The Expression of Endothelin Converting Enzyme-1 Insoforms in Murine Cells

Abstract

Endothelin-1 (ET-1) has been demonstrated to play a role in many physiological processes including vasoconstriction, cardiovascular homeostasis, inflammation, angiogenesis, and tissue regeneration. A critical step in the production of ET-1 is the conversion of the propeptide form, bigET-1, to ET-1 via the action of endothelin converting enzyme-1 (ECE-1). There are four major ECE-1 isoforms: ECE1a, 1b, 1c, and 1d, which differ only in their NH2 terminus and subcellular locations. Here we describe our analysis of the expression of ECE-1 isoforms in murine bone marrow derived macrophages (BMM0), macrophage-like cell lines (RAW264, J774) and an endothelial cell line (bEND.3). For analysis, reverse transcription-polymerase chain reaction (RT-PCR) was performed using forward primers, unique to each major isoforms, and a common reverse primer. bEnd.3, J774 and BMM0 express mRNA encoding all four major isoforms of ECE-1, as well as previously reported splice variants (SV) of ECE-1b, 1c, and 1d. A novel splice variant, KC150, was identified in isoform 1c. These splice variants all lack exon 3, which encodes the trans-membrane domain of ECE-1. The absence of a trans-membrane domain could be indicative of a secreted or cytosolic form of ECE-1. Our results also indicate that the RAW264 macrophage cell line expresses very low levels of ECE-1 encoding mRNA. This verifies our previous finding that RAW264 cells secrete only bigET-1, suggesting a deficiency in ECE-1 expression. We hope to use these ECE-1 deficient cells as a model for over expressing individual ECE-1 isoforms and splice-variants, and examine their function in regulating macrophage ET-1 production.