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

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 k...

Full description

Saved in:
Bibliographic Details
Published in:Molecular cancer 2015-05, Vol.14 (1), p.101-101, Article 101
Main Authors: Liu, Changchang, DeRoo, Elise P, Stecyk, Catherine, Wolsey, Margaret, Szuchnicki, Mateusz, Hagos, Engda G
Format: Article
Language:English
Subjects:
Animals
Apoptosis
Apoptosis - drug effects
Autophagy - drug effects
Comparative analysis
Culture Media, Serum-Free - pharmacology
DNA Damage
Embryo, Mammalian - cytology
Fibroblasts - cytology
Fibroblasts - drug effects
Fibroblasts - metabolism
Gene Knockdown Techniques
Genetic transcription
Kruppel-Like Transcription Factors - metabolism
Mice, Inbred C57BL
Models, Biological
Rapamycin
Sirolimus - pharmacology
Starvation
TOR Serine-Threonine Kinases - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 prevent
ISSN:1476-4598
1476-4598
DOI:10.1186/s12943-015-0373-6