Regulation of proteolysis by cytokines in the human intestinal epithelial cell line HCT–8: role of IFNγ

Protein metabolism contributes in the regulation of gut barrier function, which may be altered during inflammatory states. There are three major proteolytic pathways in mammalian cells: lysosomal, Ca2+-activated and ubiquitin–proteasome. The regulation of proteolytic activities during inflammation remains unknown in intestine. Intestinal epithelial cells, HCT-8, were stimulated by IL-1β, IFNγ and TNFα each alone or in combination (Cytomix). Proteolytic activities were assessed using fluorogenic substrates and specific inhibitors, protein expressions by Western blot. Lysosomal and Ca2+-activated pathways were not significantly altered by any treatment. In contrast, the activity of ubiquitin–proteasome system was stimulated by IFNγ and Cytomix (155, 160 versus 100, P < 0.05, respectively) but remained unaffected by IL-1β and TNFα. Free ubiquitin expression, but not ubiquitinated proteins, was enhanced by IFNγ and Cytomix. The expression of proteasome 20S α1 subunit, a constitutive proteasome 20S subunit, was not altered, β5 subunit expression was weakly decreased by Cytomix and inducible β5i subunit expression was markedly increased in response to IFNγ and to Cytomix (202, 206 versus 100, P < 0.05, respectively). In conclusion, lysosomal, Ca2+-activated and constitutive proteasome activities were not affected by IL-1β, IFNγ and TNFα alone or in combination, in HCT-8 cells. These results suggest that IFNγ, but not IL-1β and TNFα, increases immunoproteasome, which might contribute to enhanced antigen presentation during inflammatory bowel diseases.