Abstract
The activation of latent proenzymes is an important mechanism for the regulation of localized proteolytic activity. Human meprin-alpha, an astacin-like zinc metalloprotease expressed in normal colon epithelial cells, is secreted as a zymogen into the intestinal lumen. Here, meprin is activated after propeptide cleavage by trypsin. In contrast, colorectal cancer cells secrete meprin-alpha in a non-polarized way, leading to accumulation and increased activity of meprin-alpha in the tumor stroma. We have analyzed the activation mechanism of promeprin-alpha in colorectal cancer using a co-culture model of the intestinal mucosa composed of colorectal adenocarcinoma cells (Caco-2) cultivated on filter supports and intestinal fibroblasts grown in the companion dish. We provide evidence that meprin-alpha is activated by plasmin and show that the presence of plasminogen in the basolateral compartment of the co-cultures is sufficient for promeprin-alpha activation. Analysis of the plasminogen-activating system in the co-cultures revealed that plasminogen activators produced and secreted by fibroblasts converted plasminogen to active plasmin, which in turn generated active meprin-alpha. This activation mechanism offers an explanation for the observed meprin-alpha activity in the tumor stroma, a prerequisite for a potential role of this protease in colorectal cancer.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Cell culture media and all supplements were obtained from Invitrogen (Basel, Switzerland)
Human meprin is a metalloprotease of the astacin family of proteases [1,2,3]
Several previous observations led us to investigate the role of the plasminogen-activating system in promeprin-␣ activation in human colorectal cancer cells. (i) Colon adenocarcinoma cells (Caco-2) endogenously express meprin-␣ and, in contrast to normal colonocytes, secrete the protein in a non-polarized way from the apical and basolateral membrane domains when grown on Transwell filters [20]. (ii) Increased meprin-␣ activity was measured in colorectal cancer tissue homogenates compared with the normal colon mucosa of the same patients [20], suggesting activation of meprin-␣ by a hitherto unknown protease. (iii) An increase in the expression of components involved in plasminogen activation was described in colorectal adenocarcinomas [21, 28, 41]
Summary
Materials—Cell culture media and all supplements were obtained from Invitrogen (Basel, Switzerland). Madin-Darby canine kidney (MDCK) cells stably transfected with human meprin-␣ cDNA [32] were grown in HEPES-buffered minimal essential medium supplemented with 5% (v/v) fetal calf serum, 400 g/ml Geneticin, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM glutamine. Inserts with Caco-2 cells were assembled with the fibroblasts grown on the companion plates and incubated for 20 h in 2 ml of serum-free medium in each compartment. Meprin-␣ activity was measured in the culture media after zymogen activation with trypsin or plasmin using N-benzoyl-L-tyrosyl-p-aminobenzoic acid (PABA peptide) as a substrate and analyzed as described previously [8]. Recombinant promeprin-␣ (500 g), purified by gel filtration from overexpressing High Five insect cell culture medium, was incubated with 10 nM human plasmin in 0.1 M Tris-HCl, 0.1 M NaCl, 0.1% polyethylene glycol 8000 (pH 7.5) for 5 h at room temperature. Following transfer to a polyvinylidene difluoride membrane, the Coomassie Blue-stained meprin-␣ band was excised and subjected to N-terminal amino acid sequencing (SeqLab)
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