vitro, but its therapeutic effect and underlying mechanism on Alzheimer's disease (AD) remain elusive. PurposeThis study aimed to investigate the anti-AD effect and mechanism of PB. Study designThe therapeutic effect and mechanism of PB were investigated in APP/PS1 mice and lipopolysaccharide (LPS)-induced BV-2 cells. MethodsAfter 8 weeks of oral administration of PB or donepezil, the cognitive function was assessed using behavioral tests. Pathological damage was evaluated using histological analysis and immunohistochemical staining. Flow cytometry was applied to detect M1/M2 polarization. The expression levels of glycolysis- and oxidative phosphorylation-related proteins as well as enzyme activities were determined using Western blot and biochemical kits, respectively. The levels of inflammatory cytokines and Kruppel-like factor 5 (Klf5) were measured using enzyme-linked immunosorbent assay. AD biomarkers in serum were analyzed using single-molecular array. RNA sequencing identified the downstream molecules of Klf5, and interaction was evaluated using dual-luciferase reporter assay. ResultsOur findings demonstrated that PB effectively ameliorated cognitive impairment and reduced pathological damage in APP/PS1 mice. Furthermore, PB facilitated the transition of the phenotype of LPS-induced BV-2 cells from M1 to M2 by modulating metabolic reprogramming. Additionally, Klf5 had high expression levels in the serum of patients with AD, which strongly correlated with cognitive performance and AD biomarkers. PB downregulated Klf5 expression both in vitro and in vivo. Subsequently, poly-ADP ribosyl polymerase 14 (Parp14) was identified as a downstream molecule of Klf5 involved in regulating metabolic reprogramming, and PB regulated microglia M1/M2 polarization by inhibiting the Klf5/Parp14 pathway. ConclusionThe findings suggested that PB ameliorated cognitive dysfunction in AD by modulating microglia M1/M2 polarization via inhibiting Klf5/Parp14 pathway.