This result strongly indicates the anti inflammatory potency of HAK compounds in vivo for possible treatment selleck chemicals Inhibitors,Modulators,Libraries of central nervous system diseases. To get more information about the underlying molecu lar mechanism of HAK bioactivity, the signal transduc tion pathways involved in OSM mediated IL 6 expression were dissected in more detail. Interestingly, LPS and OSM induced signal pathways are based on the same molecular mechanism such as STAT3 or NF B activation, indicating that HAK compounds may target a common cellular event. Two major signaling cas cades the JAK STAT as well as the MAPK pathways are switched on by binding of OSM to the receptor heterodi mers OSMR gp130 or LIFR gp130. Subsequent acti vation of signal tyrosine kinases of the JAK family leads to phosphorylation of pivotal signal molecules such as STAT3 and Erk1 and 2 respectively.

The essential role of receptor subunits as well as of downstream signal ing molecules as STAT3, Erk1 and p65 for OSM trig gered IL 6 expression in U343 cells was confirmed by siRNA based knock down experiments. Furthermore, Erk1 2 and STAT3 were phosphorylated 6 h post OSM treatment, which was identified as the criti Inhibitors,Modulators,Libraries cal time point for the HAK bioactivity. Inhibitors,Modulators,Libraries Immunoblotting and immunofluorescence experiments revealed that neither OSM induced pErk1 2T202 Y204 phosphorylation nor pSTAT3Y705 phosphorylation were modified by HAK compounds. However, HAK treatment led to a significant reduction of OSM stimulated pSTAT3S727 phosphoryla tion. Importantly, the HAK based inhibition profiles for IL 6 expression and pSTAT3S727 phosphorylation are strongly correlating with each other.

Thus, Inhibitors,Modulators,Libraries suppression of OSM induced phosphorylation of pSTAT3S727 is most likely the relevant molecular mechanism of the HAK compound bioactivity to suppress IL 6 expression. In contrast to pSTAT3Y705, which is essential for dimeriza tion, nuclear translocation and DNA binding, the physiological role of pSTAT3S727 is discussed controver sially. Depending on the specific promoter Inhibitors,Modulators,Libraries and or the cellular context pSTAT3S727 can influence tran scriptional activity of target genes. However, in the case of the IL 6 promoter, where acti vated NF B binds directly to DNA, no cis regulatory elements for STAT3 binding were identified so far. Based on these observations, we hypothesize that pSTAT3S727 may regulate IL 6 gene expression by an alternative pathway.

It is known that STAT3 is com plexed with transcription factors such as c Jun, c Fos, forkhead and endothelial cell derived zinc finger protein, respectively. Furthermore, it was shown that physical interaction of the STAT3 DNA binding domain with the NF B subunit p65 led to a reduced promoter activity of inducible nitric oxide synthase gene. Together, these findings strongly thereby suggest that physical interaction between STAT3 and p65 may result in a functional coupling important for the STAT3 dependent regulation of p65 responsive genes.