| Cyclic adenosine monophosphate(cAMP)-mediated pathway is one of the most important signal pathways. It was found might play a roll in the anti-inflammatory modification by hypoxia precondiaitoning (HPC), which is kown for its protection for cells in the following hypoixa procedure. During HPC, extra cellular adenosine (ADO) increases due to the chang of oxygen concentration, this may enhance the simulation to adenylyl cyclase (AC), which will lead to an increase of cAMP in turn. On the other hand, it was recently found that cAMP inhibits the activation of p38 mitogen-activated protein kinase (p38MAPK) via cAMP response element-binding protein (CREB)-induced dynein light chain (DLC). Taken together, we hypothesize that cAMP may be increased by HPC treatment, and inhibits the activation of p38MAPK, therefore, attenuates the NF-κB mediated inflammation. However, the mechnism relates cAMP to HPC is still unknown. In general, cAMP is produeced from 5'-AMP when AC is stimulated by some G-protein coupled receptors (GPCRs), such as ADO receptor 2A, 2B, and degrades to 5'-AMP when Phosphodiesterase (PDEs) is activated. To confirm our hypothesize, we studied the intracellular cAMP level after the treatment of HPC vesus hypoxia and normoxia control, and further tried to explore the mechnism responsible to this change.MethodsMurine preconditioning model.C57BL/6 (Charles River Laboratories) male mice of 8–10 weeks of age were used in this study. The Animal Care and Use Committee of Brigham and Women's Hospital approved all procedures. Whole-body preconditioning was performed in a manner similar to that previously described. Briefly, mice were placed in humidified environmental chambers and subjected to Fi O2 8% for 10 minutes followed by Fi O2 21% for 10 minutes for 3 cycles. The O2 concentrations in the chambers were continuously measured by an O2 analyzer. Following preconditioning, mice were subjected to FiO2 21% for 120 minutes. Preconditioned and nonpreconditioned animals were subsequently placed in the chambers and subjected to FiO2 5% for 10 minutes. Animals were then removed from the chamber and sacrificed, and pulmonary tissue was collected. For each experiment, 3 animals were treated in triplicate.Cell culture, and in vitro preconditioning.HeLa cells (ATCC) were maintained in DMEM plus 10% FBS, Calu-3 cells (ATCC) in MEM plus 10%FBS, T84(ATCC) cells in a mixture of Ham's F12 and DMEM (1:1), plus 10% FBS, HMEC cells in MCDB131 plus 10% FBS at 37_C in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells. Hela or Calu-3 cells were placed in a hypoxia chamber (Coy Laboratory Products Inc.) in pre-equilibrated hypoxic media at 2% O2 for 45 minutes, then re-oxygenated in normoxic conditions (21% O2) for 20 minutes. When cells were returned to hypoxia, media was once again replaced with fresh hypoxic medium in order to minimize the effects of oxygen present. This protocol was followed for 3 cycles.ELISA assay.Calu-3 or T84 cells were plated in BD culture dishes (10 cm in diameter) and allowed to grow to approximately 70-80% confluence, treated in hypoxia chamber following the in vitro preconditioning protocol, then stimulated with 10uM adenosin for 30 minutes or not, cyclic AMP concentration of cell lysates were measured immediately using cAMP assay kit (R&D Systems, Inc. #KGE002) following steps instructed by the manufacturer: cells were lysed by cell lysis buffer, applied to the 96-well plates coated with goat anti-mouse antibody in triplicate, incubated at room temperature with primary antibody and cAMP conjugate on a orbital shaker at a speed of 500rpm for 3 hours, washed 3 times with wash buffer, optical density were then measured sequentially at 450nm and 570nm using a spectrophotometer (Bio-Rad). All reagents and the coated plates were provided in the kit.Transfections and NF-κB reporter assays.Transfection of Hela cells was carried out using Fugene 6 transfection reagent, with