Expression and localization of endothelin-converting enzyme-1 isoforms in human endothelial cells.
Endothelin-converting enzyme (ECE)-1 is a membrane-bound metalloprotease responsible for production of vasoactive endothelin (ET)-1 from inactive big ET-1. ECE-1 exists as four separate isoforms, ECE-1a, b, c, and d, which differ only in their amino-terminal regions. We investigated the expression and localization of the ECE-1 isoforms in primary human umbilical vein endothelial cells (HUVECs) and EAhy926 cells. Reverse transcriptase polymerase chain reaction showed expression of all four isoforms in both cell lines, with ECE-1d seeming, at least qualitatively, to be the predominant isoenzyme. Isoform-specific polyclonal antibodies were used to investigate isoform protein expression. ECE-1a, b, and c protein was detected in EAhy926 cells by immunoblotting; only ECE-1a and ECE-1c were detected in HUVECs. Using immunofluorescence microscopy analysis, both HUVEC and EAhy926 cells showed nuclear immunoreactivity with a monoclonal antibody recognizing all ECE-1 isoforms. The ECE-1a antibody also showed nuclear immunoreactivity in both cell lines; this seemed to colocalize with nucleolin. The ECE-1b antibody showed nuclear immunoreactivity in EAhy926 cells, but no overlap with nucleolin was seen. Intracellular immunoreactivity was seen in both cell lines using the ECE-1c antibody; this showed some colocalization with concanavalin A (an endoplasmic reticulum marker). von Willebrand Factor was used as a marker for Weibel-Palade bodies in HUVECs, but no colocalization with ECE-1 was seen during this study. The data presented here sheds new light on the localization of ECE-1a, b, and c in cultured human endothelial cells, which may further understanding of the ET system and aid design of therapeutic ECE inhibitors.
Hunter
Alison R
AR
Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
Turner
Anthony J
AJ
D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D002466:Cell Nucleolus / Q000201:enzymology / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology* / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000166:cytology; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology
D007527:Isoenzymes; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes