Krüppel-like Factor Research Articles

KLF5/MDM2 Axis Modulates Oxidative Stress and Epithelial-Mesenchymal Transition in Human Lens Epithelial Cells: The Role in Diabetic Cataract

Diabetic cataract (DC) is a common cause of visual loss in older diabetic subjects. Krüppel-like factor 5 (KLF5) plays an essential role in migration and the epithelial-mesenchymal transition (EMT) in diverse cells and is involved in oxidative stress. However, the effects of KLF5 on DC remain unknown. This study aimed to examine the biological function of KLF5 in DC and its underlying mechanism. The expression patterns of KLF5 were detected in vivo and in vitro. Then, KLF5 was knocked down in human lens epithelial cells (HLECs) to explore its functional roles and underlying mechanisms. Dual-luciferase reporter assay and chromatin immunoprecipitation analysis were used to detect whether KLF5 could bind the promoter of E3 ubiquitin ligase mouse double minute 2 (MDM2), a key regulator of EMT. Lastly, the regulation of KLF5 in the biological behaviors of HLECs via MDM2 was analyzed. We found a significant increase of KLF5 in the DC lens anterior capsular, diabetic rat lens, and high glucose (HG)-stimulated HLECs. Knockdown of KLF5 inhibited oxidative stress, inflammation, migration, and EMT of HG-stimulated HLECs. KLF5 silencing impeded MDM2 expression and restricted the activation of MARK1/FAK and NF-κB signaling pathways in HLECs under HG condition. Additionally, KLF5 was found to bind the MDM2 promoter and enhance the transcriptional activity of MDM2. The protective effects by silencing KLF5 on HG-cultured HLECs could be offset by MDM2 overexpression. We demonstrated that knockdown of KLF5 alleviated oxidative stress, migration, and EMT of HG-cultured HLECs by regulating MDM2, suggesting a potential therapeutic strategy for DC.

Krüppel-like factor 15 counteracts endoplasmic reticulum stress and suppresses lung fibroblast proliferation and extracellular matrix accumulation

The incidence of pulmonary fibrosis is on the rise, and existing treatments have limited efficacy in improving patient survival. The purpose of this study was to reveal the potential of Krüppel-like factor (KLF)15 activation in alleviating pulmonary fibrosis. Transforming growth factor beta (TGF-β) was utilized to induce lung fibroblasts to establish an in vitro model of pulmonary fibrosis. The impacts of TGF-β and KLF15 level on cell proliferation, migration, extracellular matrix (ECM) accumulation, and endoplasmic reticulum stress (ERS) were assessed. Additionally, tunicamycin, an ERS agonist, was used to investigate the role of ERS in KLF15 regulation. The results showed that KLF15 was dropped in response to TGF-β treatment. However, KLF15 overexpression reduced cell proliferation, migration, ECM accumulation, and ERS, alleviating the effects of TGF-β stimulation. Subsequent treatment with tunicamycin diminished the effects of KLF15 overexpression, demonstrating that ERS mediated the modulation of KLF15. KLF15 acts against ERS and suppresses excessive proliferation and ECM accumulation in lung fibroblast. These findings suggest that activating KLF15 is a promising strategy for alleviating pulmonary fibrosis.

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

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

PRMT5 activates KLF5 by methylation to facilitate lung cancer.

The highly expressed oncogenic factor Krüppel-like factor 5 (KLF5) promotes various cancerous processes, such as cell growth, survival, anti-apoptosis, migration and metastasis, particularly in lung cancer. Nevertheless, the modifications to KLF5 after translation are poorly understood. Protein arginine methyltransferase 5 (PRMT5) is considered as an oncogene known to be involved in different types of carcinomas, including lung cancer. Here, we show that the expression levels of PRMT5 and KLF5 are highly expressed lung cancer. Moreover, PRMT5 interacts with KLF5 and facilitates the dimethylation of KLF5 at Arginine 41 in a manner that depends on methyltransferase activity. Downregulation or pharmaceutical suppression of PRMT5 reduces the expression of KLF5 and its downstream targets both in vitro and in vivo. Mechanistically, the dimethylation of KLF5 by PRMT5 promotes the maintenance and proliferation of lung cancer cells at least partially by stabilising KLF5 via regulation of the Akt/GSK3β signalling axis. In summary, PRMT5 methylates KLF5 to prevent its degradation, thereby promoting the maintenance and proliferation of lung cancer cells. These results suggest that targeting PRMT5/KLF5 axis may offer a potential therapeutic strategy for lung cancer.

