Expression Of KLF4 Research Articles

Enhancing postmenopausal osteoporosis: a study of KLF2 transcription factor secretion and PI3K-Akt signaling pathway activation by PIK3CA in bone marrow mesenchymal stem cells.

Mesenchymal stem cells can develop into osteoblasts, making them a promising cell-based osteoporosis treatment. Despite their therapeutic potential, their molecular processes are little known. Bioinformatics and experimental analysis were used to determine the molecular processes of bone marrow mesenchymal stem cell (BMSC) therapy for postmenopausal osteoporosis (PMO). We used weighted gene co-expression network analysis (WGCNA) to isolate core gene sets from two GEO microarray datasets (GSE7158 and GSE56815). GeneCards found PMO-related genes. GO, KEGG, Lasso regression, and ROC curve analysis refined our candidate genes. Using the GSE105145 dataset, we evaluated KLF2 expression in BMSCs and examined the link between KLF2 and PIK3CA using Pearson correlation analysis. We created a protein-protein interaction network of essential genes involved in osteoblast differentiation and validated the functional roles of KLF2 and PIK3CA in BMSC osteoblast differentiation in vitro. We created 6 co-expression modules from 10 419 differentially expressed genes (DEGs). PIK3CA, the key gene in the PI3K-Akt pathway, was among 197 PMO-associated DEGs. KLF2 also induced PIK3CA transcription in PMO. BMSCs also expressed elevated KLF2. BMSC osteoblast differentiation involved the PI3K-Akt pathway. In vitro, KLF2 increased PIK3CA transcription and activated the PI3K-Akt pathway to differentiate BMSCs into osteoblasts. BMSCs release KLF2, which stimulates the PIK3CA-dependent PI3K-Akt pathway to treat PMO. Our findings illuminates the involvement of KLF2 and the PI3K-Akt pathway in BMSC osteoblast development, which may lead to better PMO treatments.

Modulation of angiopoietin-2 and Tie2: Organ specific effects of microvascular leakage and edema in mice

IntroductionCritical illness is associated with organ failure, in which endothelial hyperpermeability and tissue edema play a major role. The endothelial angiopoietin/Tie2 system, a regulator of endothelial permeability, is dysbalanced during critical illness. Elevated circulating angiopoietin-2 and decreased Tie2 receptor levels are reported, but it remains unclear whether they cause edema independent of other critical illness-associated alterations. Therefore, we have studied the effect of angiopoietin-2 administration and/or reduced Tie2 expression on microvascular leakage and edema under normal conditions. MethodsTransgenic male mice with partial deletion of Tie2 (heterozygous exon 9 deletion, Tie2+/−) and wild-type controls (Tie2+/+) received 24 or 72 pg/g angiopoietin-2 or PBS as control (n = 12 per group) intravenously. Microvascular leakage and edema were determined by Evans blue dye (EBD) extravasation and wet-to-dry weight ratio, respectively, in lungs and kidneys. Expression of molecules related to endothelial angiopoietin/Tie2 signaling were determined by ELISA and RT-qPCR. ResultsIn Tie2+/+ mice, angiopoietin-2 administration increased EBD extravasation (154 %, p < 0.05) and wet-to-dry weight ratio (133 %, p < 0.01) in lungs, but not in the kidney compared to PBS.Tie2+/− mice had higher pulmonary (143 %, p < 0.001), but not renal EBD extravasation, compared to wild-type control mice, whereas a more pronounced wet-to-dry weight ratio was observed in lungs (155 %, p < 0.0001), in contrast to a minor higher wet-to-dry weight ratio in kidneys (106 %, p < 0.05).Angiopoietin-2 administration to Tie2+/− mice did not further increase pulmonary EBD extravasation, pulmonary wet-to-dry weight ratio, or renal wet-to-dry weight ratio. Interestingly, angiopoietin-2 administration resulted in an increased renal EBD extravasation in Tie2+/− mice compared to Tie2+/− mice receiving PBS. Both angiopoietin-2 administration and partial deletion of Tie2 did not affect circulating angiopoietin-1, soluble Tie2, VEGF and NGAL as well as gene expression of angiopoietin-1, −2, Tie1, VE-PTP, ELF-1, Ets-1, KLF2, GATA3, MMP14, Runx1, VE-cadherin, VEGFα and NGAL, except for gene and protein expression of Tie2, which was decreased in Tie2+/− mice compared to Tie2+/+ mice. ConclusionsIn mice, the microvasculature of the lungs is more vulnerable to angiopoietin-2 and partial deletion of Tie2 compared to those in the kidneys with respect to microvascular leakage and edema.

