Expression Of KLF4 Research Articles

Defining the Role of the miR-145-KLF4-αSMA Axis in Mitral Valvular Interstitial Cell Activation in Myxomatous Mitral Valve Prolapse Using the Canine Model.

Mitral valve prolapse (MVP) is a common valvular disease, affecting 2-3% of the adult human population and is a degenerative condition. A total of 5-10% of the afflicted will develop severe mitral regurgitation, cardiac dysfunction, congestive heart failure, and sudden cardiac death. Naturally occurring myxomatous MVP in dogs closely resembles MVP in humans structurally, and functional consequences are similar. In both species, valvular interstitial cells (VICs) in affected valves exhibit phenotype consistent with activated myofibroblasts with increased alpha-smooth muscle actin (αSMA) expression. Using VICs collected from normal and MVP-affected valves of dogs, we analyzed the miRNA expression profile of the cells and their associated small extracellular vesicles (sEV) using RNA sequencing to understand the role of non-coding RNAs and sEV in MVP pathogenesis. miR-145 was shown to be upregulated in both the affected VICs and sEV, and overexpression of miR-145 by mimic transfection in quiescent VIC recapitulates the activated myofibroblastic phenotype. Concurrently, KLF4 expression was noted to be suppressed by miR-145, confirming the miR-145-KLF4-αSMA axis. Targeting this axis may serve as a potential therapy in controlling pathologic abnormalities found in MVP valves.

Tumor suppressor let-7 acts as a key regulator for pluripotency gene expression in Muse cells

In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the expression of an RNA-binding pluripotency-relevant protein, LIN28, and the absence of its antagonist, the tumor-suppressor microRNA (miRNA) let-7, play a key role in maintaining pluripotency. Muse cells are non-tumorigenic pluripotent-like stem cells residing in the bone marrow, peripheral blood, and organ connective tissues as pluripotent surface marker SSEA-3(+). They express pluripotency genes, differentiate into triploblastic-lineage cells, and self-renew at the single cell level. Muse cells do not express LIN28 but do express let-7 at higher levels than in iPSCs. In Muse cells, we demonstrated that let-7 inhibited the PI3K-AKT pathway, leading to sustainable expression of the key pluripotency regulator KLF4 as well as its downstream genes, POU5F1, SOX2, and NANOG. Let-7 also suppressed proliferation and glycolysis by inhibiting the PI3K-AKT pathway, suggesting its involvement in non-tumorigenicity. Furthermore, the MEK/ERK pathway is not controlled by let-7 and may have a pivotal role in maintaining self-renewal and suppression of senescence. The system found in Muse cells, in which the tumor suppressor let-7, but not LIN28, tunes the expression of pluripotency genes, might be a rational cell system conferring both pluripotency-like properties and a low risk for tumorigenicity.

LncRNA SNHG1 alleviates myocardial ischaemia-reperfusion injury by regulating the miR-137-3p/KLF4/TRPV1 axis.

Myocardial ischaemia-reperfusion injury (MIRI) contributes to serious myocardial injury and even death. Long non-coding RNAs (lncRNAs) have been reported to play pivotal roles in the occurrence and development of MIRI. Here, the detailed molecular mechanism of lncRNA SNHG1 in MIRI was explored. A cell model of MIRI was established through hypoxia/reoxygenation (H/R) stimulation. Cell viability and pyroptosis were evaluated utilizing MTT, PI staining, and flow cytometry. Interleukin (IL)-1β and IL-18 secretion levels were examined by ELISA. The gene and protein expression were detected by RT-qPCR and western blot, respectively. Dual luciferase reporter gene, RIP and ChIP assays were performed to analyse the molecular interactions. The results showed that lncRNA SNHG1 overexpression alleviated H/R-induced HL-1 cell pyroptosis (all P<0.05). LncRNA SNHG1 promoted KLF4 expression by sponging miR-137-3p. miR-137-3p silencing alleviated H/R-induced pyroptosis in HL-1 cells (all P<0.05), which was abolished by KLF4 knockdown (all P<0.05). KLF4 activated the AKT pathway by transcriptionally activating TRPV1 in HL-1 cells (all P<0.05). TRPV1 knockdown reversed the alleviation of SNHG1 upregulation on H/R-induced pyroptosis in HL-1 cells (all P<0.05). These results showed that lncRNA SNHG1 assuaged cardiomyocyte pyroptosis during MIRI progression by regulating the KLF4/TRPV1/AKT axis through sponging miR-137-3p. Our findings may provide novel therapeutic targets for MIRI.