KLF5‑mediated expression of CARD11 promotes the progression of gastric cancer.

Caspase recruitment domain-containing protein 11 (CARD11) has been reported as functioning in multiple types of cancers. In the present study, the role and mechanism of CARD11 in gastric cancer was investigated. First, CARD11 expression in gastric cancer tissues and the association of CARD11 with overall survival were analyzed by the encyclopedia of RNA interactomes database. CARD11 expression in gastric cancer cells was detected by western blotting and reverse transcription-quantitative PCR analyses. After CARD11 silencing, cell proliferation was evaluated by Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine staining and flow cytometry analysis. Wound healing and Transwell assays were used to measure the capacities of cell migration and invasion. Additionally, the expression levels of epithelial-mesenchymal transition (EMT)-related proteins and mTOR-related proteins were detected by western blot analysis. HumanTFDB predicted the binding of the transcription factor Krüppel-like factor 5 (KLF5) to the CARD11 promoter, which was confirmed by dual luciferase reporter and chromatin immunoprecipitation assays. To explore the regulatory effects between KLF5 and CARD11, KLF5 was overexpressed to perform the rescue experiments in gastric cancer cells with CARD11 silencing. Results revealed that CARD11 was highly expressed in gastric cancer and was associated with poor prognosis. CARD11 interference inhibited the proliferation of gastric cancer cells and induced cell cycle arrest. Additionally, CARD11 silencing suppressed the migration, invasion and EMT of gastric cancer cells, accompanied by upregulated E-cadherin expression and downregulated N-cadherin and vimentin expression. Moreover, the transcription factor KLF5 positively regulated the transcription of CARD11 in gastric cancer. KLF5 overexpression reversed the effects of interference of CARD11 expression in gastric cancer cells to promote their proliferation, migration, invasion and EMT. KLF5 overexpression also led to a reduction in cell cycle arrest. Finally, interference of CARD11 expression suppressed the mTOR pathway, which was activated by KLF5. In conclusion, KLF5-mediated CARD11 promoted the proliferation, migration and invasion of gastric cancer cells.

KLF4 targets RAB26 and decreases 5-FU resistance through inhibiting autophagy in colon cancer

ABSTRACT Background Accumulating studies demonstrated that resistance of colon cancer (CC) to 5-fluorouracil (5-FU) contributes to adverse prognosis. We investigated how Kruppel-like factor 4 (KLF4) affected 5-FU resistance and autophagy in CC cells. Methods KLF4 expression and its downstream target gene RAB26 in CC tissues was analyzed by bioinformatics analysis, and the effect of abnormal KLF4 expression on prognoses of CC patients was predicted. Luciferase reporter assay detected the targeted relationship between KLF4 and RAB26. The viability and apoptosis of CC cells were analyzed by CCK-8 and flow cytometry. The formation of intracellular autophagosomes was detected by confocal laser scanning microscopy and immunofluorescence staining. The mRNA and protein levels were assayed by qRT-PCR and western blot. A xenograft animal model was constructed to verify the function of KLF4. Rescue assay was employed to verify whether KLF4/RAB26 could affect 5-FU resistance in CC cells through autophagy. Results KLF4 and RAB26 were lowly expressed in CC. KLF4 correlated with patients’ survival. KLF4 was down-regulated in 5-FU resistant CC cells. KLF4 overexpression suppressed the proliferation and 5-FU resistance of CC cells, and inhibited LC3 II/I expression and autophagosome formation. Autophagy activator Rapamycin or sh-RAB26 treatment reversed the impact of KLF4 overexpression on 5-FU resistance. In vivo assay verified that KLF4 inhibited 5-FU resistance in CC cells. Rescue experiments revealed that KLF4 targeted RAB26 to inhibit CC cell autophagy, resulting in decreasing the resistance to 5-FU. Conclusion KLF4 strengthened the sensitivity of CC cells to 5-FU by targeting RAB26 to restrain autophagy pathway.

Cxxc finger protein 1 maintains homeostasis and function of intestinal group 3 innate lymphoid cells with aging.

