title: Impaired autophagy in mouse embryonic fibroblasts null for Krüppel-like Factor 4 promotes DNA damage and increases apoptosis upon serum starvation.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology, Colgate University, 13 Oak Dr., Olin Hall 205A, Hamilton, NY, 13346, USA. cliu1@colgate.edu.
authors.forename: Changchang
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Biology, Colgate University, 13 Oak Dr., Olin Hall 205A, Hamilton, NY, 13346, USA. elise_deroo@hms.harvard.edu.|Program in Cellular and Molecular Medicine, Boston Childrens Hospital, Boston, MA, 02115, USA. elise_deroo@hms.harvard.edu.|School of Medical School, Harvard University, Boston, MA, 02115, USA. elise_deroo@hms.harvard.edu.
authors.forename: Elise P
authors.initials: EP
authors.lastname: DeRoo
authors.affiliation: Department of Biology, Colgate University, 13 Oak Dr., Olin Hall 205A, Hamilton, NY, 13346, USA. catherine.stecyk@tufts.edu.|Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA, 01536, USA. catherine.stecyk@tufts.edu.
authors.forename: Catherine
authors.initials: C
authors.lastname: Stecyk
authors.affiliation: Department of Biology, Colgate University, 13 Oak Dr., Olin Hall 205A, Hamilton, NY, 13346, USA. mwolsey@colgate.edu.
authors.forename: Margaret
authors.initials: M
authors.lastname: Wolsey
authors.affiliation: Department of Biology, Colgate University, 13 Oak Dr., Olin Hall 205A, Hamilton, NY, 13346, USA. mszuchnicki@colgate.edu.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Szuchnicki
authors.affiliation: Department of Biology, Colgate University, 13 Oak Dr., Olin Hall 205A, Hamilton, NY, 13346, USA. ehagos@colgate.edu.
authors.forename: Engda G
authors.initials: EG
authors.lastname: Hagos
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004249:DNA Damage; D004622:Embryo, Mammalian / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D055785:Gene Knockdown Techniques; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D016895:Culture Media, Serum-Free; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus