Caspase1-mediated Nuclear Truncated HO-1 Activates Autophagy And Apoptosis Independent Of Its Enzymatic Activity

Background:UVR exerts many effects on the skin,including tanning,DNA damage,photoaging,carcinogenesis and immunomodulation.One of the most obvious acute effects of UVR on the skin is the induction a cascade of mediators and cytokines resulting in inflammatory response and sunburn.When the dose of UVR exceeds a threshold damage response,epidermal dendritic cells can activate apoptotic pathways and cell death.However,molecular mechanisms underlying UVR-induced apoptosis of epidermal cells are not well understood.Heme oxygenase-1?HO-1?,a 32-kDa protein,induced by UVR and other stresses is the rate-limiting enzyme in degradation of heme.HO-1 has long been thought to play important biological roles in cytoprotection attributed to the enzymatic action of HO-1 in heme degradation and the catalytic byproducts.Nevertheless,by-products of the HO reaction are also cytotoxic and inactive HO-1 mutant protein has cytoprotective roles against chemically induced oxidative stress.Thus,the mechanisms of HO-1 cytoprotective function are still under debate.More recent studies have suggested HO-1 dual effects and multifunction,and it is showed that HO-1 regulates many biological responses including autophagy,apoptosis,cell cycle progression,cell proliferation,and angiogenesis.However,the molecular mechanisms of the production HO-1 dual effects are little known.More recently subcellular localization of HO-1 has been studied,and It can localize to the nucleus,mitochondria,and caveolae.A mouse 28-kDa HO-1 isoform missing the C-terminal 53 amino acids was reported.C-terminal truncation can reduce HO activity but promote the protein nuclear auccumulation.Moreover,nuclear truncated HO-1 modulates the activation of transcription factor AP-1 and Nrf2 playing more roles in cellular signaling.The studies have revealed the C terminus of HO-1 is tend to more proteolytic cleavages,and it may exist more isoforms conferring HO-1 multifunction.However,very limited isoforms are found by far.Here,we found under UVR HO-1 can be cleaved by caspase 1/CASP1 after D224 in the C-terminus to produce a 26-kDa truncated form lacking 64 amino acids?named 26tHO-1?.Objects:To analyze the interaction of CASP1 and HO-11.To analyze the way of 26tHO-1 generation,and to clarify that HO-1 is processed by post-translational modification and cleaved to produce 26tHO-1.2.To study the interaction of CASP1 and HO-1.3.To analyze the effect of HO-1 knocked out by different targeting sites on producing of endogenous 26tHO-1.To explore the function of 26tHO-11.To study the role of 26tHO-1 in the cell apoptosis induced by UV radiation,and the cellular distribution of the isoform.2.To study the roles of 26tHO-1 in autophagy and apoptosis.3.To analyze the interaction of 26tHO-1 and nuclear transcription factor Bmal1,and their function in regulating the cell autophagy and apoptosis.Methods:The effect of UV radiation on apoptosis of Ha CaT cells was detected by flow cytometry.Through separating the nuclear and cytoplasmic fractions,the cellular distribution of 26tHO-1 was studied by western blot.We constructed HA-HO-1 and HO-1-HA overexpression vectors to study the cleavage of HO-1.By the CRISPR/Cas9 system,CASP1 and HO-1 were knocked out.The mutant HO-1without the CASP1 recognized sites was constructed.The cellular distribution of HO-1,26tHO-1 and Bmal1 were detected by immunofluorescence.The interaction of HO-1/CASP1 or 26tHO-1/Bmal1 were detected by immunoprecipitation.Results:1.UV radiation Induces HO-1,leading to nuclear localization of a 26 kDa isoform.CASP1 knockout inhibited nuclear accumulation of 26 kDa HO-1 isoform.2.32 kDa HO1 is cleaved by CASP1 at c-terminal region to produce 26 kDa isoform.The mutant HO1 with CASP1 recognized sites deletion inhibits the product of 26-kDa HO1 both in A375 HO1^-/-cells and HaCat cells.3.Comparing with HO-1,26tHO-1 has stronger effect on autophagy and apoptosis.4.HO-1 knockout affects the production of 26tHO-1.HO-1^-/-#1 and#2 express false HO1 proteins with mismatched C-termini.As expected,in cell line#1 which reserves CASP1 recognized site the protein band of 26tHO-1 could be detected by western blot.While 26tHO-1 could not be detected in cell line#2 without the recognized site.Compared with control,the two HO-1^-/-cell lines undergo inverse morphological changes.5.Nuclear 26tHO-1 interacts with transcription factor Bmal1 to mediate autophagy and apoptosis.Conclusion:We concluded that under UVR HO1 can be cleaved by caspase 1 after D224 in the C terminus to produce a 26 kDa isoform?26tHO-1?lacking 64 amino acids.26 kDa HO1 accumulates nucleus and interacts with transcription factor Bmal1 to activate autophagy and apoptosis.