KLF4 Research Articles

Identification of a Digenic Inheritance of Pklr and KLF1 Mutations Resulting in Hemolytic Anemia in a Neonate from a Study of Genotype-Phenotype Correlation of Globin, Pklr and KLF1 in Thai Patients with Pyruvate Kinase (PK) Deficiency

Introduction: Pyruvate kinase deficiency (PK def) is an inherited disorder that affects red blood cells metabolism and causes a wide range of clinical manifestation from fetal anemia, severe neonatal jaundice, and chronic transfusion dependent anemia to fully compensated hemolytic anemia. This condition is rarely reported in Southeast Asian population due to a lack of knowledge and recognition by clinicians and unavailability of laboratory for confirmation. The majority of patients with PK def result from mutations of the PKLR genes encoding this enzyme. Our group has described for the first time (Viprakasit V et al., Blood 2014) that mutations in trans of an erythroid specific transcription factor; KLF1 that regulate PKLR expression, could result in clinical presentation mimic that of classical PK def. Moreover, α and β globin mutations are highly prevalent in our region (up to 40% carrier rate) but their roles as a genetic modifier in PK def remain unknown. In addition, mutations in KLF1 seems to be highly prevalent in our population (14.6 in 1000, n= 2464 chr.), it is of interest to determine the epistatic role of α and β globin, PKLR and KLF1 mutations in patients with classical presentation of PK def.Objectives: To characterize α and β globin, PKLR and KLF 1 mutations and correlate these with clinical presentation and outcome(s) of them in patients with PK def and their family members.Methods: After obtaining informed consent, 9 index PK def patients with their available family members were evaluated for PKLR and KLF1 mutations by direct genomic sequencing as previously described. Hematological parameters and hemoglobin analyses were performed using standard techniques. DNA analysis for all common α and β globin mutations leading to thalassemia and hemoglobinopathies found in this region was performed using our standard protocol.Results: We successfully identified PKLR mutations in 7 out of 9 index patients as shown in Table 1. All patients were diagnosed with clinical PK def following several measurement of biochemical activity. We found 11 different mutations in 6 compound heterozygotes (5 of two nucleotide mutations and one with 1 missense and a large PKLR deletion) and 1 homozygote. One mutation (c.941T>C =p.I314T) seemed to be recurrent since it was found in two families; one homozygous and one compounded with N118S. We failed to identified PKLR and KLF1 in only one patient (PK-9) suggesting that this patient might have a novel molecular mechanism that remains to be found. We found only one family with globin abnormality (Hb E trait in PK-1) without any deleterious effect on patient's clinical severity. Moreover, most of PK patients (except one) have no mutation in KLF1 genes. Interestingly, the PK-3 patient (who is an offspring of the PK-2) was subsequently found to be double heterozygous of PKLR (p.V548fs) and KLF1 (G176RfsX179 (+7bp)) mutations. This patient was previously diagnosed with PK def at birth when he had serious hemolytic anemia (Hb 10 g/dL, haptoglobin < 10 IU/L) with hyperbilirubinemia requiring phototherapy, based on a very low PK enzyme activity (6.5 IU/g Hb, 20% of normal level) and the PK level from his mother was also low (11 IU/g Hb). Since then, he had an uneventful history for 6 years, except mild anemia from iron deficiency anemia during infant period. Physical examination showed no hepatosplenomegaly. Currently his baseline Hb levels were around 12-12.5 g/dL with normal reticulocyte count (1%) and hypochromic microcytosis. There is no globin abnormality in this family. The PK enzyme activity at 6-year-of age showed an improved PK activity (41% of normal activity) without further hemolysis. Our finding showed for the first time a possible interaction between PKLR and KLF1 mutations in man and this digenic inheritance seems to cause a more serious phenotype than each of these carriers alone. However, since the clinical presentation of our index case (PK-3) appeared to be apparent only at the early age, this might suggest the possible impact of KLF1 mutations that were more serious at the early age (similar to those of patients with two KLF1 mutations; KLF1 disease) and might be further compensated by other hitherto mechanisms during erythroid development.Conclusions: We found no effects of globin mutations on the clinical phenotypes of patients with classical PK def. However, a mutation in KLF1 could deteriorate clinical presentations even in a carrier of PKLR mutation at the early age. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Compound Heterozygosity for KLF1 Mutations Causing Hemolytic Anemia in Children: A Case Report and Literature Review

BackgroundAnemia is one of the most common diseases affecting children worldwide. Hereditary forms of anemia due to gene mutations are difficult to diagnose because they only rely on clinical manifestations. In regions with high prevalence of thalassemia such as southern China, pediatric patients with a hereditary hemolytic anemia (HHA) phenotype are often diagnosed with β-thalassemia. However, HHA can be caused by other gene defects. Here, a case previously diagnosed with thalassemia in a local hospital was sent to our laboratory for further genetic diagnosis. Preliminary molecular testing did not identify any mutations in globin genes.MethodsAll blood samples were collected after informed consent had been obtain from the proband’s parents. Both clinical and genetic analyses were conducted for the patient and her family members, including clinical data collection and sequencing of the KLF1 gene. Relevant literature was reviewed, including genetically confirmed cases with well-documented clinical summaries.ResultsBased on the detailed clinical data for this case, we diagnosed the patient with severe HHA. Sanger sequencing confirmed that there was a mutation on each KLF1 allele in the proband, which is missense mutation c.892G > C (p.Ala298Pro) inherited from father and frameshift mutation c.525_526insCGGCGCC (p.Gly176Argfs∗179) from the mother, respectively. A summary of the KLF1 mutation spectrum and a clarification of genotype–phenotype correlation were performed through a combined analysis of the case and literature studies.ConclusionThis study corrected the misdiagnosis and identified the etiology in a Chinese patient with HHA. Identification of the disease-causing gene is important for the treatment and care of the patient and prevention of another affected childbirth in her family. In addition, this study provided insight to better distinguish HHA patients with β-thalassemia mutations from those with KLF1 mutations.

