R. Tsuboi et al., ENDOTHELIN-1 ACTS AS AN AUTOCRINE GROWTH-FACTOR FOR NORMAL HUMAN KERATINOCYTES, Journal of cellular physiology, 159(2), 1994, pp. 213-220
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.