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.
Keywords: graves’ orbitopathy, orbital fibroblasts, serpinA3, inflammation, adipogenesis
Graves’ orbitopathy (GO) is an ocular autoimmune thyroid disease and the most common extrathyroidal manifestation of Graves’ disease.1 Its clinical symptoms include exophthalmos, eyelid swelling and retraction, proptosis, blurred or double vision, conjunctiva, and corneal ulceration.2 The primary pathogenesis of GO involves targeting thyroid-stimulating hormone receptor (TSH-R) in orbital fibroblasts, resulting in the intensive secretion of proinflammatory cytokines, proliferation and adipogenic differentiation of orbital fibroblasts, deposition of extracellular matrix components such as hyaluronan, and fibrosis.3–5 Additionally, insulin-like growth factor 1 receptor (IGF1-R) plays a significant role in GO pathogenesis by regulating hyaluronan synthesis and adipogenesis in orbital fibroblasts.2,6 A monoclonal antibody for human IGF1-R, teprotumumab, has been approved as a novel medication for GO, highlighting its potential for other biological therapies in addition to conventional glucocorticoid treatments.7,8
SerpinA3, or α-1-antichymotrypsin, is a secretory serine protease inhibitor with highly conserved secondary structures.9 It is primarily secreted by hepatocytes and inhibits proteases secreted from immune cells, such as leukocytes, neutrophils, and basophils.10 SerpinA3 is involved in cell proliferation, antiapoptotic response, wound healing, inflammation, and complement activation.9,11,12 Its role in inflammation has been widely studied, elucidating its regulatory function and potential as an inflammatory biomarker in various inflammatory diseases, such as inflammatory bowel disease, cerebral small vessel disease, and ischemic cardiomyopathy.13–15 Additionally, SerpinA3 promotes adipogenesis by activating the extracellular signal–regulated kinase (ERK) and Akt signaling pathways or by facilitating adipogenic transcription factors, such as peroxisome proliferator–activated gamma (PPAR-γ).16,17 However, the role of SerpinA3 in thyroid diseases remains largely unexplored. While the association between SerpinA3 and the inflammatory response has been reported in thyroid carcinoma, its precise role in autoimmune thyroid disease remains unclear.18 Moreover, no studies have investigated the correlation between SerpinA3 and GO, despite the significance of inflammatory responses and excessive hyaluronan production in GO.
Given the significance of SerpinA3 in inflammatory response and adipogenesis, this study investigates the role of SerpinA3 in GO pathogenesis. We examined SerpinA3 expression in the orbital tissues of patients with GO and normal participants and explored the effect of SerpinA3 inhibition on inflammation, hyaluronan secretion, and adipogenic differentiation.
Methods
Reagents
Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12), penicillin-streptomycin, phosphate-buffered saline (PBS), and trypsin/EDTA were purchased from Welgene (Daegu, Gyeongsangbuk-do, Korea). Fetal bovine serum (FBS) was obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Recombinant IL-1β was obtained from R&D Systems (Minneapolis, MN, USA), and SerpinA3 antibody was obtained from Novus (St. Louis, MO, USA). Antibodies for IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), cyclooxygenase 2 (COX-2), CCAAT/enhancer-binding protein (C/EBP)—α, phosphorylated (p)—Akt, total (t)—Akt, p-Jun N-terminal kinase (JNK), t-JNK, p-nuclear factor kappa B (NF-κB), total (t)—NF-κB, p-ERK, t-ERK, p-p38, and t-p38 were purchased from Cell Signaling (Danvers, MA, USA). PPAR-γ, C/EBP-β, adiponectin, leptin (Ob), adipocyte protein 2 (aP2), and actin antibodies were obtained from Santa Cruz Biotechnology (CA, USA). Fibronectin antibody was obtained from BD Biosciences (Franklin Lakes, NJ, USA). SerpinA3 small interfering RNA (siRNA) and control siRNA were purchased from Ambion (Carlsbad, CA, USA).
