Trx1-mediated reducing activity interrupts agonist-induced CD30 signaling in YT cells
Given its influence on ligand binding, we reasoned that Trx1 might interfere with CD30-mediated signaling. To test whether Trx1-mediated conformational alteration of CD30 affects CD30-dependent cellular responses, we made use of the YT large granular lymphoma line that has previously been used to study signals emanating from CD30 and to define the genes regulated by such signals (Muta et al, 2000). Consistent with previous results (Bowen et al, 1993), we observed that stimulation of CD30 with either agonistic antibodies or sCD30L led to upregulation of the IL-2Rα chain (CD25) within 24 h (Figure 7A, lower panel, compare columns 3, 4 and 7). Treatment of YT cells with Trx1(CCAAA) (but not with redox-inactive Trx1) prevented CD25 upregulation (Figure 7A, lower panel, columns 5 and 8), concomitant with the redox change in CD30 (Figure 7A, upper panel, column 4), thus demonstrating that the redox interaction between Trx1 and CD30 has a pronounced influence on CD30-mediated gene expression.
YT cells respond to CD30 signals by downregulating the expression of cytotoxic effector molecules, including FasL, thus decreasing their cytotoxicity against Fas-expressing target cells (Bowen et al, 1993; Muta et al, 2000). To test if Trx1 influences CD30-mediated suppression of cytotoxicity, we treated YT cells with Trx1(CCAAA) or Trx1(SSAAA) before stimulation with agonistic anti-CD30 antibody and quantified cytotoxicity against Cr-labeled Raji cells. Upon CD30 stimulation, cytotoxicity was markedly reduced (Figure 7B, compare columns 1 and 4). The decrease in cytotoxicity was completely reversed by Trx1(CCAAA) but not the catalytically inactive mutant (SSAAA) (Figure 7B, columns 5 and 6). As judged by RT–PCR, changes in cytotoxicity correlated with changes in FasL mRNA expression (Figure 7B, lower panel). These results confirm that the catalytic activity of Trx1 modulates CD30-dependent changes in cellular behavior and function.