Overexpression Of KLF4 Research Articles

MicroRNA-7 Protects Against 1-Methyl-4-Phenylpyridinium Iodide-Induced Cell Apoptosis in SH-SY5Y Cells by Directly Targeting Krüpple-Like Factor 4.

This study intended to investigate the role and underling mechanism of microRNA-7 (miR-7) on neuronal death in Parkinson's disease (PD). Human neuroblastoma cell line SH-SY5Y was employed and 1-methyl-4-phenylpyridinium iodide [MPP(+)] was used to generate PD model in vitro. Furthermore, an upregulation of miR-7 was performed in SH-SY5Y by transfection with miR-7 mimics. Cell viability and cell apoptosis were determined. Moreover, the target and the mechanism of miR-7 in MPP(+)-induced cell death were also investigated. The upregulation of miR-7 promoted cell viability and suppressed cell apoptosis in MPP(+)-treated SH-SY5Y cells. Furthermore, miR-7 could directly bind to the 3'-untranslated region of Krüppel-like factor 4 (KLF4, positions 574-580). Moreover, knockdown of KLF4 by the specific siRNA inhibited SH-SY5Y apoptosis under MPP(+) treatment. In addition, KLF4 overexpression apparently attenuated the protective effect of miR-7 in MPP(+)-induced SH-SY5Y apoptosis. This study indicated that miR-7 protects from MPP(+)-induced cell apoptosis in SH-SY5Y by directly targeting KLF4.

Deregulated KLF4 Expression in Myeloid Leukemias Alters Cell Proliferation and Differentiation through MicroRNA and Gene Targets.

Acute myeloid leukemia (AML) is characterized by increased proliferation and blocked differentiation of hematopoietic progenitors mediated, in part, by altered myeloid transcription factor expression. Decreased Krüppel-like factor 4 (KLF4) expression has been observed in AML, but how decreased KLF4 contributes to AML pathogenesis is largely unknown. We demonstrate decreased KLF4 expression in AML patient samples with various cytogenetic aberrations, confirm that KLF4 overexpression promotes myeloid differentiation and inhibits cell proliferation in AML cell lines, and identify new targets of KLF4. We have demonstrated that microRNA 150 (miR-150) expression is decreased in AML and that reintroducing miR-150 expression induces myeloid differentiation and inhibits proliferation of AML cells. We show that KLF family DNA binding sites are necessary for miR-150 promoter activity and that KLF2 or KLF4 overexpression induces miR-150 expression. miR-150 silencing, alone or in combination with silencing of CDKN1A, a well-described KLF4 target, did not fully reverse KLF4-mediated effects. Gene expression profiling and validation identified putative KLF4-regulated genes, including decreased MYC and downstream MYC-regulated gene expression in KLF4-overexpressing cells. Our findings indicate that decreased KLF4 expression mediates antileukemic effects through regulation of gene and microRNA networks, containing miR-150, CDKN1A, and MYC, and provide mechanistic support for therapeutic strategies increasing KLF4 expression.

Direct Conversion of Human Preadipocytes into Hematopoietic, Neuronal, and Pancreatic α Cells by Oct4 and Klf4 Overexpression

Cellular plasticity obtained by natural adaptation or by forced expression of developmental stage and lineage-specific transcription factors converts differentiated cells into three different levels of potency: pluripotent, multipotent (dedifferentiation), and oligo- or unipotent (transdifferentiation). These processes hold promise for regenerative medicine to replace damaged cells and organs through the identification of transcription factors that determine the fate of cellular plasticity. In this study, we report that ectopic expression of Oct4 and Klf4 induces transdifferentiation of human preadipocytes into three different lineages: hematopoietic, neuronal, and pancreatic endocrine cells. Furthermore, ectopic expression of Oct4 is sufficient to induce transdifferentiation of preadipocytes into glucagon-expressing pancreatic α cells. This will provide a simple and efficient method to produce functionally competent cells for therapeutic regenerative medicine.