Resveratrol attenuates high glucose-induced inflammation and improves glucose metabolism in HepG2 cells

Diabetes mellitus (DM) is characterized by impaired glucose and insulin metabolism, resulting in chronic hyperglycemia. Hyperglycemia-induced inflammation is linked to the onset and progression of diabetes. Resveratrol (RES), a polyphenol phytoalexin, is studied in diabetes therapeutics research. This study evaluates the effect of RES on inflammation and glucose metabolism in HepG2 cells exposed to high glucose. Inflammation and glucose metabolism-related genes were investigated using qPCR. Further, inflammatory genes were analyzed by applying ELISA and Bioplex assays. High glucose significantly increases IKK-α, IKB-α, and NF-kB expression compared to controls. Increased NF-kB expression was followed by increased expression of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-β, and COX2. RES treatment significantly reduced the expression of NF-kB, IKK-α, and IKB-α, as well as pro-inflammatory cytokines. High glucose levels reduced the expression of TGFβ1, while treatment with RES increased the expression of TGFβ1. As glucose levels increased, PEPCK expression was reduced, and GCK expression was increased in HepG2 cells treated with RES. Further, HepG2 cells cultured with high glucose showed significant increases in KLF7 and HIF1A but decreased SIRT1. Moreover, RES significantly increased SIRT1 expression and reduced KLF7 and HIF1A expression levels. Our results indicated that RES could attenuate high glucose-induced inflammation and enhance glucose metabolism in HepG2 cells.

Long non-coding RNA ACTA2-AS1 suppresses metastasis of papillary thyroid cancer via regulation of miR-4428/KLF9 axis

BackgroundMetastasis is the primary cause of recurrence and death in patients with papillary thyroid carcinoma (PTC). LncRNA ACTA2-AS1, a long non-coding RNA, acts as a tumor suppressor in multiple types of human malignancies, while the role of ACTA2-AS1 in PTC metastasis remains unclear.MethodsThe ACTA2-AS1 expression in PTC tissues was analyzed. The sponged roles of ACTA2-AS1 via miR-4428/KLF9 axis were identified using starBase tool. The function of ACTA2-AS1 in PTC was performed with in vitro and in vivo experiments. The correlation between DNA methylation and mRNA expressions of these gene in the TCGA dataset was explored.ResultsACTA2-AS1 expression was downregulated in PTC tissues without metastasis and further decreased in PTC tissues with lymph node metastasis compared with that in normal tissues. Functionally, the overexpression of ACTA2-AS1 inhibited the growth, proliferation, and invasion of PTC cells, whereas its depletion exerted opposite effect. In vivo, ACTA2-AS1 expression inhibited PTC metastasis. Furthermore, ACTA2-AS1 acted as a competing endogenous RNA for miR-4428, thereby positively regulating the expression of miR-4428 target gene, KLF9. Finally, miR-4428 overexpression enhanced invasive potential of PTC cells and significantly weakened the effects of ACTA2-AS1 on promotion and inhibition of KLF9 expression as well as invasive ability of PTC cells, respectively. In the TCGA dataset, the methylation level of ACTA2-AS1 was significantly correlated with its mRNA expression (r = 0.21, p = 2.1 × e−6).ConclusionsOur findings demonstrate that ACTA2-AS1 functions as a tumor suppressor in PTC progression at least partly by regulating the miR-4428-dependent expression of KLF9.Graphical abstract

Achaete-scute family bHLH transcription factor 2 activation promotes hepatoblastoma progression.

Achaete-scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet-On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, migration, invasion, and sphere-formation assays. Quantitative reverse-transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial-mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal-epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non-induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway-, EMT-, and stemness-related genes. ASCL2 activation may therefore be involved in the progression of HB.

