These results indicate that PAI 1 does not affect microglial activation following LPS stimulation. Plasminogen activator inhibitor type full read 1 promotes microglial migration through the low density lipoprotein receptor related protein 1 Janus kinase signal transducer and activator of transcription 1 pathway LRP1 has been previously implicated in the biological functions of PAI 1. LRP1 is a cell surface protein that has been shown to bind to a variety of ligands in cluding apolipoprotein E, lipoprotein lipase, uPA, tPA, and PAI 1. To determine the role of LRP1 in the PAI 1 mediated microglial cell migration, we used LRP1 siRNA and RAP protein to inhibit LPR1 pathway. RAP has been shown to bind LRP1 and block its interactions with all known ligands including PAI 1.

LRP1 gene silen cing using siRNA abolished the PAI 1 promoted BV 2 microglial cell migration as determined by the wound healing assay and the Boyden chamber assay. Knockdown Inhibitors,Modulators,Libraries of LRP1 expression was shown by RT PCR, dot blotting analysis, and western blotting analysis Inhibitors,Modulators,Libraries using an LRP1 specific anti body. The addition of RAP protein alone did not affect wound closure, but it completely blocked the migration enhancing effect of PAI 1 in the wound healing Inhibitors,Modulators,Libraries assay. RAP was also able to block the effect of PAI 1 in the Boyden chamber assay. These results show that PAI 1 stimulates microglial migration Inhibitors,Modulators,Libraries via LRP1. We next addressed intracellular signaling pathways associated with the PAI 1 activity. The JAK STAT path way has been previously implicated in cell migration, and a previous study has shown that PAI 1 stimulates STAT1 activation in rat smooth muscle cells.

Thus, we evaluated the role of JAK STAT1 pathway in the PAI 1 promoted microglial cell migration after LRP1 binding. PAI 1 alone induced STAT1 phosphorylation as determined by western blotting in BV 2 microglial cells. IFN was used for comparison purposes. Inhibitors,Modulators,Libraries LRP1 gene silencing diminished PAI 1 induced STAT1 phosphorylation. LRP siRNA did not re duce IFN induced STAT1 phosphorylation, indicat ing that LRP siRNA did not cause cell toxicity. Thus, LRP1 knockdown inhibited PAI 1 induced STAT1 expression and activation. These results indicate that PAI 1 promotes microglial migration through the JAK STAT1 pathway, and that LRP1 may reside in the upstream of the JAK STAT1 signaling pathway in microglia.

Indeed, the addition of AG490, a pharmacological inhibitor of JAK kinase, significantly attenuated the PAI 1 induced BV 2 microglial cell migration in the wound healing assay. These data indicate that PAI 1 enhances microglial cell migration via LRP1 and the JAK STAT1 pathway. Plasminogen activator inhibitor type 1 is overnight delivery an inducer of microglial migration in vivo To determine whether PAI 1 promotes microglial motil ity in vivo, microglial accumulation was investigated after intrastriatal injection of human PAI 1 protein.