Transduced viral IL-10 is exocytosed from lacrimal acinar secretory vesicles in a myosin-dependent manner in response to carbachol.
The purpose of this study was to determine the intracellular trafficking and release pathways for the therapeutic protein, viral IL-10 (vIL-10), from transduced acinar epithelial cells from rabbit lacrimal gland. Primary cultured rabbit lacrimal gland acinar cells (LGACs) were transduced with adenovirus serotype 5 containing viral interleukin-10 (AdvIL-10). The distribution of vIL-10 was assessed by confocal fluorescence microscopy. Carbachol (CCH)-stimulated release of vIL-10 was quantified by ELISA. vIL-10 localization and exocytosis was probed in response to treatments with agents modulating actin- and myosin-based transport. vIL-10 immunoreactivity was detected in large intracellular vesicles in transduced LGAC. vIL-10 was partially co-localized with biosynthetic but not endosomal compartment markers. vIL-10 release was sensitive to CCH, and the kinetics of release showed an initial burst phase that was similar but not identical to that of the secretory protein, beta-hexosaminidase. Disassembly of actin filaments with latrunculin B significantly increased CCH-stimulated vIL-10 secretion, suggesting that vIL-10 was released from stores sequestered beneath the subapical actin barrier. That release required the activity of actin-dependent myosin motors previously implicated in secretory vesicle exocytosis was confirmed by findings that CCH-stimulated vIL-10 release was reduced by inhibition of non-muscle myosin 2 and myosin 5c function, using ML-7 and overexpression of dominant negative myosin 5c, respectively. These results suggest that the majority of vIL-10 transgene product is packaged into a subpopulation of secretory vesicles that utilize actin-dependent myosin motors for aspects of actin coat assembly, compound fusion and exocytosis at the apical plasma membrane in response to CCH stimulation.
Xie
Jiansong
J
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, USA.
Marchelletta
Ronald R
RR
Thomas
Padmaja B
PB
Jacobs
Damon T
DT
Yarber
Francie A
FA
Cheney
Richard E
RE
Hamm-Alvarez
Sarah F
SF
Trousdale
Melvin D
MD
D008841:Actin Cytoskeleton / Q000502:physiology; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D005260:Female; D005822:Genetic Vectors; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism*; D007765:Lacrimal Apparatus / Q000378:metabolism*; D018613:Microscopy, Confocal; D009218:Myosins / Q000502:physiology; D011817:Rabbits; D022142:Secretory Vesicles / Q000378:metabolism*; D015398:Signal Transduction; D014161:Transduction, Genetic
D016753:Interleukin-10; D002217:Carbachol; D009218:Myosins