The mda-7gene localizes to chromosome 1q32, a region within an IL-10 family cluster that also contains the genes for IL-10, IL-19, and IL-20 (13). Although mda-7 has little sequence homology with IL-10, the encoded protein has approx 19% amino acid identity with other IL-10 family members (14,15). Based on its chromosomal location, the presence of an IL-10 signature motif, limited amino acid identity with other IL-10 cytokines, its translational regulation and predicted structural features, mda7 has been renamed IL-24, and categorized as an IL-10 family cytokine (16).
Chada S. et al. reported that exposure of melanoma cells to MDA-7/IL-24, induced secretion of interferon (IFN)-y and IL-6, but not of IL-4 or IL-5; in contrast, Ad-luc treatment did not induce IFN-y or IL-6, suggesting an MDA-7-specific effect (15). Transduction of these cells with Ad-mda7 induced increases in mRNA that mirrored the cytokine induction observed with exposure to the MDA-7/IL-24 protein. The report suggested that the effect was also specific to some types of tumor cells, because similar treatment of lung and breast cancer cells did not induce release of cytokines. Microarray analysis of non-small-cell lung carcinoma (NSCLC) cells transduced with Ad-mda7 corroborated the cytokine-like activity of this gene, and confirmed that MDA-7 can activate IFN-y and NF-kB signaling pathways. Given that IL-10 functions as an immunosupressive cytokine, and that it significantly inhibited MDA-7/IL-24 activity in human peripherral blood mononuclear cells (PBMCs), it is possible that mda7/IL-24 acts as an antagonist of IL-10 (16).
Exposure of melanoma and PBMC cells to MDA-7/IL-24 protein also activated Stat3 pathways, which have previously been associated with cellular transformation. The specificity of STAT3 activation in these cells was demonstrated by addition of anti-MDA-7 antibodies, but was not affected by addition of anti-IL-6 antibodies. In this study, exposure of melanoma cells to bacterially expressed protein failed to induce similar results, and the authors hypothesized that post-translational modifications to the MDA-7 protein, in particular glycosylation, were important for its functional activity (14).
Although the organization of the mda-7 gene is better understood now in comparison with the other members of the IL-10 family cytokines, the crystal structure of the MDA-7 protein has not yet been solved. The IL-10 family of cytokines functions through the JAK/STAT signaling pathway and comprises six members: IL-10, IL-19, IL-20, IL-22, MDA-7/IL-24, and IL-26. In spite of the substantial degree of sharing of the receptor sub-units, specificity of signaling is based on selective expression of either the receptors or of the cytokines themselves. Although the IL-10 family members share limited homology in some of their conserved domains, they are distinguished by their similar helical structure and IL-10 signature motif. There are three human IL-10 family proteins whose structures have been solved (IL-10, IL-19, and IL-22): all possess six a helices (A, B, C, D, E, and F) and at least one disulfide bond. The V-shaped complex is comprised of four helices (A through D) from one monomer and two helices (E and F) from the other. The classical four-helix bundle that is representative of all helical cytokines is formed by helices A, C, D, and F. Of the IL-10 family, mda-7 seems to be the only member to posses an extensive (49 amino acids) leader sequence; but is otherwise organized into a similar consistent pattern of six predicted alpha helices (A through F) based on the structure of IL-10. Helix F is conserved in all the family members, whereas the N^-terminus is variable (15). The regions in IL-10 corresponding to helix A, the A-B loop, and helix F are important in binding to its receptors. There are three potential glycosylation sites in MDA-7/IL-24; two are located on the top part of the V-shaped complex and seem to be readily accessible to modifications, the first is shared with IL-22 (Asn85-Ile-Thr), the second site maps to a unique loop between helices C and D. The third site maps to the base of helix B (aa99), but it is not likely to be used because glycosylation of this site would interfere with the structural integrity of the molecule. An in-depth review of the predicted structural features of IL-24 and how these relate to other IL-10 cytokines was recently presented (15).
A combination of structural data, homology to known cytokines, chromosomal localization, a predicted N-terminus secretion signal peptide, and evidence of its regulation of cytokine secretion, all support classification of MDA-7/IL-24 as a IL-10 family cytokine (4,9,16). A 49 amino acid leader sequence predicts it is a secreted protein; recent studies confirm this prediction and report that Ad-mda7 transduced cells release high levels of a 40-kDa form of the MDA-7 protein (see Fig. 1), which can bind to heterodimeric receptors IL-20R1/IL-20R2 and IL-22R2/IL-20R1 (9,17,18). The intracellular form of the protein (23-30 kDa) is cleaved, and extensively modified (primarily by glycosylation) before its release into the extracellular compartment (see Fig. 1B,C) (9,16,19). Expression of MDA-7 is rare in tumors, and correlates inversely with progressive stages of melanoma, as has also been shown of the expression of a novel mda-7 splice variant (mda-7s) (6,7,20). Because its initial characterization as a differentiation factor in IFN-^ and mez-erein-treated human melanoma cells, MDA-7 has attracted interest because of its unique tumor-selective antiangiogenic and proapoptotic activities, which are reviewed below.
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