Aging is accompanied by homeostatic and functional dysregulation of multiple immune cell subsets. Group 3 innate lymphoid cells (ILC3s) constitute a heterogeneous cell population that plays pivotal roles in intestinal immunity. In this study, we found that ILC3s in aged mice exhibited dysregulated homeostasis and function, leading to bacterial and fungal infection susceptibility. Moreover, our data revealed that the enrichment of the H3K4me3 modification in effector genes of aged gut CCR6+ ILC3s was specifically decreased compared to young mice counterparts. Disruption of Cxxc finger protein 1 (Cxxc1) activity, a key subunit of H3K4 methyltransferase, in ILC3s led to similar aging-related phenotypes. An integrated analysis revealed Kruppel-like factor 4 (Klf4) as a potential Cxxc1 target. Klf4 overexpression partially restored the differentiation and functional defects seen in both aged and Cxxc1-deficient intestinal CCR6+ ILC3s. Therefore, these data suggest that targeting intestinal ILC3s may provide strategies to protect against age-related infections.

Influences of lysine-specific demethylase 1 inhibitors on NO synthase-Kruppel-like factor pathways in human endothelial cells in vitro and zebrafish (Danio rerio) larvae in vivo.

Lysine-specific demethylase 1 (LSD1) inhibitors are being developed for cancer therapy, but their bioeffects on vasculatures are not clear. In this study, we compared the influences of ORY-1001 (an LSD1 inhibitor being advanced into clinical trials) and 199 (a novel LSD1 inhibitor recently developed by us) to human umbilical vein endothelial cells (HUVECs) in vitro and further verified the bioeffects of ORY-1001 to zebrafish (Danio rerio) larvae in vivo. The results showed that up to 10μM ORY-1001 or 199 did not significantly affect the cellular viability of HUVECs but substantially reduced the release of inflammatory interleukin-8 (IL-8) and IL-6. The signaling molecule in vasculatures, NO, was also increased in HUVECs. As the mechanism, the protein levels of endothelial NO synthase (eNOS) or p-eNOS, and their regulators Kruppel-like factor 2 (KLF2) or KLF4, were also increased after drug treatment. In vivo, 24 h treatment with up to 100 nM ORY-1001 reduced blood speed without changing morphologies or locomotor activities in zebrafish larvae. ORY-1001 treatment reduced the expression of il8 but promoted the expression of klf2a and nos in the zebrafish model. These data show that LSD1 inhibitors were not toxic but capable to inhibit inflammatory responses and affect the function of blood vessels through the up-regulation of the NOS-KLF pathway.

A negative feedback loop between KLF9 and the EMT program dictates metastasis of hepatocellular carcinoma.

Metastasis is the primary cause of death of hepatocellular carcinoma (HCC), while the mechanism underlying this severe disease remains largely unclear. The Kruppel-like factor (KLF) family is one of the largest transcription factor families that control multiple physiologic and pathologic processes by governing the cellular transcriptome. To identify metastatic regulators of HCC, we conducted gene expression profiling on the MHCC97 cell series, a set of subclones of the original MHCC97 that was established by in vivo metastasis selection therefore harbouring differential metastatic capacities. We found that the expression of KLF9, a member of the KLF family, was dramatically repressed in the metastatic progeny clone of the MHCC97 cells. Functional studies revealed overexpression of KLF9 suppressed HCC migration in vitro and metastasis in vivo, while knockdown of KLF9 was sufficient to promote cell migration and metastasis accordingly. Mechanistically, we found the expression of KLF9 can reverse the pro-metastatic epithelial-mesenchymal transition (EMT) program via direct binding to the promoter regions of essential mesenchymal genes, thus repressing their expression. Interestingly, we further revealed that KLF9 was, in turn, directly suppressed by a mesenchymal transcription factor Slug, suggesting an intriguing negative feedback loop between KLF9 and the EMT program. Using clinical samples, we found that KLF9 was not only downregulated in HCC tissue compared to its normal counterparts but also further reduced in the HCC samples of whom had developed metastatic lesions. Together, we established a critical transcription factor that represses HCC metastasis, which is clinically and mechanically significant in HCC therapies.

