Figure 1.

AG3340 inhibits MT1-MMP and the shedding of CD44 in IS-CD8⁺ T cells. (Upper panel) Gelatin zymography of MMP-2. To analyze the activation of MMP-2 by cellular MT1-MMP, adherent (A) and non-adherent (NA) IS-CD8⁺ cells were each incubated for 18 h in serum-free medium. Purified MMP-2 (20 ng) was added to the cells. The activation of MMP-2 was analyzed by gelatin zymography of the medium aliquots to observe the conversion of the 68 kDa MMP-2 proenzyme into the 62 kDa MMP-2 mature enzyme. Where indicated, AG3340, SB-3CT or EGCG were added to the cells for 18 h. (Middle panel) Western blotting of CD44. IS-CD8⁺ cells were surface-biotinylated and were then either allowed to adhere, in serum-free medium, to plastic coated with type I collagen/gelatin (adherent, A) or remained in suspension (non-adherent, NA). Where indicated, AG3340, SB-3CT or EGCG were added to the cells. Cell lysate and medium samples were captured with streptavidin-agarose beads. CD44 was analyzed in the captured sample aliquots (50 mg total protein each) by western blotting with an antibody to the CD44 ectodomain. (Bottom panel) MMP-2 is inhibited by low concentrations of SB-3CT. α1-Antitrypsin was incubated with MMP-2. The digest samples were analyzed by reducing SDS-gel electrophoresis. Where indicated, SB-3CT was added to the samples. AG3340, 3(S)-2,2-dimethyl-4[4-pyridin-4-yloxy-benzenesulfonyl]-thiomorpholine-3-carboxylic acid hydroxamate; MT1-MMP, membrane type-1 matrix metalloproteinase; MMP-2, matrix metalloproteinase-2; SB-3CT, 2-(4-phenoxyphenylsulfonylmethyl)thiirane; EGCG, epigallocatechin-3-gallate.