Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy.
To determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs null for Klf4 (Klf4−/−), we transfected Klf4−/−MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4+/+) and untransfected or mock-transfected Klf4−/−MEFs.
We show that overexpression of Klf4 in Klf4−/−MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4−/−MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.
Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.
KLF4; Genetic instability; DNA damage responses; Aneuploidy; Centrosome amplification; Mouse embryonic fibroblasts
Autophagy 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.
To 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.
Here 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.
Taken 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 material
The online version of this article (doi:10.1186/s12943-015-0373-6) contains supplementary material, which is available to authorized users.
Krüppel-like Factor 4; Autophagy; Apoptosis; Mammalian target of rapamycin; DNA damage; Mouse embryonic fibroblasts; Autophagy related genes
Krüppel-like factor KLF4 plays a crucial role in the development and maintenance of the mouse cornea. Here, we have compared the wild type (WT) and Klf4-conditional null (Klf4CN) corneal gene expression patterns to understand the molecular basis of the Klf4CN corneal phenotype.
Expression of more than 22,000 genes in 10 WT and Klf4CN corneas was compared by microarrays, analyzed using BRB ArrayTools and validated by Q-RT-PCR. Transient cotransfections were employed to test if KLF4 activates the aquaporin-3, Aldh3a1 and TKT promoters.
Scatter plot analysis identified 740 and 529 genes up- and down-regulated by more than 2-fold, respectively, in the Klf4CN corneas. Cell cycle activators were upregulated while the inhibitors were downregulated, consistent with the increased Klf4CN corneal epithelial cell proliferation. Desmosomal components were downregulated, consistent with the Klf4CN corneal epithelial fragility. Downregulation of aquaporin-3, detected by microarray, was confirmed by immunoblot and immunohistochemistry. Aquaporin-3 promoter activity was stimulated 7–10 fold by cotransfection with pCI-KLF4. Corneal crystallins Aldh3A1 and TKT were downregulated in the Klf4CN cornea and their respective promoter activities were upregulated 16- and 9-fold by pCI-KLF4 in co-transfections. Expression of epidermal keratinocyte differentiation markers was affected in the Klf4CN cornea. While the cornea specific keratin-12 was downregulated, most other keratins were upregulated, suggesting hyperkeratosis.
We have identified functionally diverse candidate KLF4 target genes, revealing the molecular basis of the diverse aspects of the Klf4CN corneal phenotype. These results establish KLF4 as an important node in the genetic network of transcription factors regulating the corneal homeostasis.
Cornea; Development; KLF4; Microarray
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with tumor suppressive activity in colorectal cancer. Here, we investigated whether KLF4 is involved in maintaining genetic stability in mouse embryonic fibroblasts (MEFs) isolated from mice wild type (+/+), heterozygous (+/−), or homozygous (−/−) for the Klf4 alleles. Compared to Klf4+/+ and Klf4+/− MEFs, Klf4−/− MEFs had both a higher level of apoptosis and rate of proliferation. Quantification of chromosome numbers showed that Klf4−/− MEFs were aneuploid. A higher number of Klf4−/− MEFs exhibited γ-H2AX foci and had higher amounts of γ-H2AX compared to controls. Cytogenetic analysis demonstrated the presence of numerous chromosome aberrations including dicentric chromosomes, chromatid breaks, and double minute chromosomes in Klf4−/− cells but in few, if any, Klf4+/+ or Klf4+/− MEFs. Approximately 25% of Klf4−/− MEFs exhibited centrosome amplification in contrast to the less than 5% of Klf4+/+ or Klf4+/− MEFs. Finally, only Klf4−/− MEFs were capable of anchorage-independent growth. Taken together, these findings demonstrate that MEFs null for the Klf4 alleles are genetically unstable, as evidenced by the presence of aneuploidy, chromosome aberration and centrosome amplification. The results support a crucial role for KLF4 in maintaining genetic stability and as a tumor suppressor.
