Gene Regulation Immunology
The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit

https://doi.org/10.1074/jbc.M112.436675Get rights and content
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IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ∼1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

Cancer Therapy
Cytokine
DNA
Gene Expression
Immunology
Interleukin
Intracellular Trafficking
Protein-Protein Interactions
Export
Posttranslational Regulation

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* This work was supported, in whole or in part, by the National Institutes of Health, NCI, Intramural Research Program.

    2 The abbreviations used are:

    IL-15Rα

    IL-15 receptor α

    huCMV and siCMV

    human and simian cytomegalovirus, respectively

    CHX

    cycloheximide

    nt

    nucleotides

    Endo F and Endo H

    endoglycosidase F and H, respectively

    ER

    endoplasmic reticulum

    TGN

    trans-Golgi network

    BisTris

    2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.