Dysregulation of the Long Noncoding RNA X-Inactive–Specific Transcript Expression in Male Patients with Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a sex-biased disease with female gender as a significant risk factor. Recently, we reported that increased expression of the long non-coding (lnc)RNA Xist, as induced by an intersectin-1s protein fragment with proliferative potential (EHITSN), may explain the sexual dimorphism of female pulmonary artery endothelial cells (ECs) and at least in part, the imbalance sex/ratio of PAH.Xist is essential for X-chromosome inactivation and dosage compensation of X-linked genes. Increased Xist expression was also detected in a subset of ECs and lung tissue samples of male PAH patients. The role of different Xist expression levels in ECs of male PAH patients (ECPAH) was studied in several lines of male ECPAH in conjunction with molecular, biochemical, morphological, and functional approaches. Male ECPAH showed on average 10.3-fold increase in high Xist vs. low Xist, a significant association between Xist levels and their proliferative potential, and a heterogeneous methylation of the Xist/Tsix locus. Interestingly, Xist up-regulation in male ECPAH decreases the expression of Klf2, via EHITSN interaction with EZH2, the catalytic subunit of the polycomb repressive complex 2. Moreover, the studies demonstrate that EHITSN-triggered p38/Elk1/c-Fos signaling is a pathological mechanism central to ECPAH proliferation and the dynamic crosstalk with cell cycle regulatory proteins ccna1/ccnd2, and Xist-EZH2-Klf2 interaction participate directly and differentially in establishing the proliferative profile of male ECPAH.

Cognate Antigen-Independent Differentiation and Activation of Resident Memory T cells in Chronic Kidney Disease

Resident memory T cells (TRMs) are a memory subset of T cells which are retained locally within barrier and epithelial tissues such as the kidney and have also been noted for their role in perpetuating chronic inflammation. While T cells have gained recognition for their ability to modulate kidney inflammation in various models of kidney injury TRMs are still poorly understood and their contributions to sterile models of chronic kidney disease (CKD) are unknown. We hypothesized that kidney TRMs are activated and modulate kidney inflammation in the setting of CKD. We utilized an aristolochic acid (AA) model of murine CKD to investigate the presence of kidney TRMs via flow cytometry and characterized their transcriptional profile via single cell RNA sequencing (scRNAseq). We also measured kidney cytokine levels with an MSD cytokine plate. Six weeks after AA-induced CKD, kidney CD8+ T cells were significantly increased compared to control mice (29.6 ± 1.6% vs. 4.7 ± 0.6% of CD45+, respectively; p&lt;0.0001), as were CD4+ T cells (34.4 ± 1.4% vs. 8.2 ± 0.7% of CD45+, respectively; p&lt;0.0001). 53.6 ± 2.5% of CD8+ T cells expressed CD103, a TRM marker, in AA mice compared to 4.8 ± 1.2% in control mice, and 36.0 ± 1.7% vs 1.6 ± 0.3% of CD4+ T cells. Kidney protein lysates of AA mice had significantly higher levels of IFNγ than control kidneys (18.9 ± 1.6 vs. 2.9 ± 0.7 pg/mg, respectively; p=0.0003) as well as TNF (98.6 ± 8.9 pg/mg vs. 6.9 ± 0.8 pg/mg, respectively; p=0.0002) and IL-6 (247.6 ± 39.1 pg/mg vs. 75.4 ± 22.5 pg/mg, respectively; p=0.0226). scRNAseq of immune cells revealed T cells bearing TRM markers CD103 and CD49a which lacked expression of tissue egress molecules S1pr1, Sell, Ccr7, and Klf2. scRNAseq analysis of this TRM population also revealed downregulation of Eomes and Tbx21 transcription factors and upregulation of Runx3 transcription factor, all of which correspond with previously described TRM transcriptional profiles. We next evaluated signaling pathways involved with the survival and transcriptional profile of TRMs, namely IL-15 and TGF-β. While Il2rb and Il2rg (encoding IL-15 receptor subunits) were upregulated in TRMs, IL-15 protein lysate levels were not elevated in AA kidneys compared to controls (2,600 ± 145 pg/mg protein vs. 2,281 ± 96 pg/mg protein, respectively; p=0.0831). Tgfbr1 and Tgfbr2 (encoding TGF-β receptor subunits) were also upregulated in TRMs, but TGF-β protein lysate levels were not significantly elevated in AA kidneys compared to controls (2,158 ± 159 pg/mg protein vs. 1,924 ± 202 pg/mg protein, respectively; p=0.3886). Gene ontology pathway analysis showed enrichment of pathways involved in T cell receptor signaling. Because of this we assessed the kidney immune profile of transgenic OT-1 mice, which can only recognize specific ovalbumin residues in the context of MHC class 1 H-2Kb. Six weeks after AA-induced CKD, injured OT-1 mice still had a significant increase in the proportion of CD8+ T cells that were CD103+ compared to vehicle control OT-1 mice (47.3 ± 2.9% vs. 9.6 ± 1.6% of CD45+, respectively; p&lt;0.0001) and CD4+ T cells that were CD103+ (26.7 ± 3.1% vs. 6.4 ± 2.4% of CD45+, respectively; p=0.0040). Here we demonstrate the differentiation and activation of TRMs during AA-induced CKD independent of cognate antigen interaction and highlight their activated profile, as well as an increase in kidney IFNγ, TNF, and IL-6, all proinflammatory cytokines typical of TRMs. Future studies will aim to determine the contributions of TRMs to CKD progression. F32 DK136187, T32 DK007545, R01 DK 118932, U54 DK137301. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2121: SUV39H1 Mediated Regulation Of KLF4 And KDM4a Coordinate Smooth Muscle Cell Phenotypic Plasticity