Kruppel benzeri faktör 2 polimorfizmlerinin koroner arter hastalığı riski ile ilişkisi

Purpose: Coronary artery disease is defined as complications that develop in proportion to the prevalence of ischemia due to occlusion of the coronary arteries and the resulting cell death. The development of atherosclerosis is significantly influenced by endothelial cell dysfunction. Kruppel-like factor 2, a transcription factor, has been shown to regulate critical biological events in endothelial biology, such as vascular tone, migration, proliferation, vasoreactivity, and angiogenesis. In our study, it was aimed to clarify the relationship between coronary artery disease and Kruppel-like factor 2 protein levels and C1239A polymorphisms.
 Materials and Methods: 191 individuals who underwent coronary angiography at Mersin University Faculty of Medicine, Department of Cardiology, Health, Research and Application Center were included in the study. Measurements of serum fasting blood glucose, HDL cholesterol, total cholesterol, and triglyceride levels were performed on the AU5800 (Beckman Coulter, United States) autoanalyzer. Serum LDL levels were calculated using the Friedwald equation. Serum Kruppel-like factor 2 protein levels were measured by sandwich enzyme-linked immunoassay method on the Multiskan GO (Thermo Scientific, Finland) device. Kruppel-like factor 2 C1239A variations were detected on the Applied Biosystem VIIA™ 7 Real-Time PCR (Life Technologies Co., United States) device by TaqMan® single nucleotide polymorphism (SNP) genotyping method.
 Results: Men had a 3.8-fold higher risk of CAD than women. (Odd’s ratio 3.83, 95% Confidence interval 1.98-7.39; p

Transcription factor Krüppel-like factor 4 upregulated G protein-coupled receptor 30 alleviates intestinal inflammation and apoptosis, and protects intestinal integrity from intestinal ischemia-reperfusion injury.

Intestinal ischemia/reperfusion (I/R) injury is a common clinical event occurring during multiple clinical pathological processes. Here, we designed this paper to discuss the role of G protein-coupled receptor 30 (GPR30) playing in intestinal I/R injury. An oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate the pathological process of I/R injury. With the application of enzyme-linked immunosorbent assay, TUNEL, and transepithelial electrical resistance (TEER) assays, the levels of inflammatory cytokines, cell apoptosis, and intestinal integrity were estimated. The corresponding proteins were estimated by applying western blot. Immunofluorescence was conducted to examine N-terminal Gasdermin D (GSDMD-N) expression. The interplay between KLF4 and GPR30 was demonstrated by dual-luciferase reporter assay and chromatin immunoprecipitation. The results showed that GPR30 was downregulated in Caco-2 cells exposed to OGD/R. GPR30 overexpression reduced the production of TNF-α, IL-6, IL-1β, and IL-18, the TUNEL-positive cells, as well as the contents of p-p65, Cox-2, Inos, Bax, and cleaved-PARP, but elevated the expression of Bcl-2 in OGD/R-induced Caco-2 cells. In addition, OGD/R-induced the reduction of TEER value and reduced expression of tight junction proteins in Caco-2 cells, which was partially restored by GPR30 overexpression. Furthermore, GPR30 suppressed nod-like receptor pyrin 3inflammasome and GSDMD-N expression. It was evidenced that Krüppel-like factor 4 (KLF4) could directly bind to GPR30 promoter and positively regulate GPR30 expression. The regulation of GPR30 overexpression above was weakened by KLF4 knockdown. Collectively, our findings suggested that KLF4 could transcriptionally upregulate GPR30, and GPR30 prevented intestine I/R injury by inhibiting inflammation and apoptosis, and maintaining intestinal integrity that provides potential targets for mitigating the I/R injury.

KLF2 regulates stemness of human mesenchymal stem cells by targeting FGFR3

ABSTRACT Mesenchymal stem cells (MSCs) are an attractive source of pluripotent cells for regenerative therapy; however, maintaining stemness and self-renewal of MSCs during expansion ex vivo is challenging. For future clinical applications, it is essential to define the roles and signaling pathways that regulate the fate of MSCs. Based on our earlier finding that Krüppel-like factor 2 (KLF2) participates in maintaining stemness in MSCs, we examined further the role of this factor in intrinsic signaling pathways. Using a chromatin immunoprecipitation (ChIP)-sequence assay, we found that the FGFR3 gene is a KLF2 binding site. Knockdown of FGFR3 significantly decreased the levels of key pluripotency factors, enhanced the expression of differentiation-related genes and down-regulated colony formation of human bone marrow MSCs (hBMSCs). Using alizarin red S and oil red O staining, we found that knockdown of FGFR3 inhibited the osteogenic and adipogenic ability of MSCs under conditions of differentiation. The ChIP-qPCR assay confirmed that KLF2 interacts with the promoter regions of FGFR3. Our findings suggest that KLF2 promotes hBMSC stemness by direct regulation of FGFR. Our findings may contribute to enhanced MSC stemness by genetic modification of stemness-related genes.

