To determine the role of LRP1 in the PAI 1 mediated microglial selleck bio 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 Inhibitors,Modulators,Libraries siRNA abolished the PAI 1 promoted BV 2 microglial cell migration as determined by the wound healing assay and the Boyden chamber assay. Knockdown of LRP1 expression was shown by RT PCR, dot blotting analysis, and western blotting analysis 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 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 via LRP1. We next addressed intracellular signaling pathways associated with the PAI 1 activity. The JAKSTAT path Inhibitors,Modulators,Libraries way has been previously implicated in cell migration, and a previous study has shown that PAI 1 stimulates STAT1 activation in rat smooth Inhibitors,Modulators,Libraries muscle cells. Thus, we evaluated the role of JAKSTAT1 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. 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 JAKSTAT1 pathway, and that LRP1 may reside in the upstream of the JAKSTAT1 signaling pathway in microglia. Indeed, the addition of Inhibitors,Modulators,Libraries 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 an inducer of microglial Inhibitors,Modulators,Libraries 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.

Oligomycin A Ve hicle, denatured wild type human PAI 1, wild type human PAI 1, or the R346A human PAI 1 protein mu tant were stereotaxically injected into the striatum of the mouse brain. Accumulation of microglia was immuno histochemically evaluated by counting Iba 1 positive cells around the injected area. At 48 hours after intras triatal injection of wild type human PAI 1 protein, there were large numbers of Iba 1 positive microglia accumu lated around the PAI 1 injection site. The R346A mutant protein, which is not capable of inhibiting PA, similarly induced microglial accumulation around the injection site.