| Fas ligand (FasL), TNF-α and NGF are ligands for the TNF receptor superfamily that induce apoptosis under certain conditions. Astrocytes are resistant to apoptosis induced by these ligands even though they express appropriate receptors for them. In order to determine alternative astrocyte responses, we examined the regulation of chemokine and cytokine expression in cultured mouse astrocytes.; Treatment with these cell death-inducing ligands induced the expression of chemokines MIP-2, IP-10 and MCP-1 via an MEK-dependent pathway. However, none of these ligands affected the expression of 20 selected cytokines. Electrophoretic mobility shift assays revealed the activation of NF-κB, AP-1, and HSF-1 after a 1-hour treatment with FasL or TNF-α. Pretreatment with sulfasalazine, an inhibitor of NF-κB activation, while adversely affecting cell survival, did not affect the expression of chemokines. This suggests a role for NF-κB in cell survival, but not chemokine induction. By contrast, heat shock, which activates HSF-1, induced chemokine production, and use of siRNA to knock down HSF-1 inhibited death-ligand-induced chemokine production. Taken together, these data suggest that HSF-1 is a major transcription factor responsible for chemokine induction. SN-50, an inhibitor of transcription factors containing a nuclear localization signal (NLS), was a strong chemokine inducer, indicating the existence of constitutive intercellular transcription inhibitor(s). Moreover, chemokines induced by TNF-α and FasL were completely inhibited following prolonged pre-treatment with IFN-γ. In order to investigate possible mediators of IFN-γ inhibition, we measured the DNA binding of NF-IL6 and GATA-3. NF-IL6 was chosen because it is constitutively activated in untreated astrocytes but declines in chemokine-inducing conditions. However, activation of NF-IL6 was decreased with a prolonged pre-treatment with IFN-γ suggesting that this transcription factor was not involved in mediating the IFN inhibition. GATA-3, on the other hand, was significantly increased following prolonged treatment with IFN-γ, suggesting its possible involvement in mediating the inhibition.; In order to investigate the upstream events mediating death-receptor induction of chemokines, we investigated possible roles for FLIP and PEA-15. No changes in FLIP mRNA or protein were detected following death-receptor ligation. Similarly, elimination of PEA-15 with siRNA did not affect chemokine expression. |