MicroRNA-130b May Induce Cerebral Vasospasm after Subarachnoid Hemorrhage via Modulating Kruppel-like Factor 4.

Recently, the diverse functions of microRNAs (miRNAs) in brain diseases have been demonstrated. We intended to uncover the functional role of microRNA-130b (miR-130b) in cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH). SAH was induced by injecting the autologous blood into the cisterna magna of Sprague Dawley rats. The cerebral vascular smooth muscle cells (cVSMCs) were extracted for invitro experimentation. In vitro and invivo assays were implemented with transfection of miR-130b mimic/inhibitor, sh-Kruppel-like factor 4 (KLF4), oe-KLF4 plasmids or p38/MAPK signaling pathway agonist (anisomycin), respectively, to elaborate the role of miR-130b in CVS following SAH. Elevated miR-130b and reduced KLF4 were found in SAH patients and rat models of SAH. KLF4 was the target gene of miR-130b. miR-130b promoted the proliferation and migration of cVSMCs through the Inhibition of KLF4. Besides, KLF4 inhibited the proliferation and migration of cVSMCs through blockage of the p38/MAPK pathway. Furthermore, invivo assay confirmed the inhibitory effect of decreased miR-130b in CVS following SAH. In conclusion, miR-130b may activate the p38/MAPK signaling pathway through targeted inhibition of KLF4, thereby contributing to some extent to the development of cerebral vasospasm after SAH.

Induction of KLF2 by Exercise Activates eNOS to Improve Vasodilatation in Diabetic Mice.

Exercise and statins restore the endothelial expression of Krüppel-like factor 2 (KLF2), which is diminished in diabetic db/db mice. Endothelium-specific overexpression of KLF2 improves endothelium-dependent relaxation and flow-mediated dilation through increasing nitric oxide bioavailability. KLF2 promotes endothelial nitric oxide synthase (eNOS) coupling and phosphorylation in addition to its known role in eNOS transcription. KLF2 upregulates the expression of several panels of genes that regulate eNOS activity.

Bioinformatic and systems biology approach revealing the shared genes and molecular mechanisms between COVID-19 and non-alcoholic hepatitis.

Introduction: Coronavirus disease 2019 (COVID-19) has become a global pandemic and poses a serious threat to human health. Many studies have shown that pre-existing nonalcoholic steatohepatitis (NASH) can worsen the clinical symptoms in patients suffering from COVID-19. However, the potential molecular mechanisms between NASH and COVID-19 remain unclear. To this end, key molecules and pathways between COVID-19 and NASH were herein explored by bioinformatic analysis. Methods: The common differentially expressed genes (DEGs) between NASH and COVID-19 were obtained by differential gene analysis. Enrichment analysis and protein-protein interaction (PPI) network analysis were carried out using the obtained common DEGs. The key modules and hub genes in PPI network were obtained by using the plug-in of Cytoscape software. Subsequently, the hub genes were verified using datasets of NASH (GSE180882) and COVID-19 (GSE150316), and further evaluated by principal component analysis (PCA) and receiver operating characteristic (ROC). Finally, the verified hub genes were analyzed by single-sample gene set enrichment analysis (ssGSEA) and NetworkAnalyst was used for the analysis of transcription factor (TF)-gene interactions, TF-microRNAs (miRNA) coregulatory network, and Protein-chemical Interactions. Results: A total of 120 DEGs between NASH and COVID-19 datasets were obtained, and the PPI network was constructed. Two key modules were obtained via the PPI network, and enrichment analysis of the key modules revealed the common association between NASH and COVID-19. In total, 16 hub genes were obtained by five algorithms, and six of them, namely, Kruppel-like factor 6 (KLF6), early growth response 1 (EGR1), growth arrest and DNA-damage-inducible 45 beta (GADD45B), JUNB, FOS, and FOS-like antigen 1 (FOSL1) were confirmed to be closely related to NASH and COVID-19. Finally, the relationship between hub genes and related pathways was analyzed, and the interaction network of six hub genes was constructed with TFs, miRNAs, and compounds. Conclusion: This study identified six hub genes related to COVID-19 and NASH, providing a new perspective for disease diagnosis and drug development.

Krüppel-like factor 10 modulates stem cell phenotypes of pancreatic adenocarcinoma by transcriptionally regulating notch receptors

BackgroundPancreatic adenocarcinoma (PDAC) is well known for its rapid distant metastasis and local destructive behavior. Loss of Krüppel-like factor 10 (KLF10) contributes to distant migration of PDAC. The role of KLF10 in modulating tumorigenesis and stem cell phenotypes of PDAC is unclear.MethodsAdditional depletion of KLF10 in KC (LSL: KrasG12D; Pdx1-Cre) mice, a spontaneous murine PDAC model, was established to evaluate tumorigenesis. Tumor specimens of PDAC patients were immune-stained of KLF10 to correlate with local recurrence after curative resection. Conditional overexpressing KLF10 in MiaPaCa and stably depleting KLF10 in Panc-1 (Panc-1-pLKO-shKLF10) cells were established for evaluating sphere formation, stem cell markers expression and tumor growth. The signal pathways modulated by KLF10 for PDAC stem cell phenotypes were disclosed by microarray analysis and validated by western blot, qRT-PCR, luciferase reporter assay. Candidate targets to reverse PDAC tumor growth were demonstrated in murine model.ResultsKLF10, deficient in two-thirds of 105 patients with resected pancreatic PDAC, was associated with rapid local recurrence and large tumor size. Additional KLF10 depletion in KC mice accelerated progression from pancreatic intraepithelial neoplasia to PDAC. Increased sphere formation, expression of stem cell markers, and tumor growth were observed in Panc-1-pLKO-shKLF10 compared with vector control. Genetically or pharmacologically overexpression of KLF10 reversed the stem cell phenotypes induced by KLF10 depletion. Ingenuity pathway analysis and gene set enrichment analysis showed that Notch signaling molecules, including Notch receptors 3 and 4, were over-expressed in Panc-1-pLKO-shKLF10. KLF10 transcriptionally suppressed Notch-3 and -4 by competing with E74-like ETS transcription factor 3, a positive regulator, for promoter binding. Downregulation of Notch signaling, either genetically or pharmacologically, ameliorated the stem cell phenotypes of Panc-1-pLKO-shKLF10. The combination of metformin, which upregulated KLF10 expression via phosphorylating AMPK, and evodiamine, a non-toxic Notch-3 methylation stimulator, delayed tumor growth of PDAC with KLF10 deficiency in mice without prominent toxicity.ConclusionsThese results demonstrated a novel signaling pathway by which KLF10 modulates stem cell phenotypes in PDAC through transcriptionally regulating Notch signaling pathway. The elevation of KLF10 and suppression of Notch signaling may jointly reduce PDAC tumorigenesis and malignant progression.

Identification and validation of G protein-coupled receptors modulating flow-dependent signaling pathways in vascular endothelial cells

Vascular endothelial cells are exposed to mechanical forces due to their presence at the interface between the vessel wall and flowing blood. The patterns of these mechanical forces (laminar vs. turbulent) regulate endothelial cell function and play an important role in determining endothelial phenotype and ultimately cardiovascular health. One of the key transcriptional mediators of the positive effects of laminar flow patterns on endothelial cell phenotype is the zinc-finger transcription factor, krüppel-like factor 2 (KLF2). Given its importance in maintaining a healthy endothelium, we sought to identify endothelial regulators of the KLF2 transcriptional program as potential new therapeutic approaches to treating cardiovascular disease. Using an approach that utilized both bioinformatics and targeted gene knockdown, we identified endothelial GPCRs capable of modulating KLF2 expression. Genetic screening using siRNAs directed to these GPCRs identified 12 potential GPCR targets that could modulate the KLF2 program, including a subset capable of regulating flow-induced KLF2 expression in primary endothelial cells. Among these targets, we describe the ability of several GPCRs (GPR116, SSTR3, GPR101, LGR4) to affect KLF2 transcriptional activation. We also identify these targets as potential validated targets for the development of novel treatments targeting the endothelium. Finally, we highlight the initiation of drug discovery efforts for LGR4 and report the identification of the first known synthetic ligands to this receptor as a proof-of-concept for pathway-directed phenotypic screening to identify novel drug targets.