pNF-κB-Luc plasmid(pNRE; Clontech) at a concentration of 1.6μg per well along with 0.08μg of Renilla for 24 hours and then either left at normoxia or subjected to a protocol of preconditioning. Following treatments, cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).Transcriptional analysis.cDNA was collected Using"Clonetech sprint powerscript"after extraction of total RNA. Real-time PCR (Taqman, Apllied Biosystems) was employed to examine PDE4B in Calu-3 cells and IL-6 expression in HeLa cells following the instructions from the manufacturer. PDE4B primers:forward 5'-ATGGGCAGATTTGGTACAGC-3'; reverse 5'–TAGCC GTCCAGAAATGGTTT-3'. Ready-to-use IL-6 primer was ordered from Applied Biosystems. Samples were controlled forβ-actin using the following primers: forward, 5'-GGTGGCTTTTAATGGCAAG-3'; reverse, 5'-ACTGGAACGGTGAAGGTGACAG-3' Western blot.Total protein was isolated from cells using cell lysis buffer (Cell signaling Technology, #9803), or extracted from lungs of mice with RIPA buffer (Cell signaling Technology, #9806), both containing protease inhibitor cocktail (Roche Diagnostics, #04693124001) and PMSF 1mM. Protein concentrations were measured by DC protein assay (Bio-Rad). An equal amount of protein was boiled in SDS loading buffer (Bio-Rad), then resolved on 8% polyacrylamide denaturing gels and transferred to nitrocellulose (Bio-Rad). After transfer, the membranes were stained with ponceau S stain in order to verify equal loading. Antibodies used for Western blotting included rabbit anti–PDE4B (1:1000; Abcam), rabbit anti-p38 and P-p38 (1:1000; Cell signaling Technology), rabbit anti-actin (1:2,000; Cell signaling Technology). Blots were washed, and species-matched peroxidase-conjugated secondary antibody was added. Labeled bands from washed blots were detected by enhanced chemiluminescence (Thermol Scientific).Immunofluorescence study.For p65 nuclear translocation studies, HeLa cells were plated on 4-well glass chamber slides (Nalgene Nunc International) and allowed to grow to approximately 50-70% confluence, treated in hypoxia chamber following in vitro preconditioning protocol. Cells were fixed in 1% paraformaldehyde/PBS at 4℃for 10 minutes and permeabilized with prechilled 0.2% Triton X-100/PBS/2% BSA. Cells were incubated with rabbit anti-p65 (1:200; Rockland Immunochemicals) in 1% normal goat serum in PBS for 1 hour followed by anti-rabbit Oregon Green 488 (1:100, Molecular Probes; Invitrogen) in the same buffer for 30 minutes. Cell images were captured on a fluorescence microscope.ResultsIntracellular cyclic AMP level was obviously lifted by HPC treatmentInitially, Calu-3 cells and T84 cells were treated in nomoxia, hypoxia and HPC respectively, right after the treatment, cyclic AMP was extracted using the lysis buffer provieded in the kit, then measured through ELISA assay (see Metholds for protocol). An increase of cyclic AMP was found in cells subjected to HPC, compared with hypoxia and normoxia groups (2-fold increase compared with normoxia control; P < 0.05), indicating cyclic AMP may play a roll in the anti-inflammatory modification caused by HPC treatment.Recruitment ofβ-arrestin to lipid raft was blocked by HPC treatmentTo define the mechnism accont for cyclic AMP increase, we first studied the recruitment ofβ-arrestin to lipid raft, which is reponsive to the desensitization of GPCRs, including A2AR and A2BR. Protein was extracted from HeLa cells had been treated in nomoxia, hypoxia or HPC, in two steps: first step, with lysis buffer containing triton x-100 to get cytoplasmic and most membrane proteins; second with lysis buffer containing OCG to extract lipid raft and nuclei proteins. Result of western blot assay revealed an sharp decrease ofβ-arrestin protein level in the lipid raft extraction of cells subjected to HPC, compared with nomoxia and hypoxia control groups, indicating the recruitment ofβ-arrestin to lipid raft was blocked by HPC. As an