The Ability of a 3-Gene Host Signature in Blood to Distinguish Tuberculous Meningitis From Other Brain Infections.

Tuberculous meningitis (TBM) is difficult to diagnose. We investigated whether a 3-gene host response signature in blood can distinguish TBM from other brain infections. The expression of 3 genes (dual specificity phosphatase 3 [DUSP3], guanylate-binding protein [GBP5], krupple-like factor 2 [KLF2]) was analyzed by RNA sequencing of archived whole blood from 4 cohorts of Vietnamese adults: 281 with TBM, 279 with pulmonary tuberculosis, 50 with other brain infections, and 30 healthy controls. Tuberculosis scores (combined 3-gene expression) were calculated following published methodology and discriminatory performance compared using area under a receiver operator characteristic curve (AUC). GBP5 was upregulated in TBM compared to other brain infections (P < .001), with no difference in DUSP3 and KLF2 expression. The diagnostic performance of GBP5 alone (AUC, 0.74; 95% confidence interval [CI], .67-.81) was slightly better than the 3-gene tuberculosis score (AUC, 0.66; 95% CI, .58-.73) in TBM. Both GBP5 expression and tuberculosis score were higher in participants with human immunodeficiency virus (HIV; P < .001), with good diagnostic performance of GBP5 alone (AUC, 0.86; 95% CI, .80-.93). The 3-gene host signature in whole blood has the ability to discriminate TBM from other brain infections, including in individuals with HIV. Validation in large prospective diagnostic study is now required.

Puerarin attenuates myocardial ischemic injury and endoplasmic reticulum stress by upregulating the Mzb1 signal pathway.

This study investigated the role of Mzb1 in puerarin protection against heart injury and dysfunction in acute myocardial infarction (AMI) mice. C57BL/6 mice were pretreated with and without puerarin at doses of 50mg/kg and 100mg/kg for 14days before establishing the AMI model. An AMI model was induced by ligating the left descending anterior coronary artery, and AC16 cardiomyocytes were treated with H2O2 in vitro. Echocardiography was performed to measure cardiac function. DHE staining, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assay, and DCFH-DA oxidative fluorescence staining were used to determine reactive oxygen species (ROS) production in vivo and in vitro. Bioinformatics analysis was used to predict potential upstream transcription factors of Mzb1. Puerarin dose-dependently reduced myocardial infarction area and injury, accompanied by the improvement of cardiac function in AMI mice. AMI mice manifested an increase in myocardial oxidative stress, endoplasmic reticulum (ER) stress, apoptosis, and mitochondrial biogenesis dysfunction, which were inhibited by pretreatment with puerarin. Puerarin also prevented Mzb1 downregulation in the hearts of AMI mice or H2O2-treated AC16 cells. Consistent with the in vivo findings, puerarin inhibited H2O2-induced cardiomyocyte apoptosis, ER stress, and mitochondrial dysfunction, which were attenuated by siRNA Mzb1. Furthermore, the JASPAR website predicted that KLF4 may be a transcription factor for Mzb1. The expression of KLF4 was partially reversed by puerarin in the cardiomyocyte injury model, and KLF4 inhibitor (kenpaullone) inhibited Mzb1 expression and affected its function. These results suggest that puerarin can protect against cardiac injury by attenuating oxidative stress and endoplasmic reticulum stress through upregulating the KLF4/Mzb1 pathway and that puerarin may expand our armamentarium for the prevention and treatment of ischemic heart diseases.