Krüppel-like factor 5 promotes apoptosis triggered by tumor necrosis factor α in LNCaP prostate cancer cells via up-regulation of mitogen-activated protein kinase kinase 7

ObjectivesKrüppel-like factor 5 (KLF5) modulates multiple cell processes in different cancers. It is frequently deleted and inactivated in prostate cancer and may exert a tumor suppressor function. However, how KLF5 inhibits the progression of prostate cancer is still not clear. In the present study, we identified how KLF5 and tumor necrosis factor α (TNFα) pathway, which can induce apoptosis in cancer, regulate each other in LNCaP prostate cancer cells. Material and methodsThe expression of messenger RNA and protein was detected by real-time polymerase chain reaction assay and western blot analysis, respectively. To identify whether KLF5 regulates the activity of TNFα downstream pathway, we constructed a stable KLF5 knockdown or KLF5 overexpressing cell line with lentivirus-containing short hairpin RNA targeting KLF5 or full-length KLF5 in LNCaP cells. Cell apoptosis was determined through flow cytometry assay. In addition, the regulation of KLF5 on target gene transcription was detected by reporter luciferase activity assay, and the binding of KLF5 on target promoter was detected through oligonucleotides pull-down analysis. ResultsWe found that TNFα induced the expression of KLF5 at both messenger RNA and protein levels; moreover, TNFα up-regulated KLF5 through TNF receptor 1 but not through TNF receptor 2 in LNCaP cells. Knockdown of KLF5 decreased apoptosis induced by TNFα, whereas cell apoptosis was increased by KLF5 overexpression. Consistently, expression of cleaved PARP and caspase-3 induced by TNFα was decreased by KLF5 knockdown, whereas it was increased by overexpressed KLF5. JNK activity is essential for the apoptosis induced by TNFα. We found that knockdown of KLF5 not only decreased the phosphorylation of JNK induced by TNFα, but also down-regulated the transcription of mitogen-activated protein kinase kinase 7 (MKK7), an upstream kinase of JNK, by binding to the MKK7 promoter. ConclusionsOur results indicate that KLF5 is an essential transcription regulator of MKK7 kinase and promotes the apoptosis induced by TNFα in LNCaP cells. Loss of KLF5 in prostate cancer may decrease cell response to TNFα-inducing apoptosis and facilitate cancer initiation and progression; moreover, KLF5 could be a potential molecular marker for predicting the effect of high-dose TNFα on tumor growth inhibition in prostate cancer.

MicroRNA-30 mediates anti-inflammatory effects of shear stress and KLF2 via repression of angiopoietin 2

MicroRNAs are endogenously expressed small noncoding RNAs that regulate gene expression. Laminar blood flow induces atheroprotective gene expression in endothelial cells (ECs) in part by upregulating the transcription factor KLF2. Here, we identified KLF2- and flow-responsive miRs that affect gene expression in ECs.Bioinformatic assessment of mRNA expression patterns identified the miR-30-5p seed sequence to be highly enriched in mRNAs that are downregulated by KLF2. Indeed, KLF2 overexpression and shear stress stimulation in vitro and in vivo increased the expression of miR-30-5p family members. Furthermore, we identified angiopoietin 2 (Ang2) as a target of miR-30. MiR-30 overexpression reduces Ang2 levels, whereas miR-30 inhibition by LNA-antimiRs induces Ang2 expression. Consistently, miR-30 reduced basal and TNF-α-induced expression of the inflammatory cell–cell adhesion molecules E-selectin, ICAM1 and VCAM1, which was rescued by stimulation with exogenous Ang2.In summary, KLF2 and shear stress increase the expression of the miR-30-5p family which acts in an anti-inflammatory manner in ECs by impairing the expression of Ang2 and inflammatory cell–cell adhesion molecules. The upregulation of miR-30-5p family members may contribute to the atheroprotective effects of shear stress.

KLF2 is downregulated in pancreatic ductal adenocarcinoma and inhibits the growth and migration of cancer cells.

Members of the Kruppel-like factor (KLF) family have been considered as the tumor suppressors for their inhibitory effects on cell proliferation. Dysregulation of KLF2, a member of KLF family, has been observed in various cancer types. However, its expression pattern and functions in the pancreatic ductal adenocarcinoma (PDAC) are unknown. In this study, we examined the expression of KLF2 in PDAC clinical samples and evaluated the functions of KLF2 in the progression of PDAC. KLF2 is shown to be downregulated in PDAC clinical samples and overexpression of KLF2 inhibits the growth, migration, and metastasis of PDAC cancer cells. KLF2 interacts with beta-catenin and negatively regulates the beta-catenin/TCF signaling. Taken together, this study suggests the suppressive functions of KLF2 in PDAC.

Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

Abstract 1386: KLF5 inhibits angiogenesis in PTEN-deficient prostate cancer by attenuating AKT activation and subsequent HIF1α accumulation

Abstract Background: KLF5 is a basic transcriptional factor that regulates multiple physiopathological processes. Our recent study showed that deletion of Klf5 in mouse prostates promotes tumorigenesis initiated by the deletion of Pten. While molecular characterization of Klf5-null tumors suggested that angiogenesis was partially responsible for tumor promotion, the precise function and mechanism of KLF5 deletion in prostate tumor angiogenesis remain unclear. Results: Using histological staining of Pten-null mouse prostates, we observed that deletion of Klf5 significantly increased the number of microvessels, which was accompanied by the upregulation of multiple angiogenesis-related genes based on microarray analysis with MetaCore software. In human umbilical vein endothelial cells (HuVECs), tube formation and migration, both of which are indicators of angiogenic activities, were decreased by conditioned media from PC-3 and DU 145 human prostate cancer cells with KLF5 overexpression, but increased by media from cells with KLF5 knockdown. HIF1α, a key angiogenesis inducer, was upregulated by KLF5 loss at the protein but not the mRNA level in both mouse tissues and human cell lines, as examined by immunohistochemical staining, real-time RT-PCR and Western blotting. Consistently, KLF5 loss also upregulated VEGF and PDGF, two pro-angiogenic mediators of HIF1α function, as analyzed by immunohistochemical staining in mouse tissues and ELISA in conditioned media. Mechanistically, AKT activity, which caused the accumulation of HIF1α, was increased by KLF5 knockout or knockdown but decreased by KLF5 overexpression. Consistently, PI3K/AKT inhibitors abolished the effects of KLF5 knockdown on angiogenic activity, HIF1α accumulation, and VEGF and PDGF expression. Conclusion: KLF5 loss enhances tumor angiogenesis by attenuating PI3K/AKT signaling and subsequent accumulation of HIF1α in PTEN deficient prostate tumors. Citation Format: Xinpei Ci, Changsheng Xing, Baotong Zhang, Zhiqian Zhang, Jenny Jianping Ni, Wei Zhou, Jin-Tang Dong. KLF5 inhibits angiogenesis in PTEN-deficient prostate cancer by attenuating AKT activation and subsequent HIF1α accumulation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1386. doi:10.1158/1538-7445.AM2015-1386

Abstract 3617: Inhibition of KLF4 expression in resistant B-NHL cell lines inhibited cell growth and sensitized the cells to drug-induced apoptosis

