摘要：目的研究类风湿性关节炎(rheumatoidarthritis,RA)成纤维样滑膜细胞(fibroblast-like

synoviocyte,FLS)信号转导中，IL-1β介导的丝裂原活化的蛋白激酶(mitogen-activated-proteinkinaseMAPKs)的激活情况。方法原代培养RFFLS；应用Westernblot检测IL-1β对于RAFLS蛋白质酪氨酸磷酸化状态的影响，及其对MAPKs家族成员活化的浓度效应和时相特点。结果IL-1β可以瞬时增加RAFLS蛋白质酪氨酸磷酸化的程度，并在短时间内激活MAPKs通路(以ERK2，JNK2和P38为主)。不同浓度的IL-1β对MAPKs的活化无明显差异，但ERK2，JNK2和P38的活化分别在IL-1β作用后5min，15min和1min最明显。结论IL-1β在RAFLS信号转导中，可以瞬时导致蛋白质酪氨酸化的程度增加，并同时激活MAPKs3条通路；但

MAPKs3个亚家族成员的活化具有异质性。

Activation of mitogen-activated protein kinae induced by IL-1β in fibroblast- like synoviocytes of rheumatoid arthritis

SUN Tie- zheng,YAO Li- bo,LU Hou- shan

Abstract:Aim To study activation of mitogen-activated protein kinase(MAPKs)in fibroblast-like synoviocytes(FLS)of rheumatoid arthritis(RA)under the stimulation of IL- 1β . Methods RA FLS wereprimarily cultured. Western blots were used to examine transient change of protein tyrosine phosphorylation status andMAPKs activation in RA FLS stimulated with various doses and periods of IL- 1β . Results IL- 1β transiently increasedprotein tyrosine phosphorylation, and activated MAPKs cascades (mainly ERK2, JNK2 and P38) in RA FLS. There was noobvious difference of MAPKs' activation among different doses of IL- 1β (1× 103 IU/L,1× 104 IU/L and 1× 105IU/L), but the peak activations of ERK2,JNK2 and P38 took place at 5 min,15 min and 1 min, respectively after stimulationwith IL- 1β . Conclusion During signal transduction of IL- 1β in RA FLS, tyrosine phoshorylation was increasedtransiently, MAPKs cascades were activated in a few minutes, and there was heterogenicity of activation among threesubfamily members.

Keywords: arthritis; synovial membrane; fibroblasts; signal transduction; IL- 1beta; MAPK

Rheumatoid arthritis(RA) is a chronic inflammatory cytokine-mediated disease, characterized by uncontrolled proliferation of synoviocytes, infiltration of abundant inflammatory cells and progressive joint erosion. In the pathogenesis of RA , the cytokine network mainly including IL-1,TNF-α and other cytokines contributs to activation of fibroblast-like synoviocytes(FLS)and further leads to erosion of cartilage and bone. IL-1β can increase production of collagenase，stinulate secretion of prostaglandin E2 and secondary cytokines such as IL- 6 and IL- 8; induce expression of adhesion moleculars such as ICAM-1 and VCAM-1,etc〔1〕. IL-1β receptors don't contain any intrinsic kinase domain.However, IL-1β activates a kinase which phosphorylates protein substrates such as the EGF receptor,the heat shock protein of relative molecule mass(M_r)27 000,L- plastin and IL-1β receptor of Mr 80 000. Although cAMP,diacylglycerol, PKC and the arachidonic acid metabolites have been reported to participate in IL- 1β signal transduction, the status of their involvement in different cell lines remains uncertain and controversial^[2]. So signal transduction pathway of IL-1 is still not very clear. The mitogen-activated protein kinase(MAPK) family is a group of evolutionary conservative proline- directed serine/threonine kinase, converting extracellular stimuli to intracellular signals and controlling the expression of genes essential for many cellular processes including cell growth and differentiation^[3]. The purpose of our study is to investigate MAPKs cascade activation in RA FLS stimulated with IL-1β.

