ENDOTHELIN-1 ACTS AS AN AUTOCRINE GROWTH-FACTOR FOR NORMAL HUMAN KERATINOCYTES

Endothelin-1 (ET-1) is an endothelium-derived 21 amino acid vasoconstr ictor peptide possessing two intrachain disulfide bridges. Recently it has become evident that isoforms of ET (ET-1, -2, and -3) have a wide range of pharmacological effects in various tissues and act as autocr ine/paracrine 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 keratinocyt es were cultured in serum-free MCDB 153 medium. Cell growth and [H-3] thymidine incorporation in low and high Ca++ concentration media was s timulated by ET-1, -2, and -3 with similar potencies. The strongest re sponse was observed at 10 nM ETs, whereas stimulatory activity was red uced at 100 nM. ETs suppressed keratinocyte differentiation as measure d by reactivity with involucrin antibody. Plasminogen activator activi ty (mainly urokinase) in the medium was also stimulated by the additio n of 10 nM ETs. ET-1-like immunoreactivity measured by radioimmunoassa y was 1.4 fmol/day/10(6) cells in non-treated condition medium. Among the various cytokines, tumor necrosis factor-alpha (TNF-alpha), interl eukin-1 alpha, and transforming growth factor-beta stimulated ET-1 sec retion in a dose-dependent manner. The strongest response (ten-fold) w as observed upon the addition of 10 ng/ml TNF-alpha. Scatchard plot an alysis of [I-125] ET-1 binding to keratinocytes revealed the presence of a single class of high affinity receptors (K-D 50 pM, 9 x 10(3) sit es/cell). Binding was competitively inhibited by the addition of unlab eled ET-1 and -2 with similar affinities and by ET-3 with weaker affin ity. ET-1 mRNA expression in keratinocytes was detected by reverse tra nscription-poly-merase chain reaction and was increased by treatment w ith 10 ng/ml TNF-alpha. These results suggest that ET-1 acts as an aut ocrine growth factor for keratinocytes through a specific receptor. (C ) 1994 Wiley-Liss, Inc.