Korean J Physiol Pharmacol.  2016 Nov;20(6):573-580. 10.4196/kjpp.2016.20.6.573.

Novel functional roles of caspase-related genes in the regulation of apoptosis and autophagy

• Shin JH
• Min SH

• KMID: 2364261
• DOI: http://doi.org/10.4196/kjpp.2016.20.6.573

Abstract

Caspases, a family of cysteine proteases, cleave substrates and play significant roles in apoptosis, autophagy, and development. Recently, our group identified 72 genes that interact with Death Caspase-1 (DCP-1) proteins in Drosophila by genetic screening of 15,000 EP lines. However, the cellular functions and molecular mechanisms of the screened genes, such as their involvement in apoptosis and autophagy, are poorly understood in mammalian cells. In order to study the functional characterizations of the genes in human cells, we investigated 16 full-length human genes in mammalian expression vectors and tested their effects on apoptosis and autophagy in human cell lines. Our studies revealed that ALFY, BIRC4, and TAK1 induced autophagy, while SEC61A2, N-PAC, BIRC4, WIPI1, and FALZ increased apoptotic cell death. BIRC4 was involved in both autophagy and apoptosis. Western blot analysis and luciferase reporter activity indicated that ALFY, BIRC4, PDGFA, and TAK1 act in a p53-dependent manner, whereas CPSF1, SEC61A2, N-PAC, and WIPI1 appear to be p53-independent. Overexpression of BIRC4 and TAK1 caused upregulation of p53 and accumulation of its target proteins as well as an increase in p53 mRNA levels, suggesting that these genes are involved in p53 transcription and expression of its target genes followed by p53 protein accumulation. In conclusion, apoptosis and/or autophagy mediated by BIRC4 and TAK1 may be regulated by p53 and caspase activity. These novel findings may provide valuable information that will aid in a better understanding of the roles of caspase-related genes in human cell lines and be useful for the process of drug discovery.

Keyword

Apoptosi; Autophagy; Caspase; DCP-1

MeSH Terms

Apoptosis*
Autophagy*
Blotting, Western
Caspases
Cell Death
Cell Line
Cysteine Proteases
Drosophila
Drug Discovery
Genetic Testing
Humans
Luciferases
RNA, Messenger
Up-Regulation
Caspases
Cysteine Proteases
Luciferases
RNA, Messenger

Figure

• 

  Fig. 1 Overexpression of caspase-related genes induces apoptosis or autophagy.(A) Screening and selection of 16 cell death-related genes. (B) GFP-LC3 was co-transfected with the indicated genes. After transfection, the cells were incubated for 48 h, and cell death types were then detected using fluorescence microscopy.

• 

  Fig. 2 The effects of caspase-related genes on apoptosis and p53 signaling.(A) HeLa cells were transfected with the indicated constructs. 48 hours after transfection, cells were harvested and luminescence was measured using the caspase-glo 3/7 assay. (B) p21-luc reporter and the indicated genes were transfected into HeLa cells, and the cells were incubated for 48 h. p21 activity was measured by the luciferase reporter assay. (C) After the pRGC-luc reporter and the indicated genes were transfected in HeLa cells for 48 h, p53 activity was measured by the luciferase reporter assay. Data are presented as mean±SD (n=3). *p<0.05, **p<0.01, and ***p<0.001.

• 

  Fig. 3 TAK1 overexpression induces autophagy and p53 activation.(A) HCT116 p53-/- and 293A cells were co-transfected with GFP-LC3 and the indicated constructs for 48 h. After transfection, cells were harvested and analyzed by immunoblotting. (B) HeLa cells were transfected with a mock plasmid or the indicated plasmids for 48 h and then harvested for immunoblot analysis. p53, p27, and p21 levels were evaluated using the appropriate antibodies. (C) p53 transcript levels in HeLa cells were analyzed by RT-PCR. GAPDH was used as an internal control. Data are presented as mean±SD (n=3). *p<0.05, **p<0.01, and ***p<0.001.

• 

  Fig. 4 The effects of BIRC4 and TAK1 on p53 activity.(A) HeLa cells were transfected with the pRGC-Luc and BIRC4 plasmids. After 48 h, cell lysates were collected and assessed for luciferase activity. p53 activity increased in a BIRC4 dose-dependent manner. (B) HeLa cells were transfected with pRGC-Luc and TAK1 plasmids. After 48 h, cell lysates were collected and assessed for luciferase activity. p53 activity increased in a TAK1 dose-dependent manner. (C) HeLa cells were transfected with the indicated TAK1 plasmids. p53 protein expression level increased in a TAK1 dose-dependent manner. Data are presented as the mean±SD (n=3). *p<0.05, **p<0.01, and ***p<0.001..

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