Anoikis effector Bit1 negatively regulates Erk activity

1. Anoikis effector Bit1 negatively regulates Erk activity 陈洪栋

2. Bcl2-inhibitor of transcription 1 (Bit1) is a mitochondrial protein evolutionarily conserved from bacteria to humans.When released into the cytoplasm, it elicits apoptosis(anoikis). Function: Release tRNA from a peptidyl-tRNA complex. Hence, the protein also is known as peptidyl-tRNA hydrolase 2  Acts as a proapoptotic factor when released into the cytosol or when experimentally expressed there

3.  Bit1-induced apoptosis is uniquely regulated byintegrinmediatedcell attachment. Various antiapoptotic signaling molecules,such Bcl-2, Bcl-xL, PI-3K, and Akt, can block the release of Bit1from mitochondria, but are unable to inhibit apoptosis inducedby cytosolic Bit1. To examine the role of Bit1 in vivo and the relationship of Bit1 and Erk.

4. Expenrimental procedure & Results Bit1-Null Mice Cre-LoxP recombination system

5. Bit1-Null Mice Are Postnatal-Lethal with a Runting Syndrome Bit1 wild-type, heterozygous mice, and KO mice histological examination Embryos: collected by C-section, then fixed ---- preserved---- histological sectioning---- staining---- examination.

6. Bit1-Null Mice Exhibit Delayed Development and Neutropenia kidney:the subcapsular glomeruli issmaller than control; muscle: had myfibers with smaller diameter. blood:had an 80% reduction in neutrophil counts relative to controls

7. Bit1 KO Cells Are Resistant to Anoikis Anoikis assay Multiple independent MEF strains were isolatedfrom the carcasses of E12.5 embryos and cultured in DMEM. For anoikis assay,cells were treated with staurosporine(星形孢菌素). cell viability was by staining cells by annetin-FITC and propidium iodide(碘化丙啶),followed by analysis with flow cytometry.

8. the Bit1 KO MEFs are selectively resistant to anoikis.

9. Erk activation cell survival promote cell attachment Erk Activation Is Increased in Bit1 KO MEFs and Tissues This method relies on detecting dephosphorylation ofa purified, phosphorylated, His6-tagged Erk upon incubation with total celllysate. immunoblotting

11. Bit1 Regulates Erk Activation RNA Interference Three Bit1-specific siRNAs and control siRNAs were used to transfected into Hela cells. 3 days after,cells were harvested andthe resulting total cell lysate was subjected to immunoblotting or in vitroErk-directed phosphatase assay. stable cell lines and Bit1 knockdown clones were generated.

12. Anoikis Assay After RNA Interference Stable control and Bit1 knockdownclones were plated in PolyHEMA-coated 96-well plates for48 h. Cells were collected and subjected to an apoptosis assay by using the celldeath detection ELISA kit to detect for thepresence of cytosolic nucleosomal fragments. To examine the effect of Erk2 knockdown on the anoikis resistanceof Bit1 knockdown cells, stable control and Bit1 RNAiclones weretransfected with control or Erk2-specific siRNAs; 48 h after siRNA transfection,attached cells were resuspended in PolyHEMA-coated wells for another 48 hand subjected to apoptosis assay cell death detection ELISA

13. Transient knockdown ofBit1 in HeLa cells resulted in a robust Erk activationcompared with the control siRNA-transfected cells

14. Enforced overexpression of Bit1 in the Bit1 siRNAtreated cells attenuated Erk activation

15. All three Bit1 knockdown clones exhibited increased levels ofphospho-Erkanddecreased sensitivity to anoikis .

16. Bit1 knockdown cells to contain less Erkdirected phosphatase activity

17. siRNAmediateddown-regulation of endogenous Erk2 partially restored the sensitivity to anoikis in a Bit1 knockdown clone

18. 1.deleting Bit1 from mice results in a runting syndrome that culminates in early postnatal death. 2. MEFs from the KO mice wereless susceptible to anoikis and displayed more Erk activationthan wild-type MEFs. 3. Bit1 expression and Erk activation in cultured tumor cells were inversely correlated. This mouse KO study has yielded the beginnings of a molecular pathway through which Bit1 regulates anoikis

19. 谢谢大家！