The Roles Of TIPE2 Protein In Cell Proliferation And Inflammation

TNFAIP8L2 (TIPE2), the tumor necrosis factor (TNF)-a-induced protein 8 (TNFAIP8, TIPE) like 2, is a member of the TNFAIP8 family. TIPE2 was originally identified as a gene abnormally expressed in the inflamed spinal cord of mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, using a high-throughput gene microarray technology. Initial studies demonstrated that TIPE2 is a new negative regulator of inflammation which plays important role in maintaining immune homeostasis. Its deficiency in mice causes fetal inflammatory diseases and abnormal expression in humans is associated with many kinds of diseases, such as systemic lupus erythematosus (SLE), hepatitis B, transplantation rejection, childhood asthma and atherosclerosis. Recent studies revealed that anti-inflammatory TIPE2 is a negative regulator of carcinogenesis. Forced expression of TIPE2 significantly promotes cell death and attenuates Ras-induced tumorigenesis in mice. Importantly, TIPE2 expression was either completely lost or significantly down-regulated in human hepatic cancer, gastric cancer, non-small cell lung cancer, which may be responsible for these tumor cells to escape apoptosis. TIPE2 functions as a negative regulator of both inflammation and carcinogenesis through Rac or Ras signaling. It directly binds to Rac and RalGDS, thus inhibits downstream activation of Rac and Ras cascades.Both Ras and Rac signaling cascades are involved in various physiological and pathological conditions. As a novel regulator of Ras and Rac signaling, TIPE2 may participate in regulating other diseases. In fact, our previous studies demonstrated that murine TIPE2 plays an atheroprotective action through two different mechanisms:1) negatively regulating ox-LDL-induced inflammatory responses and iNOS expression in macrophages; 2) attenuating the phenotypic switching of VSMCs (vascular smooth muscle cells) in response to ox-LDL stimuli. Based on these discoveries, we hypothesize that 1) TIPE2 may regulate arginine metabolism through regulating iNOS expression; 2) TIPE2 may play important roles in vascular proliferative diseases such as restenosis.Objectives1. To investigate the role and potential mechanisms of TIPE2 in experimental Restenosis.2. To study the role and underlying mechanisms of TIPE2 in arginine metabolism.MethodsI. To study the role and mechanism of TIPE2 in experimental restenosis.1. In vivo1.1 Injury-induced restenosisTIPE2-’-(n=15) or C57BL/6J mice (n=45) were used to perform carotid artery ligation to induce restenosis as described previously. The left carotid artery was completely ligated just proximal to the carotid bifurcation. The right carotid artery served as an uninjured control. Both the left and right carotid arteries were harvested at 7,14, and 21 days after injury. The carotid artery was dissected and fixed in 4% paraformaldehyde overnight, then embedded in optimal cutting temperature (OCT) compound. Cross-sections about 5 μm were prepared and subjected to hematoxylin and eosin staining, and the areas of the intima and media were measured by Image-Pro Plus software.1.2 EDU incorporation in vivoAll mice received intraperitoneal injection of the thymidine analogue 5-ethynyl-2’-deoxyuridine (EDU, Invitrogen) at 500 μg/100 g body weight at 12 hrs before sacrifice. Some sections were washed in PBS and permeabilized using 0.5% Triton X-100, and incorporated EDU was conjugated to Alexa Fluor 488-azides using the Click-IT reagent kit, then stained with McAb anti-SmαA and rhodamine-conjugated second Abs. After the cell nuclei were stained with 4’, 6-diamino-2-phenylindole (DAPI), the sections were visualized by fluorescent microscopy (Olympus BX51) and percentage of EDU-positive VSMCs was analyzed by Image-Pro Plus software.1.3 Recombinant Adenovirus InfectionRecombinant adenovirus encoding murine TIPE2 (mTIPE2) and GFP were prepared by Sinogenomax corporation (Beijing, China). WT mice (n=20) with complete ligation in left carotid artery were in situ infected with mTIPE2-expressed adenovirus or GFP-expressed adenovirus (1×10 pfu). Mice were subjected to a second infection by tail vein injection at day 21 and sacrificed at day 42. Cross-sections preparation and morphometric analysis