Metadichol, a Natural Ligand for the Expression of Yamanaka Reprogramming Factors in Human Cardiac, Fibroblast, and Cancer Cell Lines

The reprogramming of somatic cells into embryonic-like stem cells requires the activation of four essential transcription factors, referred to as the Yamanaka factors: Sox2, Oct4, Klf4, and c-Myc. However, the utilization of these genes and viral vectors for their delivery presents a risk of carcinogenesis, which renders induced pluripotent stem cell (iPSC) lines unsuitable for clinical applications. Typically, direct reprogramming involves the use of viral vectors to induce the expression of these factors. In contrast, metadichol, a novel approach, enhances the expression of Yamanaka factors in cells using a dose ranging from 1 pg to 100 ng, thereby preventing the need for viral vectors. This non-viral method renders cellular reprogramming safer and more clinically viable. Treatment with metadichol within the specific dose range resulted in a substantial augmentation of the fibroblast expression of OCT4, SOX2, KLF4, KLF2, and Nanog, which was confirmed through qRT-PCR and western blot analyses. The expression levels of OCT4, KLF4, Nanog, and Sox2, exhibited an increase of 4.01-, 3.51-, 1.26-, and 2.5-fold, respectively, compared to the controls. Notably, A549 and Colo-205 cancer cells demonstrated a marked elevation in expression levels. The reprogramming of primary human cancer cells presents considerable challenges. However, in triple-negative primary breast cancer cells, metadichol treatment led to the substantial upregulation of the expression levels of OCT4, KLF4, Nanog, and Sox2 by 19.6-, 8.07-, 2.45-, and 6.91-fold, respectively, across the dose range of 1 pg to 100 ng. Klotho, an antiaging gene that is modulated by metadichol, down regulates TP53, a crucial factor for the production of somatic cell iPSCs. Additionally, metadichol enhances the availability of vitamin C, which is essential for generating iPSCs from somatic cells. Increased levels of alkaline phosphatase, indicative of cellular differentiation, were observed in the treated group compared to the controls. Metadichol is a nontoxic nanoemulsion that is derived from long-chain C26–C28 alcohols from food sources and activates Yamanaka factors across various cell types without requiring the use of viral or CRISPR-based methodologies, thus substantially advancing somatic cell reprogramming.

Genetic variants and mRNA expression of KLF4 and KLF5 with hypertension: A combination of case-control study and cohort study.

Hypertension (HT) is a major risk factor for cardiovascular diseases. Krüppel-like factors (KLFs) are important transcription factors in eukaryotes. Studies have reported that KLF4 and KLF5 are correlated with several cardiovascular diseases, whereas population studies for associations between HT and KLF4 or KLF5 have been rarely reported. Thus, the current study aimed to examines the association of genetic variants and mRNA expression levels of KLF4 and KLF5 with HT, as well as the effect of antihypertensive drugs on the expression levels. The associations of one single-nucleotide polymorphism (SNP) in KLF4 and three SNPs in KLF5 with HT were investigated using a combination of case-control and cohort studies. The study population were selected from a community-based population cohort in four different regions of Jiangsu Province. Risks of HT were estimated through logistic and Cox regression analyses, respectively. In addition, mRNA expression levels of KLF4 and KLF5 were measured in 246 controls and 385 HT cases selected from the cohort study as mentioned above. Among the HT cases, 263 were not taking antihypertensive drugs [AHD(-)] and 122 were taking antihypertensive drugs [AHD(+)]. In the case-control study, SNP rs9573096 (C>T) in KLF5 was significantly associated with an increased risk of HT in the additive model (adjusted odds ratio [OR], 1.106; 95% confidence interval [CI], 1.009 to 1.212). In the cohort study of the normotensive population, rs9573096 in KLF5 was also significantly associated with an increased risk of HT in the additive model (adjusted hazards ratio [HR], 1.199; 95% CI, 1.070 to 1.344). KLF4 and KLF5 mRNA expression levels were significantly higher in the AHD(-) group than in the control group ( P < 0.05), but lower in the AHD(+) group than in the AHD(-) group ( P < 0.05). The current study demonstrated the associations of KLF4 and KLF5 genetic variants with hypertension, and the indicative discriminations of mRNA expression levels of KLF4 and KLF5 for risk of hypertension and antihypertensive treatment.

MiR-15b-3p weakens bicalutamide sensitivity in prostate cancer via targeting KLF2 to suppress ferroptosis.

