A novel isoform of pro-interleukin-18 expressed in ovarian tumors is resistant to caspase-1 and -4 processing.
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.
Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16132 Genoa, Italy.
Alessia
A
Gaggero
Alessandro
A
De Ambrosis
Delia
D
Mezzanzanica
Tiziana
T
Piazza
Anna
A
Rubartelli
Mariangela
M
Figini
Silvana
S
Canevari
Silvano
S
Ferrini
D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D020170:Caspase 1 / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism*; D053471:Caspases, Initiator; D045744:Cell Line, Tumor; D017931:DNA Primers; D005260:Female; D006801:Humans; D020382:Interleukin-18 / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D011498:Protein Precursors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D020382:Interleukin-18; D011498:Protein Precursors; D011994:Recombinant Proteins; C093516:CASP4 protein, human; D020169:Caspases; D053471:Caspases, Initiator; D020170:Caspase 1