HaCaT human keratinocytes express IGF-II, IGFBP-6, and an acid-activated protease with activity against IGFBP-6

The insulin-like growth factor (IGF) system plays an important role in skin . HaCaT human keratinocytes proliferate in response to IGFs and synthesize IGF-binding protein-3 (IGFBP-3). Recently, IGFBP-6 was also identified by N H2-terminal sequencing, but it has not been identified by Western ligand bl otting. In the present study, IGFBP-6 was detected in HaCaT-conditioned med ium by use of immunoblotting and Western ligand blotting with I-125-labeled IGF-II. Proteolytic activity against IGFBPs, an important mechanism for re gulation of their activity, was then studied. An acid-activated, cathepsin D-like protease that cleaved both IGFBP-6 and IGFBP-3 was detected. Althoug h proteolysis did not substantially reduce the size of immunoreactive IGFBP -6, it greatly reduced the ability of IGFBP-6 to bind I-125-IGF-II as deter mined by Western ligand blotting and solution assay. HaCaT keratinocytes do not express IGF-I mRNA, but IGF-II mRNA and protein expression was detecte d. These observations suggest the possibility of an autocrine IGF-II loop t hat is regulated by the relative expression of IGF-IT, IGFBP-3, and IGFBP-6 , and IGFBP proteases in these keratinocytes, although demonstration of thi s loop requires further study.