Engda G. Hagos

Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish

BackgroundThe vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.ResultsWe show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffers vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated.ConclusionWe conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.

Mouse embryonic fibroblasts null for the Krüppel-like factor 4 gene are genetically unstable

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.

Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts

BackgroundKrüppel-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.MethodsTo 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.ResultsWe 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.ConclusionTaken 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.

The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Krüppel-like Factor 5 Protein

Background: The pro-proliferative Krüppel-like factor 5 (KLF5) is posttranslationally regulated. Results: SMAD ubiquitination regulatory factor 2 (SMURF2) interacts with, ubiquitinates and degrades KLF5. Conclusion: SMURF2 negatively regulates KLF5. Significance: The findings increase the understanding of the mechanisms by which KLF5 is regulated posttranslationally. The zinc finger transcription factor Krüppel-like factor 5 (KLF5) is regulated posttranslationally. We identified SMAD ubiquitination regulatory factor 2 (SMURF2), an E3 ubiquitin ligase, as an interacting protein of KLF5 by yeast two-hybrid screen, coimmunoprecipitation, and indirect immunofluorescence studies. The SMURF2-interacting domains in KLF5 were mapped to its carboxyl terminus, including the PY motif of KLF5 and its zinc finger DNA-binding domain. KLF5 protein levels were reduced significantly upon overexpression of SMURF2 but not catalytically inactive SMURF2-C716A mutant or SMURF1. SMURF2 alone reduced the protein stability of KLF5 as shown by cycloheximide chase assay, indicating that SMURF2 specifically destabilizes KLF5. In contrast, KLF5(1–165), a KLF5 amino-terminal construct that lacks the PY motif and DNA binding domain, was not degraded by SMURF2. The degradation of KLF5 by SMURF2 was blocked by the proteasome inhibitor MG132, and SMURF2 efficiently ubiquitinated both overexpressed and endogenous KLF5. In contrast, knocking down SMURF2 by siRNAs significantly enhanced KLF5 protein levels, reduced ubiquitination of KLF5, and increased the expression of cyclin D1 and PDGF-A, two established KLF5 target genes. In consistence, SMURF2, but not the E3 ligase mutant SMURF2-C716A, significantly inhibited the transcriptional activity of KLF5, as demonstrated by dual luciferase assay using the PDGF-A promoter, and suppressed the ability of KLF5 to stimulate cell proliferation as measured by BrdU incorporation. Hence, SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by targeting it for proteasomal degradation.

Oxidative DNA damage causes premature senescence in mouse embryonic fibroblasts deficient for Krüppel‐like factor 4

Krüppel‐like factor 4 (KLF4) is a zinc‐finger‐containing transcription factor with tumor suppressor activity in various cancer types. Cells that sustain double strand breaks (DSBs) in their DNA due to high levels of reactive oxygen species (ROS) can develop genomic instability, which can result in cancer formation. One protective response to increased levels of ROS is the induction of cellular senescence. Recently, we found that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by the presence of DNA DSBs. However, it is yet unknown whether KLF4 is involved in regulating oxidative stress‐induced DNA damage. Therefore, we sought to determine the mechanisms by which ROS induce genomic instability in Klf4‐deficient MEFs. With SA‐β‐Gal staining, we show that Klf4−/− MEFs enter senescence earlier than Klf4+/+ MEFs, and western blot shows accumulation of p21 and p53 with increasing passages. In addition, immunostaining against γ‐H2AX indicates that the increased level of DNA damage in Klf4−/− MEFs positively correlates with ROS accumulation. Consistent with ROS as a source of DSB in Klf4−/− MEFs, treatment with NAC, reduces the accumulation of DNA damage. Our RT‐PCR result demonstrates that Klf4−/− MEFs have decreased expression of the antioxidant gene, Gsta4. The downregulation of the Gsta4 correlates with significant levels of ROS accumulation, as shown by DCFDA and FACS analysis, and thus the oxidative stress‐induced premature senescence. Together these findings suggest a mechanism by which KLF4 protects against DNA damage and oxidative stress at least in part through the regulation of Gsta4 and likely related genes.

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

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

Krüppel-like factor 4 mediates cellular migration and invasion by altering RhoA activity

Abstract Kru¨ppel like factor 4 (KLF4) is a transcription factor that regulates genes related to differentiation and proliferation. KLF4 also plays a role in metastasis via epithelial to mesenchymal transition. Here, we investigate the function of Klf4 in migration and invasion using mouse embryonic fibroblasts and the RKO human colon cancer cell line. Compared to wild-type, cells lacking Klf4 exhibited increased migration-associated phenotypes. In addition, overexpression of Klf4 in Klf4−/− MEFs attenuated the presence of stress fibers to wild-type levels. An invasion assay suggested that lack of Klf4 resulted in increased invasive capacity. Finally, analysis of RhoA showed elevated RhoA activity in both RKO and MEF cells. Taken together, our results strongly support the novel role of KLF4 in a post-translational regulatory mechanism where KLF4 indirectly modulates the actin cytoskeleton morphology via activity of RhoA in order to inhibit cellular migration and invasion.

Abstract 2259: Oxidative DNA damage causes premature senescence in mouse embryonic fibroblasts deficient for krüppel-like factor 4

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Kruppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor with tumor suppressor activity in various cancer types. Cells that sustain double strand breaks (DSBs) in their DNA due to high levels of reactive oxygen species (ROS) can develop genomic instability, which can result in cancer formation. One protective response to increased levels of ROS is the induction of cellular senescence. Recently, we found that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by the presence of DNA DSBs. However, it is yet unknown whether KLF4 is involved in regulating oxidative stress-induced DNA damage. Therefore, we sought to determine the mechanisms by which ROS induce genomic instability in Klf4-deficient MEFs. With SA-β-Gal staining, we show that Klf4-/- MEFs enter senescence earlier than Klf4+/+ MEFs, and western blot shows accumulation of p21 and p53 with increasing passages. In addition, immunostaining against γ-H2AX indicates that the increased level of DNA damage in Klf4-/- MEFs positively correlates with ROS accumulation. Consistent with ROS as a source of DSB in Klf4-/- MEFs, treatment with NAC, reduced the accumulation of DNA damage. Our RT-PCR result demonstrates that Klf4-/- MEFs have decreased expression of the antioxidant gene, Gstα4. The downregulation of the Gstα4 correlates with significant levels of ROS accumulation, as shown by DCFDA and FACS analysis, and thus the oxidative stress-induced premature senescence. Together these findings suggest a mechanism by which KLF4 protects against DNA damage and oxidative stress at least in part through the regulation of Gsta4 and likely related genes. Note: This abstract was not presented at the meeting. Citation Format: Changchang Liu, Stephen La Rosa, Engda Hagos. Oxidative DNA damage causes premature senescence in mouse embryonic fibroblasts deficient for kruppel-like factor 4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2259. doi:10.1158/1538-7445.AM2014-2259