Autophagy Deficiency Augment HSF1 Mediated Heat Shock Response

BackgroundAutophagy is a physiological process by which the damaged organelles and long half-life proteins are degradated at stress conditions, e.g. starvation. It is one of the cytoprotective responses in maitianing cellular homeostasis. There are three types of Autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy（CMA）. Macroautophagy is most common autophagy that is catalyzed by autophagy related genes（ATGs） amount of them. ATG7 is a key regulator of the autophagy. It works as E1, catalyzing the association between ATG5 and ATG12, and LC3 conjugation. The activation and inactivation of autophagy is dynamically regulated at physiological as well as stress conditions（e.g. nutrient deprivation, oxidative stress, toxic compounds, as well as the invasion of pathogenic agents）, activation of autophagy provides the nutrients for cell by degradation of non-functional organelles and long-life proteins.Heat shock response is another important cytoprotective process in regulating cell homeostasis against not only environmental stresses（e.g. heat shock, H₂O₂ and pathogens infection） but also the intracellular stresses（cell proliferation and differentiation）. One chararcteristic of heat shock response is the induction of HSPs, which works molecular chaperone to modulate protein folding or denatured protein degradation. The expression of heat shock protein is regulated by a small family of heat shock transcription factors that consist of HSF1, HSF2, HSF3 and HSF4. HSF1 is the domiant heat shock respons regulator. At physiological condition, HSF1 forms a heterodimer with HSP90 without transcription activity, upon stress, HSF1 is dissociated from HSP90, and translocated into nucleus forming homotrimmer, which results in HSF1 activation. The circulation of HSF1 activation and inactivation is dynamically regulated by phosphorylation, sumoylation and its associated factors（e.g. HSF2, HSP90, HSP70, RalBP1 and Daxx）.Recently researches indicate that the stress transcription factors, such as p53, NF-κB and STAT3, play an important role in regulating autophagy. HSF1 is found to regulate ATG7 directly mediating heat shock-independent autophagy to protect tumor cells against from chemotherapy. However, the function of HSF1 mediating heat shock response in autophagy is unclear in MEFs and MPTCs. According to the previous reports, defective autophagy is associated with many diseases, such as muscle dystrophy, neurodegenerative diseases, cancer incidence and aging. Both heat shock response and autophagy are important cellular protective mechanisms in response to external stressors. However, the regulatory role of autophagy on heat shock response is not clear. ObjectivesOur aim is to study how HSF1-mediated heat shock response is regulated in the condition of defective autophagy by knockingout ATG7 gene. Methods1.We established MEF/ATG7^F/F and MEF/ATG7^-/- stable cell lines by NIH3T3, the levels of autophagy and heat shock response in two kinds cells were detected by Western blot;2.MEF/ATG7^F/F and MEF/ATG7^-/- cells were treated by stress stimulations, e.g. heat shock and MG132, detecting the express of heat shock proteins by Western blot and Real time PCR;3.Western blot detected autophagy and heat shock response levels under control and heat shock stimuli in MPTC/ATG7^F/F and MPTC/ATG7^-/- cells;4.We checked the binding ability of HSF1 and heat shock genes’ promoter by chromatin immunoprecipitation（Ch IP） under 43 ℃ heat shock stimulation in two stable cell lines;5.We explored the activated molecular mechanism of HSF1 transcriptional activity by nuclear and cytoplasmic extraction assay and HSF1 trimerization experiment under 43 ℃ heat shock stimulation in MEF/ATG7^F/F and MEF/ATG7^-/- cells;6.EBSS and cisplatin which can induce apoptosis respectively treated MEF/ATG7^F/F and MEF/ATG7^-/- cells, then Western blot was performed to explore the biological function of increased HSP25 after knockout ATG7; ResultsKnocking down ATG7 gene in MEF/ATG7^F/F and MPTC/ATG7^F/F cells can block autophagy, resulting in alternative HSF1 activation by facilitating HSF1 homotrimerization. Active HSF1 is response to the HSP25 expression, which may protect cell homeostasis at stress of autophagy deficiency. ConclusionsAfter ATG7 knockout, the expression of HSP25 induced by HSF1 is activated, which maintains the homeostasis of cells that block autophagy.