Background: The phenotypic plasticity of vascular smooth muscle cells is central to growth and remodeling processes, but also underlies many cardiovascular pathologies, including atherosclerosis, restenosis, and intimal hyperplasia. This ability of VSMCs to reversibly differentiate and dedifferentiate is incompletely understood. SUV39H1, a histone methyltransferase, that generates the H3K9Me3 mark results in epigenetic transcriptional repression. We hypothesized that SUV39H1 plays a key role in VSMC plasticity. Methods: We applied knockdown, qPCR, western blotting, chromatin immunoprecipitation (ChIP) assays, RNA-Seq, and murine vascular injury to determine the role of SUV39H1 in VSMC plasticity. Results: A qPCR array screen of epigenetic regulators revealed that SUV39H1 is upregulated with PDGF-induced dedifferentiation but downregulated with rapamycin-induced differentiation in hCASMCs. Knockdown of SUV39H1 induces differentiation-specific hCASMC contractile genes and functional contractility while decreased hCASMC migration, proliferation, and de-differentiation-associated gene expression. RNA-seq transcriptomics confirmed changes in multiple pathways consistent with a role for SUV39H1 in promoting hCASMC dedifferentiation. Mechanistically, SUV39H1 knockdown suppressed expression of KLF4, a master dedifferentiation factor, decreasing KLF4 mRNA stability and upregulating miRNA143. SUV39H1 knockdown also increased expression of KDM4a, an enzyme that removes H3K9me3 marks. In vivo, we noted a significant increase in SUV39H1 and H3K9me3 expression in murine carotid artery ligation-induced intimal hyperplasia compared to uninjured vessels. Conclusion: We identify SUV39H1 as an epigenetic regulator of VSMC phenotype whose expression and activity increase with dedifferentiation in vitro and in vivo . PDGF promotes H3K9me3 repressive marks at contractile genes by promoting expression of SUV39H1 (writer), which also inhibits the KDM4a (eraser) and SUV39H1 also regulates KLF4 expression. Understanding the role of SUV39H1 in VSMC plasticity may reveal new therapeutic strategies for treating vascular diseases.

Abstract 2149: Smooth Muscle-derived Resident Vascular Adventitial Progenitor Cell Contribution To Cardiac Fibrosis And Inflammation Is Dependent On KLF4

Cardiac fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) material resulting in cardiac tissue scarring and dysfunction. While it is commonly accepted that myofibroblasts are the major contributors to ECM deposition in cardiac fibrosis, their origin remains debated. By combining lineage tracing and RNA sequencing, our group made the paradigm-shifting discovery that a subpopulation of resident vascular stem cells residing within the vascular adventitia (termed AdvSca1-SM cells) originate from mature vascular smooth muscle cells (SMCs) through an in situ reprogramming process. SMC-to-AdvSca1-SM reprogramming and AdvSca1-SM cell maintenance is dependent on induction and activity of the transcription factor, KLF4. In contrast, loss of Klf4 expression promotes the transition of AdvSca1-SM cells to a myofibroblast phenotype in vascular injury and remodeling. However, the molecular mechanism whereby KLF4 regulates AdvSca1-SM phenotype remains unclear. Our recent preliminary data confirm the existence of perivascular cardiac AdvSca1-SM cells. Leveraging a highly specific AdvSca1-SM cell reporter system, single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomic approaches, we demonstrate the profibrotic differentiation trajectory of AdvSca1-SM cells in the setting of Angiotensin II (AngII)-induced cardiac fibrosis. Differentiation is characterized by loss of stemness-related genes, including Klf4 , but gain of expression of a profibrotic phenotype. Importantly, these changes are recapitulated in human cardiac hypertrophic tissue, supporting the translational significance of profibrotic transition of AdvSca1-SM-like cells in human cardiomyopathy. Very surprisingly and paradoxically, AdvSca1-SM-specific genetic knockout of Klf4 prior to AngII treatment protects against cardiac inflammation and fibrosis, indicating that Klf4 is essential for the profibrotic response of AdvSca1-SM cells. Overall, our data reveal the contribution of AdvSca1-SM cells to myofibroblasts in the setting of cardiac fibrosis. KLF4 not only maintains the stemness of AdvSca1-SM cells, but also orchestrates their response to profibrotic stimuli, and may serve as a therapeutic target in cardiac fibrosis.

LncRNA KTN1-AS1 Drives Tumor Progression in Non-Small Cell Lung Cancer through microRNA-153-3p/KLF5 Axis

IntroductionThe most typical kind of lung cancer is non-small cell lung cancer (NSCLC). Surgery, targeted therapy, chemotherapy, and immunotherapy are all options for the treatment of NSCLC. LncRNA KTN1-AS1 is significantly increased in NSCLC, and it modulates the expression of microRNAs and downstream genes, promoting NSCLC progression.Material and methodsTCGA was employed to predict lncRNA KTN1-AS1 expression in NSCLC. The starBase was utilized to predict downstream microRNAs of KTN1-AS1. The RNA22 database was employed to predict corresponding binding sites. Subcellular localization of KTN1-AS1 was forecasted using the lncLocator database, and the results were validated by FISH. qRT-PCR was used to test KTN1-AS1, microRNA-153-3p, and KLF5 expression. CCK-8, flow cytometry, and Transwell were used to determine cell viability, proliferation, migration, and invasion. Western blot was used to test KLF5 and Ki67 protein levels, and dual-luciferase assay was used to assess bindings of KTN1-AS1 with microRNA-153-3p, and KLF5 with microRNA-153-3p.ResultsKTN1-AS1 was significantly upregulated in NSCLC cells. Silencing KTN1-AS1 significantly repressed the proliferation, migration, and invasion of NSCLC cells. KTN1-AS1 bound to microRNA-153-3p, and KLF5 was a direct target of microRNA-153-3p. Inhibition of microRNA-153-3p or overexpression of KLF5 restored the stimulatory impact of KTN1-AS1 knockdown on NSCLC cell proliferation and migration.ConclusionsKTN1-AS1 drove proliferation, migration, and invasion of NSCLC cells by regulating microRNA-153-3p/KLF5 axis. By studying the role of the KTN1-AS1/microRNA-153-3p/KLF5 axis in NSCLC, the possibility of targeting this axis as a therapeutic target in NSCLC can be explored.

Krüppel-like Factor-4-Mediated Macrophage Polarization and Phenotypic Transitions Drive Intestinal Fibrosis in THP-1 Monocyte Models In Vitro.

Background and Objectives: Despite the fact that biologic drugs have transformed inflammatory bowel disease (IBD) treatment, addressing fibrosis-related strictures remains a research gap. This study explored the roles of cytokines, macrophages, and Krüppel-like factors (KLFs), specifically KLF4, in intestinal fibrosis, as well as the interplay of KLF4 with various gut components. Materials and Methods: This study examined macrophage subtypes, their KLF4 expression, and the effects of KLF4 knockdown on macrophage polarization and cytokine expression using THP-1 monocyte models. Co-culture experiments with stromal myofibroblasts and a conditioned medium from macrophage subtype cultures were conducted to study the role of these cells in intestinal fibrosis. Human-induced pluripotent stem cell-derived small intestinal organoids were used to confirm inflammatory and fibrotic responses in the human small intestinal epithelium. Results: Each macrophage subtype exhibited distinct phenotypes and KLF4 expression. Knockdown of KLF4 induced inflammatory cytokine expression in M0, M2a, and M2c cells. M2b exerted anti-fibrotic effects via interleukin (IL)-10. M0 and M2b cells showed a high migratory capacity toward activated stromal myofibroblasts. M0 cells interacting with activated stromal myofibroblasts transformed into inflammatory macrophages, thereby increasing pro-inflammatory cytokine expression. The expression of IL-36α, linked to fibrosis, was upregulated. Conclusions: This study elucidated the role of KLF4 in macrophage polarization and the intricate interactions between macrophages, stromal myofibroblasts, and cytokines in experimental in vitro models of intestinal fibrosis. The obtained results may suggest the mechanism of fibrosis formation in clinical IBD.

CD226 promotes renal fibrosis by regulating macrophage activation and migration.

It has been found that CD226 plays an important role in regulating macrophage function, but its expression and function in macrophages during renal fibrogenesis have not been studied. Our data demonstrated that CD226 expression in macrophages was obviously upregulated in the unilateral ureteral obstruction model, while CD226 deficiency attenuated collagen deposition in renal interstitium along with fewer M1 within renal cortex and renal medulla and a lower level of proinflammatory factors compared to that of control littermates. Further studies demonstrated that Cd226-/- bone marrow-derived macrophages transferring could significantly reduce the tubular injury, collagen deposition, and proinflammatory cytokine secretion compared with that of Cd226+/+ bone marrow-derived macrophages transferring in the unilateral ureteral obstruction model. Mechanistic investigations revealed that CD226 promoted proinflammatory M1 macrophage accumulation in the kidney via suppressing KLF4 expression in macrophages. Therefore, our results uncovered a pathogenic role of CD226 during the development of chronic kidney disease by promoting monocyte infiltration from peripheral blood into the kidney and enhancing macrophage activation toward the inflammatory phenotype by suppressing KLF4 expression.

