Study On The Mechanism Of Hh Pathway In Promoting Cell Apoptosis Through Ci-Rdx-Diap1 Axis

Drosophila melanogaster,as a classic model organism,is a common zoological model to study cell apoptosis due to its advantages of easy breeding,short life cycle,strong reproductive capacity,simple chromosomes and mature genetic research techniques.Apoptosis is a process in which the body automatically removes unwanted or damaged cells under the strict regulation of multiple genes.A variety of signaling pathways are involved in the regulation of apoptosis.More and more studies have shown that abnormal apoptosis can lead to birth defects and various diseases.The Hedgehog(Hh)signaling pathway is an important proto-cancer signaling pathway that is highly conserved during evolution.Previous studies have shown that Hh signaling plays an important role in physiological and pathological processes such as embryonic development,cell fate determination,tissue damage repair,stem cell maintenance,and tumorigenesis.Studies have shown that anti-apoptotic protein Bcl2 is an important target gene of Hh pathway,which inhibits apoptosis by activating Bcl2.Although there are two Bcl2 homologues buffy and debcl in Drosophila genome,they do not play a significant role in cell apoptosis.Therefore,it is not clear whether and how Hh pathway regulates apoptosis in Drosophila.Diap1 is an important inhibitor of apoptosis in Drosophila.It has E3 ubiquitin ligase activity and inhibits abnormal apoptosis by binding to apoptotic proteins and promoting ubiquitination and degradation of apoptotic proteins.In addition,when cells were stimulated by death signals,the antiapoptotic activity of Diap1 was inhibited by upstream antagonists including Head involution defective(Hid),Reaper(Rpr),and Grim.These three proteins negatively regulate Diap1 through different mechanisms,either by decreasing Diap1 levels or by inhibiting Diap1-Dronc interaction.This study focuses on the regulation of Hedgehog signaling pathway on apoptosis.Experimental techniques and methods such as Drosophila genetic screening,immunofluorescence staining of Drosophila larva wing discs and eye discs,transgenic Drosophila injection,gene cloning,protein immunoimprinting,and somatic cloning were used.We explored the effect of Hh signaling pathway on cell apoptosis in Drosophila and further explored the specific regulatory mechanism,and obtained the following experimental results:(1)Through genetic screening of Drosophila,we found that overexpression of smo and ci,upstream of Hedgehog signaling pathway elements,exacerbated the phenotype of Hid-induced diminutive eye size in Drosophila,and the phenotype of Hid-induced diminutive eye could be partially rescued by ci knockdown.Because the proapoptotic factor Hid promotes apoptosis by inhibiting Diap1,our results suggest that the Hedgehog signaling pathway may affect eye size by regulating apoptosis.(2)In order to further confirm our conjecture,immunofluorescence staining of PH3,a marker protein for proliferation in Drosophila eye discs,and Caspase3,a marker protein for apoptosis in Drosophila eye discs,showed that activation of Hh signaling pathway indeed increased apoptosis signal.Similarly,knockdown ci reduced Hid induced apoptosis.(3)It is known that there are four classical target genes of Hh signaling pathway,namely dpp,en,ptc and rdx.We found that overexpression of rdx increased the phenotype of Hid induced small eyes,which was consistent with overexpression of ci.Knockdown of rdx and loss of a copy of rdx were consistent with the phenotype of ci knockdown,both of which partially rescued the phenotype of Hid induced Drosophila small eyes,demonstrating that Hh pathway induced apoptosis through its target gene rdx.(4)Since Hid induced cell apoptosis through Diap1,we wanted to test if Rdx regulates apoptosis through Diap1,we cloned the genes of rdx and diap1 respectively,and proved the direct interaction between the two proteins through cell experiments.(5)Our results indicate that Rdx affects eye size through its E3 ubiquitin ligase activity,we wanted to prove whether Rdx promotes Diap1 ubiquitin modification.Through cell ubiquitin experiments,it was proved that Rdx indeed promoted Ub-K63 linked polyubiquitin modification on Diap1.(6)Through protein competitive binding assay,we found that Rdx-mediated of K63-linked polyubiquitination affects the interaction between Diap1 and apoptotic protein Dronc,which stabilizes Dronc and then induces apoptosis.In summary,our results reveal that proto-cancer signaling pathway Hh pathway can promote apoptosis through Ci-Rdx-Diap1 interaction.In this study,we found that Hh signaling pathway plays a positive role in regulating cell apoptosis,which not only further expands the biological function of Rdx,but also raising a concern to choose Hh pathway inhibitors as tumor therapy in clinical practice.