result, this may lead to an decrase of the desensitization of A2AR and A2BR, make the recptors"keeping open", in turn, cells keep produce more cyclic AMP.HPC treated cells has a higher efficency in producing cAMP when stimulated with ADOTo test if HPC affects the ability of cells to produce cAMP, after the HPC/Hypoxia/ Normoxia treatment, we stimulated cells with ADO for 30minutes, measured cAMP immediately. A higher increase of cAMP in HPC group after ADO stimulation was noticed, compared with other two groups, however, in hypoxia group, cAMP was not lifted, actualy, even lower than that of non-stimulated cells, confirming the hypothesis from the result ofβ-arrestin.HPC treatment transciptionaly down-regulates PDE4B both in vitro and in vivoTotal protein of Calu-3,Hela,T84 and HMEC cells treated in either nomoxia, hypoxia or HPC were extracted, then, PDE4B, a well-known mediator of cyclic AMP degradation, was measured using western blot respectively. Consistent to the increase of cyclic AMP level, PDE4B was found to be much lower in HPC group compared with normoxia and hypoxia treated cells. Western blot assay of total protein of lung tissue from mice subjected to normoxia, hypoxia or HPC shows similar result with in vitro experiment. Further more, we measured the mRNA level of PDE4B in Calu-3 through RT-PCR, result shows a similar change with PDE4B protein, indicating the change is transcriptional. These data give us the information that the degradation of cyclic AMP was inhibited during HPC treatment.HPC treatment inhibits p38 phosphoralytion both in vitro and in vivoSequentially, to confirm if HPC treatment can inhibit p38 phosphoralytion, which is thought to be a down-stream activity to cyclic AMP while up-stream to NF-κB translocation, we meseaured both p38 and phospho-p38 in cell whole lysates at the same time using western blot. In all 4 cell lines, phospho-p38 dropped dramatically to a lower level in HPC group compared with normoxia and hypoxia, wihle no difference of p38 was found, suggesting an inhibition of p38 avtivation caused by HPC treatment.HPC treatment inhibits NF-κB activityTo define the functional attributes of NF-κB inhibition by HPC, we used an NF-κB luciferase reporter. HeLa cells were transfected pNRE-Luc vector followed by exposure to normoxia, hypoxia (2% O2, 24 hours), or HPC followed by hypoxia (see HPC protocol in Methods). These studies revealed that cells subjected to HPC displayed a significant attenuation of NF-κB activation compared with those exposed to hypoxia alone.More over, we still visualized the inhibition of NF-κB by showing the translocated NF-κB in nucleis through immunoflurecence stain. Consistent to the luciferase data, Hela cells subjected to HPC has a much less NF-κB translocation rate than hypoxia control, while close to the low level of normoxia group.HPC treatment inhibits IL-6 activityTo get more evidence of the inhibition of NF-κB inflammatory, we further meseaured the mRNA level of IL-6, which is a well-established reporter gene for NF-κB activation. As shown by the result, hypoxia was a strong stimulus for induction of IL-6 mRNA in Hela cells. Parallel examination in HeLa cells subjected to HPC revealed a loss of hypoxia-induced IL-6, thus confirming our findings from the NF-κB experiments.ConclusionsIn conclusion, HPC up-regulates intracellar cyclic AMP by both decreasing the desensitization of AC stimulators through inhibition on the cruitment ofβ-arrestin to lipid raft and blocking the degradation of cAMP through the transcriptional down-regulation PDE4B. Secondly, the lift of cyclic AMP plays a key roll in the anti-inflammatory activity through the cAMP- PKA-CREB-p38MAPK pathway during HPC treatment. A SUMO-1 modified IκBαform has been described to actively participate in the regulation of NFκB metabolism. After proteosomal degradation of IκBα, an auto-regulatory loop consisting of transcriptional activation of IκBαgene