Biochemical Characterization of Endothelin-converting Enzyme-1α in Cultured Skin-derived Cells and Its Postulated Role in the Stimulation of Melanogenesis in Human Epidermis

Abstract

The vasoconstrictive peptide endothelin-1 (ET-1) is expressed in human epidermis at the gene and protein levels and plays an important role in stimulating pigmentation via its increased secretion by keratinocytes following ultraviolet B (UVB) irradiation. However, one or more biological mechanisms underlying the secretion of ET-1 by keratinocytes in human skin have never been evaluated. In mammalian endothelial cells, a membrane-bound neutral metalloproteinase, termed endothelin-converting enzyme (ECE), catalyzes the specific cleavage of the inactive precursor Big ET to produce mature active ET, which leads in turn to the secretion of ET by those cells. To clarify the potential involvement of ECE in the processing and secretion of ET-1 by human keratinocytes, we synthesized the N-terminal peptide of human ECE-1alpha and generated a rabbit polyclonal antibody (alphaPEPT6) that specifically recognizes ECE-1alpha. Reverse transcription PCR and Western blotting analysis revealed that significant expression of ECE-1 transcripts and ECE-1alpha protein occurs in human keratinocytes. When ECE activity was assayed in extracts of human keratinocytes at pHs ranging from 5.0 to 8.0, the enzymatic profile had an optimal neutral pH of 7.0 and was sharply pH-dependent. Furthermore, when extracts of human keratinocytes were treated with alphaPEPT6, ECE activity was significantly reduced compared with extracts treated with the prebleed serum of alphaPEPT6, which supports the notion that ECE-1alpha is a major metalloproteinase with ECE activity in human keratinocytes. The exogenous addition of the pro-inflammatory cytokine interleukin-1alpha significantly increased expression of ECE-1 transcripts in cultured human keratinocytes, which suggests an association with post-inflammatory hyperpigmentation. Collectively, our results demonstrate for the first time that ECE-1alpha is expressed at significant levels in various types of human skin cells (including keratinocytes) and that it plays a constitutive role in the processing and UVB-inducible secretion of ET-1 by human keratinocytes, which leads to the stimulation of pigmentation in the epidermis.