Bicalutamide (BIC) resistance impedes the treatment of prostate cancer (PCa) and seems to involve ferroptosis; however, the underlying mechanism remains unclear. Our study aimed to explore how miR-15b-3p modulates ferroptosis in response to BIC resistance and determine whether the miRNA is suitable for early screening of PCa. Here, we found that PCa tissues had significantly higher miR-15b-3p expression than adjacent normal tissues. Analysis of blood samples in patients who underwent prostate-specific antigen (PSA) screening revealed that miR-15b-3p was a more accurate diagnostic than PSA (miR-15b-3p area under the curve [AUC] = 0.941, PSA AUC = 0.815). In vitro experiments then demonstrated that miR-15b-3p expression was markedly higher in LNCaP, PC-3, and DU145 cells than in RWPE-1 cells. Treatment with BIC decreased miR-15b-3p expression and progressive ferroptosis. Mechanistically, we identified KLF2 as the downstream target of miR-15b-3p. Overexpressing KLF2 facilitated ferroptosis via augmenting MDA and iron concentrations, in turn inhibiting the SLC7A11/GPX4 axis and decreasing GSH concentration. Through modulating ferroptosis, miR-15b-3p mimic and inhibitor weakened and enhanced BIC sensitivity, respectively. Furthermore, BIC treatment limited xenograft tumor volume in vivo, whereas agomir-15b-3p promoted tumor growth, indicating that miR-15b-3p attenuated the tumor-suppressive effects of BIC. Taken together, our results suggested that miR-15b-3p is crucial to BIC resistance, specifically via targeting KLF2 and thereby suppressing ferroptosis. High miR-15b-3p expression in early PCa screening should reflect a higher probability of cancer. In conclusion, miR-15b-3p has strong potential as a screening and diagnostic biomarker with reliable prospects for clinical application. Furthermore, because patients with high miR-15b-3p and low KLF2 expression have a greater risk of BIC resistance and malignant progression, targeting the miRNA and its downstream protein may be a new treatment strategy.

KLF4 is an epigenetically modulated, context-dependent tumor suppressor.

The epigenetic layer of regulation has become increasingly relevant in the research focused on tumor suppressors. KLF4 is a well-described zinc-finger transcription factor, mainly known for its role in the acquisition of cell pluripotency. Here we report and describe the most relevant epigenetic regulation mechanisms that affect KLF4 expression in tumors. CpG island methylation emerges as the most common mechanism in several tumors including lung adenocarcinoma, hepatocellular carcinoma, non-Hodgkin lymphomas, among others. Further layers of regulation represented by histone methylation and acetylation and by non-coding RNAs are described. Overall, KLF4 emerges as a crucial target in the fight against cancer.

Repressor elements provide insights into tissue development and phenotypes in pigs.

Repressor elements significantly influence economically relevant phenotypes in pigs; however, their precise roles and characteristics are inadequately understood. In the present study, we employed H3K27me3 profiling, assay for transposase-accessible chromatin with highthroughput sequencing (ATAC-seq), and RNA sequencing (RNA-seq) data across six tissues derived from three embryonic layers to identify and map 2 034 super repressor elements (SREs) and 22 223 typical repressor elements (TREs) in the pig genome. Notably, many repressor elements were conserved across mesodermal and ectodermal tissues. SREs exhibited tight regulation of their target genes, affecting a limited number of genes within a specific genomic region with pronounced effects, while TREs exerted broader but weaker regulation over a wider range of target genes. Furthermore, in neuronal tissues, genes regulated by repressor elements started to be repressed during the differentiation of stem cells into progenitor cells. Notably, analysis showed that many repressor elements exhibited cooperative and additive effects on the modulation of KLF4 expression. This research provides the first comprehensive map of pig repressor elements, serving as an essential reference for future studies on repressor elements.

Hypertensive Pressure Mechanosensing Alone Triggers Lipid Droplet Accumulation and Transdifferentiation of Vascular Smooth Muscle Cells to Foam Cells.

