CREB and NF-κB sites are critical to activation of the IL-6 gene promoter by bTSH.

<p>(A). Schematic demonstrating <i>cis</i>-acting regulatory elements for CREB (TGACGA, −213 to −208 nt) and NF-κB (GGGATTTTCCCA, −73 to −62 nt) relative to the transcription start site (+1). These are underlined and base substitutions resulting from site-directed mutagenesis appear above those in the corresponding wild-type sequences (emboldened). (B) Orbital fibroblasts and (C) fibrocytes were transiently transfected with empty luciferase vector or one containing the 1171 nt fragment spanning −1168 to +3 nt of the human IL-6 gene promoter, or that fragment harboring a 3 base mutation in the CREB binding site (designated “m1”), or a 4 base mutation in the NF-κB binding site (designated “m2”). Sub-confluent cultures were then treated with nothing (control) or bTSH (5 mIU/mL) for 1 h., cell layers harvested and luciferase reporter activity assessed in a luminometer. Data are expressed as the mean ± SD of triplicate determinations from a single experiment, representative of three performed. ***, P<0.001 versus TSH-treated cells transfected with wt promoter fragment. In 3 separate experiments, the m1 mutation resulted in an 86±4% and 79±4% reduction in TSH-dependent promoter activity in fibroblasts and fibrocytes respectively compared to wild type. The m2 mutation resulted in an 87±4% and 84±4% reduction, respectively.