aneuploidy; centrosome amplification; cell cycle; chromosome aberrations; γ-H2AX; KLF4
Krüppel-like factor 4 (Klf4, GKLF) was originally characterized as a zinc finger transcription factor essential for terminal differentiation and cell lineage allocation of several cell types in the mouse. Mice lacking Klf4 die postnatally within hours due to impaired skin barrier function and subsequent dehydration. Recently, KLF4 was also used in cooperation with other transcription factors to reprogram differentiated cells to pluripotent embryonic stem cell-like cells. Moreover, involvement in oncogenesis was also ascribed to KLF4, which is aberrantly expressed in some types of tumors such as breast, gastric and colon cancer. We previously have shown that Klf4 is strongly expressed in postmeiotic germ cells of mouse and human testes suggesting a role for Klf4 also during spermiogenesis. In order to analyze its function we deleted Klf4 in germ cells using the Cre-loxP system. Homologous recombination of the Klf4 locus has been confirmed by genomic southern blotting and the absence of the protein in germ cells was demonstrated by western blotting and immunofluorescence. Despite its important roles in several significant biological settings, deletion of Klf4 in germ cells did not impair spermiogenesis. Histologically, the mutant testes appeared normal and the mice were fertile. In order to identify genes that were regulated by KLF4 in male germ cells we performed microarray analyses using a whole genome array. We identified many genes exhibiting changed expression in mutants even including the telomerase reverse transcriptase mRNA, which is a stem cell marker. However, in summary, the lack of KLF4 alone does not prevent complete spermatogenesis.
Krüppel-like factor 4; testis; spermiogenesis; spermatogenesis; germ cell; Cre-loxP; mouse
Krüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied.
Endothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos.
This study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.
KLF2; KLF4; Gene interactions; Embryonic vascular integrity; Endothelial cell development
Klf5 plays an important role in maturation and maintenance of the mouse ocular surface. Here, we quantify WT and Klf5-conditional null (Klf5CN) corneal gene expression, identify Klf5-target genes and compare them with the previously identified Klf4-target genes to understand the molecular basis for non-redundant functions of Klf4 and Klf5 in the cornea.
Postnatal day-11 (PN11) and PN56 WT and Klf5CN corneal transcriptomes were quantified by microarrays to compare gene expression in maturing WT corneas, identify Klf5-target genes, and compare corneal Klf4- and Klf5-target genes. Whole-mount corneal immunofluorescent staining was employed to examine CD45+ cell influx and neovascularization. Effect of Klf5 on expression of desmosomal components was studied by immunofluorescent staining and transient co-transfection assays. Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases and 72 concordant decreases. PN56 Klf5CN corneas shared 241 increases and 98 decreases with those previously described in Klf4CN corneas. Xenobiotic metabolism related pathways were enriched among genes decreased in Klf5CN corneas. Expression of angiogenesis and immune response-related genes was elevated, consistent with neovascularization and CD45+ cell influx in Klf5CN corneas. Expression of 1574 genes was increased and 1915 genes decreased in WT PN56 compared with PN11 corneas. Expression of ECM-associated genes decreased, while that of solute carrier family members increased in WT PN56 compared with PN11 corneas. Dsg1a, Dsg1b and Dsp were down-regulated in Klf5CN corneas and their corresponding promoter activities were stimulated by Klf5 in transient co-transfection assays.
Differences between PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in functions of Klf5 during corneal maturation. Klf5 contributes to corneal epithelial homeostasis by regulating the expression of desmosomal components. Klf4- and Klf5-target genes are largely distinct, consistent with their non-redundant roles in the mouse cornea.
Krüppel-like factor 4 (KLF4) is a transcription factor that is abundantly expressed in various organisms from bacteria to mammals. It has been demonstrated that KLF4 regulates the expression of a wide range of genes. Analysis of KLF4 target genes reveals its diverse regulatory functions in cell growth, proliferation, differentiation, embryogenesis, and inflammation. However, the regulation of the expression of inducible heat shock protein 70 (HSP72) and heat shock cognate 70 (HSP73) by KLF4 is not defined. In our previous study, a complementary deoxyribonucleic acid microarray assay showed that KLF4 overexpression led to dramatic upregulation of HSP73 messenger ribonucleic acid (mRNA) in murine C2C12 myoblast cells, suggesting that HSP73 is a potential target gene regulated by KLF4. The effect of KLF4 on the expression of HSP72 and HSP73 was further examined by reverse transcriptase polymerase chain reaction and Western blot in KLF4-overexpressing or KLF4-deficient cells. The results showed the upregulation of the HSP73 constitutive expression by KLF4 overexpression in both C2C12 cells and murine RAW264.7 macrophages; in response to heat stress, however, few changes were observed in the levels of HSP73 by KLF4 overexpression. In addition, knockdown of endogenous KLF4 expression by morpholino antisense oligonucleotides significantly decreased both HSP73 mRNA and protein levels under normal conditions. Conversely, KLF4 had no effect on the expression of HSP72. Taken together, these findings suggest an important role for KLF4 as a novel regulator of the constitutive expression of HSP73.
