Gaggero Alessia, De Ambrosis Alessandro, Mezzanzanica Delia, Piazza Tiziana, Rubartelli Anna, Figini Mariangela, Canevari Silvana, Ferrini Silvano
Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16132 Genoa, Italy.
Oncogene. 2004 Sep 30;23(45):7552-60. doi: 10.1038/sj.onc.1208036.
Interleukin-18 (IL-18) is a proinflammatory cytokine synthesized as a 24 kDa inactive precursor (pro-IL-18) by several cell types, and is processed to a bioactive molecule of 18 kDa by the proteinases caspase-1 or caspase-4. All ovarian carcinoma cell lines express pro-IL-18, only in some instances coexpress caspase-1, and always express caspase-4; in any case, they display a defective processing of IL-18. We analysed whether pro-IL-18, present in two ovarian carcinoma cell lysates, could be processed 'in vitro' by recombinant active caspase-1. While most of pro-IL-18 could be cleaved by caspase-1, a residual of pro-IL-18 appeared to be resistant. Cloning and sequence analysis of the whole pro-IL-18 open reading frame demonstrated the existence of an alternatively spliced mRNA variant, which lacked exon-3 (Delta3pro-IL-18). The 12 bp exon-3 encodes for the AEDD amino-acid sequence, which is N-terminal with respect to the cleavage site of caspase-1. Both pro-IL-18 and Delta3pro-IL-18 mRNA isoforms were detected in all ovarian cancer cell lines analysed, while Delta3pro-IL-18 mRNA was undetectable in normal ovarian epithelial cells. The Delta3pro-IL-18 cDNA induced synthesis of an alternative Delta3pro-IL-18 protein upon transfection into a murine cell line. The Delta3pro-IL-18 protein was resistant to proteolytic activation by caspase-1 and -4, although it was capable to bind caspase-1. Aternative splicing of pro-IL-18 exon-3 may represent a novel mechanism of regulation of bioactive IL-18 production in human ovarian tumors.
白细胞介素-18（IL-18）是一种促炎细胞因子，由多种细胞类型合成为24 kDa的无活性前体（前IL-18），并被蛋白酶caspase-1或caspase-4加工成18 kDa的生物活性分子。所有卵巢癌细胞系均表达前IL-18，仅在某些情况下共表达caspase-1，且始终表达caspase-4；无论如何，它们对IL-18的加工存在缺陷。我们分析了存在于两种卵巢癌细胞裂解物中的前IL-18是否能被重组活性caspase-1“体外”加工。虽然大多数前IL-18可被caspase-1裂解，但仍有一部分前IL-18似乎具有抗性。对整个前IL-18开放阅读框的克隆和序列分析表明存在一种选择性剪接的mRNA变体，其缺少外显子3（Delta3前IL-18）。12 bp的外显子3编码AEDD氨基酸序列，该序列相对于caspase-1的切割位点位于N端。在所有分析的卵巢癌细胞系中均检测到前IL-18和Delta3前IL-18 mRNA异构体，而在正常卵巢上皮细胞中未检测到Delta3前IL-18 mRNA。将Delta3前IL-18 cDNA转染到小鼠细胞系后可诱导合成另一种Delta3前IL-18蛋白。Delta3前IL-18蛋白对caspase-1和-4的蛋白水解激活具有抗性，尽管它能够结合caspase-1。前IL-18外显子3的选择性剪接可能代表了人类卵巢肿瘤中生物活性IL-18产生调控的一种新机制。
