A HUMAN KERATINOCYTE CELL-LINE PRODUCES 2 AUTOCRINE GROWTH-INHIBITORS, TRANSFORMING GROWTH-FACTOR-BETA AND INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-6, IN A CALCIUM-DEPENDENT AND CELL DENSITY-DEPENDENT MANNER

Two growth inhibitors were identified in culture medium conditioned by a human keratinocyte cell line, HaCat. TGF-beta 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+ concentratio ns. However, a considerable part of transforming growth factor beta (T GF-beta) in media conditioned at a physiological Ca2+ concentration wa s in active form, whereas most TGF-beta in media conditioned at a low Ca2+ concentration was latent. The other growth-inhibitory activity, w hich was detected only in media conditioned by confluent cells at a ph ysiological Ca2+ concentration, was purified to homogeneity by a four- step procedure. The N-terminal amino acid sequence of the 33-kDa prote in was identical with that of insulin-like growth factor binding prote in-6 (IGFBP-6). Purified IGFBP-6 inhibited the growth of HaCat and Bal b/MK keratinocyte cell lines, as well as Mv1Lu cells. The growth activ ity was also demonstrated by human recombinant IGFBP-6. In summary, Ha Cat cells secrete at least two possible autocrine growth inhibitors: T GF-beta which is secreted constitutively, but activated in a Ca2+-depe ndent manner, and IGFBP-6 which is secreted in a cell density- and Ca2 +-dependent manner.