KLF4; HSP73; HSP72; Gene expression
Krüppel-like factor 9 (KLF9) is a transcriptional regulator of uterine endometrial cell proliferation, adhesion and differentiation; processes essential for pregnancy success and which are subverted during tumorigenesis. The network of endometrial genes controlled by KLF9 is largely unknown. Over-expression of KLF9 in the human endometrial cancer cell line HEC-1-A alters cell morphology, proliferative indices, and differentiation, when compared to KLF9 under-expressing HEC-1-A cells. This cell line provides a unique model for identifying KLF9 downstream gene targets and signaling pathways.
HEC-1-A sub-lines differing in relative levels of KLF9 were subjected to microarray analysis to identify differentially-regulated RNAs.
KLF9 under-expression induced twenty four genes. The KLF9-suppressed mRNAs encode protein participants in: aldehyde metabolism (AKR7A2, ALDH1A1); regulation of the actin cytoskeleton and cell motility (e.g., ANK3, ITGB8); cellular detoxification (SULT1A1, ABCC4); cellular signaling (e.g., ACBD3, FZD5, RAB25, CALB1); and transcriptional regulation (PAX2, STAT1). Sixty mRNAs were more abundant in KLF9 over-expressing sub-lines. The KLF9-induced mRNAs encode proteins which participate in: regulation and function of the actin cytoskeleton (COTL1, FSCN1, FXYD5, MYO10); cell adhesion, extracellular matrix and basement membrane formation (e.g., AMIGO2, COL4A1, COL4A2, LAMC2, NID2); transport (CLIC4); cellular signaling (e.g., BCAR3, MAPKAPK3); transcriptional regulation [e.g., KLF4, NR3C1 (glucocorticoid receptor), RXRα], growth factor/cytokine actions (SLPI, BDNF); and membrane-associated proteins and receptors (e.g., CXCR4, PTCH1). In addition, the abundance of mRNAs that encode hypothetical proteins (KLF9-inhibited: C12orf29 and C1orf186; KLF9-induced: C10orf38 and C9orf167) were altered by KLF9 expression. Human endometrial tumors of high tumor grade had decreased KLF9 mRNA abundance.
KLF9 influences the expression of uterine epithelial genes through mechanisms likely involving its transcriptional activator and repressor functions and which may underlie altered tumor biology with aberrant KLF9 expression.
Krüppel-like factors (KLF) and specificity proteins (SP) constitute a family of zinc-finger-containing transcription factors that play important roles in a wide range of processes including differentiation and development of various tissues. The human genome possesses 17 KLF genes (KLF1–KLF17) and nine SP genes (SP1–SP9) with diverse functions. We used sequence similarity searches and gene synteny analysis to identify a new putative KLF gene/pseudogene named KLF18 that is present in most of the placental mammals with sequenced genomes. KLF18 is a chromosomal neighbor of the KLF17 gene and is likely a product of its duplication. Phylogenetic analyses revealed that mammalian predicted KLF18 proteins and KLF17 proteins experienced elevated rates of evolution and are grouped with KLF1/KLF2/KLF4 and non-mammalian KLF17. Predicted KLF18 proteins maintain conserved features in the zinc fingers of the SP/KLF family, while possessing repeats of a unique sequence motif in their N-terminal regions. No expression data have been reported for KLF18, suggesting that it either has highly restricted expression patterns and specialized functions, or could have become a pseudogene in extant placental mammals. Besides KLF18 genes/pseudogenes, we identified several KLF18-like genes such as Zfp352, Zfp352-like, and Zfp353 in the genomes of mouse and rat. These KLF18-like genes do not possess introns inside their coding regions, and gene expression data indicate that some of them may function in early embryonic development. They represent further expansions of KLF members in the murine lineage, most likely resulted from several events of retrotransposition and local gene duplication starting from an ancient spliced mRNA of KLF18.