were performed as described above.2. In vitro2.1 Cell culture of VSMCsPrimary murine VSMCs used in this study were obtained from the aorta ventralis of 8-week-old male C57BL/6J (wide type, WT) or TIPE2-/-mice by enzymatic digestion (type Ⅰ collagenase and type Ⅲ elastase, Sigma). Immunostaining with anti-SMαA and Flow cytometric analysis were performed to determine vascular smooth muscle cells (VSMCs). VSMCs from passages 3 to 7 were utilized to do experiments.2.2 To study the role of TIPE2 in cell cycle regulation of VSMCsInfluence of TIPE2 on VSMCs proliferation in normal and PDGF-BB-containing condition was measured using Cell Counting Kit-8 reagent (CCK-8) as described by the manufacturer. Furthermore, TIPE2-induced change of cell cycle distribution was analyzed by flow cytometry.At last, the expression of cell cycle related proteins was detected by both realtime PCR and western blot.2.3 To study the potential mechanism whereby TIPE2 regulates cell cycle distribution in VSMCsBoth STAT3 and ERK1/2 cascades play important role on the regulation of VSMCs proliferation and cell cycle progression. Thus the phosphorylation levels of STAT3 (at Try705) and ERK1/2 in PDGF-BB-stimulated WT and TIPE24’VSMCs were test by western blotting. Furthermore, nucleus translocation of activated STAT3 was measured by Immunofluorescence. At last, PD98059 and Stattic (specific inhibitors for ERK1/2 and STAT3, respectively) were utilized to confirm the underlying mechanism that TIPE2 controls VSMCs cell cycle progression by affecting STAT3 and ERK1/2 signaling cascades.2.4 To determine whether TIPE2 controls VSMCs cell cycle transition through Racl-STAT3 and Racl-ERK1/2 signaling cascades.TIPE2 is a newly identified inhibitor of Racl. To investigate whether TIPE2 affects cell cycle through Racl, we firstly detected the activation of Racl in WT and TIPE2-’-VSMCs, WT VSMCs transfected with 2 μg of plasmids encoding mTIPE2 or R24A mutants by PDB pull down assay following western blotting. Then NSC23766 (a specific inhibitor that blocks Racl activation) and Racl specific siRNA were utilized to study whether the role of TIPE2 in control of ERK1/2, STAT3 activation, nuclear translocation and subsequent cell cycle transition depends on Racl.Ⅱ. To determine whether TIPE2 regulate arginine metabolism in macrophage1. Microbial InfectionPeritoneal macrophages from wild type and TIPE2-/-mice were infected with E.coli (DH5α) for 30 minutes at a ratio of 1:1. Free bacterial were removed by vigorous washing and cells were incubated with 100 μM of iNOS selective inhibitor 1400w for additional 2 hours. Bactericidal activity was calculated by the colony forming unit assay both before and after the 2 hours incubation.2. Construction of TIPE2-expression stable RAW264.7 cells Murine macrophage cell line Raw264.7 was transfected with a TIPE2 expression vector (pRK5-TIPE2) or pRK5 alone using Lipofectamine 2000 according to the manufacturer’s protocol. The cells were then selected in medium with 500 u,g/mL G418 (Invitrogen) for two weeks, then the resistant clones were isolated, expanded, and used for the following experiments.3. Peritoneal Macrophage Isolation and CultureFor isolation of elicited peritoneal macrophages, age and sex-matched WT and TIPE2-deficient mice were injected intraperitoneally with 1.0 mL of 6%sterile starch solution. Four days after injection, cells were harvested by intraperitoneal lavage with 10 mL ice-cold PBS and were extensively washed using ice-cold PBS. Cells were then seeded in DMEM medium with 10% FBS,100 U/mL penicillin, and 100 μg/mL streptomycin for 4 hours and adherent cells were taken as peritoneal macrophages.4. Cell treatmentPrimary peritoneal macrophages or RAW264.7 cells incubated with DMEM medium containing 10% FBS overnight and then treated with 100ng/mL LPS. After treatment, cell supernatant was collected to detect the levels of NO and urea; cells were collect to detect the expression of iNOS and arginase by realtime PCR or western blot. Expecially, phosphorylation of IκB and MAPK at 0,15,30,60,120 min after LPS stimulation was tested by western blot.5. Treatment of Animals5.1 WT or TIPE2-/- mice were treated with 1.5 mg/kg LPS (Sigma-Aldrich, St. Louis, MO, USA) or PBS intraperitoneal administration. At 