FBXO22 Mediates the NGF/TRKA Signaling Pathway in Bone Metastases in Prostate Cancer

Prostate cancer (PC) is a malignancy with high morbidity and mortality. Bone metastasis is the main driver of short survival time and difficulties in the treatment and prevention of PC. The goal of this study was to explore the biological function of E3 ubiquitin ligase F-box only protein 22 (FBXO22) in PC metastasis and its specific regulation mechanism. According to transcriptome sequencing, FBXO22 was overexpressed in PC tissues (versus adjacent tissues) and bone tissues (versus biopsied bone tissues without bone metastases). Fbxo22 down-regulation reduced bone metastases and macrophage M2 polarization in mice. FBXO22 was down-regulated in macrophages, and polarization was observed by flow cytometry. Macrophages were co-cultured with PC cells and osteoblasts to assess PC cell and osteoblast activity. FBXO22 knockdown restored osteoblast capacity. FBXO22 ubiquitinated and degraded Krüppel-like factor 4 (KLF4), which regulated the nerve growth factor (NGF)/tropomyosin receptor kinase A pathway by repressing NGF transcription. Silencing of KLF4 mitigated the metastasis-suppressing properties of FBXO22 knockdown, whereas NGF reversed the metastasis-suppressing properties of KLF4 invitro and invivo. Cumulatively, these data indicate that FBXO22 promotes PC cell activity and osteogenic lesions by stimulating macrophage M2 polarization. It also degrades KLF4 in macrophages and promotes NGF transcription, thereby activating the NGF/tropomyosin receptor kinase A pathway.

Cardiac Fibroblast Activation Induced by Oxygen-Glucose Deprivation Depends on the HIF-1α/miR-212-5p/KLF4 Pathway.

It is widely accepted that miRNAs play an important role in the pathogenesis of myocardial fibrosis. This study aimed to identify a new pathway of miR-212-5p in the activation of human cardiac fibroblasts (HCFs) induced by oxygen-glucose deprivation (OGD). First, we found that KLF4 protein was markedly decreased in OGD-induced HCFs. Then, bioinformatics analysis and verification experiments were used to identify the existence of an interaction of KLF4 with miR-212-5p. Functional experiments indicated that OGD significantly upregulated the expression of hypoxia inducible factor-1 alpha (HIF-1α) in HCFs, which positively regulated miR-212-5p transcription by binding to its promoter. MiR-212-5p inhibited the expression of Krüppel-like factor 4 (KLF4) protein by binding to the 3' untranslated coding regions (UTRs) of KLF4 mRNA. Inhibition of miR-212-5p effectively inhibited the activation of OGD-induced HCFs by upregulating KLF4 expression and inhibited cardiac fibrosis in vivo and in vitro.

Decreased Expression of KLF4 Leading to Functional Deficit in Pediatric Patients with Intestinal Failure and Potential Therapeutic Strategy Using Decanoic Acid.

Pediatric intestinal failure (IF) is the reduction in gut function to below the minimum necessary for the absorption of macronutrients and/or water and electrolytes, such that intravenous supplementation is required to maintain health and/or growth. The overall goal in treating IF is to achieve intestinal adaptation; however, the underlying mechanisms have not been fully understood. In this study, by performing single-cell RNA sequencing in pediatric IF patients, we found that decreased Kruppel-Like Factor 4 (KLF4) may serve as the hub gene responsible for the functional deficit in mature enterocytes in IF patients, leading to the downregulation of solute carrier (SLC) family transporters (e.g., SLC7A9) and, consequently, nutrient malabsorption. We also found that inducible KLF4 was highly sensitive to the loss of certain enteral nutrients: in a rodent model of total parenteral nutrition mimicking the deprivation of enteral nutrition, the expression of KLF4 dramatically decreased only at the tip of the villus and not at the bottom of crypts. By using IF patient-derived intestinal organoids and Caco-2 cells as in vitro models, we demonstrated that the supplementation of decanoic acid (DA) could significantly induce the expression of KLF4 along with SLC6A4 and SLC7A9, suggesting that DA may function as a potential therapeutic strategy to promote cell maturation and functional improvement. In summary, this study provides new insights into the mechanism of intestinal adaptation depending on KLF4, and proposed potential strategies for nutritional management using DA.