Multiple reasons for an inefficient STAT1 response upon IL-6-type cytokine stimulation

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and fertility. We found that IL-6-type cytokine stimulation of cell lines and primary human macrophages results in a different distribution of the DNA-binding competent STAT dimer species in the cytosol and nucleus as demonstrated by electrophoretic mobility shift assays. In the absence of detergent, STAT3/STAT3, STAT1/STAT3 were the predominant species in the cytoplasm while STAT3/STAT3 was predominant in the nucleus. However, in detergent containing total cellular lysates and nuclear fractions prepared with detergent containing buffers, the STAT1/STAT1 homodimer was as prominent or even more prominent than STAT3/STAT3 and STAT1/STAT3. We were interested in the cause of this discrepancy since STAT1-regulated genes have not been described to be expressed upon IL-6-type cytokine stimulation. In addition to the more transient STAT1 activation, IL-6-type cytokines such as IL-6 and OSM lead to a much less efficient STAT1 activation compared to the potent STAT1 activators IFNγ and IFNα. Studies with STAT1-deficient cells revealed that STAT1 activation does not seem to be an important competitive process to STAT3 activation arguing again for a very inefficient STAT1 activation upon IL-6-type cytokine stimulation. We also describe that pY-STAT3 is much more efficiently shuttled into the nucleus than pY-STAT1.     

Model-driven experimental analysis of the function of SHP-2 in IL-6-induced Jak/STAT signaling

Misregulated interleukin-6 (IL-6)-induced Jak/STAT signaling contributes to many diseases. Under non-pathological conditions Jak/STAT signaling is tightly regulated by a complex network of regulators. One of these regulators is the protein tyrosine phosphatase SH2-containing phosphatase 2 (SHP2). Although SHP2 is known to be a negative regulator of IL-6-induced Jak/STAT signaling, its exact molecular function is not entirely understood. To elucidate the function of SHP2 in IL-6 signaling we followed a systems biology approach, in which modeling, stepwise model refinement, and experimental analysis are closely linked. We come up with an identifiable model of early Jak/STAT signaling that describes the data and proves to be predictive. The model-based analysis implies that (1) the stepwise association of IL-6 with gp80 and gp130 and (2) STAT3 dimerization at the receptor are essential for the dynamics of early pathway activation, and (3) phosphorylation of SHP2 is nonlinear. Furthermore, the modeling results indicate that SHP2 does not act as a feedback inhibitor in an early phase of IL-6-induced Jak/STAT signaling. However, experimental data reveal that SHP2 exhibits a basal repressory function.     

Interactions of STAT3 with caveolin-1 and heat shock protein 90 in plasma membrane raft and cytosolic complexes. Preservation of cytokine signaling during fever

Interleukin-6 (IL-6) initiates STAT3 signaling in plasma membrane rafts with the subsequent transit of Tyr-phosphorylated STAT3 (PY-STAT3) through the cytoplasmic compartment to the nucleus in association with accessory proteins. We initially identified caveolin-1 (cav-1) as a candidate STAT3-associated accessory protein due to its co-localization with STAT3 and PY-STAT3 in flotation raft fractions, and heat shock protein 90 (HSP90) due to its inclusion in cytosolic STAT3-containing 200-400-kDa complexes. Subsequent immunomagnetic bead pullout assays showed that STAT3, PY-STAT3, cav-1, and HSP90 interacted in plasma membrane and cytoplasmic complexes derived from uninduced and stimulated Hep3B cells. This was a general property of STAT3 in that these interactions were also observed in alveolar epithelial type II-like cells, lung fibroblasts, and pulmonary arterial endothelial cells. Exposure of Hep3B cells to the raft disrupter methyl-beta-cyclodextrin for 1-10 min followed by IL-6 stimulation for 15 min preferentially inhibited the appearance of PY-STAT3 in the cav-1-enriched sedimentable cytoplasmic fraction, suggesting that these complexes may represent a trafficking intermediate immediately downstream from the raft. Because IL-6 is known to function in the body in the context of fever, the possibility that HSP90 may help preserve IL-6-induced STAT3 signaling at elevated temperature was investigated. Geldanamycin, an HSP90 inhibitor, markedly inhibited IL-6-stimulated STAT3 signaling in Hep3B hepatocytes cultured overnight at 39.5 degrees C as evaluated by DNA-shift assays, trafficking of PY-STAT3 to the nucleus, cross-precipitation of HSP90 by anti-STAT3 polyclonal antibody, and reporter/luciferase construct experiments. Taken together, the data show that IL-6/raft/STAT3 signaling is a chaperoned pathway that involves cav-1 and HSP90 as accessory proteins and suggest a mechanism for the preservation of this signaling during fever.