Abstract Kruppel-like factor 4 (KLF4) is a member of the KLF4 zinc-finger containing transcription factor family. Reported studies have indicated the involvement of KLF4 in the regulation of proliferation, apoptosis, and differentiation of B-cells and B-cell malignancies. We have recently reported that KLF4 is overexpressed in pediatric Burkitt lymphomas and is a predictive biomarker for survival. In addition, overexpression of KLF4 predicted unresponsiveness to CHOP treatment (Valencia-Hipolito A et al Leuk Lymphoma. 2014;55:1806-14). Preliminary findings demonstrated that the transcription factor Yin Yang 1 (YY1) plays, in part, a role in the regulation of KLF4 expression in B-NHL. We hypothesized that (1) the chemical inhibition of KLF4 may result in the inhibition of proliferation, induction of apoptosis, and sensitization to drug-induced apoptosis in B-NHL cell lines and (2) the inhibition of YY1 would mimic the chemical inhibition of KLF4. Analysis of two B-NHL cell lines revealed that the expression of KLF4 was high in Ramos and low Raji as compared to normal B-cells. Treatment with increasing concentrations (0.5-10 μM) of Kenpaullone (a KLF4 inhibitor) induced in Ramos cells both the inhibition of proliferation and cell survival and the induction of apoptosis; however, there was no effect on the treatment of Raji cells. Treatment of Ramos cells with the combination of Kenpaullone and CDDP potentiated apoptosis as compared to treatment with either the chemical inhibitor or CDDP used alone. The finding with the chemical inhibitor was validated with the transfection of Ramos cells with siRNA-KLF4. In addition, treatment with Kenpaullone inhibited the transcriptional expression of KLF4 (using a reporter assay system with pKLF4-GFP). Additional studies demonstrated that the transfection of Ramos cells with siRNA-YY1 resulted in significant inhibition of KLF4 expression and correlated with the inhibition of proliferation and the induction of apoptosis. The latter is due, in part, as KLF4 suppresses the extrinsic apoptotic pathway by inhibiting the activation and cleavage of caspases 7, 9, and 3.. Our previous analysis in TMA of pediatric lymphomas demonstrated, in all of the tumor tissues, the presence of a positive correlation between the expression of KLF4 and YY1 and that this correlation was markedly significant in the Burkitt subtype. In conclusion, the overexpression of KLF4 may be responsible, in part, in the pathogenesis, malignancy, and drug resistance of B-NHL lymphomas. In addition, the present findings suggest that the chemical inhibition of KLF4 by Kenpaullone treatment or the inhibition of YY1 may be considered as targets for therapeutic intervention in the treatment of B-lymphoma overexpressing KLF4, when used alone or in combination with sub-toxic chemo/immune-drugs. Current studies are evaluating the role of KLF4 inhibition in vivo using B-NHL tumor xenografts models. Citation Format: Mayra R. Montecillo-Aguado, Gabriel G. Vega, Hector Mayani, Sara Huerta-Yepez, Rogelio Hernández-Pando, Otoniel Martinez-Maza, Benjamin Bonavida, Mario I. Vega. Inhibition of KLF4 expression in resistant B-NHL cell lines inhibited cell growth and sensitized the cells to drug-induced apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3617. doi:10.1158/1538-7445.AM2015-3617

MiR-375 targets KLF4 and impacts the proliferation of colorectal carcinoma.

MiR-375 has been identified as oncogenes or tumor suppressor genes which has the potential to the development and growth of cancers. However, the limited information concerning the expression and role of miR-375 in colorectal cancer (CRC) is available. In this work, we provide evidence for a function of miR-375 in the inhibition of CRC proliferation. Here, we showed that miR-375, down-modulated in human colorectal cancer tissues compared with normal human colon tissues, including several colorectal cancer cell lines. Subsequently, using the luciferase reporter assays, we found that the KLF4 untranslated region (3'UTR) carries the direct binding site of miR-375. In terms of function in vitro, CCK-8 assay, colony formation assay, and cell cycle assay demonstrated that the overexpression of miR-375 suppressed CRC cell proliferation. Inhibition of KLF4 performed similar effects with miR-375 overexpression on CRC cells, and overexpression of KLF4 could significantly reverse the tumor suppressive effects of miR-375 on CRC cells. Furthermore, we found overexpressed miR-375 effectively repressed tumor growth via KLF4 in xenograft animal experiment. Taken together, these results illustrated that miR-375 depresses proliferation of CRC through regulating 3'UTR of KLF4 mRNA, which might be a promising therapeutic target for treating colorectal cancers.

KLF4 mediates the link between TGF-β1-induced gene transcription and H3 acetylation in vascular smooth muscle cells.

Transcriptional activation by transcription factors is coupled with histone acetylation and chromatin remodeling. However, the relationship between TGF-β1-induced gene transcription by Krüppel-like factor (KLF)-4 and histone acetylation remains unknown. In our study, KLF4 overexpression or knockdown, respectively increased or decreased H3 acetylation and p300 occupancy, which is concentrated in the region containing TGF-β1 control elements (TCEs) of the genes by TGF-β1 regulation during vascular smooth muscle cell (VSMC) differentiation. Coimmunoprecipitation and glutathione S-transferase pull-down assays showed that phosphatase and tensin homolog (PTEN) formed a complex with KLF4 to inhibit the phosphorylation of the latter in basal conditions. After TGF-β1 signaling activation, PTEN was phosphorylated by p38 MAPK or PI3K/Akt signaling, phosphorylated PTEN lost its ability to dephosphorylate KLF4, and the cofactors interacting with KLF4 switched from PTEN to p300. Then, KLF4-p300 complexes were recruited to KLF4-binding sites of the gene promoter of VSMCs, to acetylate histone H3 and activate transcription. In addition, phosphorylated KLF4 enhanced p300 histone acetyltransferase (HAT) activity via the p38 MAPK pathway, which may be responsible for H3 acetylation. Taken together, the results of our study reveal a novel mechanism whereby KLF4 mediates the link between TGF-β1-induced gene transcription activation and H3 acetylation during VSMC differentiation.

