Role of the suppressor of cytokine signaling-3 in the pathogenesis of Graves' orbitopathy

Abstract

Objective: Graves' orbitopathy (GO) is characterized by increased production of proinflammatory cytokines and hyaluronic acid by fibroblasts and their differentiation into adipocytes in response to immunologic stimuli. The suppressor of cytokine signaling-3 (SOCS3) is an inducible negative regulator of the JAK/STAT pathway, implicated in various inflammatory diseases. In this study, we investigated the role of SOCS3 in the inflammatory and adipogenic pathogenesis of GO.

Methods: Transcriptome profiling of orbital tissues obtained from five patients with GO who underwent orbital decompression surgery and four healthy subjects was performed using RNA-sequencing. Among the top-ranked differentially expressed genes, we identified 24 hub genes and found SOCS3 to be the most significantly upregulated gene in GO samples compared with that in healthy tissue based on quantitative real-time polymerase chain reaction. SOCS3 expression was analyzed in IL-1β-, and IGF-1-stimulated orbital fibroblasts using quantitative real-time polymerase chain reaction and western blot analysis. Knockdown of SOCS3 using siRNA transfection was performed to assess the effect of SOCS3 on the production of proinflammatory cytokines and adipogenic phenotype.

Results: We identified 184 consistently differentially expressed genes-120 upregulated and 64 downregulated- in GO tissues compared to the control. SOCS3 mRNA expression was significantly higher in GO tissues (n = 17) compared with that in control (n = 15). IL-1β and IGF-1 enhanced the expression of SOCS3 at mRNA and protein levels. Silencing of SOCS3 suppressed the levels of IL-1β-induced proinflammatory cytokines, including IL-6, IL-8, and ICAM-1. Phosphorylation of NF-kB and Akt was suppressed and adipogenic differentiation was significantly attenuated by SOCS3 knockdown.

Conclusions: SOCS3 was remarkably expressed in the adipose tissues of patients with GO and was induced by IL-1β and IGF-1 in orbital fibroblasts. SOCS3 inhibition attenuated the production of proinflammatory cytokines and adipogenesis, suggesting that SOCS3 may be a therapeutic target for controlling the inflammatory and adipogenic mechanisms in GO.

Keywords: Graves’ orbitopathy; SOCS3; adipogenesis; inflammation; orbital fibroblast; suppressor of cytokine signaling 3.

Figure 1

RNA sequencing analysis of GO orbital tissues. Orbital tissues were obtained from patients with GO (n = 17) and healthy individuals (n = 15). RNA sequencing was performed to identify genes associated with GO. (A) A volcano plot showing differentially expressed genes in GO and normal samples. Significantly upregulated genes are marked in red, and the downregulated ones are marked in blue. (B) GSEA of gene sets upregulated in GO, including those related to TNFα, TGF-β, IL-2–STAT5, and the IL-6–JAK–STAT3 signaling pathways. DUSP1, dual specificity phosphatase 1; GO, Graves’ orbitopathy; GSEA, gene set enrichment analysis; IL-2–STAT5, interleukin 2–signal transducer and activator of transcription 5; IL-6–JAK–STAT3, interleukin 6–Janus kinase–STAT3; TGF-β, transforming growth factor-beta; TNFα, tumor necrosis factor-alpha.

Figure 2

Expression levels of SOCS3 mRNA and protein in GO. (A) Orbital tissues from patients with GO (n = 17) and healthy individuals (n = 15) were used to evaluate the mRNA levels of SOCS3 using quantitative real-time polymerase chain reaction. (B, C)SOCS3 mRNA levels were evaluated in GO (n = 2) and normal (n = 2) orbital fibroblasts after stimulation with IL-1β (10 ng/mL) and IGF-1(100 ng/mL) for 1 and 3 h. (D, E) Orbital fibroblasts from GO (n = 3) and normal (n = 3) samples were treated with IL-1β (10 ng/mL) and IGF-1 (100 ng/mL) for increasing durations (0–48 h). Western blot analysis was performed at different time points (0, 6, 24, and 48 h) to determine the SOCS3 levels. Experiments were conducted in duplicate. SOCS3 levels determined using densitometry were normalized to the b-actin levels in the same sample. Results are presented as the mean relative density ± SD for three individual samples and graphs are representative of three independent experiments (*p < 0.05 versus non-stimulated cells). GO, Graves’ orbitopathy; IGF-1, insulin-like growth factor 1; IL-1β, interleukin-1 beta; SOCS3, suppressor of cytokine signaling-3.

