PGE2 Induces IL-6 in Orbital Fibroblasts through EP2 Receptors and Increased Gene Promoter Activity: Implications to Thyroid-Associated Ophthalmopathy

BACKGROUND: IL-6 plays an important role in the pathogenesis of Graves' disease and its orbital component, thyroid-associated ophthalmopathy (TAO). Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE(2), underlying the inflammatory phenotype of these cells. METHODOLOGY/PRINCIPAL FINDINGS: Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE(2) on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP(2) receptors and increased steady-state IL-6 mRNA levels. The pre-translational up-regulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE(2)-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE(2) provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP) preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE(2) promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts. CONCLUSION/SIGNIFICANCE: These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO

Thyrotropin regulates IL-6 expression in CD34+ fibrocytes: clear delineation of its cAMP-independent actions.

IL-6 plays diverse roles in normal and disease-associated immunity such as that associated with Graves' disease (GD). In that syndrome, the orbit undergoes remodeling during a process known as thyroid-associated ophthalmopathy (TAO). Recently, CD34(+) fibrocytes were found to infiltrate the orbit in TAO where they transition into CD34(+) orbital fibroblasts. Surprisingly, fibrocytes display high levels of functional thyrotropin receptor (TSHR), the central antigen in GD. We report here that TSH and the pathogenic anti-TSHR antibodies that drive hyperthyroidism in GD induce IL-6 expression in fibrocytes and orbital fibroblasts. Unlike TSHR signaling in thyroid epithelium, that occurring in fibrocytes is completely independent of adenylate cyclase activation and cAMP generation. Instead TSH activates PDK1 and both AKT/PKB and PKC pathways. Expression and use of PKCβII switches to that of PKCµ as fibrocytes transition to TAO orbital fibroblasts. This shift is imposed by CD34(-) orbital fibroblasts but reverts when CD34(+) fibroblasts are isolated. The up-regulation of IL-6 by TSH results from coordinately enhanced IL-6 gene promoter activity and increased IL-6 mRNA stability. TSH-dependent IL-6 expression requires activity at both CREB (-213 to -208 nt) and NF-κB (-78 to -62 nt) binding sites. These results provide novel insights into the molecular action of TSH and signaling downstream for TSHR in non-thyroid cells. Fibrocytes neither express adenylate cyclase nor generate cAMP and thus these findings are free from any influence of cAMP-related signaling. They identify potential therapeutic targets for TAO

G protein, adenylate cyclase, and cAMP generation in orbital fibroblasts and fibrocytes.

<p>(A) Confluent cultures were treated with bTSH (5 mIU/mL), PGE₂ (1 µM), forskolin (20 µM) or nothing (control) for 16 h. Cell layers were analyzed for cAMP content and protein determination. (B) Adenylate cyclase mRNA levels were determined in orbital fibroblasts and fibrocytes from healthy donors (n = 5) and those with GD (n = 5). (C) RNA from orbital fibroblasts, fibrocytes, and thyroid tissue was subjected to RT-PCR for Gq and Gs using the C_T method. Data are expressed as mean±SD of triplicate determinations from three different strains of each. (D) Cultures indicated were treated with nothing, bTSH, PKA inhibitors H89 (10 µM), KT5720 (10 µM) or Rp-cAMP (1 mM) alone or in the combinations indicated for 6 h. RNA was extracted and subjected to RT-PCR for IL-6. Signals were normalized to GAPDH. Data are expressed as the mean ± SD of fold-change in three independent determinations from a single experiment, representative of three experiments performed. In 3 separate experiments, H89 failed to inhibit TSH-dependent IL-6 expression (1.62±0.23-fold and 1.06±0.3-fold increase in fibroblasts and fibrocytes, respectively vs TSH alone).</p

Divergent PKCµ and PKCβII mRNA expression in pure CD34⁺ and CD34⁻ orbital fibroblast subsets.

<p>A parental strain of TAO orbital fibroblasts (containing mixed CD34⁺ and CD34⁻ cells) was sorted into pure CD34⁺ and CD34⁻ subsets by FACS as described in Experimental Procedures. These were then cultured for 48 h., RNA was isolated, andRT-PCR performed for (left panel) PKCµ and (right panel) PKCβII using the C_T method. Ct values were normalized to their respective GAPDH levels. Data are expressed as the mean ± SD of three independent replicates from a single experiment, representative of three performed. PKCµ mRNA was undetectable in CD34⁺ subsets in all 3 studies while PKCβII mRNA levels were 44±7-fold above their respective parental fibroblast cultures.</p

bTSH induces IL-6 in orbital fibroblasts and fibrocytes.

