Activation of Type IV Procollagenases by Human Tumor-associated Trypsin-2

Abstract

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg 87 -Phe 88 . The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg 99 -Lys 100 peptide bond of proMMP-2 generating 62-65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cellderived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.

The levels of trypsinogen isoenzymes in ovarian tumour cyst fluids are associated with promatrix metalloproteinase-9 but not promatrix metalloproteinase-2 activation

“…1995; Terada et al, 1995) and tumour cell lines (Koivunen et al, 1991b;Koshikawa et al, 1994). The main tumour-associated isoenzyme, trypsin-2, activates pro-urokinase-type plasminogen activator (pro-uPA) (Koivunen et al, 1989) and in vitro it is the most efficient activator of the 92 kDa gelatinase B (MMP-9) known thus far (Sorsa et al, 1997). Therefore, trypsin may participate in cancer cell-mediated proteolysis by directly degrading the extracellular matrix or by activating other proteinases (Koivunen et al, 1991a).…”

“…This suggests that trypsin-1 and trypsin-2 may be involved in the activation of MMP-9 but not MMP-2 in vivo. Although human trypsin-2 reportedly activates MMP-2 in vitro it has been observed that MMP-2 is further fragmented in its Cterminal region during a prolonged incubation with trypsin-2 (Sorsa et al, 1997). It is possible that trypsin, a very efficient proteinase, may inactivate MMP-2 or its activator MMP-14 when occurring at high concentrations.…”

“…The blots were incubated with a 1:500 dilution of polyclonal antibodies against either human trypsinogen, MMP-2, or MMP-9, or with nonimmune control serum (DAKO A/S, Glostrup, Denmark) for 3 h at 37 ˚C followed by peroxidase-conjugated second antibodies (1:200 dilution, DAKO A/S) for 1 h at 22 ˚C. After washing, the blots were developed with a solution of 60 mg/ml diaminobenzidine tetrahydrocholoride in 50 mM Tris-HCl, pH 8.0, and 0.003% H 2 O 2 (Sorsa et al, 1997). …”

“…The urokinase type plasminogen activator (uPA) -plasmin system (Mazzieri et al, 1997) and membrane type MMP-1 (MT1-MMP, MMP-14) (Sato et al, 1994;Tokuraku et al, 1995) have been suggested to represent physiological mechanisms for the control of MMP-2 and MMP-9 activity. Recent studies have shown that human trypsin-2 is more potent than plasmin and other serine proteinases in activating several MMPs, including MMP-2 and MMP-9 (Sorsa et al, 1997), and that downregulation of trypsin-2 expression and activity in colon cancer cells is associated with decreased proMMP-9 activation (Lukkonen et al, 2000).…”

“…The concerted action of various matrix metalloproteinases (MMPs) and serine proteinases such as plasmin and plasminogen activators is believed to be a key event in this step (Mignatti et al, 1986). We have shown that tumour-associated trypsin may participate in this process (Koivunen et al, 1991a;Sorsa et al, 1997). Two variants of the trypsinogen isoenzymes trypsinogen-1 (cationic) and trypsinogen-2 (anionic), called tumour-associated trypsinogen-1 and -2, have been shown to occur in ovarian tumour cyst fluid (Koivunen et al, 1989).…”