Arterial Vascular smooth muscle cells (VSMCs) play a central role in the onset and progression of atherosclerosis. Upon exposure to pathological stimuli, they can take on alternative phenotypes that, among others, have been described as macrophage like, or foam cells. VSMC foam cells make up >50% of all arterial foam cells and have been suggested to retain an even higher proportion of the cell stored lipid droplets, further leading to apoptosis, secondary necrosis, and an inflammatory response. However, the mechanism of VSMC foam cell formation is still unclear. Here, it is identified that mechanical stimulation through hypertensive pressure alone is sufficient for the phenotypic switch. Hyperspectral stimulated Raman scattering imaging demonstrates rapid lipid droplet formation and changes to lipid metabolism and changes are confirmed in ABCA1, KLF4, LDLR, and CD68 expression, cell proliferation, and migration. Further, a mechanosignaling route is identified involving Piezo1, phospholipid, and arachidonic acid signaling, as well as epigenetic regulation, whereby CUT&Tag epigenomic analysis confirms changes in the cells (lipid) metabolism and atherosclerotic pathways. Overall, the results show for the first time that VSMC foam cell formation can be triggered by mechanical stimulation alone, suggesting modulation of mechanosignaling can be harnessed as potential therapeutic strategy.

CircZNF609 regulates pulmonary fibrosis via miR-145-5p/KLF4 axis and its translation function

BackgroundPulmonary fibrosis is a growing clinical problem that develops as a result of abnormal wound healing, leading to breathlessness, pulmonary dysfunction and ultimately death. However, therapeutic options for pulmonary fibrosis are limited because the underlying pathogenesis remains incompletely understood. Circular RNAs, as key regulators in various diseases, remain poorly understood in pulmonary fibrosis induced by silica.MethodsWe performed studies with fibroblast cell lines and silica-induced mouse pulmonary fibrosis models. The expression of circZNF609, miR-145-5p, and KLF4 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RNA immunoprecipitation (RIP) assays and m6A RNA immunoprecipitation assays (MeRIP), Western blotting, immunofluorescence assays, and CCK8 were performed to investigate the role of the circZNF609/miR-145-5p/KLF4 axis and circZNF609-encoded peptides in fibroblast activation.ResultsOur data showed that circZNF609 was downregulated in activated fibroblasts and silica-induced fibrotic mouse lung tissues. Overexpression of circZNF609 could inhibit fibroblast activation induced by transforming growth factor-β1 (TGF-β1). Mechanically, we revealed that circZNF609 regulates pulmonary fibrosis via miR-145-5p/KLF4 axis and circZNF609-encoded peptides. Furthermore, circZNF609 was highly methylated and its expression was controlled by N6-methyladenosine (m6A) modification. Lastly, in vivo studies revealed that overexpression of circZNF609 attenuates silica-induced lung fibrosis in mice.ConclusionsOur data indicate that circZNF609 is a critical regulator of fibroblast activation and silica-induced lung fibrosis. The circZNF609 and its derived peptides may represent novel promising targets for the treatment of pulmonary fibrosis.

Sex hormone-binding globulin improves lipid metabolism and reduces inflammation in subcutaneous adipose tissue of metabolic syndrome-affected horses.

