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
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 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
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 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
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 (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.
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
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
Krüppel-like factor 6 (KLF6) has been recently identified as a MEF2D target gene involved in neuronal cell survival. In addition, KLF6 and TGFβ have been shown to regulate each other’s expression in non-myogenic cell types. Since MEF2D and TGFβ also fulfill crucial roles in skeletal myogenesis, we wanted to identify whether KLF6 functions in a myogenic context.
KLF6 protein expression levels and promoter activity were analyzed using standard cellular and molecular techniques in cell culture.
We found that KLF6 and MEF2D are co-localized in the nuclei of mononucleated but not multinucleated myogenic cells and, that the MEF2 cis element is a key component of the KLF6 promoter region. In addition, TGFβ potently enhanced KLF6 protein levels and this effect was repressed by pharmacological inhibition of Smad3. Interestingly, pharmacological inhibition of MEK/ERK (1/2) signaling resulted in re-activation of the differentiation program in myoblasts treated with TGFβ, which is ordinarily repressed by TGFβ treatment. Conversely, MEK/ERK (1/2) inhibition had no effect on TGFβ-induced KLF6 expression whereas Smad3 inhibition negated this effect, together supporting the existence of two separable arms of TGFβ signaling in myogenic cells. Loss of function analysis using siRNA-mediated KLF6 depletion resulted in enhanced myogenic differentiation whereas TGFβ stimulation of myoblast proliferation was reduced in KLF6 depleted cells.
Collectively these data implicate KLF6 in myoblast proliferation and survival in response to TGFβ with consequences for our understanding of muscle development and a variety of muscle pathologies.
Myoblasts; Krüppel-like factor 6; Transforming growth factor β; Cell proliferation
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
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 factor 5 (KLF5) regulates multiple biologic processes. Its function in tumorigenesis appears contradictory though, showing both tumor suppressor and tumor promoting activities. In this study, we examined whether and how Klf5 functions in prostatic tumorigenesis using mice with prostate-specific deletion of Klf5 and phosphatase and tensin homolog (Pten), both of which are frequently inactivated in human prostate cancer. Histologic analysis demonstrated that when one Pten allele was deleted, which causes mouse prostatic intraepithelial neoplasia (mPIN), Klf5 deletion accelerated the emergence and progression of mPIN. When both Pten alleles were deleted, which causes prostate cancer, Klf5 deletion promoted tumor growth, increased cell proliferation, and caused more severe morphologic and molecular alterations. Homozygous deletion of Klf5 was more effective than hemizygous deletion. Unexpectedly, while Pten deletion alone expanded basal cell population in a tumor as reported, Klf5 deletion in the Pten-null background clearly reduced basal cell population while expanding luminal cell population. Global gene expression profiling, pathway analysis, and experimental validation indicate that multiple mechanisms could mediate the tumor-promoting effect of Klf5 deletion, including the up-regulation of epidermal growth factor and its downstream signaling molecules AKT and ERK and the inactivation of the p15 cell cycle inhibitor. KLF5 also appears to cooperate with several transcription factors, including CREB1, Sp1, Myc, ER and AR, to regulate gene expression. These findings validate the tumor suppressor function of KLF5. They also yield a mouse model that shares two common genetic alterations with human prostate cancer—mutation/deletion of Pten and deletion of Klf5.
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 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.
Background: The somatic cell reprogramming factors do not always induce pluripotency.
Results: The optimal ratio of the reprogramming factors is Oct3/4-high, Sox2-low, Klf4-high, and c-Myc-high.
Conclusion: Among the various reprogramming transcription factor combinations, high Oct3/4 and low Sox2 produced the most efficient results.
Significance: The overall gene expression profiles between the high and low efficiency conditions provide novel insights for somatic cell reprogramming.