Tissue and Cell Preparation
GO orbital tissues were obtained from 30 patients with GO during the orbital decompression surgery. All patients were in a stable euthyroid state at the time of surgery (12 males and 18 females; age, 16–76 years). None of the patients had a history of radiation or steroid treatment 3 months before surgery. Normal orbital tissue was collected from 28 participants during upper and lower lid blepharoplasty without historical or clinical evidence of thyroid disease (13 males and 15 females; age, 13–83 years). None of the normal participants exhibited other inflammatory, autoimmune, or orbital symptoms. The study protocol was approved by the Institutional Review Board of the Severance Hospital of Yonsei University College of Medicine (4-2024-1208) and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all patients.
Orbital fibroblasts were collected from the orbital tissues and cultured as described previously.19 Tissue explants were minced and distributed in DMEM/F12 (1:1) supplemented with 20% FBS and 1% penicillin-streptomycin. After a monolayer of fibroblasts was formed, the cells were passaged by treatment with trypsin/EDTA and cultured in DMEM/F12 supplemented with 10% FBS and antibiotics. The strains were maintained in a humidified 5% CO2 at 37°C and stored in liquid nitrogen. Only cells between the third and sixth passages were used.
Silencing of SerpinA3
SerpinA3 siRNA (si-SerpinA3) and control siRNA (si-control) for the negative control were transfected into the cells using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. The cells were cultured in DMEM/F12 supplemented with 10% FBS and antibiotics for 24 or 48 hours.
Real-Time Quantitative PCR
Orbital tissues were homogenized using a tissue homogenizer (Precellys 24; Bertin Instruments, Montigeny-le-Bretonneux, France), and RNA was extracted using a Precellys Lysing Kit (Bertin Instruments) and TRIzol reagent (Invitrogen). RNA was quantified using NanoDrop (Thermo Fisher Scientific). From the extract, 1 µg mRNA was reverse-transcribed into cDNA (SensiFAST cDNA Synthesis Kit; Meridian Life Science, Memphis, TN, USA). Real-time PCR was performed using the SYBR Green PCR Master Mix (Takara Bio, Shiga, Japan) on a QuantStudio 3 Real-time PCR Thermocycler (Applied Biosystems, Carlsbad, CA, USA). The primers used for the experiments were as follows: SerpinA3, 5′-CCTGAACGACATACTTCTCCAGC-3′ (forward) and 5′-CATCAAGCACAGCCTTATGGACC-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-ATGGGGAAGGTGAAGGTCG-3′ (forward) and 5′-GGGGTCATTGATGGCAACAATA-3′ (reverse). All PCRs were performed in triplicate, and GAPDH expression levels were used for normalization. The results were obtained as relative fold-changes in the threshold cycle (Ct) value based on the control group, using the 2−ΔΔCt method.
Immunohistochemistry
Orbital tissues were fixed with 5% formalin, embedded in paraffin, and sectioned into 4-µm-thick slices. The slices were deparaffinized, rehydrated, and subjected to immunostaining using the Dako PT Link and Dako Autostainer 48S Link platform (Dako, Glostrup, Denmark). High-temperature antigen retrieval was achieved by heating the samples in FLEX Target Retrieval Solution Low pH Buffer (Dako k8004) for 20 minutes at 95°C. To inactivate endogenous peroxidase, the slices were immersed in 3% H2O2 for 10 minutes and incubated with a 1:1000 dilution of primary antibody against SerpinA3 for 1 hour at room temperature. Following incubation, the samples were washed with Tris-buffered saline and incubated with horseradish peroxidase (HRP)–labeled polymer for 20 minutes at room temperature, following the manufacturer's instructions. A color reaction was developed using 3,3′-diaminobenzidine tetrachloride chromogen solution for 5 minutes. All slides were counterstained with hematoxylin, and the stained images were taken using an Olympus BX60 microscope (Olympus, Melville, NY, USA). The ratio of stained to total area was quantified using ImageJ software (National Institutes of Health, Bethesda, MA, USA).
Western Blot
Orbital fibroblasts treated with varying study conditions were washed with Dulbecco's PBS solution (Welgene) and lysed with radioimmunoprecipitation assay buffer (Biosesang, Gyeonggi-do, South Korea) containing Halt protease inhibitor cocktail (Thermo Fisher Scientific). Equal amounts of protein (30 µg) were separated by 8% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA), and incubated with primary antibodies overnight at 4°C. The membranes were probed with HRP-conjugated secondary antibodies and visualized using chemiluminescent substrates (Thermo Fisher Scientific) and an image reader (LAS-4000 mini; Fuji Photo Film, Tokyo, Japan). Band intensities were measured using the ImageJ software and standardized to β-actin in the same sample.
Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA) was conducted to investigate the role of SerinA3 on the production of hyaluronan and prostaglandin E2 (PGE2) (R&D Systems). Orbital fibroblasts were cultured to confluence in 6-well plates and subsequently treated with si-SerpinA3 or si-control (20 nM) for the indicated time periods, followed by IL-1β (10 ng/mL) treatment. The cell culture medium was centrifuged, and the supernatant was collected. Hyaluronan and PGE2 concentrations were determined using the ELISA kits, according to the manufacturer's instructions. The optical density of each sample was measured at 450 nm, and the binding percentage was calculated. The experiments were performed in duplicate for each sample.
Adipogenesis and Oil Red O Staining
Cells were plated in a 6-well plate and allowed to reach confluence. To induce adipogenic differentiation, biotin (33 µM), pantothenic acid (17 µM), rosiglitazone (10 µM), insulin (1 µM), transferrin (10 µg/mL), dexamethasone (1 µM), triiodothyronine (0.2 µM), and 3-isobutyl-1-methylxanthine (IBMX; 0.1 mM) were added. Differentiation was continued for 14 days, with the culture medium replaced every 2 to 3 days. Dexamethasone and IBMX were removed from the medium from day 4 onward. To evaluate the effect of silencing SerpinA3 on adipogenic differentiation, the cells were transfected with si-SerpinA3 during differentiation, according to the manufacturer's instructions.
For Oil Red O staining, a working solution was prepared by diluting a stock solution of Oil Red O (Sigma-Aldrich, Burlington, MA, USA) with distilled water (6:4). Subsequently, the solution was filtered through a 0.2-µm filter. After the induction of differentiation, the cells were washed with warm PBS and fixed with 10% formalin at 4°C for 1 hour. The cells were washed with PBS and dried with 60% isopropanol for 5 minutes. Isopropanol was removed and the cells were stained with Oil Red O solution for 1 to 2 hours at room temperature and washed again with distilled water. The stained cells were examined and visualized under a microscope. Once the cells had completely dried, isopropanol was added to solubilize the cell-bound Oil Red O for quantification. Absorbance was measured at 490 nm using a spectrophotometer.
Statistical Analysis
At least three strains from three patients were used. The Mann–Whitney U test or Kruskal–Wallis test was used for nonparametric data comparisons, and the Kolmogorov–Smirnov test was used for abnormally distributed data. Statistical analyses were performed using SPSS for Windows version 20.0 (IBM, Armonk, NY, USA). Statistical significance was set at P < 0.05.
Results
SerpinA3 Expression Levels Were Increased in GO Orbital Tissues
SerpinA3 mRNA levels were measured in orbital tissues obtained from patients with GO (n = 30) and normal healthy participants (n = 28). The comparison between two groups exhibited a significant elevation of mean SerpinA3 mRNA levels in the GO orbital tissues compared to those in the normal orbital tissues (Fig. 1).
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.
