MK2 Signaling Pathway Regulates The Expression Of ICAM-1 In Pulmonary Microvascular Endothelial Cells In Mice With Acute Respiratory Distress Syndrome

Background&ObjectiveARDS,known as acute respiratory distress syndrome,is an inflammatory response,and many inducements can cause ARDS.Infection is still one of the main reasons for ARDS.The morbidity and mortality of ARDS in US and Asia are very high.The effective treatments are still missing.So it is necessary and urgent to clarify the mechanism of ARDS and find new potential targets of therapy.ICAM-1(intercellular adhesion molecule-1,ICAM-1),whose main function is to mediate adhesion of neutrophils and vascular endothelial cells,plays an important role in neutrophil influx into lung.Neutrophils combine with the ICAM-1 on the surface of endothelial cells when reaching lung capillaries,which changes the structure of connection between endothelial cells and form gaps.The gaps allow neutrophils get into the lung tissues through the vascular wall.It is a normal process,but when ARDS happens,the expression of ICAM-1 in microvascular endothelial cells increases enormously,which exacerbates the development of ARDS.The mechanism above is well-documented in many ARDS models.It has been proved that,vascular permeability and neutrophil infiltration decreased about 70%in mice with ICAM-1 knocked down.Studies all above prove that ICAM-1 plays an important role in ARDS.Our study has shown that MK2,one downstream kinase of p38 MAPK,phosphorylated by the latter can regulate the expression of ICAM-1 and IL-8 post-transcriptionally without influencing NF-K B activation,ICAM-1 or IL-8 mRNA transcription in HPMEC.HuR,a kind of protein widely distributed in human and other animal,belonging to the ELAV family,stabilized target mRNAs in many cell lines.Our previous study has demonstrated that activated MK2 regulated HuR by promoting the cytoplasmic accumulation of HuR without changing the total HuR protein in HPMEC after TNF-? stimulation,then HuR increased the mRNA stability of interleukin-6 expression,which plays an important role during the development of ARDS.It is said the the mechanism by which HuR promoted the stability of mRNA is to bind the ARE motif,such as AUUUA in the 3'-UTR of mRNA.Since ICAM-1 mRNA has AREs in its 3'-UTR,and our unpublished study has proved that MK2 post-transcriptionally regulated TNF-?-induced ICAM-1 expression by altering the cytoplasmic localization of HuR in HPMEC.We hypothesize that MK2/HuR pathway also regulates the expression of ICAM-1 post-transcriptionally in vivo.However,in animal experiments,the regulation effection of MK2/HuR pathway on ICAM-1 has not been proved powerfully,especially in the pulmonary micro vascular endothelial cells.In this study,we observe the expression of MK2,HuR and ICAM-1 in mouse pulmonary micro vascular endothelial cells in ARDS mice stimulated by LPS.Then we study the expression of MK2,HuR and ICAM-1 in mouse pulmonary microvascular endothelial cells after inhibiting MK2 with MK2 inhibitor-MMI-0100.Methods1L The mouse model of LPS-induced ARDS(1)C57BL/6 mice weighing abuout 20g were obtained from the Animal Facility of the Nan Jing University.All experimental procedures with animals care followed the international recommendations for the use and care of animals.All mice were housed in bright rooms with appropriate temperature(20-25?)and humidity(40-70%)and 12-12h light/dark cycle,and got food and water ad libitum.All the mice were allowed to adapt to the experimental environment at least one week before experimentation.(2)? Animals were randomly divided into 2 groups:control group(n=5)and intraperitoneal LPS treatment group(LPS group)(n=5).To induce ARDS.Mace in LPS group received LPS(5 mg/kg)diluted in 100 ?1 PBS through intra-peritoneal injection,animals in the control group were given 100 ?l PBS intraperitoneally without LPS.All mice with PBS and LPS injection were euthanized after 24 hours respectively.? Animals were randomly divided into 3 groups:control group(n=5),intraperitoneal LPS treatment group(LPS group)(n=5),and LPS +MMI-0100 group(n=5).To induce ARDS,mace in LPS group received LPS(5 mg/kg)diluted in 100 ?l PBS through intra-peritoneal injection,LPS + MMI-0100 group mice received LPS injection(5 mg/kg)and MMI-0010(0.001mg/mouse)at 0 hour and received MMI-0100(0.001mg/mouse)after 6 hours,12 hours,18hours respectively.Animals in the control group were given 100 ?l PBS intraperitoneally without LPS.All mice were euthanized after 24 hours respectively.2.The ratio of wet to dry of mouse lung24 hours after the injection of LPS or PBS or MMI-0100,the middle lobe of right lungs of the mice were collected,and weighed quickly to get the 'wet' weight,then oven dried at 75? for 24h to obtain the 'dry' weight.The ratio of wet lung to dry lung(W/D)was to quantify lung tissue edema.3.Histopathologic studyMice without any disposal were performed on histopathologic study.24 hours after the injection of LPS or PBS or MMI-0100,the upper lobe of right lungs of the mice was harvested and fixed with 10%buffered formalin for 24 h,then embedded in paraffin and sliced.Lung tissues were observed under a light microscope after stained with hematoxylin-eosin(H&E).4.The count of neutrophilsLung tissues were washed with PBS to wash the blood cells,infiltrated into collagenase(sigma)diluted in 1XHBSS buffer,cut into pieces,digested for 30minutes in 37?,then pulverized and wished with Roswell Park Memorial Institute 1640 through strainer,resuspended in 35%percoll(sigma),centrifuged at 4°C(800g)for 15minutes,fixed in Red Blood Cell Lysis Buffer(Beyotime Institute of Biotechnology;China)for 3 minutes and centrifuged at 4?