These data clearly show the strong potency of HAK compounds to modify IL 6 expression in vivo. Effect of HAK compounds on OSM mediated phosphorylation of signal transducer and activator of transcription 3 and extracellular signal regulated kinase 1 The activation of the OSM signaling cascade resulting in the stimulation useful handbook of IL 6 expression is known to involve intracellular phosphorylation events. Therefore, effects of HAK compounds on the OSM induced phos phorylation of signal transducer and activator of transcrip tion 3 and extracellular signal regulated kinase 1 were investigated. Since HAK Inhibitors,Modulators,Libraries compounds were shown to be bioactive Inhibitors,Modulators,Libraries 3 to 6 h post stimulation, U343 cells were incubated with OSM for 6 h. In contrast to non sti mulated control, OSM induced phosphorylation of Erk1 as well as STAT3 6 h post stimulation.

Interestingly, HAK compounds suppressed STAT3 phosphorylation at serine 727, but neither phosphorylation of pSTAT3Y705 nor pErk1 2T202 Y204. Compound HAK 8 showed a significantly lower effect on pSTAT3S727 phosphorylation. This Inhibitors,Modulators,Libraries observation correlates well with the missing effect of compound HAK 8 on IL 6 expression as shown in Table 1. HAK compound specific suppression of OSM induced pSTAT3S727 was confirmed by immunocytochemistry. U343 cells culti vated on cover slips were treated identically as described before. In figure Inhibitors,Modulators,Libraries 7A an example of HAK compound effi ciency to suppress nuclear pSTAT3S727 phosphorylation 6 h post OSM stimulation is shown. Nuclear localization was confirmed by DAPI staining. In fig ure 7C densitometric results of at least 3 independent experiments are summarized.

While compounds HAK1 7 significantly suppressed the OSM mediated phosphoryla tion of pSTAT3S727, compound HAK 8 did not. Results from western blot analyses and immunocytochemistry are strongly correlating with each other. Thus, it Inhibitors,Modulators,Libraries appears that the IL 6 reducing bioactivity of HAK compounds is most likely based on suppression of STAT3 phosphorylation at serine 727. STAT3 and NF B subunit p65 are forming a OSM dependent complex which is sensitive to HAK compounds Normally, STAT3 is activated by phosphorylation at tyro sine 705, which induces dimerization, nuclear transloca tion and DNA binding. In contrast to other transcription factors like NF B, Creb and c EBPb, STAT3 can not bind directly to the IL 6 promoter, because there are no STAT3 binding elements present.

It is known from lit erature that several forms of interactions neverless and cross talks between NF B and STAT3 exist. For instance, recent studies have shown a physical interaction between STAT3 and NF B. The importance of pSTAT3S727 for protein interactions are under discussion. However, there is no information so far on the role of pSTAT3S727 for the interaction with NF B. To characterize a possible OSM induced and pS727 depen dent complex formation between STAT3 and NF B, we performed co immunoprecipitation of p65 followed by western blotting for STAT3.