MATERIALS AND METHODS

Synoviocytes culture Synovial tissue was collected at the time of total knee replacement(TKR) or synovectomy from patients with RA. The diagnosis of RA conformed to the 1987 revised American College of Rheumatology Criteria^〔4〕 .Fibroblast-like synoviocytes were isolated by enzymatic dispersion of synovial tissue. Briefly, the synovial membrane was minced aseptically and then incubated with 1.0 mg/L collagenase I(Gibco-BRL) in serum-free DMEM for 4 hour at 37℃, filtered through a nylon mesh, extensively washed, and cultured in DMEM suplemented with 200 g/L fetal bovine serum(FBS), penicillin,streptomycin and L-glutamine in a humidified 50 mL/L CO₂ atmosphere. After overnight culture, nonadherent cells were removed, and adherent cells were cultured in DMEM plus 200 mL/L FBS. At 85％ confluence, cells were trypsinized, split at 1∶3 ratio and recultured in medium. Synoviocytes from passage 3-5 were used in theseexperiments.

Preparation and quantitative of protein exstracts and action of cytokines 5× 10⁵ synovial cells were grown to subconflunce for 24 hour. After starvation with 10 ml/L FBS or 3 g/L BSA, synovial cells were stimulated with IL-1β in different doses(1,10,1× 10⁴ IU/L) for 5 min,or 1× 10⁴ IU/L in different periods (1, 2, 5 and 15 min). Serum-free DMEM was used as the control. Cells were washed with cold PBS and lysed by the addition of 100 μ L lysis buffer[20 mmol Tris/Cl (pH∶ 8.0), 1％ (v/v)NP- 40, 150 mmol/L NaCl, 1 g/L(v/w)NaN3,5 mg/L aprotinin, 1 mmol/L PMSF, 1 mmol/L EDTA, 1mmol/L Na3VO4, 25 μ mol/L PNP',1 μ mol/L pepstatin A, 1 μ mol/L leupeptin], then scraped off and kept on ice for 20min. Insoluble material was removed by centrifugation at 15 000× g for 15 min at 4℃ . The supernatant was saved and protein concentration was determined using Bio-Rad DC protein assay kit.

SDS-PAGE and Western Blot An identical amount of protein(30 μ g) for each lysate was subjected to 100 g/L SDS-PAGE. Protein were transferred to a nitrocellulose membrane. The membranes were blocked overnight with 50 g/L non-fat milk in PBS at 4℃ , washed in PBS-T and incubated at room temperature for 3 hour in a 1∶2 000 dilution of mouse anti- phospho-rylated monoclonal antibody(4G10), or a 1∶ 2 000 dilution of rabbit anti-active ERK antibody, 1∶2000 dilution of mouse anti-active JNK antibody, or 1∶ 2 000 dilution of mouse anti-active P38 antibody(Promega). The membranes were then washed in PBS-T and were incubated with 1∶2 000 goat anti-mouse or rabbit IgG antibodies coupled with horseradish peroxidase. The enhanced chemiluminecence(ECL)system (Amersha-m) was used to detection.The membranes were sequentially exposed to X-Kodak film for 15 seconds and the latter was processed. Then the membranes were probed with antinon-active ERK, -JNK and -P38 antibodies to ensure that the identical amount of protein were loaded.

RESULTS

Synoviocytes of passage 3 were homogenous population of fibroblast-like synoviocytes. We analyzed the early biological events of RA FLS stimulated with IL-1β . As shown in figure 1 , stimulation with IL-1β resulted in tyrosine phosphorylation of several bands [including (M_r× 10³)110，75，65，54，46，42 and 38].Among these bands,bands of M_r 42 000 and 38 000 increased to the peak activation in 1-2 min. The bands of M_r× 10³ 110,75,65 and 54increased gradually in 15 min.

Fig 1 Transient effect of IL-1β (10 kU/L) on protein tyrosine

phosphorylation in RA FLS

1: 1 min; 2: 2 min; 3: 5 min; 4:15 min; 5: 0 min.

Because the activation of three subfamily members of MAPKs was all induced by biphosphorylation on the site