Endocrinol Metab.  2023 Dec;38(6):739-749. 10.3803/EnM.2023.1780.

Phospholipase C-γ as a Potential Therapeutic Target for Graves’ Orbitopathy

• Roh TH
• Chae MK
• Ko JS
• Kikkawa DO
• Jang SY
• Yoon JS

• KMID: 2549267
• DOI: http://doi.org/10.3803/EnM.2023.1780

Abstract

Background
Phospholipase C-γ (PLC-γ) plays a crucial role in immune responses and is related to the pathogenesis of various inflammatory disorders. In this study, we investigated the role of PLC-γ and the therapeutic effect of the PLC-specific inhibitor U73122 using orbital fibroblasts from patients with Graves’ orbitopathy (GO).
Methods
The expression of phospholipase C gamma 1 (PLCG1) and phospholipase C gamma 2 (PLCG2) was evaluated using polymerase chain reaction in GO and normal orbital tissues/fibroblasts. The primary cultures of orbital fibroblasts were treated with non-toxic concentrations of U73122 with or without interleukin (IL)-1β to determine its therapeutic efficacy. The proinflammatory cytokine levels and activation of downstream signaling molecules were determined using Western blotting.
Results
PLCG1 and PLCG2 mRNA expression was significantly higher in GO orbital tissues than in controls (P<0.05). PLCG1 and PLCG2 mRNA expression was significantly increased (P<0.05) in IL-1β, tumor necrosis factor-α, and a cluster of differentiation 40 ligand-stimulated GO fibroblasts. U73122 significantly inhibited the IL-1β-induced expression of proinflammatory molecules, including IL-6, IL-8, monocyte chemoattractant protein-1, cyclooxygenase-2, and intercellular adhesion molecule-1 (ICAM-1), and phosphorylated protein kinase B (p-Akt) and p38 (p-p38) kinase in GO fibroblasts, whereas it inhibited IL-6, IL-8, and ICAM-1, and p-Akt and c-Jun N-terminal kinase (p-JNK) in normal fibroblasts (P<0.05).
Conclusion
PLC-γ-inhibiting U73122 suppressed the production of proinflammatory cytokines and the phosphorylation of Akt and p38 kinase in GO fibroblasts. This study indicates the implications of PLC-γ in GO pathogenesis and its potential as a therapeutic target for GO.

Keyword

Phospholipase C gamma; Graves ophthalmopathy; U73122; Proinflammatory cytokines; Inflammation; Orbital fibroblasts

Figure

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  Fig. 1. The mRNA expression of (A) phospholipase C gamma 1 (PLCG1) and (B) phospholipase C gamma 2 (PLCG2) was higher in Graves’ orbitopathy (GO) orbital tissues than in normal tissues. The transcript levels of PLCG1 and PLCG2 were evaluated through realtime polymerase chain reaction using orbital tissues obtained from subjects with GO (n=15) and healthy controls (n=14). Each dot corresponds to data obtained from one subject. Results are presented as mean±standard deviation. aP<0.05.

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  Fig. 2. External stimulation upregulated phospholipase C gamma 1 (PLCG1) and phospholipase C gamma 2 (PLCG2) mRNA expression in orbital fibroblasts obtained from patients with Graves’ orbitopathy (n=3). PLCG1 and PLCG2 transcript levels were measured through realtime polymerase chain reaction for increasing length of time (0 to 24 hours). (A) Orbital fibroblasts were treated with interleukin (IL)-1β (10 ng/mL). PLCG1 and PLCG2 mRNA expression peaked at 3 and 1 hour of treatment, respectively. (B) Orbital fibroblasts were treated with tumor necrosis factor (TNF)-α (10 ng/mL). PLCG1 and PLCG2 mRNA expression peaked at 16 and 6 hours of treatment, respectively. (C) After stimulation of cluster of differentiation 40 ligand (CD40L) (1 μg/mL), PLCG1 and PLCG2 mRNA expression gradually increased and was highest at 24 hours of treatment. Results are presented as mean±standard deviation. aP<0.05; bP<0.01, compared with the control group.

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  Fig. 3. U73122 inhibits interleukin (IL)-1β-induced expression of proinflammatory molecules in orbital fibroblasts. Fibroblasts obtained from patients with Graves’ orbitopathy (GO) (n=3) and healthy controls (n=3) were treated with 1 μM U73122 with or without IL-1β (10 ng/mL) for 48 hours. (A) Western blot analyses were performed to analyze the expression of proinflammatory molecules IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), cyclooxygenase-2 (COX-2), and intercellular adhesion molecule-1 (ICAM-1). (B) U73122 treatment significantly decreased the expression of all the IL-1β-induced proinflammatory molecules in GO fibroblasts. (C) In normal orbital fibroblasts, IL-6, IL-8, and ICAM-1 expression induced by IL-1β were significantly decreased. Results are presented as the mean relative expression ratio±standard deviation and are normalized to the level of β-actin in the same sample. aP<0.05.

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  Fig. 4. U73122 inhibits interleukin (IL)-1β-induced expression of downstream signaling molecules in orbital fibroblasts. Fibroblasts obtained from patients with Graves’ orbitopathy (GO) (n=3) and healthy controls (n=3) were treated with 1 μM U73122 with or without IL- 1β (10 ng/mL) for 1 hour. (A) Western blot analyses were performed to investigate phosphorylated protein kinase B (p-Akt), phosphorylated extracellular signal-regulated kinase (p-ERK), p-p38, and phosphorylated c-Jun N-terminal kinase (p-JNK) protein expression. (B) U73122 treatment significantly decreased IL-1β-induced expression of p-Akt and p-p38 in GO fibroblasts, whereas (C) IL-1β-induced expression of p-Akt and p-JNK was significantly decreased in normal orbital fibroblasts. Results are presented as the mean relative expression ratio of each protein (phosphorylated form/total form)±standard deviation, normalized to the level of β-actin in the same sample. MAPK, mitogenactivated protein kinase. aP<0.05.

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  Fig. 5. Schematic diagram of the relationships among U73122, phospholipase C (PLC)-γ, and downstream signaling pathways of insulin-like growth factor-1 receptor (IGF-1R), thyrotropin receptor (TSHR), and interleukin-1 receptor (IL-1R) in orbital fibroblasts from patients with Graves’ orbitopathy. Ab, autoantibody; TNF-α, tumor necrosis factor-α; CD40L, cluster of differentiation 40 ligand; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; TF, transcription factors; HA, hyaluronan.

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