PAFAH1B3 is a KLF9 target gene that promotes proliferation and metastasis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies. Uncontrolled cell proliferation, invasion and migration of pancreatic cancer cells are the fundamental causes of death in PDAC patients. Our previous studies showed that KLF9 inhibits the proliferation, invasion and migration of pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In this study, we found that platelet-activating factor acetylhydrolase IB3 (PAFAH1B3) is highly expressed in pancreatic cancer tissues and cells. In vitro and in vivo studies showed that overexpression of PAFAH1B3 promoted the proliferation and invasion of pancreatic cancer cells, while downregulation of PAFAH1B3 inhibited these processes. We found that KLF9 expression is negatively correlated with PAFAH1B3 expression in pancreatic cancer tissues and cells. Western blotting revealed that KLF9 negatively regulates the expression of PAFAH1B3 in pancreatic cancer tissues and cells. Rescue experiments showed that overexpression of PAFAH1B3 could partially attenuate the suppression of pancreatic cancer cell proliferation, invasion and migration induced by KLF9 overexpression. Finally, chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were carried out, and the results showed that KLF9 directly binds to the promoter of PAFAH1B3 and inhibits its transcriptional activity. In conclusion, our study indicated that KLF9 can inhibit the proliferation, invasion, migration and metastasis of pancreatic cancer cells by inhibiting PAFAH1B3.

Hepatic Klf10-Fh1 axis promotes exercise-mediated amelioration of NASH in mice

Exercise is an effective non-pharmacological strategy for the treatment of nonalcoholic steatohepatitis (NASH), but the underlying mechanism needs further investigation. Kruppel-like factor 10 (Klf10) is a transcriptional factor that is expressed in multiple tissues including liver, whose role in NASH is not well defined. In our study, exercise induces hepatic Klf10 expression through the cAMP/PKA/CREB pathway. Hepatocyte-specific knockout of Klf10 (Klf10LKO) increases lipid accumulation, cell death, inflammation and fibrosis in NASH diet-fed mice and reduces the protective effects of treadmill exercise against NASH, while hepatocyte-specific overexpression of Klf10 (Klf10LTG) works in concert with exercise to reduce NASH in mice. Mechanistically, Klf10 promotes the expression of fumarate hydratase 1 (Fh1), thereby reducing fumarate accumulation in hepatocytes. This decreases the trimethyl (me3) levels of histone 3 lysine 4 (H3K4me3) on lipogenic genes promoters to attenuate lipogenesis, thus ameliorating free fatty acids (FFAs)-induced hepatocytes steatosis, apoptosis, insulin resistance and blunting dysfunctional hepatocytes-mediated activation of macrophages and hepatic stellate cells. Therefore, by regulating the Fh1/fumarate/H3K4me3 pathway, Klf10 acts as a downstream effector of exercise to combat NASH.

Downregulation of GPX8 in hepatocellular carcinoma: impact on tumor stemness and migration

PurposeGPX8, which is found in the endoplasmic reticulum lumen, is a member of the Glutathione Peroxidases (GPXs) family. Its role in hepatocellular carcinoma (HCC) is unknown.MethodsImmunohistochemical staining was used to detect the protein levels of GPX8 in HCC tissue microarrays. A short hairpin RNA lentivirus was used to knock down GPX8, and the main signaling pathways were investigated using transcriptome sequencing and a phosphorylated kinase array. The sphere formation assays, cloning-formation assays and cell migration assays were used to evaluate the stemness and migration ability of HCC cells. Identifying the GPX8-interacting proteins was accomplished through immunoprecipitation and protein mass spectrometry.ResultsThe GPX8 protein levels were downregulated in HCC patients. Low expression of GPX8 protein was related to early recurrence and poor prognosis in HCC patients. GPX8 knockdown could enhance the stemness and migration ability of HCC cells. Consistently, Based on transcriptome analysis, multiple signaling pathways that include the PI3K-AKT and signaling pathways that regulate the pluripotency of stem cells, were activated after GPX8 knockdown. The downregulation of GPX8 could increase the expression of the tumor stemness markers KLF4, OCT4, and CD133. The in vivo downregulation of GPX8 could also promote the subcutaneous tumor-forming and migration ability of HCC cells. MK-2206, which is a small-molecule inhibitor of AKT, could reverse the tumor-promoting effects both in vivo and in vitro. We discovered that GPX8 and the 71-kDa heat shock cognate protein (Hsc70) have a direct interaction. The phosphorylation of AKT encouraged the translocation of Hsc70 into the nucleus and the expression of the PI3K p110 subunit, thereby increasing the downregulation of GPX8.ConclusionThe findings from this study demonstrate the anticancer activity of GPX8 in HCC by inactivating the Hsc70/AKT pathway. The results suggest a possible therapeutic target for HCC.

