M. Yang1, Q. Du1, P. R. Varley1, Z. Liang1, B. Chen1, C. Heres1, D. B. Stolz2, S. C. Watkins2, D. A. Geller1 1Thomas E. Starzl Transplantation Institure,Department Of Surgery, University Of Pittsburgh School Of Medicine,Pittsburgh, PA, USA 2Center Of Biologic Imaging,University Of Pittsburgh,Pittsburgh, PA, USA
Introduction: Exosomes play an important role in cell communication, including tumorigenesis, cancer metastases, and immune regulation. Rab27a is a GTPase that has been shown to promote exosome secretion. However, mechanisms controlling cell exosome secretion in response to pathologic conditions are not well-defined. Interferon regulatory factor 1 (IRF-1) is a transcription factor regulating immunity, carcinogenesis, and hepatic I/R injury. The role of IRF-1 in regulating Rab27a and exosome secretion is unknown. Since IRF-1 can regulate expression of some GTPases, we hypothesized that IRF-1 might regulate Rab27a expression and increase exosome secretion.
Methods: Primary human hepatocytes (hHC) and human hepatoma cell line Huh-7 were treated with Interferon-γ (IFNγ). PCR and Western blot were used to detect IRF-1 and Rab27a mRNA and proteins. Exosomes secreted by hHC and Huh-7 cells were isolated with ultracentrifugation. Isolated exosomes were confirmed by transmission electron microscopy (TEM) and surface markers. Exosome proteins secreted by these cells were quantitated with bicinchoninic acid assay (BCA). The sizes of the exosomes were measured by nanoparticle tracking analysis (NTA). The binding sites of IRF-1 in promoter region of Rab27a were predicted with PROMO bioinformatics software. IRF-1 expression plasmid and Rab27a promoter-driven luciferase reporter were used to determine the effect of IRF-1 on Rab27a transcription. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) were used to verify IRF-l binding in the Rab27a gene promoter region in vivo and in vitro.
Results: IFNγ increased IRF-1 and Rab27a mRNA and protein in primary hHC in a dose-dependent manner (Fig. A). Deflated soccer-ball like exosomes, which were verified by TEM (Fig. B), were secreted more by hHC and Huh7 after IFNγ treatment (Fig. C, p < 0.05). IFNγ did not alter the exosome size (data not shown). Multi-vesicle bodies (MVB) are precursors of exosomes and were markedly increased by IFNγ stimulation in Huh7 cells (Fig. D, CD63 staining red). Transfection of HepG2 cells with IRF-1 expression plasmid increased Rab27a promoter activity 4.9-fold. Using PROMO software, we found ten putative IRF-1 binding motifs upstream in the Rab27a promoter region. ChIP and EMSA verified five IRF-1 binding sites in the Rab27a promoter region (data not shown).
Conclusion: These novel findings indicate that IFNγ induces IRF-1 which promotes exosome secretion through increasing the expression of Rab27a. These results provide important insights into fundamental cellular signaling pathways regulating exosome secretion under inflammatory conditions in hepatocytes and hepatocellular carcinoma.