0 h,3 h, and 24 h after treatment, mice were euthanized for blood sampling, and then the lung and liver tissues were collected and stored at -80℃ until use.5.2 Nitrite levels in sera were determined by the Griess assay, while Urea levels were determined with the use of standard enzymatic methods and commercial kits. The expression of iNOS and arginase in liver/lung was also tested in this model.ResultsI. TIPE2 attenuates injury-induced Restenosis by negatively regulating VSMCs proliferation1. TIPE2 suppresses injury-induced Restenosis in mice.a. In injury-induced restenosis models, neointimal area was significantly larger in TIPET-/- mice than WT controls,1.9-fold larger at day 14, and 3.7-fold larger at day 21 after injury;b. An increased I/M (Intimal/Media) ratio in TIPE2-/- mice was observed at day 14 (TIPE2-/- vs. WT,0.739±0.1075 (n=5) vs.0.416±0.0601 (n=5), P=0.0252) and day 21 (TIPE2-/-vs. WT,2.422±0.2101 (n=5) vs.0.517±0.0812 (n=5), P< 0.0001) after injury.c. Recombinant adenovirus-mediated overexpression of mTIPE2 significantly delayed injury-induced neointimal formation in WT mice.2. TIPE2 deficiency significantly promotes VSMCs proliferation in vivo. We examined the cellular proliferation in carotid arteries by EDU staining. Intriguing, EDU-positive cells in arteries were significantly increased in TIPE2-/-mice. At day 7 after injury, a higher percentage of EDU-positive cells was observed in TIPE2-/-mice than WT mice controls (Figure IF, TIPE2-/- vs. WT, 21.69±2.639%(n=5) vs.9.58±1.625%(n=5), P=0.0175).3. Both injury and PDGF-BB markedly upregulated TIPE2 expression in VSMCs.a. TIPE2 protein was upregulated in response to injury in vascular media of WT miceb. Both the mRNA and protein levels of TIPE2 in WT but not TIPE2-/- VSMCs were increased significantly following PDGF-BB (10 ng/mL) stimulation.4. TIPE2 inhibits VSMCs proliferation in both normal and PDGF-BB-stimulated conditions.a. TIPE2 deficiency increased the proportion of VSMCs in S phase (Untreated: TIPE2-/-vs. WT,12.17 ± 0.436%(n=5) vs.6.00 ± 0.351%(n=5), P= 0.0004; PDGF-BB-treated:41.61 ± 2.138%(n=5) vs.19.12 ± 0.944%(n=5), P= 0.0007).b. TIPE2 ablation increased expression of Cyclin D1 and D3, which are essential for the G1/S phase transition, in PDGF-BB-treated VSMCsc. TIPE2 overexpression inhibits D type cyclins expression and thus sequesters VSMCs in Go/G1 phase.5. TIPE2 control VSMCs proliferation through Racl-STAT3, Racl-ERK signaling cascades.a. TIPE2 deficiency in VSMCs markedly enhanced Racl activation in response to PDGF-BB stimulation. Whereas overexpression of TIPE2 significantly inhibited Racl activationb. The phosphorylation levels of STAT3 (at Try705) and ERK1/2 were strikingly enhanced in TIPE2-deficient VSMCs, but were markedly decreased by TIPE2 overexpression.c. The enhanced activation of ERK1/2 and STAT3 in TIPE2-deficient VSMCs could be eliminated by NSC23766, a specific inhibitor that blocks Racl activation.d. The different mRNA expression levels between WT and TIPE2-’-VSMCs was eliminated by PD98059 and Stattic (specific inhibitors for ERK1/2 and STAT3, respectively).6. TIPE2 blocks STAT3 nuclear translocation in a Racl dependent mannera. The levels of intranuclear STAT3 were significantly increased in TIPE2-/-VSMCs compared with WT cells treated with or without PDGF-BB.b. The spontaneously nuclear translocation of STAT3-C (a dominant-active mutant of STAT3) could be blocked by Racl-siRNA.c. Overexpression of TIPE2 could significantly suppress nucleus translocation of STAT3-C.d. The inhibitory effect of TIPE2 and Rac 1-siRNA in the nucleus translocation of STAT3-C could almost completely be abolished by Racl-Q61L, a constitutively active form of Rac 1.II. TIPE2 Negatively Regulates Inflammation by Switching Arginine Metabolism from Nitric Oxide Synthase to Arginase1. TIPE2 deficiency enhances NO-mediated bactericidal activity in macrophagesCompared with WT, TIPE2-/- macrophages exhibited increased bacterial clearance with E.coli infection. However, the addition of iNOS inhibitor partially reversed the enhanced killing activity seen in TIPE2-/- macrophages. Thus, the increased NO production in TIPE2-/- macrophages correlates with increased E.coli killing in vitro.2. TIPE2 overexpression attenuates LPS-induced iNOS