The protein expression of SerpinA3 in orbital tissues from GO (n = 2) and normal participants (n = 2) was assessed through immunohistochemistry (IHC). The immunostaining results demonstrated enhanced expression of SerpinA3 in GO orbital tissues compared to that in normal healthy participants (Fig. 2A). Quantitative analysis of IHC results indicated that SerpinA3 protein expression was increased by 2.3- to 5.4-fold in GO orbital tissues compared to that in normal tissues (Fig. 2B).
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.
Silencing SerpinA3 Attenuates the IL-1β–Induced Expression of Proinflammatory Mediators
To determine the role of SerpinA3 in the inflammatory response, SerpinA3 was silenced by transfection of si-SerpinA3 transfection in GO (n = 3) and normal (n = 3) orbital fibroblasts. After 72 hours of IL-1β (10 ng/mL) stimulation, the expression of proinflammatory mediators was measured in si-SerpinA3–transfected GO and normal orbital fibroblasts. Western blotting revealed that the enhanced production of IL-6, IL-8, MCP-1, ICAM-1, and COX-2 in response to IL-1β stimulation was significantly suppressed by si-SerpinA3 transfection in both GO and normal orbital fibroblasts (Fig. 3).
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).
As another proinflammatory mediator, the protein secretion level of PGE2 was quantified with ELISA in orbital fibroblasts obtained from patients with GO (n = 4) and normal participants (n = 3). After 48 hours of IL-1β (10 ng/mL) stimulation, PGE2 concentration in GO and normal cells was remarkably elevated (P < 0.05; Fig. 4A). When transfected with si-SerpinA3, IL-1β–stimulated elevation of PGE2 was significantly suppressed in both GO and normal cells. In IL-1β–treated GO cells, the mean PGE2 level was 5109.9 pg/mL, with diminution to 693.5 pg/mL when transfected with si-SerpinA3 (P = 0.014). In normal cells, the mean PGE2 level reduced from 4862.2 ng/mL to 806.2 ng/mL upon SerpinA3 inhibition (P = 0.036). The mean reductions were 86.4% and 83.4% in the GO and normal cells, respectively. PGE2 levels of each orbital fibroblast are presented in Supplementary Table S1.
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.
Silencing SerpinA3 Inhibits Hyaluronan Synthesis
The effect of SerpinA3 inhibition on hyaluronan production was assessed in GO (n = 4) and normal (n = 3) orbital fibroblasts after 48 hours of IL-1β (10 ng/mL) stimulation. ELISA results revealed that IL-1β treatment significantly elevated hyaluronan production in GO and normal orbital fibroblasts, while si-SerpinA3 transfection notably attenuated IL-1β–stimulated hyaluronan synthesis (P < 0.05; Fig. 4B). Upon si-SerpinA3 treatment, the mean hyaluronan levels in IL-1β–treated GO and normal cells reduced from 1698.0 to 281.5 ng/mL and 1281.7 to 273.1 ng/mL, respectively (P = 0.014, 0.036). The mean reductions were 83.4% and 78.7% in the GO and normal cells, respectively. Individual hyaluronan levels are presented in Supplementary Table S1.
Silencing SerpinA3 Attenuates the Activation of Proinflammatory Signaling Molecules
To assess the signaling pathways involved in SerpinA3—induced proinflammatory responses, the phosphorylation of NF-κB, Akt, and mitogen-activated protein kinase (MAPK) family members, including ERK, p38, and JNK, was analyzed in GO and normal orbital fibroblasts transfected with si-SerpinA3 or si-control. According to Western blot analyses, the inhibition of SerpinA3 significantly suppressed the IL-1β–induced phosphorylation of NF-κB, ERK, p38, and JNK in both GO and normal orbital fibroblasts. The downregulation of Akt phosphorylation was observed only in GO orbital fibroblasts (Fig. 5).
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).
