Characterization of serum adhesive proteins that block tumor necrosis factor-mediated cell death

Previously we have shown that TGF-beta1 protects murine L929 fibroblasts from TNF/ActD-mediated cell death by inducing the expression of an extracellular matrix TNF-resistance triggering (TRT) protein. TRT promotes TNF-resistance via activation of tyrosine and serine/threonine kinases in L929 cells. To examine the presence of TRT activity in serum (designated STRT), human sera were diluted, treated with or without PMSF and subjected to sequential ammonium sulfate precipitation (ASP). Aliquots of the ASP protein fractions were coated onto 96-well plates, followed by thorough washing. When L929 cells were seeded and cultured on the wells coated with STRT proteins, these cells resisted killing by TNF, TNF/ActD, doxorubicin and serum deprivation, but not by anti-Fas/ActD, staurosporine and ActD. STRT activity was found at the 15% ASP fraction of untreated sera, but shifted to the 20% ASP fraction of PMSF-treated sera. Two likely STRT proteins of approximately 226 and 265 kDa were found in these fractions, compared to the corresponding nonfunctional ASP fractions. Functionally, STRT was inactivated by trypsin, but not by 5 M salt, various serine and/or cysteine protease inhibitors, and antibodies against fibronectin, vitronectin, C1q, histidine-rich glycoprotein, CD44, chondroitin sulfate and hyaluronic acid. STRT failed to alter the expression of proteins involved in apoptosis such as RIP, ICH-1L, BCL-X, TIAR and IkappaBalpha, and could not induce IkappaBalpha degradation. The induced TNF-resistance could be reversed by treatment of STRT-stimulated cells with testicular hyaluronidase, as well as with tyrosine kinase inhibitors tyrophostin, lavendustin A and AG-490 (a selective inhibitor of JAK2 kinase). However, the STRT function could not be blocked by the MEK kinase inhibitor PD98059 and the NF-kappaB inhibitors curcumin and a synthetic inhibitor peptide for NF-kappaB translocation. Together, our data suggest that tyrosine kinase activation is involved in the STRT-mediated resistance to TNF and TNF/ActD in L929 cells.