but increases arginase I expression in RAW264.7 cells.Upon LPS stimulation, both the mRNA and protein levels of iNOS in TIPE2 overexpression cells were much lower than controls, suggesting that enhanced TIPE2 expression inhibited the induction of iNOS by LPS challenge. However,the mRNA level of ARG1 but not ARG2 was increased significantly in cellsoverexpressing TIPE2 compared to vector controls.3. TIPE2 overexpression decreases LPS-induced NO production but increases urea levels in RAW264.7 cellsLPS stimulation resulted in increased NO production in both TIPE2 overexpression cells and vector controls. However, the levels of NO in TIPE2 overexpression cells were much lower than vector controls, indicating that TIPE2 overexpression attenuates LPS-induced NO production. Consistent with the change of ARG1, TIPE2 overexpression in Raw264.7 significantly decreases the urea concentration in the culture medium.4. TIPE2-deficiency increases LPS-induced NO production but decreases urea production in primary macrophages.After treatment with LPS, the levels of iNOS mRNA and protein in TIPE2-deficient macrophages from TIPE2-/- mice were significantly higher than macrophages from WT controls. As a consequence, the levels of NO in TIPE2-deficient macrophages were significantly increased compared to WT controls. In addition, TIPE2 deficiency attenuates LPS-induced ARG1 transcription thus decreases the urea production.5. TIPE2-deficient mice exhibit increased iNOS expression and NO production following LPS challenge.The levels of iNOS in liver and lung in TIPE2-deficient mice were markedly higher than those in WT mice following LPS challenge, which results in the enhanced induction of serum NO in TIPE2-/-mice, while the expression of arginase I mRNA in liver and lung obtained from TIPE2’’’mice was lower than WT controls. Rather to our surprise, TIPE2 deficiency enhances expression of ARG2 in liver and lung, thus results in increased levels of serum urea.6. LPS challenge increases IκB, JNK and p38 phosphorylation in TIPE2-deficient macrophages.TIPE2 deficiency in macrophages promotes the phosphorylation and degradation of IκB. Meanwhile, significant enhanced activation of JNK and p38 in TIPE2-deficient macrophages in response to LPS was also observed. It has been reported that both NF-κB and MAPK play important role in LPS-induced iNOS expresson, thus these results suggest that TIPE2 may negatively regulates iNOS expression and iNOS-mediated inflammatory responses by inhibiting NF-κB, JNK, and p38 pathways.Conclusions1. TIPE2 attenuates experimental restenosis by negatively regulating VSMCs proliferation. The main mechanisms base on the inhibitory effects of TIPE2 on Racl activation and downstream signaling transduction toward to ERK1/2 and STAT3.2. TIPE2 inhibits LPS-induced iNOS expression in macrophage, resulting in arginine metabolism skewing from iNOS to arginase-mediated metabolic pathways, by which TIPE2 negatively regulates NO production and NO-mediated inflammatory responses.Innovation and significancesThe innovation and significance of this study are:i) TIPE2 is a newly identified anti-inflammation factor that regulates immune functions. However, little is known about the role of TIPE2 outside the immune field. Here we report that TIPE2 inhibits VSMCs proliferation and subsequent restenosis, providing new insights into TIPE2 functions; ii) our study is the first to report that TIPE2 skews arginine metabolism from iNOS to arginase-mediated metabolic pathway and thus inhibits NO-related inflammatory responses; this provides a new mechanism of TIPE2 action in inflammation. Below is the detailed description.1. It is the first to report the crucial roles of TIPE2 in restenosis, and the possibilty of treating experimental restenosis with TIPE2.2. It is the first to investigate the regulatory role of TIPE2 in STAT3 activation and nuclear translocation, which is helpful to further explore therapeutic strategies targeting STAT3.3. The studies in Part I elaborate the mechanisms of TIPE2 action in VSMCs proliferation, which may provide a new therapeutic strategy for vascular hyperplasia diseases.4. The studies in Part Ⅱ uncover the role of TIPE2 in arginine metabolism, providing a new mechanism for TIPE2 action in inflammation.