ERp57 is present in STAT3-DNA complexes

STAT3 has been found constitutively activated in M14 melanoma cell line, as previously found in other melanoma cells. Using EMSA, DNA affinity experiments, and chromatin immunoprecipitation, STAT3 was found in M14 to bind the alpha2-macroglobulin gene enhancer in association with the protein disulfide isomerase isoform ERp57. The two proteins have also been found to be associated when bound to the SIE sequence in HepG2 cells stimulated by IL-6. In both cases an anti-ERp57 antibody hinders the binding of STAT3 to its consensus sequence on DNA, indicating that ERp57 is a necessary component of the DNA-bound STAT3 complex. Considering the functional association of the two proteins, the overexpression of ERp57 observed in a variety of transformed cells might be relevant to the oncogenic properties of STAT3.     

Stat3 mediates interleukin-6 [correction of interelukin-6] inhibition of human endothelial nitric-oxide synthase expression

Chronic activation of the acute phase response (APR) is associated with atherosclerosis. Elevated levels of interleukin-6, the major inducer of the APR, are associated with an increased risk of cardiovascular events. One of the clinical hallmarks of atherogenesis is endothelial dysfunction, characterized by a decrease in endothelial production of nitric oxide (NO). We hypothesized that interleukin-6 (IL-6) decreases endothelial NO synthase (eNOS) expression. We now show that IL-6 treatment of human aortic endothelial cells (HAEC) decreases steady-state levels of human eNOS mRNA and protein. This decrease in eNOS expression is caused in part by IL-6 inhibition of transactivation of the human eNOS promoter. To explore the mechanism by which IL-6 affects eNOS expression, we examined activation of signal transducer and transactivator-3 (Stat3). The IL-6 receptor (IL-6R) is expressed in HAEC, and Stat3 is phosphorylated in response to IL-6 stimulation of the IL-6R. We identified four consensus sequences for Stat3 binding (SIE) in the eNOS promoter at positions -1520, -1024, -840, and -540. Transfection of eNOS promoter mutants revealed that the SIE at -1024 mediates Stat3 inhibition of eNOS promoter activity. Gel-shift analysis of nuclear extracts from HAEC treated with IL-6 confirms that Stat3 binds to a complex containing the SIE at -1024. RNA silencing of STAT3 blocks the inhibitory effect of IL-6 on eNOS expression. Our data show that IL-6 has direct effects upon endothelial cells, inhibiting eNOS expression in part through Stat3. Decreased levels of eNOS may be an important component of the pro-atherogenic effect of the APR.