Figure 3

Effect of SOCS3 suppression on the expression of proinflammatory proteins in GO. Orbital fibroblasts obtained from patients with GO (n = 3) and healthy individuals (n = 3) were transfected with 20 nM si-SOCS3 or si-con for 48 h, followed by treatment with or without IL-1β (10 ng/mL). Western blot analysis was performed to compare the levels of proinflammatory cytokines, IL-6, IL-8, ICAM-1, and COX-2. Protein levels determined using densitometry were normalized to the β-actin levels in the same sample. Results are presented as the mean relative density ± SD for three individual samples and graphs are representative of three independent experiments (*p < 0.05 between si-con and si-SOCS3; si-con + IL-1c and si-SOCS3 + IL-1β). COX-2, cyclooxygenase-2; GO, Graves’ orbitopathy; IL, interleukin; ICAM-1, intercellular adhesion molecule 1; SOCS3, suppressor of cytokine signaling-3.

Figure 4

Effects of SOCS3 suppression on the activation of NF-κB and AKT signaling proteins following IL-1β treatment. Orbital fibroblasts derived from patients with GO (n = 3) and healthy individuals (n = 3) were transfected with 20 nM si-SOCS3 or si-con and cultured for 48 h, followed by IL-1β treatment (10 ng/mL) for 1 h, which resulted in an increase in the level of phosphorylated forms of NF-κB and AKT. Protein levels determined using densitometry were normalized to the β-actin levels in the same sample. Results are presented as the mean relative density ± SD for three individual samples and graphs are representative of three independent experiments (*p < 0.05 between si-con and si-SOCS3; si-con + IL-1β and si-SOCS3 + IL-1β). AKT, protein kinase B; GO, Graves’ orbitopathy; IL-1β, interleukine-1 beta; ICAM-1, intercellular adhesion molecule 1; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SOCS3, suppressor of cytokine signaling-3.

Figure 5

Effects of SOCS3 silencing on the activation of MAP kinase and STAT3 signaling molecules following IL-1β treatment. Orbital fibroblasts derived from patients with GO (n = 3) and healthy individuals (n = 3) were transfected with 20 nM si-SOCS3 or si-con and cultured for 48 h, followed by IL-1β treatment (10 ng/mL) for 1 h. Total and phosphorylated forms of ERK, p38, JNK, and STAT3 were assayed using western blotting. Protein levels determined using densitometry were normalized to the β-actin levels in the same sample. Results are presented as the mean relative density ± SD for three individual samples and graphs are representative of three independent experiments (*p < 0.05 between si-con and si-SOCS3; siCon + IL-1β and siSOCS3 + IL-1β). ERK, extracellular signal-regulated kinase; GO, Graves’ orbitopathy; IL-1β, interleukine-1 beta; ICAM-1, intercellular adhesion molecule 1; JNK, c-Jun N-terminal kinases; SOCS3, suppressor of cytokine signaling-3; Stat3, Signal transducer and activator of transcription 3.

Figure 6

Effect of SOCS3 inhibition on adipogenesis of GO. Orbital fibroblasts from patients with GO (n = 3) were incubated in adipogenic medium for 14 days with or without pretreatment with si-con or si-SOCS3 for 48 h. Orbital fibroblasts were stained with Oil Red O at day 14 of adipogenesis and were photographed at ×100 magnification. Cell-bound Oil Red O was solubilized using 100% isopropanol, and optical density was measured at 490 nm to quantify lipid accumulation at days 0, 5, 10, and 14. Data are represented as the mean relative fold compared to si-control cells. Western blot analysis of PPARγ, C/EBPα, C/EBPβ, ap2, adiponectin, and leptin was performed during 14 days of adipogenic differentiation of orbital fibroblasts. Representative bands are shown (*p < 0.05 between siCon and siSOCS3; siCon + IL-1β and siSOCS3 + IL-1β). GO, Graves’ orbitopathy; PPARγ; peroxisome proliferator-activated receptor gamma, C/EBPα, CCAAT/enhancer-binding protein alpha; C/EBPβ, CCAAT/enhancer-binding protein beta; ap2, Apetala 2; IL-1β, interleukine-1 beta; SOCS3, suppressor of cytokine signaling-3.