The Effect And Mechanism Of TRIM22 On Monocyte Apoptosis

Part 1 The effect and mechanism of TRIM22 on monocyte apoptosisObjective:TRIM22,a member of the TRIM protein family,is constitutively expressed in peripheral blood leukocytes and lymphoid tissues,especially expressed at high levels in monocytes.Increasing evidence suggests that TRIM22 plays important roles in antiviral response and inflammation.Additionally,TRIM22 is a target gene of p53,which is a well-known regulator of cell growth and cell death.Overexpression of TRIM22 interferes with clonogenic growth of U937 cells.Thus,we speculated that TRIM22 may involve in inflammatory response through controlling monocyte fate.This part of the study was designed to investigate the effect and mechanism of TRIM22 on monocyte apoptosis.Method:The recombinant replication-deficient adenoviral vectors encoding TRIM22 gene were constructed and transfected into a monocytic cell line THP-1 cell.Apoptosis of transfected cells was induced by staurosporine and assayed using flow cytometry both in absence and presence of lipopolysaccharide.The levels of cleaved caspase-3,cleaved caspase-9 and cytoplasmic cytochrome c were assayed using immunoblot.The caspase signaling pathways involved were illustrated by inhibitory experiments used a caspase inhibitor z-VAD-fink.Pro-/anti-apoptotic protein levels and protein oligomerization were measured using immunoblot.The correlation of TRIM22 and Bak during apoptosis was further confirmed in primary human monocytes exposed to inflammatory stimuli.Results:Recombinant adenoviral vectors expressing TRIM22 were constructed and infected THP-1 cells.Seventy-two hours after transduction,efficient infection was confirmed by real-time PCR and western blot analysis.Overexpression of TRIM22 in THP-1 cells led to extensive apoptosis both in the absence(87.8%±6.4%vs.23.1%±9.4%,P<0.01)and presence(91.1%±6.3%vs.25.5%±14.3%,P<0.01)of lipopolysaccharide.This function was associated with activation of caspase-9 and caspase-3,and release of cytochrome c,while inhibiting caspase activity using a caspase inhibitor z-VAD-fmk prevented TRIM22-induced apoptosis(10.9%±3.5%vs.82.6%± 22.3%,P<0.01).Overexpression of TRIM22 in monocytes also enhanced pro-apoptotic protein Bak expression and oligomerization.In primary human monocytes exposed to inflammatory stimuli,TRIM22 level positively correlated with Bak level after induction of apoptosis.Conclusion:TRIM22 plays a critical role in monocyte apoptosis via upregulating the expression level and oligomerization of Bak.Part 2 The domain in TRIM22-mediated apoptosisObjective:The RING domain and the B30.2/SPRY domain are functionally important structures of TRIM22,in which the former possesses E3 ubiquitin ligase activity,and the latter is required for nuclear localization and the formation of nuclear bodies.The aim of this study was to identify which domain is critical for the pro-apoptotic role of TRIM22.Method:The recombinant replication-deficient adenoviral vector expressing wild-type TRIM22(Ad.TRIM22),its RING domain deletion mutants(Ad.TRIM22-ARING),and its SPRY domain deletion mutants(Ad.TRIM22-ASPRY)was constructed and transfected into a monocytic cell line THP-1 cell.Their effects on apoptosis were evaluated by flow cytometry.Bak expression and Bak oligomerization were analized using western blot.Results:Deletion of either RING domain or SPRY domain in TRIM22 molecule moderately increased cell apoptosis(65.1%±6.6%vs.32%±11.3%,P<0.05;65.1%±6.6%vs.31.3%±10.5%,P<0.05).Furthermore,deletion of either RING or SPRY domain in TRIM22 molecule partially blocked up-regulation of Bak expression and oligomerization.Conclusion:Both RING and SPRY domains are crucial for the effect of TRIM22 on monocyte apoptosis.Part 3 Expression level of TRIM22 in monocytes of septic patients and its relationship with Bak levelObjective:Monocyte apoptosis is a key mechanism that orchestrates host immune response during sepsis.We have found TRIM22 plays a critical role in monocyte apoptosis via regulating Bak oligomerization,suggesting TRIM22 may impact on monocyte fate in sepsis.This part of the study was designed to observe TRIM22 expression level in monocytes from adult and pediatric septic patients,and analyzed its relationship with Bak expression level.Method:We enrolled 15 septic and 8 non-septic critically ill adult patients admitted to university hospital ICU from October 2014 to Feberury 2015,as well as 10 septic and 14 non-septic surgical pediatric patients admitted to university hospital PICU from November 2014 to March 2015.The monocytes were isolated from peripheral whole blood of the enrolled patients which was drawn within 24 hours after diagnosis of sepsis.The expression levels of TRIM22 and Bak in monocytes were measured by real-time PCR.Results:The present study showed that the monocytic mRNA levels of TRIM22 in adult septic patients was significantly lower compared to that in controls(P<0.05),and positively correlated with Bak level(R=0.6064,P=0.0006).In monocytes from pediatric septic patients,TRIM22 levels were also down-regulated(P<0.05).The monocytic mRNA levels of Bak in those pediatric septic patients who have lower TRIM22 level showed a declining trend(R=0.5801,P=0.0787).Conclusion:TRIM22 expression was downregulated in patients with sepsis,which is positively correlated with Bak expression,suggesting TRIM22 may play a critical role in monocyte apoptosis during sepsis.