The proteasome inhibitor carfilzomib exerts anti-inflammatory and antithrombotic effects on the endothelium

BackgroundCarfilzomib (CFZ) is a second-generation proteasome inhibitor used to treat multiple myeloma. Potent inhibition of the proteasome results in chronic proteotoxic endoplasmic reticulum (ER) stress, leading to apoptosis. While CFZ has improved survival rates in multiple myeloma, it is associated with an increased risk of cardiovascular adverse effects. While this has been putatively linked to cardiotoxicity, CFZ could potentially also exhibit adverse effects on the endothelium. ObjectivesTo investigate the effects of CFZ on the endothelium. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with CFZ, and expression of relevant markers of ER stress, inflammation, and thrombosis was measured and functionally assessed. ResultsCFZ failed to induce ER stress in HUVECs but induced the expression of Kruppel-like factor 4, endothelial nitric oxide synthase, tissue plasminogen activator, and thrombomodulin and reduced tumor necrosis factor alpha (TNFα)-mediated intercellular adhesion molecule 1 and tissue factor expression, suggesting a potential protective effect on the endothelium. Consistent with these observations, CFZ reduced leukocyte adhesion under shear stress and reduced factor Xa generation and fibrin clot formation on the endothelium following TNFα treatment and inhibited von Willebrand factor (VWF) and angiopoietin-2 exocytosis from Weibel–Palade bodies. Subsequently, CFZ inhibited the formation of VWF-platelet strings, and moreover, media derived from myeloma cell lines induced VWF release, a process also inhibited by CFZ. ConclusionThese data demonstrate that CFZ is unable to induce ER stress in confluent resting endothelial cells and can conversely attenuate the prothrombotic effects of TNFα on the endothelium. This study suggests that CFZ does not negatively alter HUVECs, and proteasome inhibition of the endothelium may offer a potential way to prevent thrombosis.

Exploring the Mechanism of KLF15 on the Biological Activity and Autophagy of Gastric Cancer Cells based on PI3K/Akt/Mtor Signaling Pathway.

To explore the mechanism of KLF15 on the biological activity and autophagy of gastric cancer cells based on the PI3K/Akt/mTOR signaling pathway. The gastric cancer AGS cells were divided into the Con group, pcDNANC group, pcDNA-KLF15 group, LY294002 group and IGF-1 group. RT-PCR was used to detect the expression of KLF15 in human gastric mucosal cells and gastric cancer cells; MTT method to detect cell proliferation; Transwell method to detect cell invasion; flow cytometry to detect cell apoptosis; Western blotting to detect PI3K, Akt, mTOR in cells, LC3, Beclin1, p62 protein expression.P<0.05 was used to indicate statistical significance. Compared with the human gastric mucosal cell line GES-1 cells, the expression of KLF15 in human gastric cancer cell lines MKN-28, MFC, SCG-7901 and AGS cells was significantly decreased, And the expression of KLF15 in AGS cells, was the lowest (P=0.006). Compared with the Con group, The expression of KLF15 in the cells of the PCDNA-KLF15 group was significantly increased (P=0.018); There was no significant difference in the expression of KLF15 between the Con group and the PCDNA-NC group (P=0.225). Compared with the Con group, the proliferation and invasion abilities of the cells in the pcDNA-KLF15 group were significantly reduced, And the apoptosis ability was significantly increased (P=0.019). The ratio of LC3II/LC31 and the expression of Beclin1 Protein in the control group were significantly higher than those in the Con group (P=0.017). Overexpression of KLF15 can inhibit the proliferation and invasion of Gastric cancer cells and promote cell apoptosis and autophagy, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

Thrombomodulin reduces α-synuclein generation and ameliorates neuropathology in a mouse model of Parkinson’s disease

The neurotoxic α-synuclein (α-syn) oligomers play an important role in the occurrence and development of Parkinson’s disease (PD), but the factors affecting α-syn generation and neurotoxicity remain unclear. We here first found that thrombomodulin (TM) significantly decreased in the plasma of PD patients and brains of A53T α-syn mice, and the increased TM in primary neurons reduced α-syn generation by inhibiting transcription factor p-c-jun production through Erk1/2 signaling pathway. Moreover, TM decreased α-syn neurotoxicity by reducing the levels of oxidative stress and inhibiting PAR1-p53-Bax signaling pathway. In contrast, TM downregulation increased the expression and neurotoxicity of α-syn in primary neurons. When TM plasmids were specifically delivered to neurons in the brains of A53T α-syn mice by adeno-associated virus (AAV), TM significantly reduced α-syn expression and deposition, and ameliorated the neuronal apoptosis, oxidative stress, gliosis and motor deficits in the mouse models, whereas TM knockdown exacerbated these neuropathology and motor dysfunction. Our present findings demonstrate that TM plays a neuroprotective role in PD pathology and symptoms, and it could be a novel therapeutic target in efforts to combat PD.Schematic representation of signaling pathways of TM involved in the expression and neurotoxicity of α-syn. A TM decreased RAGE, and resulting in the lowered production of p-Erk1/2 and p-c-Jun, and finally reduce α-syn generation. α-syn oligomers which formed from monomers increase the expression of p-p38, p53, C-caspase9, C-caspase3 and Bax, decrease the level of Bcl-2, cause mitochondrial damage and lead to oxidative stress, thus inducing neuronal apoptosis. TM can reduce intracellular oxidative stress and inhibit p53-Bax signaling by activating APC and PAR-1. B The binding of α-syn oligomers to TLR4 may induce the expression of IL-1β, which is subsequently secreted into the extracellular space. This secreted IL-1β then binds to its receptor, prompting p65 to translocate from the cytoplasm into the nucleus. This translocation downregulates the expression of KLF2, ultimately leading to the suppression of TM expression. By Figdraw.