Impaired autophagy in mouse embryonic fibroblasts null for Krüppel-like Factor 4 promotes DNA damage and increases apoptosis upon serum starvation.

BackgroundAutophagy is a major cellular process by which cytoplasmic components such as damaged organelles and misfolded proteins are recycled. Although low levels of autophagy occur in cells under basal conditions, certain cellular stresses including nutrient depletion, DNA damage, and oxidative stress are known to robustly induce autophagy. Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor activated during oxidative stress to maintain genomic stability. Both autophagy and KLF4 play important roles in response to stress and function in tumor suppression.MethodsTo explore the role of KLF4 on autophagy in mouse embryonic fibroblasts (MEFs), we compared wild-type with Klf4 deficient cells. To determine the levels of autophagy, we starved MEFs for different times with Earle’s balanced salts solution (EBSS). Rapamycin was used to manipulate mTOR activity and autophagy. The percentage of cells with γ-H2AX foci, a marker for DNA damage, and punctate pattern of GFP-LC3 were counted by confocal microscopy. The effects of the drug treatments, Klf4 overexpression, or Klf4 transient silencing on autophagy were analyzed using Western blot. Trypan Blue assay and flow cytometry were used to study cell viability and apoptosis, respectively. qPCR was also used to assay basal and the effects of Klf4 overexpression on Atg7 expression levels.ResultsHere our data suggested that Klf4−/− MEFs exhibited impaired autophagy, which sensitized them to cell death under nutrient deprivation. Secondly, DNA damage in Klf4-null MEFs increased after treatment with EBSS and was correlated with increased apoptosis. Thirdly, we found that Klf4−/− MEFs showed hyperactive mTOR activity. Furthermore, we demonstrated that rapamycin reduced the increased level of mTOR in Klf4−/− MEFs, but did not restore the level of autophagy. Finally, re-expression of Klf4 in Klf4 deficient MEFs resulted in increased levels of LC3II, a marker for autophagy, and Atg7 expression level when compared to GFP-control transfected Klf4−/− MEFs.ConclusionTaken together, our results strongly suggest that KLF4 plays a critical role in the regulation of autophagy and suppression of mTOR activity. In addition, we showed that rapamycin decreased the level of mTOR in Klf4−/− MEFs, but did not restore autophagy. This suggests that KLF4 regulates autophagy through both mTOR-dependent and independent mechanisms. Furthermore, for the first time, our findings provide novel insights into the mechanism by which KLF4 perhaps prevents DNA damage and apoptosis through activation of autophagy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0373-6) contains supplementary material, which is available to authorized users.

Abstract 8: RNA-binding Protein Quaking Maintains Endothelial Barrier Function Through β-catenin and VE-cadherin

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is determined largely by adherens junctions. Remarkably little is known about their regulation at the post[[Unable to Display Character: ‐]]transcriptional level. We find that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent adult endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression. Subsequently we aimed to investigate the role of QKI in EC vascular integrity. Silencing of QKI markedly impaired (0.65 fold ±0.13; p<0.05) the capacity to form a high resistance endothelial monolayer, as measured using Electric cell-substrate impedance sensing. To confirm a role for QKI in maintaining EC barrier function in vivo, we measured Bradykinin-induced vascular leakage in QKI viable mice (QKIv), which express decreased levels of the QKI protein. Indeed, QKIv mice displayed a 20% (p<0.05) increase in extravascular accumulation of Evans blue-labeled albumin compared to wild type littermates. Interestingly, the mRNA of both β-catenin and VE-cadherin, the prime adhesion proteins in EC adherens junctions, contain conserved QKI-binding sites. Moreover the targeted reduction of QKI resulted in a reduction of β-catenin and VE-cadherin protein expression. Importantly, we identified a direct role for QKI in regulating mRNA biology of β-catenin and VE-cadherin, as RNA immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the mRNA and induces transcript translation, respectively. This effects was perturbed upon reduced QKI expression. In conclusion, we show that QKI functions as a critical regulator of β-catenin and VE-cadherin in endothelial cells, and the modulation of QKI expression affects endothelial monolayer integrity, in vitro and in vivo. These studies provide novel insight into the importance of post-transcriptional regulation of components of the endothelial adherens junction, and may have wide ranging implications for the preservation of vascular integrity in disease.

