title: Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.
abstract: Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.
authors.affiliation: From the Jules Stein Eye Institute, Geffen School of Medicine.
authors.forename: Rajendra K
authors.identifier: 0000-0002-8746-0266
authors.initials: RK
authors.lastname: Gangalum
authors.affiliation: From the Jules Stein Eye Institute, Geffen School of Medicine.
authors.forename: Ankur M
authors.initials: AM
authors.lastname: Bhat
authors.affiliation: Brain Research Institute, UCLA, Los Angeles, California 90095.
authors.forename: Sirus A
authors.initials: SA
authors.lastname: Kohan
authors.affiliation: From the Jules Stein Eye Institute, Geffen School of Medicine, Brain Research Institute, UCLA, Los Angeles, California 90095 Molecular Biology Institute and bhat@jsei.ucla.edu.
authors.forename: Suraj P
authors.initials: SP
authors.lastname: Bhat
mesh_terms: D002460:Cell Line; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D055354:Exosomes / Q000378:metabolism*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D059666:Lysosomal-Associated Membrane Protein 3 / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000166:cytology; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure; D038203:alpha-Crystallin B Chain / Q000235:genetics* / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D059666:Lysosomal-Associated Membrane Protein 3; D034741:RNA, Small Interfering; D038203:alpha-Crystallin B Chain; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D020691:rab GTP-Binding Proteins
keywords: CD63; LAMP1; Rab5; Rab7; endosome; exosome complex; lysosome; retinal pigment epithelium; secretion; small heat shock protein (sHsp)