and SUMO-1 modification of newly synthesized IκBαexists. The SUMOylated IκBαform is resistant to signal induced degradation consequently halting NFκB activation. We describe a physiologic environment by which rapid induction and deactivation of NFκB results in significant accumulation of SUMO-1 modified IκBα, with subsequent decreased NFκB mediated gene expression. Initially we demonstrated that the relative protein levels of IκBα/SUMO were significantly increased after hypoxia preconditioning (HPC). To confirm that in fact IκBα/SUMO control NFκB activation in the presence of hypoxia, assays targeting shRNA and over-expression of SUMO-1 were designed. With the use of a NF-κB-luciferase reporter plasmid, cells were co-transfected with shRNA plasmid and the full-length cDNA for SUMO-1. A significant increase in NF-κB activation was noted during hypoxia in shRNA-transfected cells, compared to wild type cells. Conversely, over-expression of SUMO-1 resulted in a significant reduction in NF-κB activation despite hypoxia. As proof of principle for the physiologic significance of SUMO-1 contribution to NF-κB activation, p65 subunit in nucleis and IL-6 mRNA levels of wild type compared to shRNA and over-expressing cells were also studied. In summary, we present an endogenous mechanism by which cells acquire anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NF-κB activation.Methods:Murine model.(See part one)Cell culture, in vitro hypoxia and chemical treatmentsHeLa cells (ATCC) were maintained in DMEM plus 10% FBS, at 37℃in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells (See part one)Transfections and NF-κB reporter assays.Transfection of Hela cells was carried out using Fugene 6 transfection reagent (Roche Diagnostics) as directed by the manufacturer. Plasmids used in transfections were SAE-1(SUMO activating enzyme-1, SAE-1) plasmid, shRNA for SUMO-1 and control plasmids (Origen). On the second day of the transfection, HeLa cells in 6-well plates were transfected for the second time with pNF-κB-Luc (pNRE; Clontech) at a concentration of 1.6μg per well along with 0.08μg of Renilla for 24 hours and then either left at normoxia or subjected to a protocol of hypoxia preconditioning. Following treatments: cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).Transcriptional analysis.cDNA was collected Using"Clonetech sprint powerscript"after extraction of total RNA. Real-time PCR (Taq-man) was employed to examine IL-6 expression levels in HeLa cells. IL-6 Primer was ready-to-use, ordered from Applied Biosystems. Samples were controlled forβ-actin using the following primers: sense , 5'-GGTGGCTTTTAGGATGGCAA G-3'; antisense , 5'-ACTGGAACGGTGA AGGTGACAG-3'; 162 bp).Western blot assay(See part one for method)Antibodies used for Western blotting included rabbit anti-SUMO-1 (1:1000), rabbit anti–IκBα(1:1000), rabbit anti–Phospho-IκBα(1:1000), all from Cell Signaling Technology, Inc. Blots were washed, and species-matched peroxidase-conjugated secondary antibody was added. Labeled bands from washed blots were detected by enhanced chemiluminescence (Thermol Scientific).Immunoprecipitation.Total protein was isolated from HeLa cells and measured as described above. After pre-cleared with 60ul of Immobilized Protein A/G (Thermol Scientific) for 2 hours, rabbit anti-SUMO-1 antibody or normal rabbit serum (Sigma-Aldrich) were added into the protein, gently shaked for overnight. 