IL6 and CNTF itself induce CNTF e pression, suggesting a potential role of STAT3, which is downstream of their gp130 receptor. selleck kinase inhibitor We set out to identify the CNTF repressing signaling pathway from neuronal ligand to astroglial transcription factor, and whether its pharmacological inhibition would increase functional CNTF using adult SVZ neurogenesis as an outcome measure. Results Glial CNTF is repressed through vB5 integrin To identify which integrins repress CNTF, we first tested various ECM ligands with known differential integrin binding partners in rat C6 astroglioma cells which e press CNTF. The advantage of the C6 cell is the purity, consistency and ease of the cultures compared to primary astrocytes.

Moreover, the low CNTF e pres sion by C6 cells makes them a good cell model to study changes in CNTF e pression whereas the very high levels in cultured primary astrocytes combined with the half life of 7 hours of the CNTF mRNA make it more difficult to detect modest changes under acute conditions. CNTF mRNA was decreased by 25% when cells were cul tured for 4 hours on laminin, fibronectin or vitronectin. CNTF e pression was not affected by fibrino gen, thrombospondin and collagen. We therefore e cluded their integrin binding partners from further study. We also e cluded leukocyte specific integrins from further consideration as well as 7, 8, B6 whose pres ence in astrocytes is currently unknown. Finally, we did not test B8 antibodies as mature astrocytes have down regulated vB8 integrin and we could not obtain a suitable function blocking antibody against rat.

Having narrowed down potential integrins that might affect CNTF e pression, function blocking antibodies were used against 6, v, B1 and B5 integrin subunits. Freshly plated C6 cells incubated for 4 hours with v and B5 Carfilzomib in tegrin antibodies had 28% and 38% more CNTF mRNA, respectively, compared to no antibody or purified isotype specific IgG. In contrast, 6 and B1 integrin antibodies did not significantly alter CNTF e pression. Interestingly, the only integrin with a B5 subunit is vB5, suggesting that it may be specifically involved in inhibiting CNTF e pression. Astroglial CNTF is repressed by neuronal Thy 1 The surface protein Thy 1 is enriched in neurons through out the CNS and binds vB5 integrin, but its role in the brain is unknown.

Primary cortical neurons were incubated with Thy 1 blocking or IgG control anti bodies prior to seeding onto primary astrocyte monolayers. Thy 1 antibody increased CNTF e pression by 40%. This suggests that neuronal Thy 1 is an inhibitor of astroglial CNTF e pression. We did not test antibodies against laminin because the integrin binding motif is unknown. GW786034 Vitronectin and fibronectin are not present in neurons. Glial Focal Adhesion Kinase represses CNTF mRNA and protein FAK is the best known kinase associated with integrin sig naling.