KLF5 inhibits angiogenesis in PTEN-deficient prostate cancer by attenuating AKT activation and subsequent HIF1α accumulation.

BackgroundKLF5 is a basic transcriptional factor that regulates multiple physiopathological processes. Our recent study showed that deletion of Klf5 in mouse prostate promotes tumorigenesis initiated by the deletion of Pten. While molecular characterization of Klf5-null tumors suggested that angiogenesis was partially responsible for tumor promotion, the precise function and mechanism of KLF5 deletion in prostate tumor angiogenesis remain unclear.ResultsApplying histological staining to Pten-null mouse prostates, we observed that deletion of Klf5 significantly increased the number of microvessels, accompanied by the upregulation of multiple angiogenesis-related genes based on microarray analysis with MetaCore software. In human umbilical vein endothelial cells (HuVECs), tube formation and migration, both of which are indicators of angiogenic activities, were decreased by conditioned media from PC-3 and DU 145 human prostate cancer cells with KLF5 overexpression, but increased by media from cells with KLF5 knockdown. HIF1α, a key angiogenesis inducer, was upregulated by KLF5 loss at the protein but not the mRNA level in both mouse tissues and human cell lines, as determined by immunohistochemical staining, real-time RT-PCR and Western blotting. Consistently, KLF5 loss also upregulated VEGF and PDGF, two pro-angiogenic mediators of HIF1α function, as analyzed by immunohistochemical staining in mouse tissues and ELISA in conditioned media. Mechanistically, AKT activity, which caused the accumulation of HIF1α, was increased by KLF5 knockout or knockdown but decreased by KLF5 overexpression. PI3K/AKT inhibitors consistently abolished the effects of KLF5 knockdown on angiogenic activity, HIF1α accumulation, and VEGF and PDGF expression.ConclusionKLF5 loss enhances tumor angiogenesis by attenuating PI3K/AKT signaling and subsequent accumulation of HIF1α in PTEN deficient prostate tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0365-6) contains supplementary material, which is available to authorized users.

Role of Pneumococcal Autolysin for KLF4 Expression and Chemokine Secretion in Lung Epithelium.

In severe pneumococcal pneumonia, the delicate balance between a robust inflammatory response necessary to kill bacteria and the loss of organ function determines the outcome of disease. In this study, we tested the hypothesis that Krueppel-like factor (KLF) 4 may counter-regulate Streptococcus pneumoniae-related human lung epithelial cell activation using the potent proinflammatory chemokine IL-8 as a model molecule. Pneumococci induced KLF4 expression in human lung, in primary human bronchial epithelial cells, and in the lung epithelial cell line BEAS-2B. Whereas proinflammatory cell activation depends mainly on the classical Toll-like receptor 2-mitogen-activated protein kinase or phosphatidylinositide 3-kinase and NF-κB pathways, the induction of KLF4 occurred independently of these molecules but relied, in general, on tyrosine kinase activation and, in part, on the src kinase family member yamaguchi sarcoma viral oncogene homolog (yes) 1. The up-regulation of KLF4 depended on the activity of the main pneumococcal autolysin LytA. KLF4 overexpression suppressed S. pneumoniae-induced NF-κB and IL-8 reporter gene activation and release, whereas small interfering RNA-mediated silencing of KLF4 or yes1 kinase led to an increase in IL-8 release. The KLF4-dependent down-regulation of NF-κB luciferase activity could be rescued by the overexpression of the histone acetylase p300/cAMP response element-binding protein-associated factor. In conclusion, KLF4 acts as a counter-regulatory transcription factor in pneumococci-related proinflammatory activation of lung epithelial cells, thereby potentially preventing lung hyperinflammation and subsequent organ failure.

Krüppel-like factor 4 is a transcriptional regulator of M1/M2 macrophage polarization in alcoholic liver disease.