Silencing SerpinA3 Suppresses the Adipogenesis of Orbital Fibroblasts
The effect of SerpinA3 suppression on adipogenic differentiation of orbital fibroblasts was investigated. GO orbital fibroblasts were transfected with si-SerpinA3 or si-control for 48 hours and incubated for 14 days in adipogenic media with or without IL-1β stimulation. Oil Red O staining revealed that adipogenic differentiation was markedly enhanced on days 10 and 14 in si-control–transfected cells, whereas si-SerpinA3–transfected cells exhibited no significant enhancement in adipogenesis (Fig. 6). Additionally, silencing SerpinA3 reduced the levels of adipogenic transcriptional factors—PPAR-γ, C/EBP-α and -β, aP2, adiponectin, and leptin—throughout the differentiation process (Fig. 7).
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).
Discussion
In this study, we investigated the role of SerpinA3 in GO pathogenesis. SerpinA3 transcript and protein expression levels were upregulated in GO orbital tissues, suggesting that SerpinA3 is correlated with GO pathogenesis. Silencing SerpinA3 blunted the IL-1β–induced production of proinflammatory cytokines and signaling pathway molecules. Additionally, SerpinA3 inhibition resulted in a significant reduction in hyaluronan production. Moreover, increased adipogenesis and adipogenic marker expression were attenuated by siRNA transfection against SerpinA3. Our results demonstrate that SerpinA3 significantly correlates with GO pathogenesis, including inflammation, adipogenesis, and hyaluronan secretion.
SerpinA3, a member of the acute-phase protein family, is associated with various inflammatory diseases. Increased SerpinA3 levels are associated with the severity or prolongation of inflammatory responses in chronic inflammatory diseases, including ulcerative colitis, lupus nephritis, and cerebral small vessel disease.13,14,20,21 In models of acetaminophen—induced hepatic injury, SerpinA3 deficiency reduced hepatic inflammation by reducing the expression of M1 macrophage markers, including IL-1β, IL-6, and TNF-α.22 Additionally, IL-6 stimulation upregulated the expression levels of SerpinA3, along with ICAM-1 and MCP-1, in retinal endothelial cells.23 Consistent with previous results, our data demonstrated that SerpinA3 induces the production of proinflammatory cytokines, as silencing SerpinA3 resulted in the downregulation of IL-1β–induced expression of IL-6, IL-8, MCP-1, ICAM-1, and COX-2. Given that SerpinA3 transcript levels were increased in GO orbital tissue, these results suggest that SerpinA3 plays a crucial role in the inflammatory pathogenesis of GO.
Despite the significance of SerpinA3 in inflammatory regulation, the precise effect of SerpinA3 in inflammation remains unclear. Additionally, SerpinA3 may exhibit anti-inflammatory properties under certain inflammatory conditions. In dry eye disease, SerpinA3 alleviated disease severity by inhibiting TNF-α expression.24,25 Similarly, in diabetic retinopathy, SerpinA3 was reported to reduce TNF-α and ICAM-1 expression, indicating its protective effect.26 Given its dual roles in inflammation, SerpinA3 may act as either a pro- or anti-inflammatory mediator depending on the disease context, its cellular source, and subcellular distribution.27 To our knowledge, no study has focused on the correlation between SerpinA3 and GO pathogenesis. However, taking into account the results of this study, SerpinA3 appears to play a proinflammatory role in GO.