(800g)for 8 minutes to remove red blood cells,resuspended again and stained with a CDllb+-labeled and Ly6G+-labeled antibodies for 30 min at 25?,neutrophils were counted using a flow cytometer with the antibodies CD11b+ and Ly6G+.5.The isolation of mouse lung microvascular endothelial cellsMice were euthanized 24h after the injection of LPS,PBS or MMI-0100,lung tissues were dealed with collagenase(sigma)diluted in 1XHBSS buffer,cut into pieces,digested for 30minutes in 37?,then pulverized and washed with Roswell Park Memorial Institute 1640 through strainer,resuspended in 35%percoll(sigma),centrifuged at 4?(800g)for 15minutes,fixed in Red Blood Cell Lysis Buffer(Beyotime Institute of Biotechnology;China)for 5 minutes and centrifuged at 4?(800g)for 8 minutes to remove red blood cells,resuspended again and stained with a CD45-PE-labeled and CD31-APC-labeled antibodies for 30 min at 25?,the mouse lung microvascular endothelial cell was colletcted CD45-and CD31+immunomagnetic beads positive selection.6.The test on mRNAs of HuR and ICAM-1 with Real-Time PCRRNA was isolated from lung tissues and MLMEC,then cDNA was synthesized standardly.The expression of mRNA was analyzed with a two-step quantitative real-time RT-PCR.Melting curve analyses were used to confirm the specificity of primers.Relative expression values were normalized using an internal GAPDH control.7.The proteion expression of MK2,HuR and ICAM-1 in mouse lung microvascular endothelial cellsThe total proteins and cytoplasmic and nuclear proteins of mouse lung microvascular endothelial cells were extracted using Total and Cytoplasmic Protein Extraction Kits(Beyotime Institute of Biotechnology;China)according to the manufacturer's protocol.Then the pretein expression of MK2,HuR and ICAM-1 was tested with western blot.8.Immunohistochemical studyTo evaluate the effects of MK2/HuR on the expression of ICAM-1,the lung tissues were collected,fixed in 10%formalin and performed to immunohistochemical routine following the manufacturer's protocol.Results1.The establishment of LPS induced ARDS in mouseCompared with control group and LPS + MMI-0100 group,the lung wet/dry ratio of LPS group changed differently(p<0.05).The count of neutrophil in LPS group increased a lot compared with control group and LPS + MMI-0100 group(p<0.05).Histopathologic study showed that control mice had no abnormal tissues;the lung tissue structure was severely damaged in LPS group,there was severe interstitial edema more neutrophil infiltration too;LPS + MMI-0100 group showed slight interstitial lung edema and neutrophil infiltration.2.The test of mRNA expression of HuR and ICAM-1 in mouse lung microvascular endothelial cellsThe mRNA expression of HuR in control group,LPS group and LPS+MMI-0100 group had no difference(p>0.05).Compared with the mRNA expression of ICAM-1 in control group and LPS + MMI-0100 group,LPS group changed differently(p<0.05).3.The test of protein expression of MK2,HuR and ICAM-1 in mouse lung microvascular endothelial cells with western blotThe protein expression of total MK2 in control group and LPS group had no difference in mouse lung microvascular endothelial cells,and LPS + MMI-0100 group decreased a little.The phosphorylated MK2 in LPS group increased compared with control group,while LPS + MMI-0100 group decreased a lot.Total HuR and nucleus HuR had no significant difference in three groups,but HuR in cytoplasm in LPS group increased significantly.ICAM-1 increased a lot in LPS group both in mouse lung microvascular endothelial cells and on the surface of mouse lung microvascular endothelial cells.The expression of HuR and ICAM-1 in mouse lung cells except microvascular endothelial cells had no difference in three groups.4.The test of protein expression of p-MK2 and ICAM-1 in mouse lung with immunohistochemical studyThe immunohistochemistry results showed that p-MK2 and ICMA-1 protein in LPS stimulation mouse lung tissues was higher than the control group and LPS+MMI-0100 group lung tissues.Conclusions1.In mice with ARDS induced by LPS,MK2/HuR pathway was activated,shown as MK2 phosphorylation and HuR shift from nucleus to cytoplasm.2.ICAM-1 expression increased dramaticlly in mice with ARDS,especially in the mouse lung microvascular endothelial cells,which indicates that PMVECs play a key role in ARDS.3.When MMI-0100 inhibitted the activation of MK2,HuR in cytoplasm reduced,resulting in the lower-expression of ICAM-1.It suggested that MK2/HuR pathway regulates the expression of IC AM-1 in mouse PMVEC by affecting the nuclear-cytoplasmic transfer of HuR.