Equine metabolic syndrome (EMS) is a steadily growing endocrine disorder representing a real challenge in veterinary practice. As a multifactorial condition, EMS is characterized by three main metabolic abnormalities including insulin resistance, increased adiposity or obesity and hoof laminitis. Adipose tissue dysfunction is recognized as a core pathophysiological determinant of EMS, as it strongly participates to lipotoxicity and systemic metaflammation, both of which have been closely linked to the development of generalized insulin resistance. Besides, sex hormone binding globulin (SHBG) is an important sex steroids transporters that has been recently proposed as an important metabolic mediator. Therefore, the aim of this study was to verify whether SHBG treatment may ameliorate subcutaneous adipose tissue metabolic failure under EMS condition in terms of lipidome homeostasis, lipid metabolism programs, insulin signalling and local inflammation. Subcutaneous adipose tissue (SAT) biopsies were collected post-mortem from healthy (n = 3) and EMS (n = 3) slaughtered horses. SHBG protein has been applied to SAT samples from EMS horses for 24h at a final concentration of 50nM, while control groups (healthy and untreated EMS) were cultured in the presence of SHBG-vehicle only. Tissues from all groups were afterwards secured for downstream analysis of gene expression using RT-qPCR, protein levels by Western blot and ELISA assay and lipidomics through GC-MS technique. Obtained results showcased that SHBG intervention efficiently normalized the altered fatty acids (FAs) profiles by lowering the accumulation of saturated and trans FAs, as well as the pro-inflammatory arachidonic and linoleic acids. Moreover, SHBG showed promising value for the regulation of adipocyte lipolysis and engorgement by lowering the levels of perilipin-1. SHBG exerted moderated effect toward SCD1 and FASN enzymes expression, but increased the LPL abundance. Interestingly, SHBG exhibited a negative regulatory effect on pro-adipogenic stimulators and induced higher expression of KLF3, IRF3 and β-catenin, known as strong adipogenesis repressors. Finally, SHBG protein showed remarkable ability in restoring the insulin signal transduction, IR/IRS/Pi3K/AKT phosphorylation events and GLUT4 transporter abundance, and further attenuate pro-inflammatory response by lowering IL-6 tissue levels and targeting the PDIA3/ERK axis. Overall, the obtained data clearly demonstrate the benefice of SHBG treatment in the regulation of adipose tissue metabolism in the course of EMS and provide new insights for the development of molecular therapies with potential translational application to human metabolic disorders.

Multiscale analysis of Klf10's impact on the passive mechanical properties of murine skeletal muscle

Skeletal muscle is a hierarchical structure composed of multiple organizational scales. A major challenge in the biomechanical evaluation of muscle relates to the difficulty in evaluating the experimental mechanical properties at the different organizational levels of the same tissue. Indeed, the ability to integrate mechanical properties evaluated at various levels will allow for improved assessment of the entire tissue, leading to a better understanding of how changes at each level evolve over time and/or impact tissue function, especially in the case of muscle diseases.Therefore, the purpose of this study was to analyze a genetically engineered mouse model (Klf10 KO: Krüppel-Like Factor 10 knockout) with known skeletal muscle defects to compare the mechanical properties with wild-type (WT) controls at the three main muscle scales: the macroscopic (whole muscle), microscopic (fiber) and submicron (myofibril) levels. Passive mechanical tests (ramp, relaxation) were performed on two types of skeletal muscle (soleus and extensor digitorum longus (EDL)).Results of the present study revealed muscle-type specific behaviors in both genotypes only at the microscopic scale. Interestingly, loss of Klf10 expression resulted in increased passive properties in the soleus but decreased passive properties in the EDL compared to WT controls.At the submicron scale, no changes were observed between WT and Klf10 KO myofibrils for either muscle; these results demonstrate that the passive property differences observed at the microscopic scale (fiber) are not caused by sarcomere intrinsic alterations but instead must originate outside the sarcomeres, likely in the collagen-based extracellular matrix. The macroscopic scale revealed similar passive mechanical properties between WT and Klf10 KO hindlimb muscles.The present study has allowed for a better understanding of the role of Klf10 on the passive mechanical properties of skeletal muscle and has provided reference data to the literature which could be used by the community for muscle multiscale modeling.

4-amino-2-trifluoromethyl-phenyl retinate alleviates lipopolysaccharide-induced acute myocardial injury through activation of the KLF4/p62 axis

Ferroptosis plays a pivotal role in the pathological process of sepsis-induced cardiomyopathy (SIC). All-trans retinoic acid (ATRA) enhances the host immune response to lipopolysaccharides (LPS). This study investigated the role of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a derivative of ATRA, in myocardial injury caused by sepsis. Male C57BL/6 mice were intraperitoneally injected with LPS to establish a sepsis model. H9c2 cells were stimulated by LPS to establish an injury model. We observed that ATPR improved myocardial injury in mice, which was presented in terms of an increased glutathione (GSH) level and reduced production of malondialdehyde (MDA), as well as an increased number of mitochondrial cristae and maintenance of the mitochondrial membrane integrity. ATPR improved cardiac function in the LPS-injured mice. It inhibited the inflammatory response as evidenced by the decreasing mRNA levels of TNF-α and IL-6. The elevated protein expression levels of Nrf2, SLC7A11, GPX4, and FTH1 in mice and H9c2 cells showed that ATPR inhibited ferroptosis. Immunoprecipitation of LPS-stimulated H9c2 cells demonstrated that ATPR increased the interaction between p62 and Keap1. ATPR upregulated the KLF4 and p62 protein expression. However, the inhibition of Nrf2 by ML385 reduced the protective effect of ATPR in LPS-treated H9c2 cells. Furthermore, we used siRNA to knock down KLF4 in H9c2 cells and found that the KLF4 knockdown eliminated the inhibition of ferroptosis by ATPR in H9c2 cells. Therefore, ATPR alleviates LPS-induced myocardial injury by inhibiting ferroptosis via the KLF4/p62 axis.

Abstract B127: Novel anti-CD147 antibody DS-1471a exerts antitumor effect in hepatocellular carcinoma Patient Derived Xenograft models and its efficacy correlates with the expression of CD147

Abstract Background: CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we developed a novel humanized IgG4-formatted monoclonal anti-CD147 antibody, DS-1471a. Our previous studies revealed this antibody showed potent antitumor efficacy superior to standard of care drugs in various cell line xenograft mouse models such as hepatocellular carcinoma (HCC), pancreatic invasive ductal adenocarcinoma, and chronic myelogenous leukemia. In this study, we further examined antitumor efficacy and biomarker expression in 18 patient derived xenograft (PDX) models established from HCC patients. Methods: DS-1471a or vehicle was administered intravenously once per week at 10 mg/kg doses in HCC-PDX models and tumor volumes were measured every 2 or 3 days. After tumors were harvested, samples were analyzed by simple western blotting (sWB, capillary based western blotting system) and immunohistochemistry (IHC). H-scores were calculated from IHC results and correlation between H-score, protein expression by sWB, and tumor growth inhibition (TGI) ratio were examined. Results: Seven out of 18 PDX models showed over 50% TGI with DS-1471a treatment in comparison to vehicle, regardless of gender, tumor grade, and hepatitis virus type. Protein expression analysis by sWB showed good correlation of several biomarker candidates with efficacy of DS-1471a. Among the biomarker candidates tested, CD147, the DS-1471a target molecule, showed the strongest correlation with efficacy (r = 0.78). SMAD4, which regulates the downstream signaling of the CD147 complex, and FBXO22, reported to contribute to CD147 degradation, also showed good correlation with DS-1471a efficacy (r = 0.66 and r = 0.77, respectively). By both sWB and IHC analysis, all PDX models showed negative or weak KLF5 expression, another biomarker candidate for DS-1471a that is known to be a suppressor for SMAD signaling pathway. A clear correlation between CD147 H-score and TGI was observed (r= 0.74) in these patient derived PDX models. Correlation between CD147 H-score and protein expression levels by sWB was also observed (r = 0.71). Of note, all PDX models showed moderate to high expression of CD147. Conclusion: These pre-clinical study results suggest DS-1471a may be potentially beneficial for HCC treatment. In addition, our results show correlation of DS-1471a efficacy with expression of CD147. Further studies are needed to further understand the full potential benefit of DS-1471a in clinical trials. Citation Format: Hiroshi Yuita, Ryuichi Nakamura, Jun Tsukada, Hideo Yukinaga, Huynh The Hung, Keisuke Fukuchi. Novel anti-CD147 antibody DS-1471a exerts antitumor effect in hepatocellular carcinoma Patient Derived Xenograft models and its efficacy correlates with the expression of CD147 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B127.

Association of DDX5/p68 protein with the upstream erythroid enhancer element (EHS1) of the gene encoding the KLF1 transcription factor

EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. Regulation of KLF1 is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the DDX5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We further show that this protein associates with DEK and CTCF. We postulate that the range of interactions of DDX5/p68 with these and other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1 and enables the formation of a proper chromatin configuration at the Klf1 locus. These individual interactions provide quantitative contributions that, in sum, establish the high-level activity of the Klf1 promoter and suggest they can be selectively manipulated for clinical benefit.