URINARY TRYPSIN-INHIBITOR, A KUNITZ-TYPE PROTEASE INHIBITOR, MODULATES TUMOR NECROSIS FACTOR-STIMULATED ACTIVATION AND TRANSLOCATION OF PROTEIN-KINASE-C IN U937 CELLS

Urinary trypsin inhibitor (UTI) is a Kunitz-type protease inhibitor. W e have reported that UTI inhibited TNF-induced urokinase (uPA) product ion via a protein kinase C (PKC)-dependent mechanism. It is likely tha t UTI suppresses tumor cell invasion and metastasis by a mechanism, po ssibly by inhibiting uPA production. In the present study, we attempte d to determine how UTI is associated with PKC, and how UTI is involved in uPA-dependent tumor cell invasion and metastasis. The increments o f membrane-associated PKC activity by TNF were subsequently accompanie d by a rapid loss of cytosol-associated PKC activity in U937 leukemia cells. Semi-quantitative immunoblotting of membrane and cytosol fracti ons showed that the translocation of PKC-alpha, -beta, and -epsilon we re blocked by the addition of UTI in cells stimulated with TNF but not in cells stimulated with PMA, demonstrating that PKC itself is not se nsitive to UTI. This effect was dependent on the carboxyl-terminus of UTI. In addition, UTI neither inhibited TNF binding to cellular recept ors nor inactivate PKC and uPA activities directly. Taken together, th ese experiments suggest that the carboxyl-terminus of UTI may inhibit the PKC-signalling pathways upstream of diacylglycerol by a mechanism, possibly by interrupting the coupling of receptor and effector system s. UTI was shown to have an interesting new function besides being a p rotease inhibitor. This is the first report that UTI has a selective i nhibition of TNF-activated PKC. We conclude that UTI suppresses tumor cell invasion and metastasis by a mechanism that UTI inhibits TNF-stim ulated uPA production via a PKC-dependent mechanism.