Role of SerpinA3 in the Pathogenesis of Graves' Orbitopathy in Orbital Fibroblasts

Abstract

Purpose: We investigated the implications of SerpinA3, a secretory serine protease inhibitor, in inflammation and adipogenesis of Graves' orbitopathy (GO). To identify its precise function in GO pathogenesis, we evaluated the role of SerpinA3 in the inflammation and adipogenesis of GO.

Methods: SerpinA3 expression was compared between GO (n = 30) and normal participants (n = 28) in orbital tissue explants using real-time PCR. Orbital fibroblasts from GO (n = 3) and normal participants (n = 3) were transfected with or without small interfering RNA against SerpinA3 before IL-1β stimulation. Western blotting assessed inflammatory cytokine and signaling molecule expression. Adipogenic differentiation was assessed using Oil Red O staining, and adipogenic marker expression was determined through Western blotting. Enzyme-linked immunosorbent assay was used to compare prostaglandin E2 (PGE2) and hyaluronan levels in GO (n = 4) and normal participants (n = 3).

Results: SerpinA3 transcript levels were significantly higher in GO orbital tissues. Silencing SerpinA3 suppressed the IL-1β-induced expression of IL-6, IL-8, monocyte chemotactic protein 1, intercellular adhesion molecule 1, cyclooxygenase 2, and PGE2 and attenuated the levels of phosphorylated nuclear factor κB, Akt, extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. Moreover, silencing SerpinA3 reduced hyaluronan production, adipogenic differentiation, and adipogenic marker expression, including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding proteins α and β, adipocyte protein 2, adiponectin, and leptin.

Conclusions: Silencing SerpinA3 attenuated the expression of proinflammatory mediators, adipogenic differentiation, and hyaluronan production. Our results indicate that SerpinA3 plays a significant role in GO and may serve as a novel therapeutic target.

Figure 1.

mRNA transcript levels of SerpinA3 in orbital tissues from patients with GO and normal participants. SerpinA3 mRNA was extracted from GO (n = 30) and normal (n = 28) orbital tissues and measured using RT-qPCR. SerpinA3 mRNA expression was elevated in GO tissues compared to normal participants (*P < 0.05). GO, Graves’ orbitopathy; RT-qPCR, real-time quantitative PCR.

Figure 2.

Comparison of SerpinA3 protein expression in GO and normal orbital tissues. (A) IHC staining was performed on orbital tissues obtained from patients with GO (n = 2) and normal participants (n = 2). Representative staining results from GO and normal orbital tissue are shown. (B) Quantitative analysis showed that SerpinA3 expression was elevated in GO orbital tissues relative to normal tissues. Data are presented as mean ratio ± SD.

Figure 3.

Effect of silencing SerpinA3 on the expression of proinflammatory mediators. Orbital fibroblasts were obtained from patients with GO (n = 3) and normal participants (n = 3) and transfected with either control (si-con) or SerpinA3 siRNA (si-SerpinA3) (20 nM, 48 hours). The cells were then treated with 10 ng/mL IL-1β for 72 hours. (A) The protein levels of proinflammatory cytokines were evaluated by Western blot analysis. (B) The elevated protein levels of IL-6, IL-8, MCP-1, ICAM-1, and COX-2 in response to the IL-1β stimulation were significantly blunted in both GO and normal orbital fibroblasts by si-SerpinA3 transfection. Representative gel images are shown. The results are presented as mean density ratio ± SD, as assessed using densitometry, normalized to the level of the β-actin in the same sample (*P < 0.05).

Figure 4.

Effect of SerpinA3 knockdown on the production of PGE2 and hyaluronan. Orbital fibroblasts from patients with GO (n = 4) and normal participants (n = 3) were transfected with either control (si-con) or SerpinA3 siRNA (si-SerpinA3) (20 nM, 48 hours) and then challenged with or without 10 ng/mL IL-1β for 48 hours. The experiments were performed in duplicate for each sample. (A) PGE2 levels in orbital fibroblasts treated with si-con and si-SerpinA3 were measured with ELISA. When transfected with si-SerpinA3, increased PGE2 levels upon IL-1β stimulation were significantly reduced in both GO and normal cells compared to si-control. (B) Hyaluronan production from orbital fibroblasts treated with si-con and si-SerpinA3 were evaluated with ELISA. In both GO and normal cells, hyaluronan levels were elevated with IL-1β treatment and significantly mitigated by si-SerpinA3 transfection.

Figure 5.

Effect of silencing SerpinA3 on the activation of proinflammatory signaling molecules. Orbital fibroblasts were obtained from patients with GO (n = 3) and normal participants (n = 3) and transfected with either control (si-con) or SerpinA3 siRNA (si-SerpinA3) (20 nM, 24 hours) followed by stimulation with or without 10 ng/mL IL-1β 30 min. (A) The levels of phosphorylated and total NF-κB, Akt, ERK, p38, and JNK were examined with Western blot analyses. Representative gel images are shown. (B) Densitometric quantification revealed that IL-1β stimulation elevated the phosphorylation of NF-κB, Akt, ERK, p38, and JNK in both GO and normal orbital fibroblasts. The transfection of si-SerpinA3 significantly diminished IL-1β–stimulated expression of p-NF-κB, p-Akt, p-ERK, p-p38, and p-JNK in GO orbital fibroblasts. In normal fibroblasts, enhanced levels of p-NF-κB, p-ERK, p-p38, and p-JNK upon IL-1β were reduced significantly. Data in the columns indicate mean density ratio ± SD, normalized to the level of β-actin in the same sample (*P < 0.05).

Figure 6.

Suppressive effect of SerpinA3 silencing on the adipogenesis of orbital fibroblasts. GO (n = 3) orbital fibroblasts were incubated in adipogenic medium for 14 days after transfection with either control (si-con) or SerpinA3 siRNA (si-SerpinA3) (20 nM, 48 hours) followed with or without IL-1β treatment (10 ng/mL). (A) Oil Red O staining demonstrated intracytoplasmic lipid accumulation over the 14-day differentiation (×100 magnification). (B) The optical density was measured at 490 nm for quantification of solubilized Oil Red O staining. On day 10 and day 14, the adipogenesis in both IL-1β–treated and untreated cells was significantly mitigated in si-SerpinA3–transfected cells. Data are presented as mean optical density ratio ± SD (*P < 0.05).

Figure 7.

Suppressed production of adipogenic marker proteins by SerpinA3 knockdown. GO (n = 3) orbital fibroblasts were transfected with either 20 nM control (si-con) or SerpinA3 siRNA (si-SerpinA3) for 48 hours, challenged with or without 10 ng/mL of IL-1β, and then incubated in adipogenic medium for 14 days to induce differentiation. (A) Protein expression levels of PPAR-γ, C/EBP-α and C/EBP-β, adipocyte protein 2, adiponectin, and leptin were measured using Western blot analysis at multiple time points (0, 5, 10, and 14 days) during differentiation. Representative gel images are shown. (B) The IL-1β–stimulated expression of adipogenic markers was significantly diminished in si-SerpinA3–transfected cells, as compared to the cells transfected with si-control. The results are presented as mean density ratio ± SD, as assessed using densitometry and normalized to the level of the β-actin in the same sample (*P < 0.05).