Epstein-Barr virus latent membrane protein 1 promotes concentration in multivesicular bodies of fibroblast growth factor 2 and its release through exosomes.
FGF-2, a potent angiogenic factor that is involved in tumor invasion, is known to be released extracellularly by a nonclassical secretory pathway. Recently it has become clear that Epstein-Barr virus, specifically its oncoprotein LMP1, can induce expression of angiogenic factors. Among these factors is FGF-2. LMP1 not only promotes expression of FGF-2, but also the release extracellularly of its 18-kDa isoform. We analyzed the mechanism of FGF-2 release induced by LMP1. Confocal immunofluorescence microscopy revealed colocalization of FGF-2 with LMP1 in small dots also stained positively for CD63 and cathepsin D, markers of late endosomes or multivesicular bodies. Biochemical analysis and immunoelectron microscopy of purified exosomal fractions from cotransfected cells demonstrated increased release of exosomes and the concentration of LMP1 and FGF-2 in these structures. Moreover, cotransfection appeared to induce partial redistribution of the Na(+)/K(+)-ATPase, which participates in FGF-2 release, from the plasma membrane to the intracellular LMP1/FGF-2 positive dots. Treatment with ouabain, which inhibits Na(+)/K(+)-ATPase activity, partially suppressed FGF-2 secretion via exosomes in a dose-dependent manner. The results suggest that exosomes may represent a previously unrecognized mechanism for FGF-2 release mediated by LMP1, and that this pathway involves the activity of Na(+)/K(+)-ATPase.
Ceccarelli
Simona
S
Dipartimento di Medicina Sperimentale, Università La Sapienza, Roma, Italy.
Visco
Vincenzo
V
Raffa
Salvatore
S
Wakisaka
Naohiro
N
Pagano
Joseph S
JS
Torrisi
Maria Rosaria
MR
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron / Q000379:methods; D010042:Ouabain / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000099177:Tsg101 Protein
D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors; D016222:Fibroblast Growth Factor 2; D010042:Ouabain; D034741:RNA, Small Interfering; D000254:Sodium-Potassium-Exchanging ATPase; D014157:Transcription Factors; D014763:Viral Matrix Proteins; D000099177:Tsg101 Protein; C037662:EBV-associated membrane antigen, Epstein-Barr virus