To assess which export pathway is

To assess which export pathway is used by the iNOS mRNA, we inhibited CRM1 activity by incubation of DLD-1 beta 2 adrenergic receptor with LMB. Since LMB covalently and exclusively binds to CRM1, it represents a potent and specific inhibitor of the CRM1 transport activity [38]. Inhibition of CRM1 activity as well as siRNA-mediated reduction of CRM1 expression leads to a marked decrease in iNOS expression and activity (Fig. 1, Fig. 2). LMB mediated inhibition of iNOS expression has also been described in murine BV2 microglial cells [11]. Therefore, CRM1 seems also to be involved in the regulation of human iNOS expression. Since we observed no influence of LMB on human 16kb iNOS promoter activity (Fig. 1D) and CRM1 is known to mediate the transport of RNAs from the nucleus to the cytoplasm, we also analyzed the amount of cytoplasmic and nuclear iNOS mRNA after LMB treatment of the cells (Fig. 3). The ratio of cytoplasmic to nuclear iNOS mRNA is clearly decreased after CRM1 inhibition, indicating that the export of the iNOS mRNA is, at least in part, accomplished by a CRM1-dependent mechanism. We observed similar data for the nucleocytoplasmic transport of the TNF-α and TTP mRNA (data not shown). In contrast to the cytokine-regulated ARE-containing iNOS mRNA, the amount of cytoplasmic and nuclear GAPDH mRNA remains unchanged after CRM1 inhibition. This indicates that CRM1-mediated mRNA export actually is only perceived by a specific class of mRNAs, most likely those that contain AREs in their 3′-UTR and whose expression is regulated cytokine-dependently. This assumption parallels the observation of Schutz et al. that CRM1-mediated RNA transport only occurs in activated T cells [13]. Since CRM1 itself does not possess any RNA binding affinity, an adapter protein is needed for efficient mRNA transport. The RNA-BP HuR has been shown to mediate CRM1-dependent transport for several mRNAs in HeLa cells [14], [15]. However, in DLD-1 cells, LMB incubation did not change the distribution of HuR between the cytoplasm and the nucleus. Therefore HuR does not seem to be involved in the CRM1-mediated nucleocytoplasmic transport of the iNOS mRNA (Fig. 4). Also the localization of other RNA-BPs regulating iNOS expression like KSRP (Fig. 4) or the poly(A)binding protein PABP (data not shown) remained unchanged after inhibition of CRM1 activity. For TTP, we observed a shift to the nucleus following LMB incubation of DLD-1 cells (data not shown), which goes in line with earlier reports that TTP is a shuttling protein transported to the cytoplasm in a CRM1-dependent manner [39]. However TTP cannot act as adapter protein for iNOS mRNA export, since it does not interact with any part of the iNOS mRNA [27]. Recent reports claim that the eukaryotic translation initiation factor eIF4E – beside its well-known translational function – also operates as a key coordinator for nuclear export of mRNAs that are involved in cellular proliferation [20]. Therefore we assessed the localization of eIF4E with and without LMB in DLD-1 cells. Surprisingly, the inhibition of CRM1 leads to decreased eIF4E levels in the cytoplasm and enhanced amounts of the protein in the nucleus. This observation served as a first hint that eIF4E may govern CRM1-dependent iNOS mRNA transport. By blocking eIF4E transport activity with ribavirin, we could confirm its function in iNOS mRNA export. Ribavirin treatment markedly inhibited CM-induced iNOS expression and activity (Fig. 5). In addition the ratio of cytoplasmic and nuclear iNOS mRNA was lowered under ribavirin treatment (Fig. 6A). The distribution of GAPDH mRNAs again remained unaltered (Fig. 6B) and no effect of ribavirin on the human 16kb iNOS promoter was observed (Fig. 6C). To further support these data we generated DLD-1 cells that stably overexpress the promyelocytic leukemia protein PML (as EGFP-PML protein). PML is known to specifically inhibit eIF4E activity and thereby to inhibit the nuclear export of eIF4E transport target mRNAs [37], [40]. As expected PML overexpression dampened cytokine induced iNOS expression in DLD-1 cells (Fig. 7). As shown in Fig. S1 inhibition of eIF4E function by ribavirin or overexpression of PML did not change iNOS mRNA stability. Therefore it seems that iNOS mRNA trapped in the nucleus by inhibition of nuclear export is degraded very fast. The stability of iNOS mRNA transported to the cytoplasm seems not to be changed under these conditions.