A Human Keratinocyte Cell Line Produces Two Autocrine Growth Inhibitors, Transforming Growth Factor-β and Insulin-like Growth Factor Binding Protein-6, in a Calcium- and Cell Density-dependent Manner

Abstract

Two growth inhibitors were identified in culture medium conditioned by a human keratinocyte cell line, HaCat. TGF-β was detected in media conditioned by growing or confluent HaCat cells, as well as in media conditioned at physiological (1 mM) or low (0.03 mM) Ca2+ concentrations. However, a considerable part of transforming growth factor β (TGF-β) in media conditioned at a physiological Ca2+ concentration was in active form, whereas most TGF-β in media conditioned at a low Ca2+ concentration was latent. The other growth-inhibitory activity, which was detected only in media conditioned by confluent cells at a physiological Ca2+ concentration, was purified to homogeneity by a four-step procedure. The N-terminal amino acid sequence of the 33-kDa protein was identical with that of insulin-like growth factor binding protein-6 (IGFBP-6). Purified IGFBP-6 inhibited the growth of HaCat and Balb/MK keratinocyte cell lines, as well as Mv1Lu cells. The growth activity was also demonstrated by human recombinant IGFBP-6. In summary, HaCat cells secrete at least two possible autocrine growth inhibitors: TGF-β which is secreted constitutively, but activated in a Ca2+-dependent manner, and IGFBP-6 which is secreted in a cell density- and Ca2+-dependent manner. Two growth inhibitors were identified in culture medium conditioned by a human keratinocyte cell line, HaCat. TGF-β was detected in media conditioned by growing or confluent HaCat cells, as well as in media conditioned at physiological (1 mM) or low (0.03 mM) Ca2+ concentrations. However, a considerable part of transforming growth factor β (TGF-β) in media conditioned at a physiological Ca2+ concentration was in active form, whereas most TGF-β in media conditioned at a low Ca2+ concentration was latent. The other growth-inhibitory activity, which was detected only in media conditioned by confluent cells at a physiological Ca2+ concentration, was purified to homogeneity by a four-step procedure. The N-terminal amino acid sequence of the 33-kDa protein was identical with that of insulin-like growth factor binding protein-6 (IGFBP-6). Purified IGFBP-6 inhibited the growth of HaCat and Balb/MK keratinocyte cell lines, as well as Mv1Lu cells. The growth activity was also demonstrated by human recombinant IGFBP-6. In summary, HaCat cells secrete at least two possible autocrine growth inhibitors: TGF-β which is secreted constitutively, but activated in a Ca2+-dependent manner, and IGFBP-6 which is secreted in a cell density- and Ca2+-dependent manner.