60ul of Immobilized Protein A/G were added into each tube, incubated for 2 hours, centrifuged at 7500rpm for 4 minutes, complex-bound gel were then collected and washed with cell lysis buffer for 3 times. Finally, 100ul of 2% SDS loading buffer was added into each tube, boiled at 95℃for 5 minutes, centrifuged at 14000rpm for 30 seconds, loaded to 12% gel and followed western blot steps mentioned above.ResultsHPC-induced SUMO-1 modification prevents phosphorylation of IκBαduring hypoxiaSUMO-1 modification prevents phosphorylation and, in turn degradation of IκBα, therefore helps to stabilize NF-κB p65/p50 subunits. This has been demonstrated in inflammatory model induced by TNF-α. However, does it work in other models, for example, hypoxia-induced inflammation? In our study, Hela cells were subjected to normoxia, hypoxia or HPC treatment for 3 cycles, at the end of each cycle; proteins were extracted for western blot assay. Result shows a decrease of IκBαalong with the dropping of SUMO-1-conjugated IκBαin hypoxia, while in HPC, when SUMO-1-conjugated IκBαgoes up, IκBαstops to decrease as well. Furthermore, HeLa cells transfected with SAE-1 plasmid or control vector were subjected to hypoxia treatment for 3-24 hours, western assay shows: IκBαand SUMO-1-conjugated IκBαdecreased dramatically in wild type cells while is stable in SUMO-1 over-expressed cells; phospho-IκBαincreased in wild type cells while kept low in SUMO-1 over-expressed cells; Iκκβ, no significant change in both groups. From above experiments, we can draw the conclusion that SUMO-1 modification prevents phosphorylation of IκBαduring hypoxia.HPC-induced SUMO-1 modification of IκBαinhibits NF-κB mediated inflammatory during hypoxiaTo confirm if the SUMO-1 modification of IκBαinhibits NF-κB mediated inflammatory induced by hypoxia, we first used the luciferise reporter assay to measure NF-κB activity and its downstream changes related to inflammatory. HeLa cells were transfected with either SAE-1 plasmid or SUMO-1 shRNA, subjected to normoxia; hypoxia or HPC treatment followed by hypoxia, and then measured NF-κB activity by luciferise reporter assay. As we can see, cells transfected with SUMO-1 plasmid showed a significant (P < 0.05, SAE-1 versus control vector) decrease in NF-κB activity, meanwhile, in HPC group, those had been transfected with shRNA for SUMO-1 showed an increase of NF-κB activity (P < 0.05, SUMO-1 shRNA versus control vector).Moreover, we still analyzed p65 protein, which reflects the translocation rate of NF-κB into nuclei, in the nuclear lysates extracted from Hela cells in parallel experiments. Data shows a similar pattern with luciferase assay: in SUMO-1 over-expressing cells, nuclear p65 was decreased despite of hypoxia treatment; while in shRNA group, HPC failed to inhibit the increase of nuclear p65.Finally, IL-6, which is the downstream target of NF-κB in the inflammatory chain, was meseaured at transcriptional level by using Real Time-PCR. The result is consistent to the change of NF-κB activity.Taken together, these changes indicate NF-κB mediated inflammation after hypoxia treatment can be inhibited by HPC induced SUMO-1 modification of IκBα.ConclusionsIn summary, we present an endogenous mechanism by which cells acquire anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NF-κB activation Adenosine (ADO) signaling has been found to play an key roll in hypoxia preconditioning (HPC), which enhancs cell anti-infalammatory ability. In our previous work, SUMO-1 modified IκBαform has been demonstrated to actively participate in the inhibition of NFκB activation. Using models of HPC, we have found a cyclic dependent increase in extra cellular Adenosine and theorize that the increase in cytoplasmic pool of IκBα/SUMO is mediated by Adenosine signaling, just like the adenosine–mediated cullin-1 deneddylation. Initially, We stimulated the ADO signaling pathway both in normoxia and hypoxia with the treatment of general Adenosine receptor agonist NECA, found an increase of IκBα/SUMO protein by western blot assay. In turn, when the stimulation was dampened by the general ADO receptor antagonist---8-PT, the increase was blocked, confirming the effect of ADO on the up-regulation of IκBα/SUMO. Further, functional evaluation of the effect was done by using luciferase assay on NFκB activity after genetically knocked SUMO-1 down by shRNA and stimualated with NECA. Result shows a loss of inhibition on NFκB activity