PGE₂ upregulates IL-6 mRNA and IL-6 gene promoter activity.

<p><b>A</b>: Divergent levels of IL-6 protein induced by PGE₂ in orbital and dermal fibroblasts are reflected in the abundance of IL-6 mRNA. Fibroblasts were treated with nothing or PGE₂ (1 µM) for 16 h. Ct values were normalized to GAPDH and expressed as fold-change. Data are expressed as the mean ± SD of triplicate independent determinations from one dermal and one orbital fibroblast cell. In 3 different strains of each, basal IL-6 mRNA levels were 3.2 fold greater in orbital fibroblasts. Following PGE₂ (1 µM) for 16 h, levels were 3.7-fold higher in orbital versus dermal fibroblasts. <b>B</b>: PGE₂ upregulated IL-6 gene promoter activity in orbital and dermal fibroblasts transiently transfected with empty luciferase vector or that construct fused to an 1171-nt fragment spanning −1168 to +3 nt of the human IL-6 gene promoter. Cultures were then treated with nothing (control) or PGE₂ (1 µM) for 1 h. Data are expressed as the mean ± SD of triplicate independent determinations. * denotes statistical difference between groups. In another study, 3 different orbital fibroblast strains demonstrated 2.6 fold greater IL-6 promoter activity compared to dermal fibroblasts following 1 h treatment with PGE₂ (1 µM).</p

Divergent CBP/pCREB complex formation and its importance to PGE₂-dependent IL-6 expression in orbital fibroblasts.

<p><b>A</b>: Pull-down studies demonstrating CBP/pCREB protein-protein interactions provoked by PGE₂. Representative Western blot demonstrates pCREB protein (arrow) in nuclear extracts from one dermal and one orbital fibroblasts (input), or following immunoprecipitation with either anti-CBP antibody (IP-CBP) or a control antibody (control). <b>B</b>: Western blot demonstrating CBP protein (arrow) in nuclear extract (input) or following immunoprecipitation with anti-pCREB antibody (IP-pCREB) or a control antibody (control). <b>C</b>: CBP knocked down by siRNA results in diminished nuclear pCREB recruitment. pCREB protein (arrow) in orbital fibroblast nuclear extracts (input) or following immunoprecipitation with anti-CBP antibody (IP-CBP) in fibroblasts transfected with CBP siRNA for 72 hr. and treated with nothing or PGE₂ for 20 min. Note the absence of detectable pCREB in the immunoprecipitate. <b>D</b>: CBP knockdown reduced PGE₂-induced IL-6 protein. Cultures were transfected with either CBP siRNA or control siRNA and treated with nothing or PGE₂ (1 µM) for 16 h. Media were collected and subjected to ELISA for IL-6. Data are expressed as the mean ± SD of three independent determinations. These results were confirmed in two other orbital fibroblast strains where IL-6 production was reduced 1.2 fold by CBP siRNA.</p

IL-6 production in orbital fibroblasts is induced by PGE₂ in a concentration- and time-dependent manner.

<p>Confluent orbital cultures, in this case from a patient with TAO, were treated <b>A</b>: with escalating concentrations of PGE₂ for 16 h. or <b>B</b>: with PGE₂ (1 µM) for graded intervals or . <b>C</b>: with or without PGE₂ (1 µM) for 16 h in three different dermal and orbital fibroblast strains, each from a different donor. Media were collected and subjected to ELISA analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015296#s4" target="_blank"><i>Materials and Methods</i></a>. Data are expressed as the mean ± SD of triplicate determinations. * denotes P<0.005 compared to PGE₂ treatment alone. <b>D</b>: Orbital and dermal cultures were treated with nothing or with PGE₂ (1 µM) for 16 h., and cell layer protein was collected and analyzed by Western blot for IL-6 protein. Membranes were then re-probed with anti-β actin antibody as a loading control. Band densities, corrected for their respective β-actin signals: Dermal control, 0.132, Dermal PGE₂, 0.146; Orbital control, 1.098; Orbital PGE₂, 5.132.</p

Knockdown of CREB with siRNA attenuates PGE₂-induced IL-6 protein expression in orbital fibroblasts.

<p><b>A</b>: siRNA specific to CREB (CREB si) or scrambled siRNA (Control si) was transfected into 80% confluent cultures. Representative Western blot analysis demonstrates the impact of the knockdown of CREB protein. <b>B</b>: Media were subjected to IL-6 ELISA. Data are expressed as the mean ± SD of three independent determinations These results were representative of those in two other orbital fibroblast strains.</p

PGE₂ provokes the phosphorylation of CREB in orbital fibroblasts, an effect blocked with H89.

