Hormonal Regulation and Cell-Specific Expression of Endothelin-Converting Enzyme 1 Isoforms in Bovine Ovarian Endothelial and Steroidogenic Cells1

Abstract

Endothelin-converting enzyme 1 (ECE-1) is a key enzyme in the biosynthesis of endothelin 1 (ET-1), a potent regulator of ovarian function. Different ECE-1 isoforms are localized in distinct intracellular compartments. Thus, the spatial and temporal pattern of ECE-1 expression determines the site of big ET-1 activation and the bioavailability of ET-1. This study was undertaken to investigate the hormonal regulation and cell-specific expression of ECE-1 isoforms in endothelial and steroidogenic cells of bovine follicles and corpora lutea (CL). Using enriched follicular and luteal cell subpopulations and in situ hybridization techniques, we showed that the ECE-1 gene is expressed by both endothelial and steroidogenic cells; however, the intracellular ECE-1a isoform was present only in ET-1-expressing endothelial cells. Steroidogenic cells in follicles or in CL, deficient in ET-1, expressed only the plasma membrane ECE-1b isoform. The intensity of antisense ECE-1 labeling in the granulosa cell layer increased with follicular size; insulin-like growth factor I and insulin upregulated ECE-1 expression when cultured with granulosa cells, suggesting that these growth factors may increase ECE-1 in growing follicles. In contrast, ET-1 and LH downregulated ECE-1 in steroidogenic cells. This effect could account for low ECE (and ET-1) levels, which characterize the early luteal phase. These findings suggest that ECE-1 is regulated during different stages of the cycle in a physiologically relevant manner. The hormonal regulation and intracellular localization of bovine ECE-1 isoforms revealed in this study may provide new insights into ET-1 biosynthesis and mode of action in different cellular microenvironments within the ovary.

Microvascular endothelial cells of the corpus luteum

“…Endothelin-1 (ET-1) is synthesized by proteolytic cleavage of a large precursor molecule, prepro endothelin-1, which is facilitated by a metalloproteinase, endothelin converting enzyme (ECE). In the bovine corpus luteum, prepro endothelin-1 mRNA appears to be expressed only in endothelial cells whereas, both the steroidogenic cells and luteal endothelial cells express ECE [87,94-96]. Thus, the steroidogenic cells and endothelial cells may differentially cooperate in the biosynthesis of mature endothelin-1.…”

“…Thus, the steroidogenic cells and endothelial cells may differentially cooperate in the biosynthesis of mature endothelin-1. The ECE is expressed in the corpus luteum throughout the normal estrous or menstrual cycle [94-96]. However, ECE mRNA and protein levels peak at mid luteal phase and are reduced during corpus luteum regression suggesting that endothelin-1 plays a role in corpus luteum development.…”

“…However, ECE mRNA and protein levels peak at mid luteal phase and are reduced during corpus luteum regression suggesting that endothelin-1 plays a role in corpus luteum development. A recent report [94] describes two ECE isoforms, ECE-1a and ECE-1b, in steroidogenic cells and cytokeratin-negative endothelial cells of the bovine corpus luteum. Based on their cellular localization, the isoforms are capable of cleaving the intra- or extra-cellular endothelin-1 precursor molecule, respectively.…”

Small Interfering RNA Molecules Targeting Endothelin-Converting Enzyme-1 Inhibit Endothelin-1 Synthesis and the Invasive Phenotype of Ovarian Carcinoma Cells

“…Therefore, the final step in synthesizing ECE-1 must take place in proximity to its site of action, which may explain why its expression is widespread even in cells that do not express ETs. ECE-1 is found in endothelial cells throughout the body, but it is also highly expressed in other cell types, such as testicular Leydig cells, adrenal cells, and ovarian granulosa cells (25,26). Four full-length, membrane-bound isoforms of ECE-1 (1a, 1b, 1c, and 1d), which only differ in their 5 ¶ end, are known and have been characterized (27,28).…”

Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain

“…Enrichment of Luteal Steroidogenic and Endothelial Cells-For enrichment of luteal cell subpopulations, mid-cycle corpora lutea were dispersed by using collagenase IV as described previously (5,32). Briefly, corpora lutea were sliced and dispersed in M-199 containing 0.5% bovine serum albumin and collagenase (420 units/ml).…”