The Role Of SIGIRR In LPS-induced Ali In Mice And The Screening And Preliminary Study Of SIGIRR Interacting Proteins

1. Background and objectiveAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical critical diseases. At the present, there are not specific therapies for ALI/ARDS. Toll-like receptors (TLRs) are one of important pathogen pattern recognition receptors in innate immune system. The excessive activation of TLR-signaling plays a key role in uncontrolled inflammatory response. Single immunoglobin IL-1 receptor related protein (SIGIRR). SIGIRR, also named Toll-like receptor/Interleukin-1 receptor 8 (TIR8), is one of recently discovered TIR superfamily members and is a negative regulator for TLR signaling. However, the precise and potential effect of SIGIRR on lung injury remains unclear. Trapping the crucial components of Toll-like receptor/Interleukin-1 receptor (TIR) signaling by SIGIRR may be one of possible mechanisms of SIGIRR negative regulation, but the detailed molecular mechanisms of SIGIRR negative regulation require further investigations. It has been proved that the intracellular TIR domain of SIGIRR can interact with IL-1R/TLR4 complexes in the TIR signaling. Yeast two hybrid system is an effective method for studying the interaction between two proteins. Using yeast two-hybrid screening, we can find novel proteins that interacted with the functionally known protein and speculate the possible roles of these novel proteins. With these in mind, the present study was performed to investigate the effect of enhanced SIGIRR expression on LPS-induced ALI in mice by constructing recombinant adenoviral vector that carries the mSIGIRR, to further identify intracellular protein that interacts with SIGIRR in human lung cDNA library by using a yeast two-hybrid screen and then to study the novel binding proteins of SIGIRR superficially. Our expected results will provide direct and precise evidences about the role of SIGIRR in LPS-induced ALI/ARDS, a novel insight into the molecular mechanisms of SIGIRR negative regulation, and new targets for the prevention and treatment of ALI/ARDS.2. Methods(1) Construction and identification of recombinant adenoviral vector (Ad.mSIGIRR) An adenovirus was generated that contained a single open reading frame encoding a murine SIGIRR (mSIGIRR). First, cDNA coding for mSIGIRR was excised from pCMV-SPORT6-mSIGIRR and ligated into the adenoviral shuttle plasmid pDC316 to generate the plasmid pDC316-mSIGIRR. And the pDC316-mSIGIRR was identified by DNA sequencing and dual-site endonuclease digestion. Recombinant viruses were then generated using HEK293 packaging cells co-transfected with pDC316-mSIGIRR and a plasmid containing cDNA for adenoviral proteins (pBHGlox) via the AdMax system. The quality and titer of Ad.mSIGIRR were identified by polymerase chain reaction (PCR), determination of the viral particle absorbance, and means of 50% tissue culture infectious dose, respectively. Ad.mSIGIRR was used to infect A549 cells and to establish respiratory adenovirus infected mice models. Then the expression activity of Ad.mSIGIRR in vitro and in vivo was detected by Western blot and immunohistochemistry. Meanwhile, the pulmonary histological changes of adenovirus infected mice models were determined by routine pathological examination.(2) Enhanced expression of SIGIRR ameliorates LPS-induced ALI in miceBALB/c mice were lightly anesthetized by ether inhalation, and inoculated intranasally with 4×10⁷ PFU (in 50?L) of Ad.mSIGIRR (group Ad.mSIGIRR) or Ad.V (group Ad.V). Forty-eight hours after intranasal administration of viruses, LPS at 15 mg/kg was injected into the peritoneal cavity of conscious mice to establish ALI mouse model. The mice were sacrificed at 0, 2, 6, 12, 24, and 48 h after LPS challenge. Blood and lung samples were harvested aseptically for subsequent experiments. The expression of SIGIRR was determined by RT-PCR, Western blot and immunohistochemistry analysis. Meanwhile, the differences of pulmonary histological changes and 7-day survival rate between two groups were compared. The levels of TNF-?in lung and serum, the concentration of NO in lung, and the activity of MPO and NF?B were also examined by using corresponding methods.(3) The screening and preliminary study of SIGIRR interacting proteinsHuman SIGIRR (480–1230bp) cDNA encoding the intracellular domain of SIGIRR was amplified from pReceiver-LV19-SIGIRR by PCR and ligated into pSos vector to generate the bait plasmid pSos-SIGIRR. And the pSos-SIGIRR was identified by DNA sequencing and dual-site endonuclease digestion. After the mutational revertant and phenotypic verification of cdc25H yeast cells and the transcriptional activation of bait plasmid were performed, the bait and the cDNA library were co-transformed into yeast cells. Yeast cultures and two-hybrid procedures were carried out according to the procedure of the protocol of CytoTrap yeast two-hybrid system (Stratagene). The screening results were analyzed by bioinformatics method and further confirmed by co-immunoprecipitation. In consideration of the subcellular localization, one clone encoding the partial cDNA fragment of paralemmin-3 (PALM3) was chosen as the object of subsequent study. The PALM3 expression in a human alveolar epithelial cell line (A549 cells) was detected by RT-PCR. The expression patterns of SIGIRR and PALM3 were compared by means of Real-time RT-PCR. To test whether PALM3 has a role in the LPS-mediated inflammatory cytokine gene expression, the expression of PALM3 expression in A549 cells was knocked down by siRNA. Then the levels of cytokines (TNF-?, IL-6 and IL-1?) in cultured supernatants were examined by ELISA.3. Results(1) The recombinant adenovirus vector carries that murine SIGIRR, Ad.mSIGIRR, was constructed successfully. The titer and the number of viral particle of Ad.mSIGIRR were 4×10^<sup>9 PFU/m and 4.65×10¹¹ VP/ mL, respectively, which met the requirement of subsequent experiments. The adenovirus delivery system had no effect on pulmonary inflammation and histological changes.(2) SIGIRR was constitutively expressed in normal murine lung and was mainly detected in the pulmonary epithelial cells. After LPS stimulation, SIGIRR protein and mRNA levels were downregulated. Compared with those in group Ad.V, SIGIRR mRNA and protein expression were enhanced in group Ad.mSIGIRR (P < 0.05). Enhanced expression of SIGIRR significantly decreased the levels of TNF-?and NO, inhibited the activation of NF-?B, attenuated pulmonary pathological changes and neutrophil infiltration, and improved the outcome of ALI (P < 0.05).(3) Using yeast two-hybrid screening, four clones were identified as the potential interaction protein of SIGIRR. They were Homo sapiens mitochondrion, complete genome; Homo sapiens cyclin H (CCNH), mRNA; Homo sapiens paralemmin-3 (PALM3), mRNA; and Homo sapiens v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), mRNA, respectively. Among these positive clones, Homo sapiens mitochondrion, complete genome was noncoding sequence. KRAS was a false positive clone in the background of Cytotrap Y2H system. The protein expression of CCNH located in cellular nucleus, while that of PALM3 located in cellular membrane. PALM3 was selected for the subsequent study. The interaction between PALM3 and SIGIRR was confirmed by co-immunoprecipitation. PALM3 was expressed in A549 cells. The expression of SIGIRR was downregulated but that of PALM3 was upregulated by LPS-stimulation in A549 cells. Silencing PALM3 by RNA interference inhibited the release of inflammatory cytokines in A549 cells after LPS-stimulation (P < 0.05).4. Conclusion(1) The expression of SIGIRR is downregulated post LPS-challenge. SIGIRR overexpression has a protective effect on LPS-induced ALI in mice by regulating the LPS-TLR4 signaling.(2) PALM3 should be a novel interacting partner of SIGIRR. The PALM3 mRNA expression is upregulated by LPS-stimulation in a human alveolar epithelial cell line (A549 cells). The interaction between SIGIRR and PALM3 may partly account for the mechanism of the negatively regulatory effect of SIGIRR.