<p>(A) Confluent cultures were shifted to medium containing 1% FBS for 20 h and then treated without or with bTSH (5 mIU/mL) for 16 h. Media were collected and subjected to IL-6-specific ELISA. Cell layers were analyzed for protein content. Data are expressed as mean ± SD of three independent determinations (***, p<0.001). In a total of 3 experiments, IL-6 induction by bTSH was 18.1±5.1-fold in fibroblasts and 24.1±4.4-fold in fibrocytes. (B) Cultures were treated without or with bTSH for 6 h. Cellular RNA from three strains each of TAO orbital fibroblasts, healthy orbital fibroblasts, fibrocytes from healthy donors, and those with GD. Real-time RT-PCR was performed using the comparative critical threshold (C_T) method. Ct values were normalized to respective GAPDH levels. Data are expressed as the mean±SD of three independent determinations. (C) IL-6 levels were determined as in panel A following treatment without or with M22 (2 µg/ml) for 16 h. (***, p<0.001). The induction of IL-6 by M22 in 3 experiments was 22.4±9.2-fold in fibroblasts and 47.1±10.1-fold in fibrocytes. (D) IL-6 levels were determined in fibroblasts from two individuals harboring a loss of function TSHR mutation (TSH-resistant) and two unaffected family members (control), treated without or with bTSH for 16 h as in panel A. Data are expressed as mean ± SD fold-change of three independent determinations (**, p<0.01; ***, p<0.001 vs untreated controls). In 3 separate experiments, TSH induced IL-6 by 12.7±1.6-fold and 14.5±1.6-fold in cultures from the healthy donors and 6.4±0.5 and 8.2±0.6-fold respectively in those from the two affected individuals.</p

Role of PKC in the induction by TSH of IL-6.

<p>(A) Confluent orbital fibroblasts and fibrocytes were treated with TSH alone or in combination with GFX (10 µM) for 16 h. Media were subjected to an IL-6 ELISA. Data are expressed as mean ± SD of triplicate independent determinations. (##, P<0.01 vs untreated controls **, P<0.01 vs TSH alone). In 3 separate experiments, GFX inhibited TSH-provoked IL-6 expression by 64.2±5.1% and 65.9±4.8% in fibroblasts and fibrocytes, respectively. (B) Cultures were treated with nothing, bTSH, 8-Br-cAMP (1 mM), PMA (50 ng/mL) or the combination indicated for 16 h. Media were analyzed for IL-6 content (**, P<0.01 vs untreated controls). (C). Cellular protein from three strains of each cell type were subjected to Western blot analysis of PKCµ and PKCβII. (D) RNA was extracted from the cell types indicated and subjected toRT-PCR for PKCµ and PKCβII mRNA by the C_T method. Signals were normalized to GAPDH. Data are expressed as the mean ± SD of fold-change in three independent determinations from a single experiment, representative of three experiments performed. (E) Cultures were treated with nothing, bTSH (5 mIU/mL), or PMA (50 ng/mL) for 30 min, harvested, and proteins analyzed by Western blot for PKCµ, pPKCµ (Ser 916), PKCβII, and pPKCβII (Ser 660). Results are from a single experiment, representative of three performed. (F) Targeting siRNAs and their scrambled counterparts were transfected into sub- confluent monolayers as described in Experimental Procedures. After 48 h, they were treated with nothing or bTSH (5 mIU/mL) for 16 h. Media were collected and subjected to an IL-6 ELISA. Data are expressed as the mean ± SD of three independent determinations from a single experiment, representative of three performed. **, p<0.01 vs TSH-treated cultures transfected with control siRNA. In 3 separate experiments, PKCµ siRNA inhibited TSH-provoked IL-6 by 48.5±1% in fibroblasts and PKCβII siRNA inhibited TSH-induced IL-6 by 59± % in fibrocytes.</p

Effects of cycloheximide on the induction of IL-6 by TSH.

<p>Evidence that TSH delays IL-6 mRNA degradation. (A) Orbital fibroblasts and fibrocytes were treated with nothing or bTSH (5 mIU/mL) in the absence or presence of cycloheximide (10 µg/mL) for 6 h. Cellular RNA was isolated, and RT-PCR performed for IL-6. Ct values were normalized to GAPDH. Data are expressed as mean ± SD of three independent determinations. In 3 separate experiments, IL-6 production increased 31.8±2.8-fold in fibroblasts. <b>(</b>B<b>)</b> (left panel) Orbital fibroblasts and (right panel) fibrocytes were pretreated with bTSH (mIU/ml) for 12 h. Some culture wells were shifted to medium without TSH while the others were continued in its presence. All cultures received DRB (50 µM) at time “0” and were harvested at the times indicated along the abscissas. RT-PCR was performed and data graphed as a best fit line. Data are expressed as percent of transcript levels at time “0” ± SD of triplicate independent determinations, each from a separate experiment. (*, P<0.05 TSH-treated vs untreated cultures).</p

Involvement of PDK1 in the induction by TSH of IL-6.

