KUPFFER CELL-DERIVED 95-KD TYPE-IV COLLAGENASE GELATINASE-B - CHARACTERIZATION AND EXPRESSION IN CULTURED-CELLS/

Release of 92-kd type IV collagenase/gelatinase, also known as gelatin ase B, by inflammatory and tumor cells is increasingly recognized and is believed to facilitate cellular migration across basement membranes . It has been implicated in the pathogenesis of many diseases, but lit tle is known of its cellular origin(s) and function in liver. In this study we have demonstrated synthesis and release of gelatinase B by hu man and rat Kupffer cells in primary culture. Northern analysis of RNA extracted from Kupffer cells stimulated with phorbol ester demonstrat ed a 2.8 kb transcript for gelatinase B. Immunoblotting and zymography of serum-free Kupffer cell-conditioned media demonstrated extracellul ar release of immunoreactive enzyme and gelatinase activity, Mr 92,000 (95,000 from rat cells). The organomercurial 4-aminophenyl mercuric a cetate (APMA) activated the enzyme in vitro, indicating secretion prim arily as a proenzyme. Stimulation of Kupffer cells by phorbol ester ma rkedly induced gelatinase B release, which was inhibited by cyclohexim ide. In contrast, cycloheximide had no effect on constitutive secretio n in culture, suggesting that there is some intracellular storage. Kup ffer cell-derived gelatinase B was also partially purified and charact erized. After separation by gelatin sepharose and gel filtration chrom atography, gelatin-degrading activities of 95, 88, 75, and 65 kd were detected, the three lower-molecular-weight species probably representi ng activated forms. Enzyme activity was inhibited by ethylenediaminete tra-acetic acid (EDTA), but not by serine- and thiol-protease inhibito rs, and was restored by zinc. Activity was also inhibited by tissue in hibitor of metalloproteinase-1 (TIMP-1) and alpha-2 macroglobulin. The partially purified enzyme rapidly degraded denatured collagens (gelat in) as well as native types III, IV, and V collagens, but had no activ ity against casein, types I and VI collagens.