Endothelin-I (ET-1) is an endothelium-derived 21 amino acid vasoconstrictor peptide possessing two intrachain disulfide bridges. Recently it has become evident that isoforms of ET (ET-l, -2, and -3) have a wide range of pharmacological effects in various tissues and act as autocrineiparacrine factors. We demonstrate here that ET-1 is secreted from normal human keratinocytes and may work as an autocrine growth factor through a specific receptor. In this study, human foreskin keratinocytes were cultured in serum-free MCDB 153 medium. Cell growth and [3H] thymidine incorporation in low and high Ca++ concentration media was stimulated by ET-I, -2, and -3 with similar potencies. The strongest response was observed at 10 nM ETs, whereas stimulatory activity was reduced at 100 nM. ETs suppressed keratinocyte differentiation as measured by reactivity with involucrin antibody. Plasminogen activator activity (mainly urokinase) in the medium was also stimulated by the addition of 10 n M ETs. ET-I-like immunoreactivity measured by radioimmunoassay was 1.4 fmol/day/l O6 cells in non-treated condition medium. Among the various cytokines, tumor necrosis factor-(TNF-a), interleukin-la, and transforming growth factor-p stimulated ET-1 secretion in a dosedependent manner. The strongest response (ten-fold) was observed upon the addition of 10 ng/ml TNF-a. Scatchard plot analysis of [12511 ET-1 binding to keratinocytes revealed the presence of a single class of high affinity receptors (K, 50 pM, 9 x 10' sitesicell). Binding was competitively inhibited by the addition of unlabeled ET-1 and -2 with similar affinities and by ET-3 with weaker affinity. ET-1 mRNA expression in keratinocytes was detected by reverse transcription-polymerase chain reaction and was increased by treatment with 10 ng/ml TNF-a. These results suggest that ET-1 acts as an autocrine growth factor for keratinocytes through a specific receptor. o 1994 Wiley-Liss, Inc.