Recently, SerpinA3 has been identified as a primary factor in adipogenesis. Knockdown of SerpinA3 demonstrated a suppressive role in 3T3-L1 adipogenesis and the expression of PPAR-γ, C/EBP-α, and C/EBP-β.28 In an in vivo study using SerpinA3c–/– mice, the expression of adipogenic regulators, including PPAR-γ, C/EBP-β, vaspin, and leptin, was diminished.17 Consistent with these results, our study demonstrated that silencing SerpinA3 blunted the expression of PPAR-γ, C/EBP-α and -β, aP2, adiponectin, and leptin and attenuated adipogenesis, as assessed by Oil Red O staining. Specifically, SerpinA3 has been suggested to proliferate adipogenesis by inhibiting the role of serine proteases, which play a suppressive role in integrin/IGF-1–mediated signaling.28 IGF-1 and its receptor IGF-1R play crucial roles in GO pathogenesis through the interaction of IGF-1R with TSH-R in GO orbital fibroblasts.2,6 Notably, teprotumumab, a monoclonal antibody against IGF-1R, serves as a second-line treatment for GO.7 Taken together, the precise role of SerpinA3 in adipogenesis in GO orbital fibroblasts requires further analysis.
SerpinA3 in GO pathogenesis may be associated with the MAPK, NK-κB, and Akt signaling pathways. In temporal lobe epilepsy, SerpinA3 facilitated neuroinflammation by activating the NF-κB signaling pathway.29 Similarly, in glioma, SerpinA3 was associated with immune cell infiltration and the enrichment of MAPK, TNF, p53, phosphoinositide 3-kinase (PI3K)–Akt, and NF-κB signaling pathways.30 Notably, PI3K-Akt, NF-κB, and MAPK family members, including ERK1/2, p38, and JNK, have been indicated to participate in inflammation and adipogenic differentiation in GO.31–33 In this study, we revealed that silencing SerpinA3 attenuates the phosphorylation of ERK, p38, JNK, Akt, and NF-κB, implying that SerpinA3 acts as a primary mediator in GO pathogenesis.
Numerous studies have established a correlation between SerpinA3 and extracellular matrix deposition. In myocardial infarction, SerpinA3 exhibited an antifibrotic role, as SerpinA3 knockout resulted in the increase of postinfarction fibrosis.34 Similarly, SerpinA3 protein levels were reduced in the myoma tissues of uterine fibroids.35 In contrast, SerpinA3 treatment in a diabetic mouse model promoted the deposition of full-length intact fibronectin and collagen.36 Although the implication of SerpinA3 in fibrosis has been widely assessed, its effect on hyaluronan secretion remains unclear despite the significance of hyaluronan deposition in chronic inflammatory diseases, including GO. In this study, SerpinA3 inhibition significantly diminished hyaluronan production in orbital fibroblasts, indicating the role of SerpinA3 in hyaluronan deposition in GO.
This study has several limitations. First, as our findings are primarily based on in vitro experiments, further in vivo studies using animal models are necessary to validate the role of SerpinA3 in GO pathogenesis. Additionally, due to the in vitro study design, serum SerpinA3 levels were not assessed. Evaluating the correlation between serum SerpinA3 levels and clinical activity in patients with GO could provide further clinical relevance. Additionally, orbital tissues and fibroblasts from the control group were obtained from the eyelid, which may not fully match the biological characteristics of GO orbital tissues from the deep orbit.
In conclusion, our study demonstrated that SerpinA3 inhibition attenuates inflammation, adipogenesis, and hyaluronan secretion, which are the primary factors in GO pathogenesis. While the role of SerpinA3 in regulating inflammation remains debated owing to its dual functions, our data revealed that in orbital fibroblasts, SerpinA3 consistently facilitates proinflammatory cytokines and signaling pathways. The enhanced expression of SerpinA3 in GO orbital tissue indicated that SerpinA3 may serve as a potential biomarker or therapeutic target of inhibition. Further in vivo studies are required to establish the therapeutic potential of SerpinA3 inhibitors as a novel treatment strategy for GO.
Supplementary Material
Acknowledgments
Supported by a grant of the MD-PhD/Medical Scientist Training Program through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea and Lassen Therapeutics (2022-31-1289).
Disclosure: M.S. Kim, None; S.H. Choi, None; H.Y. Park, None; S.Y. Jang, None; J.S. Ko, None; J. Kim, None; J.S. Yoon, None
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