Sorting soluble tumor necrosis factor (TNF) receptor for storage and regulated secretion in hematopoietic cells.
Hematopoietic cells contain secretory lysosomes that degranulate at sites of inflammation. We envisage that secretory granules can act as vehicles for targeting inflammatory sites, including malignancies, and thereafter, locally release therapeutically active agents to these sites. Exogenous proteins, such as the soluble tumor necrosis factor receptor 1 (sTNFR1), have been shown previously to be targeted to secretory lysosomes [1]. In this work, we asked whether exogenous, secretory lysosome-targeted proteins were subject to regulated secretion. sTNFR1-transmembrane (tm)-cytosol-sorting signal (Y) and sTNFR1-tm-Y-enhanced green fluorescent protein (egfp) were expressed in rat basophilic leukemia cell clones having different secretory capacities. sTNFR1-tm-Y was targeted directly from the Golgi to secretory lysosomes, followed by generation of membrane-free sTNFR1, whose secretion could be triggered by a Ca2+ ionophore or immunoglobulin E receptor activation. In contrast, sTNFR1-tm-Y-egfp was targeted to the plasma membrane and then subjected to endocytosis and presumably, secretory lysosome targeting, as judged by results from antibody ligation and cell-surface biotinylation. Activation of protein kinase C with phorbol ester promoted ectodomain shedding at the cell surface, resulting in sTNFR1 release from sTNFR1-tm-Y-egfp. These results support a concept for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.
Gao
Ying
Y
Department of Hematology, C14, BMC, Lund, Sweden.
Hansson
Markus
M
Calafat
Jero
J
Tapper
Hans
H
Olsson
Inge
I
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004705:Endocytosis; D004789:Enzyme Activation / Q000187:drug effects; D005089:Exocytosis / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006413:Hematopoietic System; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism* / Q000473:pathology; D008164:Luminescent Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D021381:Protein Transport; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured / Q000378:metabolism
D015703:Antigens, CD; D008164:Luminescent Proteins; D010703:Phorbol Esters; D021382:Protein Sorting Signals; D017455:Receptors, IgE; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C