Dusetti N J, Ortiz E M, Mallo G V, Dagorn J C, Iovanna J L
Unité 315, INSERM, Marseille, France.
J Biol Chem. 1995 Sep 22;270(38):22417-21. doi: 10.1074/jbc.270.38.22417.
Expression of the pancreatitis-associated protein I (PAP I), an exocrine pancreatic protein, increases rapidly and strongly in acinar cells during the acute phase of pancreatitis. This is reminiscent of the response to stress of acute phase proteins. We have previously demonstrated that serum factors from rats with acute pancreatitis, but not from healthy rats, could induce endogenous PAP I gene expression in the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C., Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204, 238-243). In the present work, we have evaluated the influence of several mediators of inflammation on rat PAP I gene transcription in these cells. Tumor necrosis factor alpha induced an increase in PAP I mRNA expression, and interferon gamma caused an even greater increase in PAP I mRNA level. These stimulations were antagonized by dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also ineffective. IL-6 and dexamethasone together induced a marked stimulation of PAP I gene transcription, and this effect was slightly attenuated by IL-1. To analyze the cis-regulatory elements responsible for the induction of transcription, we fused a 1.2-kilobase segment of the rat PAP I promoter to the chloramphenicol acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA was transfected into AR-42J cells. Addition of IL-6 or dexamethasone was ineffective, whereas their mixture increased the CAT activity 12 times. Progressive deletions of the PAP I promoter were then fused to the CAT gene, and the constructs were transfected to AR-42J cells. A 12-fold increase in CAT activity was seen upon IL-6/dexamethasone treatment with constructs containing more than 274 base pairs upstream from the cap site. In that region, two sequences are similar to the canonical IL-6 response element. Site-directed mutagenesis of these regions strongly decreased induction, showing that they were functional. PAP I should therefore be classified among acute phase proteins of class 2, whose expression is increased by IL-6 acting in combination with glucocorticoids.
胰腺炎相关蛋白I（PAP I）是一种胰腺外分泌蛋白，在胰腺炎急性期，腺泡细胞中其表达迅速且强烈增加。这让人联想到急性期蛋白对压力的反应。我们之前已证明，急性胰腺炎大鼠的血清因子能诱导腺泡细胞系AR - 42J中内源性PAP I基因表达，而健康大鼠的血清因子则不能（杜塞蒂，N.，马洛，G.，达戈尔恩，J.-C.，约万纳，J. L.（1994年）《生物化学与生物物理研究通讯》204，238 - 243）。在本研究中，我们评估了几种炎症介质对这些细胞中大鼠PAP I基因转录的影响。肿瘤坏死因子α诱导PAP I mRNA表达增加，干扰素γ使PAP I mRNA水平升高得更多。这些刺激作用被地塞米松拮抗。单独的白细胞介素（IL）-1、IL - 6或地塞米松无效。IL - 1与IL - 6或地塞米松的组合也无效。IL - 6和地塞米松共同作用显著刺激PAP I基因转录，且IL - 1会使这种效应略有减弱。为分析负责转录诱导的顺式调控元件，我们将大鼠PAP I启动子的1.2千碱基片段与氯霉素乙酰转移酶（CAT）基因融合作为报告基因。将所得嵌合DNA转染到AR - 42J细胞中。添加IL - 6或地塞米松无效，而它们的混合物使CAT活性增加了12倍。随后将PAP I启动子逐步缺失片段与CAT基因融合，并将构建体转染到AR - 42J细胞中。用包含帽位点上游超过274个碱基对的构建体进行IL - 6/地塞米松处理后，CAT活性增加了12倍。在该区域，有两个序列与典型的IL - 6反应元件相似。对这些区域进行定点诱变可强烈降低诱导作用，表明它们具有功能。因此，PAP I应归类为2类急性期蛋白，其表达通过IL - 6与糖皮质激素联合作用而增加。
