title: Targeting of a heterologous protein to a regulated secretion pathway in cultured endothelial cells.
abstract: The stimulation of regulated exocytosis in vascular endothelial cells (EC) by a variety of naturally occurring agonists contributes to the interrelated processes of inflammation, thrombosis, and fibrinolysis. The Weibel-Palade body (WPB) is a well-described secretory granule in EC that contains both von Willebrand factor (vWF) and P-selectin, but the mechanisms responsible for the targeting of these proteins into this organelle remain poorly understood. Through adenoviral transduction, we have expressed human growth hormone (GH) as a model of regulated secretory protein sorting in EC. Immunofluorescence microscopy of EC infected with GH-containing recombinant adenovirus (GHrAd) demonstrated a granular distribution of GH that colocalized with vWF. In contrast, EC infected with an rAd expressing the IgG(1) heavy chain (IG), a constitutively secreted protein, did not demonstrate colocalization of IG and vWF. In response to phorbol ester, GH as well as endogenously synthesized vWF were rapidly released from GHrAd-infected EC. By immunofluorescence microscopy, granular colocalization of GH with endogenous tissue-type plasminogen activator (tPA) was also demonstrated, and most of the tPA colocalized with vWF. These data indicate that EC are capable of selectively targeting heterologous proteins, such as GH, to the regulated secretory pathway, which suggests that EC and neuroendocrine cells share common protein targeting recognition signals or receptors.
authors.affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, MA 02115, USA.
authors.forename: Y H
authors.initials: YH
authors.lastname: Datta
authors.forename: H
authors.initials: H
authors.lastname: Youssoufian
authors.forename: P W
authors.initials: PW
authors.lastname: Marks
authors.forename: B M
authors.initials: BM
authors.lastname: Ewenstein
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001692:Biological Transport; D003594:Cytoplasmic Granules / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D005089:Exocytosis; D005822:Genetic Vectors / Q000235:genetics; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000032:analysis; D014471:Umbilical Veins; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D019382:Human Growth Hormone; D010959:Tissue Plasminogen Activator