Identification Of The Role Of FKBP12 In Initiating Necroptosis And The Associated Systemic Inflammatory Response Syndrome

BackgroundNecroptosis is a newly identified programmed cell death(PCD),which has a clear regulatory target,specific regulatory mechanism and exact physiological and pathological functions.Necroptosis 1s a kind of regulated cell death mode,and the dead cells show the morphological characteristics of cell necrosis(such as organelle swelling and cell membrane rupture,etc.).Previous studies have shown that death ligands,chemotherapeutic drugs,pathogenic microorganisms(bacteria and viruses)invasion and other factors can induce necroptosis,which determines that it has a wide range of biological functions,and play an important role in physiological and pathological processes such as embryonic development,ischemia-reperfusion injury and inflammation.Therefore,the identification of new targets for the regulation of necroptosis and the discussion of its regulatory mechanism can provide theoretical guidance and reference for the drug development and clinical treatment of diseases related to necroptosis.FK506 binding protein 12(FK506-bimding protein 12,FKBP12)1s a kind of 12kD immunophilim family protein,which contains peptide-proline isomerase domain and can catalyze the cis-trans conformation change of N-termimal peptide bond in polypeptide or protein proline residue,and change the activity of the substrate protein.At the same time,FKBP12 can be used as a molecular chaperone to assist the correct folding of some proteins,as ajunction protein to bind to some receptors(TGF p-RI and RAR),and regulate the activation of receptors.Therefore,FKBP12 has a variety of biological functions,such as regulating gene transcription and intracellular calcium concentration,blocking the signal transduction of TGF-Smad pathway and so on.However,no related studies have found that FKBP12 is involved in the regulation of necroptosis.ObjectiveThe purpose of this study was to identify the regulatory role and mechanism of FKBP12 in necroptosis,and explore the role of FKBP12 in diseases related to necroptosis,so as to identify new targets for regulating necroptosis.It provides a reference for the treatment of diseases related to necroptosis.Methods(1)The morphological characteristics of cell death were analyzed by transmission electron microscope,and the type and proportion of cell death were detected by Annexin V-FITC/PI staining combined with flow cytometry,so as to identify the mode of cell death induced by TNFa in L929 and HT22 cells.(2)Through lentivirus-mediated gene knockin and knockdown techniques,a cell line with stable knockdown or overexpression of FKBP12 was constructed,and the regulatory effect of FKBP12 on necroptosis was detected in combination with FKBP12 ligand drugs.(3)The apoptosis model of NIH/3T3 cells induced by TNFa and CHX was used to study the effect of FKBP12 knockdown on TNFa induced apoptosis.(4)Immunoprecipitation and Western blot were used to detect the combination of RIPK1 and RIPK3,and Duolink in situ PLA system combined with laser confocal microscope was used to verify the formation of necrosome between RIPK1 and RIPK3.(5)The interaction between RIPK1,RIPK3 and MLKL protein homologous molecules was detected by NuPAGE?electrophoresis system and Duolink in situ PLA system.(6)The effect of FKBP12 knockdown or overexpression was verified by Western blot or Real-time PCR technique.At the same time,Western blot was also used to detect the expression or phosphorylation of other proteins,and to detect the subcellular localization of proteins by cell component separation technique.(7)The animal model of systemic inflammatory response syndrome(SIRS)was established by intravenous injection of TNFa in mice.The development of SIRS was determined by monitoring the change of body temperature and mortality of mice.HE staining was used to detect the injury of liver and cecum in mice and to evaluate the target organs of SIRS.(8)FKBP12 knockdown mice were established by lentivirus-mediated RNA interference,or mice were injected intraperitoneally or intragastrically with FKBP12 ligands to detect the regulatory role and mechanism of FKBP12 in the process of systemic inflammatory response syndrome induced by TNFa.Results1.FKBP12 is a new target for regulating necroptosis,(1)The death of L929 and HT22 cells induced by TNFa combined with Z-VAD had the classical morphological and structural changes of necrotic cells,showing Annxin V-FITC/PI double positive staining,and can be blocked by Necrostatin-1(Nec-1),GSK'872 and Necrosalfonamide(NSA).,which are the target inhibitors of necroptosis.So it has been identified as classic necroptosis.(2)FKBP12 ligand compounds and FKBP12 knockdown can block the necroptosis induced by TNFa alone or in combination with other compounds.Restoring the expression of FKBP12 in FKBP12 knockdown cells can restore the sensitivity of L929 cells to necroptosis induced by TNFa.These results suggest that FKBP12 is an important target for regulating necroptosis.2.The regulatory mechanism of FKBP12 in TNFa-induced necroptosis.(1)FKBP12 knockdown and FKBP12 ligand FK506 could block the necroptosis induced by TNFa,but had no effect on the activation of mTOR signaling pathway induced by TNFa,so the regulation of FKBP12 on necroptosis was not related to mTOR signaling pathway.(2)FKBP12 knockdown could significantly inhibit the phosphorylation of RIPKl,RIPK3 and MLKL induced by TNFa and Z-VAD,and the formation and membrane transfer of MLKL polymers,indicating that FKBP12 could regulate the activation of RIPK1-RIPK3-MLKL signaling pathway.(3)FKBP12 knockdown can inhibit the formation of necrosome and RIPK1,RIPK3 homopolymer,thus affecting the phosphorylation of RIPK1 and RIPK3.So this may be the way for FKBP12 to regulate the activation of RIPK1-RIPK3-MLKL signaling pathway.(4)FKBP12 regulates the expression of RIPK1 and RIPK3,and then affects the phosphorylation level and the formation of subsequent necrosome,so as to realize the regulation of necroptosis.3.The regulatory role of FKBP12 in TNFa-induced systemic inflammatory response syndrome.(1)FKBP12 knockdown mice were constructed by intravenous injection of FKBP12 shRNA lentivirus concentrate,and the mRNA and protein levels of FKBP12 in cecum,which is the target organ of SIRS model,were detected to verify the knockdown effect of FKBP12.(2)FKBP12 knockdown could inhibit the hypothermia and death of mice induced by TNFa injection.At the same time,the administration of FK506 and Rapamycin had similar effects as FKBP12 knockdown,indicating that FKBP12 knockdown could alleviate the incidence of SIRS induced by TNFa.It is further suggested that FKBP 12 is an important target protein for the regulation of SIRS.(3)The results of HE staining showed that there was no significant change in liver tissue of mice induced by TNFa,but obvious inflammation of cecum tissue occurred,while FKBP12 knockdown alleviated the degree of cecal injury.At the same time,FKBP12 knockdown can inhibit the activation of RIPKI and RIPK3 by TNFa in cecal tissue,indicating that FKBP12 is an important target for regulating necroptosis.ConclusionFKBP12 is a new target for regulating necroptosis.It can promote the formation of RIPK1/RIPK3 heterodimers and RIPK1 and RIPK3 homopolymers by regulating the expression and phosphorylation of RIPK1 and RIPK3,so as to promote the activation of RIPK1-RIPK3-MLKL signaling pathway,and initiate the necroptosis induced by TNF?at last.At the same time,FKBP12 also plays an important role in the regulation of systemic inflammatory response syndrome caused by necroptosis,indicating that FKBP12 can also regulate the occurrence of necroptosis in vivo.Based on the above results,we believe that FKBP12 protein may be an effective target for the treatment of inflammatory diseases such as SIRS,and the ligand drugs of FKBP12 protein may be used in the clinical treatment of a variety of necroptosis related diseases,including SIRS.