KLF4 suppresses anticancer effects of brusatol via transcriptional upregulating NCK2 expression in melanoma

Brusatol (Bru), a main extract from traditional Chinese medicine Brucea javanica, has been reported to exist antitumor effect in many tumors including melanoma. However, the underlying mechanism in its anti-melanoma effect still need further exploration. Here, we reported that the protein expression of KLF4 in melanoma cells were significantly downregulated in response to brusatol treatment. Overexpression of KLF4 suppressed brusatol-induced melanoma cell apoptosis; while knockdown of KLF4 enhanced antitumor effects of brusatol on melanoma cells not only in vitro but also in vivo. Further studies on the mechanism revealed that KLF4 bound to the promoter of NCK2 directly and facilitated NCK2 transcription, which suppressed the antitumor effect of brusatol on melanoma. Furthermore, our findings showed that miR-150-3p was dramatically upregulated under brusatol treatment which resulted in the downregulation of KLF4. Our results suggested that the miR-150-3p/KLF4/NCK2 axis might play an important role in the antitumour effects of brusatol in melanoma.

Abstract LB191: A genome-wide CRISPR-based functional screen uncovers the KLF4-Survivin/BIRC5 signaling pathways as a key regulator of cell fate following endoplasmic reticulum stress: Implications for tumor growth and therapeutic targeting

Abstract The growth and spread of tumors involve intricate interactions with the surrounding tissue microenvironment. As a tumor develops, it faces various stresses from its microenvironment, including hypoxia, nutrient deficiency, and acidosis. In response to these challenges, cancer cells can hijack existing cytoprotective mechanisms, such as the Unfolded Protein Response (UPR). The UPR activates signaling pathways at the translational and later transcriptional levels to alleviate cellular stress and prevent cell death. However, under unresolved or chronic endoplasmic reticulum (ER) stress, the UPR may paradoxically promote cell death. The precise mechanisms through which the UPR influences cellular fate during ER stress remain poorly understood. To address this knowledge gap, we employed a functional CRISPR-based genetic knockout screen to identify novel regulators of the UPR. Using a lentiviral genome-wide CRISPR-Cas9 knockout library, we identified over-represented sgRNAs (associated with pro-apoptotic genes) and underrepresented sgRNAs (associated with pro-survival genes) following ER stress induced by thapsigargin and tunicamycin. One of the top candidates identified was Survivin/BIRC5, a protein with anti-apoptotic and cell cycle regulatory activities and which is overexpressed in tumor cells compared to healthy tissues. Functional studies revealed that genetic or pharmacological inhibition of Survivin expression enhanced ER stress-induced apoptosis, but not apoptosis in response to other forms of stress. These findings were validated in multiple cancer cell lines, ruling out off-target effects. Mechanistically, ER stress led to PERK-dependent downregulation of Survivin, and overexpression of Survivin blocked Thapsigargin-induced apoptosis. In vivo xenograft models showed that combining Survivin ablation with PERK inhibition significantly delayed tumor growth and improved overall survival. Through a combination of bioinformatics analysis and loss-of-function studies, we identified the transcription factor KLF4 as a mediator of PERK-dependent effects on Survivin repression. Notably, CRISPR-mediated KLF4 knockout successfully rescued Survivin levels post-ER stress. Additionally, an analysis of TCGA data across various tumor types provided supporting evidence of a negative correlation between KLF4 and Survivin expression. In summary, this study unveils a novel regulatory axis, PERK-KLF4-Survivin, as a key determinant of cell fate in the cellular responses to ER stress. Targeting Survivin in conjunction with PERK inhibition showed promising antitumor effects in vivo. Citation Format: Nektaria M. Leli, Hengxi Liu, Souvik Dey, Lauren Brady, Carlo Salas Salinas, Victoria Wu, Spiros Tastsoglou, Giorgos Skoufos, Ioannis Verginadis, Ilias Karagounis, Artemis G. Hatzigeorgiou, Constantinos Koumenis. A genome-wide CRISPR-based functional screen uncovers the KLF4-Survivin/BIRC5 signaling pathways as a key regulator of cell fate following endoplasmic reticulum stress: Implications for tumor growth and therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB191.

Krüppel-like factor 10 protects against metabolic dysfunction-associated steatohepatitis by regulating HNF4α-mediated metabolic pathways

BackgroundKrüppel-like factor 10 (KLF10), a zinc finger transcription factor, plays a pivotal role in modulating TGF-β-mediated cellular processes such as growth, apoptosis, and differentiation. Recent studies have implicated KLF10 in regulating lipid metabolism and glucose homeostasis. This study aimed to elucidate the precise role of hepatic KLF10 in developing metabolic dysfunction-associated steatohepatitis (MASH) in diet-induced obese mice. MethodsWe investigated hepatic KLF10 expression under metabolic stress and the effects of overexpression or ablation of hepatic KLF10 on MASH development and lipidemia. We also determined whether hepatocyte nuclear factor 4α (HNF4α) mediated the metabolic effects of KLF10. ResultsHepatic KLF10 was downregulated in MASH patients and genetically or diet-induced obese mice. AAV8-mediated overexpression of KLF10 in hepatocytes prevented Western diet-induced hypercholesterolemia and steatohepatitis, whereas inactivation of hepatocyte KLF10 aggravated Western diet-induced steatohepatitis. Mechanistically, KLF10 reduced hepatic triglyceride and free fatty acid levels by inducing lipolysis and fatty acid oxidation and inhibiting lipogenesis, and reducing hepatic cholesterol levels by promoting bile acid synthesis. KLF10 highly induced HNF4α expression by directly binding to its promoter. The beneficial effect of KLF10 on MASH development was abolished in mice lacking hepatocyte HNF4α. In addition, the inactivation of KLF10 in hepatic stellate cells exacerbated Western diet-induced liver fibrosis by activating the TGF-β/SMAD2/3 pathway. ConclusionsOur data collectively suggest that the transcription factor KLF10 plays a hepatoprotective role in MASH development by inducing HNF4α. Targeting hepatic KLF10 may offer a promising strategy for treating MASH.

Functional role of circRNA CHRC through miR-431-5p/KLF15 signaling axis in the progression of heart failure

Pathological myocardial hypertrophy is a common early clinical manifestation of heart failure, with noncoding RNAs exerting regulatory influence. However, the molecular function of circular RNAs (circRNAs) in the progression from cardiac hypertrophy to heart failure remains unclear. To uncover functional circRNAs and identify the core circRNA signaling pathway in heart failure, we construct a global triple network (microRNA, circRNA, and mRNA) based on the competitive endogenous RNA (ceRNA) theory. We observe that cardiac hypertrophy-related circRNA (circRNA CHRC), within the ceRNA network, is down-regulated in both transverse aortic constriction mice and Ang-II--treated primary mouse cardiomyocytes. Silencing circRNA CHRC increases cross-sectional cell area, atrial natriuretic peptide, and β-myosin heavy chain levels in primary mouse cardiomyocytes. Further screening shows that circRNA CHRC targets the miR-431-5p/KLF15 axis implicated in heart failure progression in vivo and in vitro. Immunoprecipitation with anti-Ago2-RNA confirms the interaction between circRNA CHRC and miR-431-5p, while miR-431-5p mimics reverse Klf15 activation caused by circRNA CHRC overexpression. In summary, circRNA CHRC attenuates cardiac hypertrophy via sponging miR-431-5p to maintain the normal level of Klf15 expression.

Abstract 3034: Krüppel-like factors in regulation of gastric cancer progression

Abstract Introduction: Gastric cancer (GC) is the third leading cause of cancer-associated mortality and the fifth most frequently diagnosed cancer worldwide. The development and progression of GC is a complicated multistage process subjected to dynamic changes in gene regulation. Investigating the effects of these changes is therefore critical for improving our knowledge about GC initiation and progression. Krüppel-like factors (KLFs) are evolutionarily conserved zinc finger-containing transcription factors with altered expressions and diverse regulatory functions in GC. We previously reported that there is loss of KLF4 in GC. Here we report that KLF4 is a crucial regulator of KLF5 expression and vice versa and together they regulate GC cell behavior. Methods: The Cancer Genome Atlas (TCGA) and pan cancer data of differential expressions of KLFs in GC was analyzed. TCGA RNAseq and clinical data for the differential expression status of KLF genes in normal and cancerous tissues and the clinicopathological association was accessed from the UALCAN web portal. The expressions of KLF4 and KLF5 in human normal stomach and GC and orthotopic xenografts were detected by immunohistochemistry (IHC). KLF4, KLF5 cross regulation was evaluated by gene manipulation of each gene individually and checking the expression of the other factor in GC cells. The effect of KLFs on GC cell proliferation, migration, and invasion was determined by Ki67 staining of tissues, PrestoBlue cell viability assay, scratch wound and transwell cell invasion assays. Results and Conclusion: Our results indicated that not only KLF4 and KLF5 have contrasting expressions and effects in GC but also these two factors cross regulate each other. While lower expressions of KLF4 was found in human GC and orthotopic xenografts, increased expression of KLF5 was found in both human GC tissue and orthotopic xenografts. KLF4 silencing and CRISPR/Cas9 knockdown in human GC cells resulted in increased expression of KLF5 which was associated with increased GC cell proliferation and invasiveness. Interestingly, KLF5 knockdown by CRISPR/Cas9 in human GC cells on the other hand resulted in increased expression of KLF4, which was associated with decreased GC cell growth. Our data therefore suggests that the differential expressions of KLF4 and KLF5 might serve as valuable prognostic markers in GC. Citation Format: Sooraj Kakkat, Prabhat Suman, Sandeep Goswami, Sujit Basu, Martin J. Heslin, Elba A. Turbat-Herrera, Veronica Ramirez Alcantara, Chandrani Sarkar, Debanjan Chakroborty. Krüppel-like factors in regulation of gastric cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3034.