A Potential Role for Genes within the mTORC1 Pathway in the Regulation of Embryonic Globin Expression Revealed By ChIP-Seq, RNA-Seq and eQTL Mapping in the Nan Anemia Mouse Model

The Nan mouse model carries a mutation in the gene encoding the erythroid-specific transcription factor, Klf1 (Krüppel-like factor 1). KLF1 regulates the terminal development of erythroid cells via the transcriptional regulation of a diverse set of genes. We recently showed by ChIP-seq and RNA-seq that Nan-KLF1 fails to bind a subset of normal KLF1 gene targets and also ectopically binds a large set of genes not normally engaged by KLF1. The result is a corrupted transcriptome that produces anemia and a ~100-fold increase in βh1 expression in adult Nan spleen and bone marrow. Genetic modifiers regulating βh1 expression in mice can potentially be translated to the regulation of human fetal γ-globin expression and identify new drug targets for the treatment of Sickle Cell Disease and β-thalassemia, as increased fetal globin ameliorates disease symptoms. We identified genetic loci modifying βh1 expression by eQTL (expression quantitative trait locus/loci) mapping using two congenic Nan strains intercrossed with C57BL/6J. We identified several significant and suggestive eQTL in the two crosses, indicating that multiple genes regulate βh1 expression in adult Nan spleen. Notably, no previously described repressors of γ-globin expression mapped within the eQTL 95% confidence intervals. By integrating genes falling within eQTL confidence intervals on Chr 7, 4, 11, and 17 with ChIP-seq data obtained using an immortalized Klf1 -null fetal erythroid cell line (K1) expressing either WT or Nan Klf1, RNA-seq data from sorted WT and Nan adult spleen erythroid precursors (pro-, baso-, poly- and orthochromatic erythroblasts), and available functional annotations (DAVID and Ingenuity pathway analysis), we identified several strong candidate genes. Among them, several genes encoding components of the mTORC1 pathway are located in eQTL confidence intervals and/or have altered binding by Nan-KLF1 resulting in a dramatic decrease of their expression in Nan. The mTORC1 pathway regulates various cellular functions upon activation by nutrients and growth factors. In prior studies, decreased Mtor (mechanistic target of rapamycin) or Rptor (regulatory associated protein of MTOR complex) induced increased expression of fetal globin, providing compelling rationale that additional genes in this pathway may induce a significant increase in βh1 expression in Nan . In our eQTL dataset, Rptor, Tsc2 (tuberous sclerosis 2), and Mlst8 (MTOR associated protein, LST8 homolog), three genes that belong to the mTORC1 pathway, are located near eQTL peaks on chromosomes 11 and 17. Nan-KLF1 fails to bind Rragd (Ras-related GTP binding),a target of KLF1 required for mTOR activation, resulting in a dramatic and progressive decrease in its expression in Nan terminally differentiating erythroid precursors (6.4, 169, 1910, and 290 fold decrease during maturation from pro- to orthochromatic erythroblasts). Rptor expression is decreased 4.1 and 10.6 fold in polychromatic and orthochromatic erythroblasts, respectively. Interestingly, the top gene ontology terms annotated for the differentially expressed genes include translation, ribosome biogenesis, redox mechanisms, cell cycle control and DNA repair. These functions are regulated by the mTORC1 pathway and strongly suggest that the Nan phenotype is influenced by its downregulation. We conclude that (1) several genes from the mTORC1 pathway, including genes not previously described, are strong candidates that may contribute simultaneously to increased βh1 expression in Nan and (2) functions regulated by mTORC1 are strongly dysregulated by the Nan mutation and reinforce the hypothesis that this pathway plays a key role in the Nan phenotype. The identification of additional modifiers of βh1 in Nan that can be translated to human HbF may provide new therapeutic opportunities. Moreover, given the role of nutrition in the regulation of the mTORC1 pathway, new nutritional guidelines for patients with Sickle Cell Disease and β-thalassemia may emerge. DisclosuresNo relevant conflicts of interest to declare.