Macrophages play an important role in inflammation and liver injury. In ALD, activated macrophages, including M1 (proinflammatory) and M2 (anti-inflammatory) macrophages, are present in the liver. As KLF4 has been described as a regulator of macrophage polarization, we investigated its role in ALD. Chronic alcohol feeding in C57Bl/6 mice led to increased expression of M1 (TNF-α, MCP1, and IL-1β) and M2 (Arg1, Mrc1, and IL-10) genes and the frequency of CD206+CD163+ M2 macrophages in the liver. KLF4 mRNA and protein levels were increased in the livers of EtFed compared with PF mice. In macrophages, in vivo and in vitro, EtOH increased KLF4 levels, transcriptional activity, and expression of M1 and M2 genes. KLF4 knockdown and overexpression experiments demonstrated alcohol-dependent and -independent functions of KLF4 in regulating M1 and M2 markers. KLF4 siRNA treatment, alone and in synergy with alcohol, increased the levels of M1 markers. In contrast, KLF4 overexpression increased the levels of M2 and decreased M1 markers, and this was enhanced further by alcohol. KLF4 was regulated by alcohol and its metabolites. KLF4 mRNA and activity were increased in the presence of 4-MP, an inhibitor of ADH, and CYP2E1. However, inhibition of acetaldehyde breakdown attenuated KLF4 induction and promoted M1 polarization. We conclude that KLF4 regulates M1 and M2 markers in ALD. EtOH promotes KLF4 and M2 phenotype, whereas acetaldehyde attenuates KLF4 and promotes M1 macrophage, which may explain the increased presence of M1 and M2 macrophage populations in ALD.

The Transcription Factor KLF2 Restrains CD4+ T Follicular Helper Cell Differentiation

SummaryT follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4+ T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4+ T cells led to increased Tfh cell generation and B cell priming, while KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4+ T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization, and by regulation of lineage-defining transcription factors.

Klf2 contributes to the stemness and self-renewal of human bone marrow stromal cells.

Mesenchymal stem cells (MSCs) have shown great therapeutic potential in clinical trials; however, loss of pluripotency due to culture senescence is a major factor limiting their application. Understanding the physiology of stem cell self-renewal and stemness, and identifying the molecules that regulate these processes, are critical to future advances in tissue and organ regeneration. The Krüppel-like factor (Klf) family are key transcription factors implicated in self-renewal of embryonic stem cells. Here we identify Klf2 as a crucial transcription factor in undifferentiated human bone marrow stromal cells (hBMSCs), as indicated by gene expression in three culture media. To investigate the role of Klf2 in detail, an overexpression study using a lentiviral system in hBMSCs was performed. After Klf2 overexpression, cell proliferation was increased. The expression of pluripotency-associated genes, including Oct4, Nanog, and Rex1, was also upregulated by Klf2 overexpression. In addition, quantitative RT-PCR indicated a lower level of expression of differentiation related genes in Klf2 overexpressing cells as compared to control cells. Our results identify a functionally conserved role for Klf2 in hBMSCs, in which its expression is biologically important for stemness and self-renewal. These results are the first to show a role for Klf2 in the proliferation and pluripotency of hBMSCs.

The dosage of Patz1 modulates reprogramming process.

The acquisition of pluripotent cells can be achieved by combined overexpression of transcription factors Oct4, Klf4, Sox2 and c-Myc in somatic cells. This cellular reprogramming process overcomes various barriers to re-activate pluripotency genes and re-acquire the highly dynamic pluripotent chromatin status. Many genetic and epigenetic factors are essentially involved in the reprogramming process. We previously reported that Patz1 is required for maintenance of ES cell identity. Here we report that Patz1 plays an inhibitory role in OKSM-induced reprogramming process since more iPS colonies can be induced from Patz1+/− MEFs than wild type MEFs; while the addition of Patz1 significantly repressed reprogramming efficiency. Patz1+/− MEFs can surpass the senescence barrier of Ink4a/Arf locus, thus enhancing iPS colonies formation. Moreover, Patz1+/− MEFs displayed higher levels of acetylated histone H3, H3K4me2, H3K4me3, H3K36me3 and lower levels of histone H3K9me3 and HP1α, indicating that heterozygous knockout of Patz1 results in a globally open chromatin which is more accessible for transcriptional activation. However, Patz1−/− MEFs gave the lowest reprogramming efficiency which may result from cell senescence trigged by up-regulated Ink4a/Arf locus. Together, we have demonstrated that the dosage of Patz1 modulates reprogramming process via significantly influencing cell senescence, proliferation and chromatin structure.

Krüppel-like Factors KLF4 and KLF2 Regulate microRNA-150 Expression in Myeloid Leukemias

Background: Acute myeloid leukemia (AML) is characterized by increased self-renewal of leukemia stem/progenitor cells and failure of differentiation to mature myeloid cells. MicroRNAs (miRNAs) are small single stranded non-coding RNAs 19 to 24 nucleotides in length that regulate expression of tens to hundreds of genes via mRNA degradation or translational repression. MiRNA contributions to normal hematopoiesis have been described and deletion of key miRNA processing enzymes in murine and human cells suggests that miRNA loss contributes to the cancer phenotype and aberrant differentiation in leukemia.By combining observations of miRNA expression in normal hematopoietic progenitor cells and patient AML cells and high-throughput lentiviral expression library screening approaches in AML cell lines we have identified candidate miRNAs that contribute to altered proliferation and differentiation in AML cells. We have previously established 1) that miR-150 expression is decreased in a large subset of primary patient AML samples, in particular poor risk cytogenetic groups, 2) and that miR-150 re-expression induces myeloid differentiation and decreases cell proliferation of normal hematopoietic progenitor cells and AML cell lines and primary patient cells in part through downregulation of MYB expression. MiR-150 loss is relevant in other hematopoietic and solid tumor malignancies where re-expression inhibits cell proliferation, promotes apoptosis and induces reversal of endothelial to mesenchymal transition.Transcription factors are important regulators of myeloid differentiation and cell proliferation. Moreover, as highlighted by recent sequencing of the AML genome, alterations in myeloid transcription factors through mutation, gene rearrangement, and altered expression play a significant role in leukemogenesis. Consequently, we have focused on how myeloid transcription factors regulate miRNA expression, specifically for miR-150.Results: Using 5’RACE from healthy bone marrow RNA, we identified a major transcription start site at 214 basepairs upstream of the pre-miR-150 hairpin. We identified the minimal miR-150 promoter region as -266 to +259 basepairs from the major transcription start site using miR-150 promoter truncation luciferase constructs assayed in myeloid leukemia cell lines (THP-1, K562, and KG1a) and a lymphoid leukemia cell line (Jurkat). We identified DNA binding sites for the Krüppel-like factor (KLF) family of transcription factors that are necessary for miR-150 promoter activity using site-directed DNA mutagenesis of the luciferase reporters. KLFs regulate proliferation, differentiation, pluripotency, migration and inflammation. Depending on cell type and context, KLFs can function as tumor suppressors or oncogenes. To identify which KLF isoforms regulate miR-150 expression, we assayed the ability of KLFs 2, 3, 4, 5, 6, 7, 9, and 10 to induce miR-150 promoter activity using the luciferase reporters and endogenous miR-150 expression by quantitative PCR. KLF2 and KLF4 overexpression increased miR-150 promoter activity in luciferase assays 50-fold and 450-fold respectively in K562 cells. Furthermore, KLF2 and KLF4 induced endogenous miR-150 expression 20-fold and 100-fold respectively as detected by quantitative PCR in both THP-1 and K562 cells. Prior work has established that KLF2 and KLF4 regulate the differentiation of monocytes. We then confirmed that KLF2 and KLF4 overexpression promotes myeloid differentiation of THP-1 cells by flow cytometry and gene expression that was partially reversed by inhibition of miR-150 expression.Conclusions: Previous studies have determined that KLF2 and KLF4 expression are decreased or absent in a significant subset of AML cases. Our observations suggest that loss of KLF2 and KLF4 expression contributes to decreased miR-150 expression which in turn alters cell proliferation and differentiation. Other studies have implicated the cell cycle inhibitor p21WAF1/CIP1 and altered PPAR gamma signaling downstream of KLF4. Nonetheless, our mechanistic understanding is limited. Our work suggests that the loss of miR-150 and other miRNAs downstream of these transcription factors also contributes. Understanding the interactions between KLFs, miR-150 and other miRNAs has broader significance as KLF2 and KLF4 expression is altered in other hematopoietic and solid tumors. DisclosuresNo relevant conflicts of interest to declare.