Takahashi Hidetoshi, Hashimoto Yoshio, Ishida-Yamamoto Akemi, Iizuka Hajime
Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaokahigashi, Asahikawa 078-8510, Japan.
J Dermatol Sci. 2005 Sep;39(3):175-82. doi: 10.1016/j.jdermsci.2005.03.006. Epub 2005 Apr 22.
Roxithromycin (RXM), a new 14-member macrolide antibiotic, is effective for chronic airway diseases such as diffuse panbronchiolitis and bronchial asthma. Recent study disclosed that RXM inhibits nuclear factor-kappaB (NF-kappaB)-mediated inflammation. Involucrin is one of the precursor proteins of the cornified cell envelope (CE) and is markedly increased in inflammatory skin diseases such as psoriasis. However, its molecular mechanism of action remains unknown.
To determine the effect of RXM on involucrin expression of keratinocytes.
We constructed chloramphenicol acetyltransferase (CAT)-involucrin promoter expression vector and CAT assay was performed. Furthermore, western blot and RT-PCR were performed to examine the expression of involucrin in RXM-treated cultured human keratinocytes.
The increased involucrin expression by 12-O-tetradecanoylphorbol acetate (TPA) was suppressed by 10(-6) M RXM and the maximal inhibitory effect was observed at 48 h. RXM suppressed increased CAT activity by TPA and the effect was not inhibited by H-7 or cafferic acid phenethyl ester (CAPE). Deletion of T1 region (-119 to -113) of involucrin promoter completely abolished TPA-dependent stimulatory and RXM-dependent inhibitory promoter activity. Gel shift assay showed that c-Jun (but not p65) selectively binds to the T1 region. The assay of activator protein-1 (AP-1) and NF-kappaB activities revealed that RXM decreased both transcriptional activities. Co-transfection of c-jun and c-fos expression vectors, or p65 and p50 expression vectors, rescued decreased CAT activity by RXM, respectively.
Our study demonstrated for the first time that involucrin expression of keratinocytes is suppressed by RXM through direct inhibition of AP-1 and indirect inhibition of NF-kappaB.
罗红霉素（RXM）是一种新型的14元大环内酯类抗生素，对弥漫性泛细支气管炎和支气管哮喘等慢性气道疾病有效。最近的研究表明，RXM可抑制核因子-κB（NF-κB）介导的炎症。包壳蛋白是角质化细胞包膜（CE）的前体蛋白之一，在银屑病等炎症性皮肤病中显著增加。然而，其分子作用机制尚不清楚。
确定RXM对角质形成细胞包壳蛋白表达的影响。
构建氯霉素乙酰转移酶（CAT）-包壳蛋白启动子表达载体并进行CAT分析。此外，进行蛋白质免疫印迹法和逆转录-聚合酶链反应（RT-PCR）检测RXM处理的培养人角质形成细胞中包壳蛋白的表达。
12-O-十四酰佛波醇-13-乙酸酯（TPA）诱导的包壳蛋白表达增加被10⁻⁶ M RXM抑制，在48小时观察到最大抑制作用。RXM抑制TPA诱导的CAT活性增加，且该作用不受H-7或咖啡酸苯乙酯（CAPE）抑制。包壳蛋白启动子T1区（-119至-113）缺失完全消除了TPA依赖性刺激和RXM依赖性抑制启动子活性。凝胶迁移试验表明，c-Jun（而非p65）选择性结合T1区。激活蛋白-1（AP-1）和NF-κB活性分析显示，RXM降低了两者的转录活性。共转染c-jun和c-fos表达载体，或p65和p50表达载体，分别挽救了RXM降低的CAT活性。
我们的研究首次证明，RXM通过直接抑制AP-1和间接抑制NF-κB抑制角质形成细胞的包壳蛋白表达。
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