Vitamin K3 (menadione) Induces Cytotoxicity in Chronic Myeloid Leukemia Stem Cells By Upregulating DYRK2 through the Inhibition of SIAH2 Ubiquitin Ligase

Leukemic stem cells (LSCs) generated by the transformation of normal hematopoietic stem cells (HSCs) by BCR-ABL are elusive targets that can initiate and sustain leukemia owing to their unique capacity to regenerate themselves during cell division (self-renewal) and differentiate into a bulk of leukemia cells. Although chronic myeloid leukemia (CML) can be successfully managed using tyrosine kinase inhibitors (TKIs), drug discontinuation trials have shown that 30-60% of patients, depending on the drug used for first- and second-line treatment, can remain in treatment-free remission. Thus, discontinuation is still considered experimental and there are not safety guidelines to stop treatment. A better understanding of self-renewal mechanisms in LSCs will support the development of LSC-specific drugs to induce treatment-free remission in chronic-phase patients by eradicating BCR-ABL-positive LSCs. We recently discovered in mouse models that deletion of the Klf4 gene encoding for the transcription factor KLF4 severely abrogated progression of BCR-ABL(p210)-induced CML by impairing survival and self-renewal of LSCs but not normal hematopoietic stem cells (HSCs). Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated DYRK2 protein levels, which was associated with p53-mediated apoptosis and inhibition of self-renewal via depletion of c-Myc protein in LSCs and not in normal HSCs. Here, we report that this mechanism is active in bone marrow cells from chronic-phase CML patients and that stabilization of DYRK2 protein by inhibition of the ubiquitin E3 ligase with vitamin K3 (VK3) has the potential therapeutic approach. Treatment of CML cell lines and bone marrow cells from CML patients with VK3 induced dose-dependent cytotoxicity and apoptosis. This was correlated with increased DYRK2 expression, depletion of c-Myc, and activation of p53-mediated apoptosis. Most importantly, VK3 induced DYRK2 expression and abrogated capacity of purified CD34+ CML stem cells, but not normal CD34+ cells, to generate colonies in methylcellulose assays. Cytotoxicity of VK3 largely depended on the expression of DYRK2, the reduced form menadiol inhibited cell viability similarly to menadione (VK3), and the presence of p53 enhanced cytotoxicity by inducing apoptosis in addition to inhibition of self-renewal. VK3 augmented cytotoxicity of Imatinib in CML cell lines and inhibited cell survival in Imatinib-resistant CML cells. The ability to target CML LSCs was confirmed by in vivo administration of VK3 in BCR-ABL induced mouse model followed by transplantation of leukemic bone marrow in secondary recipient mice. Altogether, our results suggest that stabilization of DYRK2 by inhibition of SIAH2 can induce apoptosis in bulk leukemia cells and LSCs by activating p53 and depleting c-Myc. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Krüppel-Like Factor 15 Expression by Herpes Simplex Virus Type 1 or Bovine Herpesvirus 1 Productive Infection.

Expression of Krüppel-like factor 15 (KLF15), a stress-induced transcription factor, is induced during bovine herpesvirus 1 (BoHV-1) reactivation from latency, and KLF15 stimulates BoHV-1 replication. Transient transfection studies revealed that KLF15 and glucocorticoid receptor (GR) cooperatively transactivate the BoHV-1-immediate-early transcription unit 1 (IEtu1), herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0), and ICP4 promoters. The IEtu1 promoter drives expression of bICP0 and bICP4, two key BoHV-1 transcriptional regulatory proteins. Based on these studies, we hypothesized infection is a stressful stimulus that increases KLF15 expression and enhances productive infection. New studies demonstrated that silencing KLF15 impaired HSV-1 productive infection, and KLF15 steady-state protein levels were increased at late stages of productive infection. KLF15 was primarily localized to the nucleus following infection of cultured cells with HSV-1, but not BoHV-1. When cells were transfected with a KLF15 promoter construct and then infected with HSV-1, promoter activity was significantly increased. The ICP0 gene, and to a lesser extent, bICP0 transactivated the KLF15 promoter in the absence of other viral proteins. In contrast, BoHV-1 or HSV-1 encoded VP16 had no effect on KLF15 promoter activity. Collectively, these studies revealed that HSV-1 and BoHV-1 productive infection increased KLF15 steady-state protein levels, which correlated with increased virus production.

FoxO-KlLF5 Pathway Switches the Flow of Macronutrients Under the Control of Insulin

KLF15 is a transcription factor that plays an important role in the activation of gluconeogenesis from amino acids as well as the suppression of lipogenesis from glucose. Although it is well known that hepatic Klf15 expression is elevated during fasting, its regulatory mechanisms have not been elucidated. Here we identified the transcription start site of liver-specific KLF15 transcript and showed that FoxO1 and FoxO3 (FoxOs) transcriptionally regulate Klf15 gene expression by directly binding to the liver-specific Klf15 promoter. To achieve this, we performed a precise in vivo promoter analysis using “in vivo Ad-luc” analytical system, combined with the genome-wide screening method “TFEL scan”, using our original cDNA library named Transcription Factor Expression Library (TFEL), which covers nearly all the transcription factors in the mouse genome. Hepatic Klf15 expression is significantly increased by the attenuation of the insulin signaling in STZ-induced insulin deficiency and insulin receptor knockdown models. In addition, we have found that FoxOs elevate the expression levels of amino acid catabolic enzymes via KLF15, resulting in accelerated amino acid metabolism during fasting. Based on these findings, we conclude that the FoxO-KLF15 pathway switches the macronutrient flow in the liver under the control of insulin.

KLF11 deficiency exacerbates renal damage in experimental unilateral ureteral obstruction

KLFs (Kruppel-like factors) comprise a family of over 17 distinct transcription factors that have been shown to regulate cell metabolism and renal function, including podocyte differentiation and maintenance of the glomerular filtration barrier. In non-renal systems, KLF11 has been shown to regulate fibrosis. However, no previous studies have established a potential role for KLF11 in renal pathophysiology. We sought to test the hypothesis that KLF11 regulates renal fibrosis in unilateral ureteral obstruction, a well-established murine model of renal fibrosis. Mice with homozygous deletion of the KLF11 gene (KO) and wild-type (WT) mice were subjected to unilateral ureteral obstruction (UUO; n=8 per group); sham controls (n=4 per group) were subjected to manipulation of the ureter without ligation. Kidneys were harvested at 9 days following surgery,. Semiquantitative histopathologic analysis of interstitial fibrosis, tubular atrophy, and interstitial inflammation was performed in a blinded fashion. Analysis of gene expression was performed on renal cortex using Pathway Detect RNA arrays and by RNASeq. Histopathologic analysis of sham controls revealed no significant alterations. However, expression of several elements of the TGF-beta signaling pathway was significantly higher in KLF11 KO sham mice, compared to WT mice, including TGF-beta receptors type I and type II, Smad2, Smad 7, Bmpr1B, and Bmpr2R, and the KLF-related transcription factor Sp1 (all p<0.05). In mice subjected to UUO, interstitial fibrosis, tubular atrophy, and interstitial inflammation were significantly greater in KLF11 KO mice compared to WT mice. Interstitial inflammation was associated with influx of alternatively activated macrophages, as assessed by quantitative analysis of CD206+ infiltrating cells (p<0.05); no significant increase in CD3+ T cell numbers were noted. Expression of several members of the TGF-beta signaling pathway were significantly increased in KLF11 KO UUO mice, compared to WT UUO mice, including TGF-beta 1, TGF-beta-2, and TGF-beta3, TGF-beta receptors type I and type II, and Smad 1,2,3,4, and 5 (all p<0.05). Conversely, expression of the transcription factor JunB was markedly increased in WT UUO mice compared to WT sham mice (12.2 -fold; p<0.0001). However, JunB expression was markedly decreased in KLF11 KO mice (both sham and UUO). Although MMP expression was not significantly different in KO versus WT mice, TIMP3 expression was higher in KO mice compared to WT mice. In conclusion, we found that renal damage is exacerbated in KLF11 KO mice subjected to UUO, compared to WT UUO mice and is associated with influx of potentially pro-fibrotic, alternatively activated M2 macrophages. KLF11 appears to be a critical regulator of TGF-beta signaling, as KLF11 deficiency is associated with increased expression of TGF-beta and signaling intermediates of this fibrogenic pathway.

Gluconeogenic signals regulate hepcidin gene expression via a CRBN-KLF15 axis

Hepcidin (HAMP) is synthesized in the liver. It is a key iron-regulatory hormone that controls systemic iron homeostasis. Cereblon (CRBN) and Kruppel-like factor 15 (KLF15) are known to regulate diverse physiological functions. In this study, we investigated the role of CRBN on hepatic hepcidin gene expression and production under gluconeogenic stimuli. Fasted mice as well as forskolin (FSK)- and glucagon (GLU)-treated mice had reduced serum iron levels but increased expression levels of hepatic Crbn and Klf15 and hepcidin secretion. MicroRNA (miRNA) expression analysis of fasted and Ad-Crbn-infected mice revealed significant reduction of microRNA-639 (miR-639). Hepatic overexpression of Crbn elevated hepcidin expression and production along with Klf15 gene expression, whereas knockdown of Crbn and Klf15 markedly decreased FSK- and fasting-mediated induction of hepcidin gene expression and its biosynthesis in mouse livers and primary hepatocytes. Moreover, expression of KLF15 significantly increased the activity of hepcidin reporter gene. It was exclusively dependent on the KLF15-binding site identified within the hepcidin gene promoter. Overall, this study demonstrates that CRBN and KLF15 are novel mediators of gluconeogenic signal-induced hepcidin gene expression and production. Thus, CRBN and KLF15 might be novel potential therapeutic targets to intervene metabolic dysfunction.

An Intact Krüppel-like factor 9 Gene Is Required for Acute Liver Period 1 mRNA Response to Restraint Stress.

The clock protein period 1 (PER1) is a central component of the core transcription-translation feedback loop governing cell-autonomous circadian rhythms in animals. Transcription of Per1 is directly regulated by the glucocorticoid (GC) receptor (GR), and Per1 mRNA is induced by stressors or injection of GC. Circulating GCs may synchronize peripheral clocks with the central pacemaker located in the suprachiasmatic nucleus of the brain. Krüppel-like factor 9 (KLF9) is a zinc finger transcription factor that, like Per1, is directly regulated by liganded GR, and it associates in chromatin at clock and clock-output genes, including at Per1. We hypothesized that KLF9 modulates stressor-dependent Per1 transcription. We exposed wild-type (WT) and Klf9 null mice (Klf9-/-) of both sexes to 1 hour restraint stress, which caused similar 2- to 2.5-fold increases in plasma corticosterone (B) in each genotype and sex. Although WT mice of both sexes showed a 2-fold increase in liver Per1 mRNA level after restraint stress, this response was absent in Klf9-/- mice. However, injection of B in WT and Klf9-/- mice induced similar increases in Per1 mRNA. Our findings support that an intact Klf9 gene is required for liver Per1 mRNA responses to an acute stressor, but a possible role for GCs in this response requires further investigation.

KLF5 Is Activated by Gene Amplification in Gastric Cancer and Is Essential for Gastric Cell Proliferation.

Gastric cancer is the third leading cause of cancer death worldwide. In this study, we tried to clarify the function of KLF5 in gastric cancer. Copy number variation (CNV) and the expression of KLF5 were interrogated in public datasets. The clinical significance of KLF5 amplification and gene expression in gastric cancer were evaluated. The function of KLF5 in cell proliferation was studied in gastric cancer cell lines and organoids. We found that KLF5 amplification mainly occurred in the chromosome instable tumors (CIN) and was significantly associated with TP53 mutation. In addition, higher KLF5 expression correlated with more locally invasive gastric cancer and higher T stage. Next, a KLF5 gene expression signature was curated. The genes in the signature were involved in cell development, cell cycle regulation, cell death, suggesting potential roles played by KLF5. Functional studies using siRNAs revealed that KLF5 was essential for the proliferation of gastric cancer cells. Finally, using gastric organoid models, we revealed that the proliferation of organoids was significantly inhibited after the down regulation of KLF5. Our study revealed that KLF5 was amplified and over-expressed in gastric cancer, and it may play an oncogene-like role in gastric cancer by supporting cell proliferation.

KLF4, a Key Regulator of a Transitive Triplet, Acts on the TGF-β Signaling Pathway and Contributes to High-Altitude Adaptation of Tibetan Pigs.

Tibetan pigs are native mammalian species on the Tibetan Plateau that have evolved distinct physiological traits that allow them to tolerate high-altitude hypoxic environments. However, the genetic mechanism underlying this adaptation remains elusive. Here, based on multitissue transcriptional data from high-altitude Tibetan pigs and low-altitude Rongchang pigs, we performed a weighted correlation network analysis (WGCNA) and identified key modules related to these tissues. Complex network analysis and bioinformatics analysis were integrated to identify key genes and three-node network motifs. We found that among the six tissues (muscle, liver, heart, spleen, kidneys, and lungs), lung tissue may be the key organs for Tibetan pigs to adapt to hypoxic environment. In the lung tissue of Tibetan pigs, we identified KLF4, BCL6B, EGR1, EPAS1, SMAD6, SMAD7, KDR, ATOH8, and CCN1 genes as potential regulators of hypoxia adaption. We found that KLF4 and EGR1 genes might simultaneously regulate the BCL6B gene, forming a KLF4–EGR1–BCL6B complex. This complex, dominated by KLF4, may enhance the hypoxia tolerance of Tibetan pigs by mediating the TGF-β signaling pathway. The complex may also affect the PI3K-Akt signaling pathway, which plays an important role in angiogenesis caused by hypoxia. Therefore, we postulate that the KLF4–EGR1–BCL6B complex may be beneficial for Tibetan pigs to survive better in the hypoxia environments. Although further molecular experiments and independent large-scale studies are needed to verify our findings, these findings may provide new details of the regulatory architecture of hypoxia-adaptive genes and are valuable for understanding the genetic mechanism of hypoxic adaptation in mammals.

Krüppel-Like Factor 15 Modulates CXCL1/CXCR2 Signaling-Mediated Inflammatory Response Contributing to Angiotensin II-Induced Cardiac Remodeling.

Inflammation is involved in cardiac remodeling. In response to pathological stimuli, activated cardiac fibroblasts (CFs) secreting inflammatory cytokines and chemokines play an important role in monocyte/macrophage recruitment. However, the precise mechanism of CF-mediated inflammatory response in hypertension-induced cardiac remodeling remains unclear. In the present study, we investigated the role of transcription factor Krüppel-like factor 15 (KLF15) in this process. We found that KLF15 expression decreased while chemokine CXCL1 and its receptor CXCR2 expression increased in the hearts of angiotensin II (Ang II)-infused mice. Compared to the wild-type mice, KLF15 knockout (KO) mice aggravated Ang II-induced cardiac hypertrophy and fibrosis. Deficiency of KLF15 promoted macrophage accumulation, increase of CXCL1 and CXCR2 expression, and mTOR, ERK1/2, NF-κB-p65 signaling activation in the hearts. Mechanistically, Ang II dose- dependently decreased KLF15 expression and increased CXCL1 secretion from cardiac fibroblasts but not cardiac myoblasts. Loss- or gain-of-function studies have shown that KLF15 negatively regulated CXCL1 expression through its transactivation domain (TAD). Intriguingly, the adenovirus-mediated full length of KLF15—but not KLF15 with TAD deletion overexpression—markedly prevented pathological change in Ang II-infused mice. Notably, the administration of CXCR2 inhibitor SB265610 reversed KLF15 knockout-mediated aggravation of cardiac dysfunction, remodeling, and inflammation induced by Ang II. In conclusion, our study identifies that KLF15 in cardiac fibroblasts negatively regulates CXCL1/CXCR2 axis-mediated inflammatory response and subsequent cardiac remodeling in hypertension.

Evaluation of miR-107, DAPK1, and KLF4 Expression in Colorectal Tumors and Effect of Oxaliplatin and 5-FU on their Levels in Colorectal Cancer Cell Lines

Background: In recent years, the role of micro-RNAs in the cancer pathophysiology has attracted a great deal of scientific attention. MiRNAs regulate a variety of cellular functions, such as apoptosis, differentiation and migration by targeting oncogenic or tumor suppressor genes. We conducted the current study to assess the expression of miR-107, Kruppel-like factor 4 (KLF4) and death-associated protein kinase (DAPK1) genes in malignant and normal colon tissues and also colorectal cancer (CRC) model cells exposed to oxaliplatin and 5-FU chemotherapy agents. Method: In this case-control study, the tissue samples from CRC patients were collected during colonoscopy process in 2013 -2016 at Imam Reza hospital. Subsequently, the expression levels of miR-107, KLF4, and DAPK1 were detected with quantitative Real-Time PCR. Furthermore, in the in vitro phase of this study, we investigated the changes in the expression level of miR-107, KLF4 and DAPK1 transcripts after oxaliplatin and 5-FU treatment. Results: Unlike miR-107, the expression levels of KLF4 and DAPK1 genes decreased in the tumor samples compared to those in the marginal specimens. In addition, both oxaliplatin and 5-FU significantly increased the expression level of miR-107. There were significant correlations between the expression levels of miR-107, KLF4, and DAPK1genes and clinicopathological features, for instance lymph node metastasis and cell differentiation. Conclusion: The current study suggested a tumor suppressor role for KLF4 and DAPK1 in CRC. The altered expression of miR-107, KLF-4, and DAPK1 genes in CRC tumors and healthy tissues could be utilized for CRC diagnosis and prognosis. Furthermore, the studied genes could be considered as potential therapeutic targets in CRC.

The Effect of hsa-miR-451b Knockdown on Biological Functions of Gastric Cancer Stem-Like Cells.

Numerous researches have extensively studied factors such as microRNAs that lead to cancer. Thus, the current study's purpose is to investigate the biological consequences of hsa-miR-451b inhibition on the properties and functions of gastric cancer stem-like cells. First, gastric cancer stem-like cells were transfected by hsa-miR-451b inhibitor then we used real-time RT-PCR to evaluate its effect on the expression of hsa-miR-451b and two of its direct target genes, Stemness markers such as KLF4, SOX2, CD44, OCT3/4 and NANOG genes and finally Akt, PI3K, Bcl-2, Bax, CASP3 and PCNA genes involved in apoptosis. Here, we conducted a DNA Laddering assay to investigate apoptosis.The level of the MMP-2 and -9 Activities and Migration were examined by Zymography and Transwell invasion assay. HUVEC cells were used to investigate angiogenesis. The outcomes revealed that the level of the MMP-2 and -9 Activities, migration and angiogenesis decreased, but apoptosis was induced by inhibiting hsa-miR-451b. Evaluating KREMEN1 and CASK expression showed that the former increased, and the latter dropped under hsa-miR-451b inhibition. Also, upregulation of the KLF4 and SOX2 and downregulation of the CD44, OCT3/4, and NANOG decreased Self-renewal ability of gastric cancer stem cells under hsa-miR-451b inhibition. Even, under hsa-miR-451b inhibition, downregulation of Akt, PI3K, Bcl-2 and PCNA as well as upregulation of Bax and CASP3 revealed a movement towards apoptosis in MKN-45 stem-like cells. In summary, hsa-miR-451b is an oncomir in the carcinogenesis of gastric cancer stem-like cells and may be suggested as an appropriate therapeutic target for future gastric cancer treatment.

KLF15 Loss-of-Function Mutation Underlying Atrial Fibrillation as well as Ventricular Arrhythmias and Cardiomyopathy

Atrial fibrillation (AF) represents the most common type of clinical cardiac arrhythmia and substantially increases the risks of cerebral stroke, heart failure and death. Accumulating evidence has convincingly demonstrated the strong genetic basis of AF, and an increasing number of pathogenic variations in over 50 genes have been causally linked to AF. Nevertheless, AF is of pronounced genetic heterogeneity, and the genetic determinants underpinning AF in most patients remain obscure. In the current investigation, a Chinese pedigree with AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy was recruited. Whole exome sequencing and bioinformatic analysis of the available family members were conducted, and a novel heterozygous variation in the KLF15 gene (encoding Krüppel-like factor 15, a transcription factor critical for cardiac electrophysiology and structural remodeling), NM_014079.4: c.685A>T; p.(Lys229*), was identified. The variation was verified by Sanger sequencing and segregated with autosomal dominant AF in the family with complete penetrance. The variation was absent from 300 unrelated healthy subjects used as controls. In functional assays using a dual-luciferase assay system, mutant KLF15 showed neither transcriptional activation of the KChIP2 promoter nor transcriptional inhibition of the CTGF promoter, alone or in the presence of TGFB1, a key player in the pathogenesis of arrhythmias and cardiomyopathies. The findings indicate KLF15 as a new causative gene responsible for AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy, and they provide novel insight into the molecular mechanisms underlying cardiac arrhythmias and hypertrophic cardiomyopathy.

