Effect of protein synthesis inhibitors on formation and degradation of tight junctions in HT 29 adenocarcinoma cells

The human colon adenocarcinoma cell line HT 29 grows in DMEM virtually without tight junctions (TJ). Fascia occludens type TJ can be induced in these cells by treatment with a variety of proteases or with hypertonic ammonium sulfate solution. The induced formation of TJ is not affected by pretreatment of the cells with cycloheximide or puromycin. The induced TJ are almost completely degraded within 2 h at 37 degrees C both in the absence and presence of the inhibitors studied. With ammonium sulfate as the initial inducing agent, it was possible to induce a second round of TJ formation as early as 2 h after the initial treatment, i.e., immediately after the degradation of the TJ formed in the first round. The same result was obtained in cells treated with cycloheximide. Similar results were also obtained when TJ were initially induced by a very mild trypsin treatment. However, if the initial induction involved a more rigorous proteolytic treatment, the cells needed a recovery period of several h before TJ could be induced again. Under these conditions, recovery from the protease treatment was impaired by the addition of protein synthesis inhibitors at any time prior to complete recovery. It follows that proteolytic treatment of cells not only induces TJ formation but also destroys cell surface proteins which must be available for the formation of TJ strands. It seems possible that these proteins mediate cell adhesion events which may be a prerequisite for, but not a part of the actual TJ formation.