Hulkower K I, Wertheimer S J, Levin W, Coffey J W, Anderson C M, Chen T, DeWitt D L, Crowl R M, Hope W C, Morgan D W
Hoffmann-La Roche, Inc., Nutley, New Jersey.
Arthritis Rheum. 1994 May;37(5):653-61. doi: 10.1002/art.1780370508.
In order to investigate potential regulatory mechanisms for the increased production of prostaglandin E2 (PGE2) in interleukin-1 beta (IL-1 beta)-stimulated rheumatoid synovial fibroblasts (RSF), this study examined the induction of phospholipase A2 (PLA2) and prostaglandin H synthase (PGHS) enzymes and the correlation of these events with PGE2 production in IL-1 beta-stimulated RSF.
Protein and messenger RNA (mRNA) levels of cytosolic PLA2 (cPLA2) and PGHS-2 enzymes in IL-1 beta-stimulated RSF were measured by Western and Northern blotting, respectively, using specific antisera and complementary DNA probes. Enzymatic activity of cPLA2 was determined in cell-free reaction mixtures utilizing mixed micelles of 14C-phosphatidylcholine and Triton X-100 as the substrate. PGE2 levels were quantitated using a commercial enzyme immunoassay kit.
Incubation of RSF with IL-1 beta increased the mRNA and protein levels for the high molecular weight cPLA2 as well as for the mitogen/growth factor-responsive PGHS (PGHS-2). The IL-1 receptor antagonist completely abolished the induction of these two enzymes and the stimulation of PGE2 production by IL-1 beta in RSF. In contrast, levels of the other known forms of these enzymes, i.e., the 14-kd secretory group II PLA2 (sPLA2) and the constitutive form of PGHS (PGHS-1), were unaffected by IL-1 beta treatment.
These are the first data to demonstrate the coordinate induction by IL-1 of cPLA2 and PGHS-2 in RSF. The time-course for the induction of these enzymes suggests that their increase contributes to the increased production of PGE2 in IL-1-treated RSF, and may help explain the capacity of RSF to produce large amounts of PGE2.
为了研究白细胞介素-1β（IL-1β）刺激的类风湿性滑膜成纤维细胞（RSF）中前列腺素E2（PGE2）产量增加的潜在调节机制，本研究检测了磷脂酶A2（PLA2）和前列腺素H合成酶（PGHS）的诱导情况，以及这些事件与IL-1β刺激的RSF中PGE2产生的相关性。
分别使用特异性抗血清和互补DNA探针，通过蛋白质印迹法和Northern印迹法检测IL-1β刺激的RSF中胞质型PLA2（cPLA2）和PGHS-2酶的蛋白质和信使核糖核酸（mRNA）水平。利用14C-磷脂酰胆碱和Triton X-100的混合胶束作为底物，在无细胞反应混合物中测定cPLA2的酶活性。使用商用酶免疫分析试剂盒对PGE2水平进行定量。
RSF与IL-1β孵育可增加高分子量cPLA2以及有丝分裂原/生长因子反应性PGHS（PGHS-2）的mRNA和蛋白质水平。IL-1受体拮抗剂完全消除了这两种酶的诱导以及IL-1β对RSF中PGE2产生的刺激。相比之下，这些酶的其他已知形式，即14-kd分泌型II组PLA2（sPLA2）和PGHS的组成型形式（PGHS-1）的水平不受IL-1β处理的影响。
这些是首次证明IL-1在RSF中协同诱导cPLA2和PGHS-2的数据。这些酶诱导的时间进程表明，它们的增加有助于IL-1处理的RSF中PGE2产量的增加，并可能有助于解释RSF产生大量PGE2的能力。