Somatic cell reprogramming is achieved by four reprogramming transcription factors (RTFs), Oct3/4, Sox2, Klf4, and c-Myc. However, in addition to the induction of pluripotent cells, these RTFs also generate pseudo-pluripotent cells, which do not show Nanog promoter activity. Therefore, it should be possible to fine-tune the RTFs to produce only fully pluripotent cells. For this study, a tagging system was developed to sort induced pluripotent stem (iPS) cells according to the expression levels of each of the four RTFs. Using this system, the most effective ratio (Oct3/4-high, Sox2-low, Klf4-high, c-Myc-high) of the RTFs was 88 times more efficient at producing iPS cells than the worst effective ratio (Oct3/4-low, Sox2-high, Klf4-low, c-Myc-low). Among the various RTF combinations, Oct3/4-high and Sox2-low produced the most efficient results. To investigate the molecular basis, microarray analysis was performed on iPS cells generated under high (Oct3/4-high and Sox2-low) and low (Oct3/4-low and Sox2-high) efficiency reprogramming conditions. Pathway analysis revealed that the G protein-coupled receptor (GPCR) pathway was up-regulated significantly under the high efficiency condition and treatment with the chemokine, C-C motif ligand 2, a member of the GPCR family, enhanced somatic cell reprogramming 12.3 times. Furthermore, data from the analysis of the signature gene expression profiles of mouse embryonic fibroblasts at 2 days after RTF infection revealed that the genetic modifier, Whsc1l1 (variant 1), also improved the efficiency of somatic cell reprogramming. Finally, comparison of the overall gene expression profiles between the high and low efficiency conditions will provide novel insights into mechanisms underlying somatic cell reprogramming.
Epigenetics; G Protein-coupled Receptors (GPCR); Induced Pluripotent Stem (iPS) Cell; Microarray; Reprogramming
Krüppel-like factor 8 (KLF8) regulates critical cellular processes including cell cycle progression, transformation, epithelial-to-mesenchymal transition, migration and invasion by either repressing or activating target gene promoters. As a repressor, KLF8 recruits the CtBP co-repressor via its PVDLS repression motif. However, how KLF8 acts as an activator has not been determined. Here we report the identification of both the KLF8 activation domain and associated co-activators. By site-directed mutagenesis and cyclin D1 promoter reporter assays using both mouse fibroblasts and human epithelial cells, we determined that deletion of residues 100–260 or mutation of Q118-Q248 abolished KLF8 transactivity. This transactivity was dramatically reduced in p300−/−, CBP−/− or PCAF−/− cells and could be restored by re-expressing p300 or PCAF, but not CBP. Co-immunoprecipitation analyses demonstrated that KLF8 interacted with these co-activators whereas the Q118N-Q248N mutant did not. Chromatin immunoprecipitation experiments showed that KLF8 promoted histone acetylation at the promoter whereas the Q118N-Q248N mutant had a dramatic loss of this function. Western blotting revealed that unlike wild-type KLF8 the Q118N-Q248N was no longer able to upregulate cyclin D1 protein level. BrdU incorporation assays showed that the Q118N-Q248N mutant also lost the ability to promote DNA synthesis. Taken together, these results identified the KLF8 activation domain located between residues 101–260 where the well-conserved Q118 and Q248 are essential for recruiting p300 and PCAF to activate target gene transcription.
krüppel-like factor 8; transcription activation; p300; creb-binding protein; p300/CBP associated factor; cyclin D1
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.
Krüppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress, such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death by mouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental role for KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF2−/− embryos die earlier, by E11.5. E9.5 FVB/N KLF2−/− hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normal single layer. By E10.5, traditional and endothelial-specific FVB/N KLF2−/− AV cushions are hypocellular, suggesting that the cells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF2−/− hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number of mesenchymal cells migrating from FVB/N KLF2−/− AV explants into a collagen matrix is reduced considerably compared to wild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/N KLF2−/− embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF2−/− hearts have largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF2−/− embryos have a delay in the formation of the atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucose dehydrogenase (Ugdh), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and Ugdh promoters in chromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. In conclusion, KLF2−/− heart phenotypes are genetic background-dependent. KLF2 plays a role in EMT through its regulation of important cardiovascular genes.