Doxorubicin-induced senescence promotes stemness and tumorigenicity in EpCAM-/CD133- nonstem cell population in hepatocellular carcinoma cell line, HuH-7.

The therapeutic induction of senescence is a potential means to treat cancer, primarily acting through the induction of a persistent growth‐arrested state in tumors. However, recent studies have indicated that therapy‐induced senescence (TIS) in tumor cells allows for the prolonged survival of a subgroup of cells in a dormant state, with the potential to re‐enter the cell cycle along with an increased stemness gene expression. Residual cells after TIS with increased cancer stem cell phenotype may have profound implications for tumor aggressiveness and disease recurrence. Herein, we investigated senescence‐associated stemness in EpCAM+/CD133+ liver cancer stem cell and EpCAM−/CD133− nonstem cell populations in HuH7 cell line. We demonstrated that treatment with doxorubicin induces senescence in both cell populations, accompanied by a significant increase in the expression of reprogramming genes SOX2, KLF4, and c‐MYC as well as liver stemness‐related genes EpCAM, CK19, and ANXA3 and the multidrug resistance‐related gene ABCG2. Moreover, doxorubicin treatment significantly increased EpCAM + population in nonstem cells indicating senescence‐associated reprogramming of nonstem cell population. Also, Wnt/β‐catenin target genes were increased in these cells, while inhibition of this signaling pathway decreased stem cell gene expression. Importantly, Dox‐treated EpCAM−/CD133− nonstem cells had increased in vivo tumor‐forming ability. In addition, when SASP‐CM from Dox‐treated cells were applied onto hİPSC‐derived hepatocytes, senescence was induced in hepatocytes along with an increased expression of TGF‐β, KLF4, and AXIN2. Importantly, SASP‐CM was not able to induce senescence in Hep3B‐TR cells, a derivative line rendered resistant to TGF‐β signaling. Furthermore, ELISA experiments revealed that the SASP‐CM of Dox‐treated cells contain inflammatory cytokines IL8 and IP10. In summary, our findings further emphasize the importance of carefully dissecting the beneficial and detrimental aspects of prosenescence therapy in HCC and support the potential use of senolytic drugs in HCC treatment in order to eliminate adverse effects of TIS.

Genome-wide identification, evolution of Krüppel-like factors (klfs) and their expressions during GCRV challenge in grass carp (Ctenopharyngodonidella)

The Krüppel-like factors (KLFs) are a family of transcription factors containing three highly conserved tandem zinc finger structures, and each member participates in multiple physiological and pathological processes. The publication of genome sequences and the application of bioinformatics tools have led to the discovery of numerous gene families in fishes. Here, 24 klf genes were re-annotated in grass carp. Subsequently, the number of klf family members were investigated in some representative vertebrate species. Then, a series of bioinformatics analysis showed that grass carp klfs in the same subfamily had similar genome structure patterns and conserved distribution patterns of motifs, which supported their molecular evolutionary relationships. Furthermore, the mRNA expression profiles showed that 24 grass carp klfs were ubiquitously expressed in 11 different tissues, and some of them displayed tissue-enriched expression patterns. Finally, the expressions of the evolutionarily expanded klf members (klf2a, 2b, 2l, 5a, 5b, 5l, 6a, 6b, 7a, 7b, 11a, 11b, 12a, 12b, 15 and 15l) during GCRV infection were also analyzed. The results suggested that grass carp klf genes with common evolutionary sources may share functional diversity and conservation. In conclusion, this study provides preliminary clues for further researches on grass carp klf members and their underlying transcriptional regulatory mechanisms during GCRV infection.

Response of Human Retinal Pigment Epithelial Cells to the Effect of the Conditioned Media of Newt Retinal Regenerates

The development of the retina and the signaling pathways involved in this process are conserved among vertebrates. Therefore, it was hypothesized that active factors stimulating the retinal regeneration in newts can cause transdifferentiation of human retinal pigment epithelial (RPE) cells. To verify this, we studied the effect of conditioned media (CM) obtained from in vitro regenerating retinas of newts on the phenotype, expression of a number of genes, and localization of cell proteins of human RPE cells of the ARPE-19 line. Within 120 h of cultivation, a change in the morphology of human RPE cells was observed, which manifested in a change in shape and an increase in cell size, which was accompanied by a decrease in proliferative activity. 24 hours after exposure to CM, ARPE-19 showed a short-term increase in the expression of TUBB3 (a panneuronal marker) and a decrease in the expression of BMP2 and BMP4 (neural diffe-rentiation inhibitors). Using immunocytochemical (ICC) methods, we observed an increase in the intensity of staining of cells with antibodies to βIII-tubulin protein and a decrease in the intensity of staining with antibodies to the Cx43 protein of gap junctions (compared to the control) during the period between 24 and 72 h after exposure. At the same time, we observed a decrease in the levels of mRNA expression of the RPE markers OTX2 and KRT18, as well as the KLF4 gene, which is responsible for proliferation and differentiation. Additionally, the shift in the distribution of β-catenin to cytoplasmic localization was recorded using the ICC method. To exclude mesenchymal (and osteogenic in particular) differentiation under the effect of CM, we studied expression of the SPP1 and RUNX2 genes by qPCR. No SPP1 transcription was found in ARPE-19 cells in the control and under the effect of CM, and a decrease in the expression level of RUNX2 mRNA under the effect of CM was observed. Therefore, an attempt to shift to neuronal differentiation was observed in ARPE-19 cells during the period between 24 and 72 h after exposure to the CM of newt retinal regenerates, as indicated by an increase in transcription and translation of βIII-tubulin, a weakening of intercellular adhesion, and a decrease in the expression of inhibitors of neural differentiation BMP2 and BMP4, as well as RPE markers OTX2 and KRT18. However, after that, due to the short-term effect of CM, during the period between 72 and 120 h after exposure, the cells returned to the initial differentiation, as evidenced by a decrease in the expression of neuronal differentiation markers (NES, TUBB3, PAX6) and an increase in the expression of RPE markers (OTX2, MITF).

