TGF-beta-induced matrix proteins inhibit p42/44 MAPK and JNK activation and suppress TNF-mediated I kappa B alpha degradation and NF-kappa B nuclear translocation in L929 fibroblasts

The role of transforming growth factor beta 1 (TGF-beta 1)-induced extracel lular matrix proteins in the modulation of cellular response to the cytotox ic effect of tumor necrosis factor (TNF) or Fas ligand was investigated. Mu rine L929 fibroblasts were prestimulated with or without TGF-beta 1 for 1-2 4 h and the resulting extracellular protein matrices were prepared. Unstimu lated control L929 cells were then cultured on these matrices. Compared to control matrix-stimulated L929 cells, the TGF-beta 1 matrix-stimulated cell s resisted TNF killing in the presence of actinomycin D (ActD), but became more susceptible to killing by anti-Fas antibodies/ActD. The induced TNF re sistance is independent of the NF-kappa B antiapoptotic effect. For example , exposure of TGF-beta 1 matrix-stimulated L929 cells to TNF failed to resu lt in I kappa B alpha degradation and NF-kappa B nuclear translocation or a ctivation. Also, control matrix stimulated the activation of p42/44 mitogen -activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in L929 cells, whereas TGF-beta 1 matrix suppressed the activation. Nonetheless, in response to TNF, JNK activation was restored in the TGF-beta 1 matrix-stim ulated cells. By metabolic labeling, ammonium sulfate precipitation and N-t erminal amino acid microsequencing, TGF-beta 1 was shown to induce a novel matrix protein of 46 kDa (p46) from L929 cells. Adsorption of p46 by peptid e antibodies against its N-terminus removed the TGF-beta 1 matrix protein-m ediated protection against TNF/ActD cytotoxicity and its enhancement of ant i-Fas/ActD killing, indicating that p46 is responsible for these effects. I mmunostaining of L929 cells revealed that the antibodies were bound to a me mbrane protein of 100 kDa (p100). Thus, the matrix p46 is likely derived fr om the released membrane p100. (C) 2000 Academic Press.