We used in situ zymography following exposure to TWEAK for 24 h. We found significantly increased gelatinolytic activity in TWEAK or TNF treated hMECD3 cells, compared with non treated cells. In some cells, gelatinolytic activity appeared to delineate cells, suggesting localization at the plasma membrane. Among proteinases BTB06584? that have gelatinolytic activity are the gelatinases MMP 2 and treatment with TWEAK and TNF. In fibroblast and skeletal muscle cells, TWEAK is reported to activate MAPKs and regulate the expression of MMP 9. To assess whether this is also the case in brain endothelial cells, cultures were incubated for 1 h before adding TWEAK in the presence GW5074 and U0126, which are respectively inhibitors for ERK12 and MEK2 involved in the MAPK signaling pathways.

Densitometric scanning of zymograms indicates that MAPK inhibitors efficiently suppressed the up regulation of MMP 9, whose expression remained at basal levels and had no effect on the expression or activity levels of MMP 2. These results suggest that in human brain endothelial cells, TWEAK induces MMP 9 up regulation via the MAPK signaling pathways. We Inhibitors,Modulators,Libraries next evaluated the effects of exogenous recombinant human MMP 9 on the permeability of the hCMECD3 monolayer. The addition of 250 ngml rhMMP 9 for 24h enhanced the permeability of the endothelial cell mono layer by 20%. Considering that TWEAK increases both Pe of the hCMECD3 monolayer and MMP 9 levels and that exogenously applied rhMMP 9 also increases Pe, we assessed whether TWEAK increased Pe could be modulated by MMP inhibitors.

TWEAK and the broadband MMP inhibitor RXPO3 were applied to the Inhibitors,Modulators,Libraries hCMECD3 monolayer for 24h and Pe was assessed. We find that inhibition of MMP activity neither prevents bar rier impairment nor leads to a significant barrier recovery. To investigate the cellular distribution of MMP 9 in non treated and TWEAK treated endothelial cells, we used an antibody for MMP 9 whose specificity had been previously validated on neuroblastoma N2a cells transfected with MMP 9 GFP constructs. In nontreated and TWEAK treated hCMECD3 cells, MMP 9 showed a punctuate, vesicular like pattern distributed throughout Inhibitors,Modulators,Libraries the cytoplasm. MMP 9 immunolabeling was clearly increased in the TWEAK and TNF treated cells. Detailed analysis of the labeled endothelial cells also indicates perinuclear Inhibitors,Modulators,Libraries accu mulation of MMP 9, presumably in the Golgi and trans Golgi network.

We show that MMP 9 is localized in part at the membrane Inhibitors,Modulators,Libraries of brain endothelial cells. In agreement with our previous findings in other cell types of the CNS, MMP 9 was also localized in the nucleus of brain endothelial cells. TWEAK down regulated expression of ZO 1, a major component of HCMEC tight junctions Because ZO 1 is exclusively located in tight junctions but also constitutes a substrate for MMP 9, we assessed expression of this protein in the hCMECD3 inhibitor EPZ-5676 cells under TWEAK exposure.