Inhibition of the HEG1-KRIT1 interaction increases KLF4 and KLF2 expression in endothelial cells.

ABSTRACTThe transmembrane protein heart of glass1 (HEG1) directly binds to and recruits Krev interaction trapped protein 1 (KRIT1) to endothelial junctions to form the HEG1–KRIT1 protein complex that establishes and maintains junctional integrity. Genetic inactivation or knockdown of endothelial HEG1 or KRIT1 leads to the upregulation of transcription factors Krüppel‐like factors 4 and 2 (KLF4 and KLF2), which are implicated in endothelial vascular homeostasis; however, the effect of acute inhibition of the HEG1–KRIT1 interaction remains incompletely understood. Here, we report a high‐throughput screening assay and molecular design of a small‐molecule HEG1–KRIT1 inhibitor to uncover acute changes in signaling pathways downstream of the HEG1–KRIT1 protein complex disruption. The small‐molecule HEG1–KRIT1 inhibitor 2 (HKi2) was demonstrated to be a bona fide inhibitor of the interaction between HEG1 and KRIT1 proteins, by competing orthosterically with HEG1 through covalent reversible interactions with the FERM (4.1, ezrin, radixin, and moesin) domain of KRIT1. The crystal structure of HKi2 bound to KRIT1 FERM revealed that it occupies the same binding pocket on KRIT1 as the HEG1 cytoplasmic tail. In human endothelial cells (ECs), acute inhibition of the HEG1–KRIT1 interaction by HKi2 increased KLF4 and KLF2 mRNA and protein levels, whereas a structurally similar inactive compound failed to do so. In zebrafish, HKi2 induced expression of klf2a in arterial and venous endothelium. Furthermore, genome‐wide RNA transcriptome analysis of HKi2‐treated ECs under static conditions revealed that, in addition to elevating KLF4 and KLF2 expression, inhibition of the HEG1–KRIT1 interaction mimics many of the transcriptional effects of laminar blood flow. Furthermore, HKi2‐treated ECs also triggered Akt signaling in a phosphoinositide 3‐kinase (PI3K)‐dependent manner, as blocking PI3K activity blunted the Akt phosphorylation induced by HKi2. Finally, using an in vitro colocalization assay, we show that HKi6, an improved derivative of HKi2 with higher affinity for KRIT1, significantly impedes recruitment of KRIT1 to mitochondria‐localized HEG1 in CHO cells, indicating a direct inhibition of the HEG1–KRIT1 interaction. Thus, our results demonstrate that early events of the acute inhibition of HEG1–KRIT1 interaction with HKi small‐molecule inhibitors lead to: (i) elevated KLF4 and KLF2 gene expression; and (ii) increased Akt phosphorylation. Thus, HKi’s provide new pharmacologic tools to study acute inhibition of the HEG1–KRIT1 protein complex and may provide insights to dissect early signaling events that regulate vascular homeostasis.

Krüppel-like factor 4 promotes survival and expansion in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy of the bone marrow that affects mostly elderly adults. Alternative therapies are needed for AML patients because the overall prognosis with current standard of care, high dose chemotherapy and allogeneic transplantation, remains poor due to the emergence of refractory and relapsed disease. Here, we found expression of the transcription factor KLF4 in AML cell lines is not silenced through KLF4 gene methylation nor via proteasomal degradation. The deletion of KLF4 by CRISPR-CAS9 technology reduced cell growth and increased apoptosis in both NB4 and MonoMac-6 cell lines. Chemical induced differentiation of gene edited NB4 and MonoMac6 cells with ATRA and PMA respectively increased apoptosis and altered expression of differentiating markers CD11b and CD14. Transplantation of NB4 and MonoMac-6 cells lacking KLF4 into NSG mice resulted in improved overall survival compared to the transplantation of parental cell lines. Finally, loss-of-KLF4 did not alter sensitivity of leukemic cells to the chemotherapeutic drugs daunorubicin and cytarabine. These results suggest that KLF4 expression supports AML cell growth and survival, and the identification and disruption of KLF4-regulated pathways could represent an adjuvant therapeutic approach to increase response.