A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1.
Antibody conjugates directed against intercellular adhesion molecule (ICAM-1) or platelet-endothelial cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles that are specifically recognized and internalized by endothelial cells. There is increasing evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization, we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells. We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis, suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C (PKC) inhibitors, agents known to inhibit macropinocytosis, reduced the internalization of clustered ICAM-1 and PECAM-1. However, expression of dominant-negative dynamin-2 constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates stimulated the formation of actin stress fibers by human umbilical vein endothelial cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho kinase (ROCK) were required for internalization. Interestingly, these kinases are part of the signal transduction pathways that are activated when circulating leukocytes engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated endocytosis is regulated using comparable signaling pathways.
Muro
Silvia
S
Department of Physiology, University of Pennsylvania School of Medicine, B-400 Richards/6085, 3700 Hamilton Walk, Philadelphia, PA 19104, USA.
Wiewrodt
Rainer
R
Thomas
Anu
A
Koniaris
Lauren
L
Albelda
Steven M
SM
Muzykantov
Vladimir R
VR
Koval
Michael
M
D000577:Amides / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000494:pharmacology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002966:Clathrin / Q000494:pharmacology; D034281:Dynamins / Q000235:genetics / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D008954:Models, Biological; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D000577:Amides; D000911:Antibodies, Monoclonal; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; D004791:Enzyme Inhibitors; D007783:Lactones; D018942:Macrolides; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011725:Pyridines; D013844:Thiazoles; D018799:Intercellular Adhesion Molecule-1; C108830:Y 27632; D000584:Amiloride; D011493:Protein Kinase C; D034281:Dynamins; C035359:monorden