<p><b>A</b>: Confluent cultures were treated with PGE₂ (1 µM) for different intervals. Cellular proteins were subjected to Western blot analysis of CREB and pCREB. Densitometric analysis of pCREB protein concentrations are expressed as the ratio to total CREB protein as a percent of the value at “0” min. Data are expressed as the mean ± SD of triplicate independent determinations. (* denotes statistical significance between treatment groups). <b>B</b>: H89 (10 µM) inhibits PGE₂-provoked CREB phosphorylation in orbital fibroblasts. The inhibitor was added for 6 hrs, followed by addition of PGE₂ (1 µM) for 15 min. Cellular proteins were subjected to Western blot analysis of pCREB protein using β-actin as a loading control.</p

PGE₂-induced IL-6 production can be attenuated by inhibitors of PKA.

<p>Confluent orbital fibroblasts, in this case from a patient with TAO, were treated with PGE₂ (1 µM) or Sp-cAMP (1 mM) in the absence or presence of H89 (10 µM). Media were collected and analyzed for IL-6 content after 16 hr incubations. Data are presented as the mean ± SD of triplicate independent determinations from a single orbital fibroblast strain. . This result was confirmed in two other orbital fibroblast strains where IL-6 production was reduced 5.6 fold by H89. * denotes P<0.005 compared to PGE₂ treatment alone; ** denotes P<0.005 compared to no treatment.</p

PGE₂ enhances DNA binding of pCREB preferentially in orbital fibroblasts.

<p>Eighty percent confluent cultures were serum starved for 20 h and then treated with PGE₂ (1 µM) for 16 h. Nuclear extracts (5 µg) were subjected to the TransAM ELISA for detecting pCREB/DNA complexes as determined at 450 nm. Data are expressed as the mean ± SD of triplicate independent determinations from a single orbital and dermal fibroblast strain. They were representative of results from 3 strains of each which demonstrated 2.1 fold greater pCREB binding in orbital versus dermal strains.</p

PGE₂ induces more robust cAMP generation in orbital compared to dermal fibroblasts resulting from higher levels of adenylate cyclase.

<p><b>A:</b> Confluent cultures were serum starved for 20 h and then treated with PGE₂ (1 µM) for 16 hrs. Cells were then lysed with 0.1 N HCl and cAMP was measured by cAMP immunoassay. Data are presented as the mean ± SD of three independent determinations. (* indicates statistical differences between groups). <b>B:</b> cAMP levels in three different dermal and orbital fibroblast strains treated with nothing or PGE₂ (1 µM) for 16 hrs. Data are presented as the mean ± SD of three independent determinations. The level of cAMP produced in orbital fibroblasts is 3 fold (p<0.05) greater than in dermal fibroblasts, <b>C:</b> Orbital fibroblasts express higher adenylate cyclase levels when compared to dermal fibroblasts. Data derived from Western blot analysis of three separate dermal and orbital fibroblast strains. These were normalized to their respective β-actin levels. They are expressed as the mean ± SD, p<0.005, n = 3.</p

TSH-induced TNFα mRNA expression involves Akt and NF-κB.

<p>AKTi (A) or MG132 (B) was added 1 hour before TSH stimulation of healthy cultured fibrocytes. RNA was isolated after 6 hours of induction. AKTi treatment leads to significant reduction in TSH-induced TNFα mRNA expression (from 23-fold to 1.3-fold expression, <i>p</i> <0.0001) (A). Similarly, MG132 treatment inhibits TSH-induced TNFα mRNA expression (from 93-fold to 16-fold expression; p< 0.001) (B).</p

TSH-induced TNFα protein production involves Akt and NF-κB.

<p>FACS analysis shows that pretreatment with either AKTi (AKT inhibitor) or MG132 (NF-κB inhibitor) reduces TSH-induced TNFα protein production in healthy circulating fibrocytes (MFI 2.92 reduced to 1.46 with addition of AKTi and 1.33 with MG132) (A). Luminex analysis shows that pretreatment with either AKTi or MG132 reduces TSH-induced TNFα production in healthy cultured fibrocytes. TSH-induced TNFα production of 1312 pg/ml is reduced by 52% to 612 pg/ml with AKTi and by 81% to 251 pg/ml with MG132 (p <0.0001) (B).</p