Members of the Krüppel-like family of transcription factors regulate diverse developmental processes in various organs. Previously, we have demonstrated the role of Klf4 in the mouse ocular surface. Herein, we determined the role of the structurally related Klf5, using Klf5-conditional null (Klf5CN) mice derived by mating Klf5-LoxP and Le-Cre mice. Klf5 mRNA was detected as early as embryonic day 12 (E12) in the cornea, conjunctiva and eyelids, wherein its expression increased during development. Though the embryonic eye morphogenesis was unaltered in the Klf5CN mice, postnatal maturation was defective, resulting in smaller eyes with swollen eyelids that failed to separate properly. Klf5CN palpebral epidermis was hyperplastic with 7-9 layers of keratinocytes, compared with 2-3 in the wild type (WT). Klf5CN eyelid hair follicles and sebaceous glands were significantly enlarged, and the meibomian glands malformed. Klf5CN lacrimal glands displayed increased vasculature and large number of infiltrating cells. Klf5CN corneas were translucent, thicker with defective epithelial basement membrane and hypercellular stroma. Klf5CN conjunctiva lacked goblet cells, demonstrating that Klf5 is required for conjunctival goblet cell development. The number of Ki67-positive mitotic cells was more than doubled, consistent with the increased number of Klf5CN ocular surface epithelial cells. Co-ablation of Klf4 and Klf5 resulted in a more severe ocular surface phenotype compared with Klf4CN or Klf5CN, demonstrating that Klf4 and Klf5 share few if any, redundant functions. Thus, Klf5CN mice provide a useful model for investigating ocular surface pathologies involving meibomian gland dysfunction, blepharitis, corneal or conjunctival defects.
Klf5; cornea; conjunctiva; meibomian glands; lacrimal glands; eyelids; goblet cells
KLF5 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.
Applying 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.
KLF5 loss enhances tumor angiogenesis by attenuating PI3K/AKT signaling and subsequent accumulation of HIF1α in PTEN deficient prostate tumors.
Electronic supplementary material
The online version of this article (doi:10.1186/s12943-015-0365-6) contains supplementary material, which is available to authorized users.
KLF5; Angiogenesis; HIF1α; AKT; PTEN; Prostate cancer
Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription.
We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3−/− mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3
erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions.
These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression.
KLF1; KLF3; Alpha globin; Globin gene regulation; Transcription factor
The zinc finger transcription factor Krüppel-like factor 4 (KLF4) regulates numerous physiologic processes including proliferation, differentiation, and development. Studies also showed that KLF4 is involved in tumorigenesis and somatic cell reprogramming. Here we aimed to assess whether KLF4 is a prognostic indicator for colon cancer.
Levels of KLF4 were measured by immunohistochemical analysis of a tissue microarray containing 367 independent colon cancer sections. Univariate data analysis was performed in addition to construction of multivariate models with several clinicopathologic factors to evaluate KLF4 as an independent predictor of survival and cancer recurrence (disease-free survival).
Colon cancer tissues had significantly overall lower KLF4 levels compared to non-cancer tissues (P < 0.0001). Using logistic regression, a trend was noted for decreased odds of KLF4 expression in higher stages of tumors. In both univariate and multivariate analyses, KLF4 was a significant predictor of survival and recurrence.
KLF4 expression is significantly down-regulated in colon cancer and loss of KLF4 is an independent predictor of survival and recurrence.
These findings suggest that KLF4 may serve as a prognostic biomarker for colon cancer.
KLF4; Colon Cancer; Tissue microarray; Survival; Recurrence
Krüppel-like factor 8 (KLF8) plays important roles in cell cycle and oncogenic transformation. On other hand, androgen receptor (AR) is crucial in development of both androgen-dependent and independent prostatic malignancies. The aim of this study is to investigate the role of KLF8 in prostate cancer (PCa) and the relationship between KLF8 and AR.
Eight human PCa cell lines, including androgen-dependent LNCap cells and androgen-independent 22Rv1 cells, as well as human PCa samples were studied. LNCap cells and 22Rv1 cells were transfected with plasmids encoding full-length wild-type KLF8 or KLF8 shRNA. The expression of KLF8 protein was detected using Western blotting or immunohistochemical staining. Cell proliferation in vitro was measured with MTT assay, and in vivo in a xenograft nude mouse model. Yeast two-hybrid screening, co-immunoprecipitation and pull down assays were used to examine the binding of KLF8 to AR. Luciferase reporter gene assay was used to measure the transcriptional activity of the genes targeted by AR.
In 133 human PCa samples, KLF8 protein staining was observed in 92.65% (63/68) of high-grade PCa, 66.15% (43/65) of low-grade PCa, and 6.82% (3/44) of adjacent normal tissues. The expression of KLF8 was significantly associated with poorer overall survival. Overexpression of KLF8 enhanced the proliferation of both LNCap and 22Rv1 cells, while knockdown of endogenous KLF8 suppressed the proliferation. These manipulations exerted similar effects on the tumor volumes in the xenograft nude mouse model. Yeast two-hybrid screening revealed that KLF8 was a novel AR-interacting protein. With pull down assay and co-immunoprecipitation assay, we demonstrated that KLF8 bound directly to AR, and KLF8 enhanced AR target gene transcription.
The results demonstrate that KLF8 is a novel AR transcriptional co-activator that is overexpressed in PCa and may play a role in progression of hormone-refractory PCa.
Krüppel-like factor 8 (KLF8); prostate cancer; Gleason score; PSA value; androgen receptor; transcriptional co-activator
KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3H275R). This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3H275R mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3H275R mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans.
Cardiac defects are among the most common malformations in humans. Most causative genetic mutations remain unknown. To discover new causative genes important in cardiovascular development and function, we examined 1770 mice with randomly mutated genes and found a mutant with aortic valvular stenosis, and increased risk of fetal and neonatal death. Using linkage analysis and sequencing, we identified a protein-altering point mutation in the gene regulatory protein KLF3. Mice that survived into adulthood with one mutant copy of the Klf3 gene had low arterial blood pressure, enlarged hearts, and increased mortality due to heart failure. When both copies of the Klf3 gene was mutant, then embryos had heart defects, and all died before birth. KLF3 had no previously known role in heart development so to confirm these findings, we (1) knocked down klf3 expression in zebrafish embryos and (2) examined mice with a mutation that effectively eliminated the KLF3 protein. In both cases, cardiovascular dysfunction was observed. In conclusion, we have discovered that KLF3 plays diverse and important roles in cardiovascular development and function in mice. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans.
The ovarian hormones estrogen and progesterone promote uterine receptivity and successful pregnancy through their cognate receptors functioning in concert with context-dependent nuclear coregulators. Previously, we showed that the transcription factor Krüppel-like factor (KLF) 9 is a progesterone receptor (PGR) coactivator in the uterus and that mice null for Klf9 exhibit subfertility and reduced progesterone sensitivity. The highly related family member KLF13 displays increased expression in uteri of pregnant and nonpregnant Klf9 null mice and similarly regulates PGR-mediated transactivation in endometrial stromal cells. However, a uterine phenotype with loss of Klf13 has not been reported. In the present study, we demonstrate that Klf13 deficiency in mice did not compromise female fertility and pregnancy outcome. Klf13 null females had litter sizes, numbers of implanting embryos, uterine morphology, and ovarian steroid hormone production comparable to those of wild-type (WT) counterparts. Further, pregnant WT and Klf13 null females at Day Postcoitum (DPC) 3.5 had similar uterine Pgr, estrogen receptor, and Wnt-signaling component transcript levels. Nuclear levels of KLF9 were higher in Klf13 null than in WT uteri at DPC 3.5, albeit whole-tissue KLF9 protein and transcript levels did not differ between genotypes. The lack of a similar induction of nuclear KLF9 levels in uteri of virgin Klf13(−/−) mice relative to WT uteri was associated with lower stromal PGR expression. In differentiating human endometrial stromal cells, coincident KLF9/KLF13 knockdown by small interfering RNA targeting reduced decidualization-associated PRL expression, whereas KLF9 and KLF13 knockdowns alone reduced transcript levels of WNT4 and BMP2, respectively. Results suggest that KLF9 and KLF13 functionally compensate in peri-implantation uterus for pregnancy success.
Krüppel-like factor (KLF) 9 may compensate for absence of KLF13 in maintaining uterine receptivity for embryo implantation.
female reproductive tract; implantation; Krüppel-like factors; pregnancy; progesterone/progesterone receptor; steroid hormone receptors
Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased (∼ 9-fold; 15–30 min) with later increases in expression of Klf4 and Klf6 (∼ 5-fold; 30–60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1–2 h (∼ 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1β or tumor necrosis factor α downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli.
Cardiac myocytes; Endothelin-1; Immediate early genes; Krüppel-like factors; Gene expression; Cytokines
Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebpα promoter. We find that C/ebpα is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebpα promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis.
KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. In KLF2-/- embryos, β-like globin gene expression is reduced, and E10.5 erythroid cells exhibit abnormal morphology. In this study, other genes regulated by KLF2 were identified by comparing E9.5 KLF2-/- and wild-type (WT) yolk sac erythroid precursor cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression between KLF2-/- and WT; eighty-nine of these are downregulated in KLF2-/-. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. The SOX2 gene, encoding a pluripotency factor, is regulated by KLF2 in both ES and embryonic erythroid cells. Previous work had identified genes with erythroid-enriched expression in the yolk sac. The erythroid-enriched genes reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT, and are therefore candidates for controlling primitive erythropoiesis. Each of these genes contains a putative KLF2 binding site(s) in its promoter and/or an intron. Reelin has an established role in neuronal development. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter in erythroid cells, validating this approach to identify KLF2 target genes.
yolk sac; embryonic erythropoiesis; KLF2; expression profiling; laser capture microdissection
Krüppel-like factor 5 (KLF5) is a pro-proliferative transcriptional regulator primarily expressed in the intestinal crypt epithelial cells. Constitutive intestine-specific deletion of Klf5 is neonatal lethal suggesting a crucial role for KLF5 in intestinal development and homeostasis. We have previously shown Klf5 to play an active role regulating intestinal tumorigenesis. Here we examine the effect of inducible intestine-specific deletion of Klf5 in adult mice. Klf5 is lost from the intestine beginning at day 3 after the start of a 5-day treatment with the inducer tamoxifen. Although the mice have no significant weight loss or lethality, the colonic tissue shows signs of epithelial distress starting at day 3 following induction. Accompanying the morphological changes is a significant loss of proliferative crypt epithelial cells as revealed by BrdU or Ki67 staining at days 3 & 5 after start of tamoxifen. We also observed a loss of goblet cells from the colon and Paneth cells from the small intestine upon induced deletion of Klf5. In addition, loss of Klf5 from the colonic epithelium is accompanied by a regenerative response that coincides with an expansion in the zone of Sox9 expression along the crypt axis. At day 11, both proliferation and Sox9 expression return to baseline levels. Microarray and quantitative PCR analyses reveal an upregulation of several regeneration-associated genes (Reg1A, Reg3G and Reg3B) and down-regulation of many Klf5 targets (Ki-67, cyclin B, Cdc2 and cyclin D1). Sox9 and Reg1A protein levels are also increased upon Klf5 loss. Lentiviral-mediated knockdown of KLF5 and exogenous expression of KLF5 in colorectal cancer cell lines confirm that Sox9 expression is negatively regulated by KLF5. Furthermore, ChIP assays reveal a direct association of KLF5 with both the Sox9 and Reg1A promoters. We have shown that disruption of epithelial homeostasis due to Klf5 loss from the adult colon is followed by a regenerative response led by Sox9 and the Reg family of proteins. Our study demonstrates that adult mouse colonic tissue undergoes acute physiological changes to accommodate the loss of Klf5 withstanding epithelial damage further signifying importance of Klf5 in colonic homeostasis.
To elucidate how krüppel-like factor 4 (KLF4) activates mitofusin 2 (mfn-2) expression in all-trans retinoic acid (ATRA)-induced vascular smooth muscle cell (VSMC) differentiation.
The mfn-2 promoter-reporter constructs and the KLF4 acetylation-deficient or phosphorylation-deficient mutants were constructed. Adenoviral vector of KLF4-mediated overexpression and Western blot analysis were used to determine the effect of KLF4 on mfn-2 expression. The luciferase assay and chromatin immunoprecipitation were used to detect the transactivation of KLF4 on mfn-2 gene expression. Co-immunoprecipitation and GST pull-down assays were used to determine the modification of KLF4 and interaction of KLF4 with p300 in VSMCs.
KLF4 mediated ATRA-induced mfn-2 expression in VSMCs. KLF4 bound directly to the mfn-2 promoter and activated its transcription. ATRA increased the interaction of KLF4 with p300 by inducing KLF4 phosphorylation via activation of JNK and p38 MAPK signaling. KLF4 acetylation by p300 increased its activity to transactivate the mfn-2 promoter.
ATRA induces KLF4 acetylation by p300 and increases the ability of KLF4 to transactivate the mfn-2 promoter in VSMCs.
Krüppel-like factor 4; all-trans retinoic acid; mitofusin 2; vascular smooth muscle cells; p300
Background and aim: The Krüppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity (LOH). However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas (HCCs). We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines. Methods: We analyzed the exon-2 of KLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting. Results: KLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression of KLF6 mRNA or protein was down-regulated in 8 (34.7%) or 9 (39.1%) of 23 HCC tissues. Wild-type KLF6 (wtKLF6) inhibited cellular proliferation and prolonged G1-S transition by inducing the expression of p21WAF1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant (mKLF6) had no effects. Conclusion: Our findings suggest that KLF6 may be involved in pathogenesis of HCC.
Tumor suppressor gene; Krüppel-like factor 6 (KLF6); Mutation; Gene expression; Hepatocellular carcinoma
A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed.
By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes.
Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype.