Abstract
The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine changes in enzyme activity, but correlate with changes in membrane fluidity as revealed by measurement of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and fluorescence recovery after photobleaching analyses. ELISA and immunoblot experiments conducted with granulocytes, endothelial cells, and keratinocytes revealed rapid increase of ectodomain shedding of ADAM10 and ADAM17 substrates upon membrane fluidization. Large amounts of unsaturated FFA may be liberated from cholesteryl esters in LDL that is entrapped in atherosclerotic lesions. Incubation of cells with thus modified LDL resulted in rapid cleavage of ADAM substrates with corresponding functional consequences on cell proliferation, cell migration, and endothelial permeability, events of high significance in atherogenesis. We propose that FFA represent critical regulators of ADAM function that may assume relevance in many biological settings through their influence on mobility of enzyme and substrate in lipid bilayers.
Highlights
During the past decade, ADAMs3 (A Disintegrin And Metalloproteinase) have emerged as the major proteinase family
We found that pro-Vibrio cholerae cytolysin (VCC) cleavage was markedly accelerated by application of unsaturated free fatty acids (FFA) both in free form and in association with low density lipoprotein (LDL), as is probably widely present in atherosclerotic lesions
ADAM10-mediated Cleavage of pVCC Leads to Hemolysis of Rabbit Erythrocytes—To distinguish whether changes in ADAM activity might be due to enhanced cell surface expression of the enzymes or to a bona fide increase in enzyme activity or to accelerated substrate cleavage occurring through biophysical alterations of the membranes, we developed a simple erythrocyte test system
Summary
ADAMs3 (A Disintegrin And Metalloproteinase) have emerged as the major proteinase family. ADAM10-mediated Cleavage of pVCC Leads to Hemolysis of Rabbit Erythrocytes—To distinguish whether changes in ADAM activity might be due to enhanced cell surface expression of the enzymes or to a bona fide increase in enzyme activity or to accelerated substrate cleavage occurring through biophysical alterations of the membranes, we developed a simple erythrocyte test system.
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