<p>(A) Cultures were untreated (control) or bTSH (5 mIU/ml) was added to medium for 30 min. Cellular protein was subjected to Western blot analysis for PDK1 and pPDK1 and re-probed for β-actin. Results are representative of three separate experiments performed. Densitometric analysis; pPDK1, control fibroblasts, 16.25±2.85 AU; plus bTSH, 42.7±6.54 AU; control fibrocytes, 64.5±2.67 AU; plus TSH, 78.5±3.8 AU. In 3 separate experiments, TSH increased pPDK1 levels by 2.6±0.3-fold and 1.2±0.3-fold in fibroblasts and fibrocytes, respectively. (B) Sub- confluent cultures were transfected with either control siRNA or one targeting PDK1, followed by 48 h incubation. Cultures were treated with nothing or bTSH (5 mIU/mL) for 16 h, media collected and subjected to IL-6-specific ELISA and cell layers analyzed for protein content. Data are expressed as the mean ± SD of three independent determinations. Inset: Cell layers were subjected to Western blot analysis for PDK1 after transfection with control siRNA or that targeting PDK1. In 3 separate experiments, PDK1 siRNA reduced TSH-induced IL-6 levels by 73±4% in fibroblasts and 73±5% in fibrocytes. (C) Orbital fibroblasts, in this case from a patient with TAO, were transfected with PDK1siRNA while fibrocytes were treated with OSU-03012 (5 µM) for 6 h. Cultures were treated as indicated (bTSH, 5 mIU/mL) for 30 min. Cellular protein was subjected to Western blot analysis of PKCµ and pPKCµ in fibroblasts (left panel) and PKCβII and pPKCβII in fibrocytes (right panel). (D) Confluent cultures were pre-treated without or with OSU-03012 (5 µM) for 6 h, then treated with nothing (control) or bTSH (5 mIU/ml) for 30 min. Cellular proteins were subjected to Western blot analysis probing with AKT and pAKT antibodies. Inhibition of TSH-dependent pAKT by OSU-03012 in 3 separate experiments was 14.4±1.2% and 2.5±0.6% in fibroblasts and fibrocytes, respectively.</p

Involvement of NF-κB in the induction of IL-6 by bTSH.

<p>(A) Confluent orbital fibroblast and fibrocyte cultures were treated without or with bTSH for 60 min. Cytosolic and nuclear protein fractions were prepared as described in Experimental Procedures. Nuclear protein extract was probed with anti-NF-κB-p65 Abs by Western blot analysis. Densitometry: nuclear p65, control vs bTSH-treated fibroblasts, 11.72 AU vs 58.5 AU; fibrocytes, 10.2 AU vs 75.3 AU, respectively. Cytosolic extracts were subjected to Western blot analysis of IκBα, pIKK, and IKKβ. (B) Orbital fibroblasts, in this case from a patient with TAO, and fibrocytes were treated with nothing or bTSH (5 mIU/mL) in the absence or presence of PDTC (100 µM) for 16 h. Media were analyzed for IL-6 content by ELISA. Data are presented as the mean ± SD of 3 independent determinations. (##, P<0.01 vs untreated controls; **, P<0.01 vs TSH alone). (C) Sub-confluent orbital fibroblast and fibrocyte cultures were transfected with control siRNA or that targeting Rel A, incubated for 48 h. and then treated with nothing or bTSH (5 mIU/mL) for 16 h. Media were analyzed for IL-6. Inset: Western blot confirming knockdown of Rel A. Data are expressed as the mean ± SD of three independent determinations. ***, p<0.001 vs control siRNA. (D) Orbital fibroblasts and fibrocytes were treated with nothing or bTSH (5 mIU/mL) in the absence or presence of GFX (10 µM) or Akti (1 µM) for 30 min. Nuclear protein fractions were prepared as described in Experimental Procedures and probed with anti-NF-κB-p65 Abs by Western blotting. Densitometry: Fibroblasts bTSH vs bTSH plus GFX, 0.826±0.196 AU vs 0.485±0.06 AU, bTSH vs bTSH plus AKTi, 0.253±0.02 AU vs 0.0 AU; Fibrocytes, 0.575±0.072 AU vs 0.391±0.056 AU and 0.485±0.03 AU vs 0.043±0.012 AU respectively. In 3 separate experiments, GFX reduced TSH-dependent p65 levels by 42±7% and 32±8% in fibroblasts and fibrocytes, respectively. AKTi reduced these levels by 91±1% in fibrocytes.</p