after NECA stimulation when SUMO-1 had been knocked down, indicating SUMO-1 is crucial in the ADO pathway. More over, in cells over-expressing A2AR or A2BR have a higher level of IκBα/SUMO. Finally, in vivo experiments were conducted with cd73-knocked out (cd73-/-) mice, which were lack of ability to generate endogenous ADO. Mice were subjected to normoxia/hypoxia/HPC, and then total proteins of mice lung were analyzed by western blot. A signifcantly drop of IκBα/SUMO was found in cd73-/- mice compared with wild type mice. It is more important that, for cd73-/- mice, there was no increase of IκBα/SUMO in HPC group over normoxia and hypoxia groups, indicating the block the endogenous ADO pathway caused the failure for HPC to increas IκBα/SUMO, in another word, ADO is critical in the regulation of IκBα/SUMO.MethodsMurine model.CD73-/- mouse model was generated as described in our previous work. C57BL/6 (Charles River Laboratories) male mice of 8–10 weeks of age were used as control in this study. The Animal Care and Use Committee of Brigham and Women's Hospital approved all procedures. Whole-body preconditioning was performed in a manner similar to that previously described. (See part one) Cell culture, in vitro hypoxia and chemical treatmentsHeLa cells (ATCC) were maintained in DMEM plus 10% FBS, at 37℃in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells (See part one). Cells were treated with NECA in DMSO at a rang from 100nM to 100μM or pretreatment with 8-phenyltheophylline (8-PT) in ddH2O at concentration of 100nM. Treatment time is indicated in results.Transfections and NF-κB reporter assays.Transfection of Hela cells was carried out using Fugene 6 transfection reagent (Roche Diagnostics) as directed by the manufacturer. Plasmids used in transfections SUMO-1 shRNA or control plasmids (Origen). On the second day of the transfection, HeLa cells were transfected for the second time with pNF-κB-Luc (See part one) for 24 hours and then treated with NECA for 30 minutes. Following treatments: cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).Western blot assay(See part two)ResultsAdenosine regulates SUMO-1 modification of IκBαin vitroTo define the functional attributes of SUMO-1 modification of IκB by Ado signaling, NECA stimulation on hela cells was conducted to get direct evidence of Ado's up-regulation on SUMO-1 modification of IκB . Data showed a dose-dependent increase of IκBα/SUMO after hela cells were treated with NECA for about 30 minutes in normoxia, expectedly, this increase can be blocked by pre-treatment with 8-PT, a general inhibitor for Ado receptors. Not only in normal condition, but also in hypoxia, a similar pattern of the change of IκBα-SUMO after cells was treated with NECA or 8-PT/NECA was noticed. More over, when transfected with A2AR or A2BR, a higher of IκBα-SUMO was found compared with wild type cells, confirming above results that ADO pathway was involved in regulation of SUMO-1 modification of IκB.SUMO-1 modification of IκBαis mediated by Ado in mouse modelsTo make clear if SUMO-1 modification of IκBαis mediated by Ado, we used a CD73 (-/-) mouse model which lacks the ability to generate Ado endogenously in our study. Wild type C57BL/6 mice were used as control. Both types of mice were subjected to normoxia, hypoxia or HPC treatment, total protein of mice lung was analyzed by western blot. SUMO-1-conjugated IκB in both groups shows a similar pattern with cell model. Down-regulated in hypoxia, while up-regulated in HPC treated mice. What is more important, in CD73 (-/-) mice, SUMO-1-conjugated IκB level is much less than that in wild type mice, indicating Ado is crucial in SUMO-1 modification of IκBα. ConclusionsIn summary, we demonstrated that ADO signaling mediateds SUMO-1 modificaion of IκBα, which in turn induces anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NFκB activation.
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