Role of Proprotein Convertase Subtilisin/Kexin Type 9 in the Pathogenesis of Graves' Orbitopathy in Orbital Fibroblasts

Abstract

Background: The proprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated in the pathogenesis of inflammatory diseases. We sought to investigate the role of PCSK9 in the pathogenesis of Graves' orbitopathy (GO) and whether it may be a legitimate target for treatment.

Methods: The PCSK9 was compared between GO (n=11) and normal subjects (n=7) in orbital tissue explants using quantitative real-time PCR, and in cultured interleukin-1β (IL-1β)-treated fibroblasts using western blot. Western blot was used to identify the effects of PCSK9 inhibition on IL-1β-induced pro-inflammatory cytokines production and signaling molecules expression as well as levels of adipogenic markers and oxidative stress-related proteins. Adipogenic differentiation was identified using Oil Red O staining. The plasma PCSK9 concentrations were compared between patients with GO (n=44) and healthy subjects (n=26) by ELISA.

Results: The PCSK9 transcript level was higher in GO tissues. The depletion of PCSK9 blunted IL-1β-induced expression of intercellular adhesion molecule 1 (ICAM-1), IL-6, IL-8, and cyclooxygenase-2 (COX-2) in GO and non-GO fibroblasts. The levels of activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphorylated forms of Akt and p38 were diminished when PCSK9 was suppressed in GO fibroblasts. Decreases in lipid droplets and attenuated levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein β (C/EBPβ), and leptin as well as hypoxia-inducible factor 1α (HIF-1α), manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1) were noted when PCSK9 was suppressed during adipocyte differentiation. The plasma PCSK9 level was significantly higher in GO patients and correlated with level of thyrotropin binding inhibitory immunoglobulin (TBII) and the clinical activity score (CAS).

Conclusions: PCSK9 plays a significant role in GO. The PCSK9 inhibition attenuated the pro-inflammatory cytokines production, oxidative stress, and fibroblast differentiation into adipocytes. PCSK9 may serve as a therapeutic target and biomarker for GO.

Keywords: Graves’ orbitopathy; PCSK9; adipogenesis; inflammation; oxidative stress; proprotein convertase subtilisin/kexin type 9; thyroid eye disease.

Figure 1

Expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), low density lipoprotein receptor (LDLR), and hypoxia-inducible factor 1a (HIF-1a) mRNAs in Graves’ orbitopathy (GO) and non-GO orbital tissues. The RNA extracted from GO (n=9) and non-GO (n=7) orbital tissues was reverse-transcribed by real-time PCR and quantified. Experiments were performed in triplicate for each donor. The results showed elevated transcript levels of PCSK9 (A), LDLR (B), and HIF-1a (C) in GO tissues than in non-GO tissues. Data in the column indicate the mean ± SD fold elevation relative to the control.

Figure 2

Western blot of proprotein convertase subtilisin/kexin type 9 (PCSK9) and low density lipoprotein receptor (LDLR) after interleukin-1β (IL-1β) treatment. Confluent orbital fibroblasts obtained from Graves’ orbitopathy (GO) (n=3) and non-GO subjects (n=3) were treated with 10 ng/ml of IL-1β for increasing lengths of time (0–24 h). Western blot analyses were performed to investigate the levels of PCSK9 and LDLR. The treatment with IL-1β increased the levels of PCSK9 and LDLR in both GO and non-GO tissues in a time-dependent manner. Representative gel images are shown. Data in the columns indicate the mean density ratio ± SD, normalized to the level of β-actin in the same sample (*p < 0.05 vs. 0 h in each group).

Figure 3

Effect of silencing proprotein convertase subtilisin/kexin type 9 (PCSK9) on the expression of pro-inflammatory cytokines protein. Confluent fibroblasts obtained from Graves’ orbitopathy (GO) patients (n=3) were treated with either control siRNA or PCSK9 siRNA (50 nM, 24 h). Then, they were challenged with 10 ng/ml of interleukin-1β (IL-1β) and compared to non-IL-1β-treated counterparts. Western blot analyses were conducted to compare the levels of pro-inflammatory cytokines, intercellular adhesion molecule 1 (ICAM-1), IL-6, IL-8, and cyclooxygenase-2 (COX-2). The same experiment was repeated with fibroblasts obtained from non-GO subjects (n=3). Representative gel images are shown. The mean density ratio ± SD from fibroblasts were normalized to the level of β-actin in the same sample (*p < 0.05 between sicon + IL-1β and siPCSK9 + IL-1β; sicon, control siRNA; siPCSK9, PCSK9 siRNA).

Figure 4

Effect of silencing proprotein convertase subtilisin/kexin type 9 (PCSK9) on the activation of signal molecules by interleukin-1β (IL-1β) treatment. Confluent orbital fibroblasts obtained from Graves’ orbitopathy (GO) patients (n=3) were treated with or without 10 ng/ml of IL-1β after transfection with control siRNA or PCSK9 siRNA (50 nM, 24 h). Treatment with IL-1β (10 ng/ml, 60 min) resulted in an increase in the levels of nuclear NF-κB p65 and phosphorylated forms of Akt and p38. The treatment with PCSK9 siRNA in GO cells significantly blunted the increases in the transcription factors. However, in fibroblasts from non-GO subjects (n=3), the PCSK9 inhibition only suppressed the phosphorylated Akt. Representative gel images are shown. Data in the columns indicate the mean density ratio ± SD of the bands obtained from the GO patients, normalized to the level of β-actin in the same sample (*p < 0.05 between sicon + IL-1β and siPCSK9 + IL-1β).

Figure 5

Proprotein convertase subtilisin/kexin type 9 (PCSK9) siRNA suppresses adipogenesis and oxidative stress in Graves’ orbitopathy (GO) fibroblasts. Orbital fibroblasts from GO (n=3) patients were cultured in adipogenic medium to induce differentiation into adipocytes for 7 days. (A) Oil red O staining showed treatment with PCSK9 siRNA (50 nM, 24 h) attenuated adipogenesis. (B) Quantification by measurements of optical density of cell lysates at 490nm echoed the histochemical results. The results are presented as the mean optical ratio ± SD (*p < 0.05 between sicon and siPCSK9). (C) Western blot analyses showed that throughout the 7-day period of adipogenesis, the cell lysates of fibroblasts collected at different time points showed a gradual increase in production of PCSK9, which was markedly decreased when PCSK9 siRNA (50 nM, 24 h) was treated. The levels of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein β (C/EBPβ), were substantially curtailed in fibroblasts transfected with PCSK9 siRNA. The levels of mature adipocyte marker, leptin, were also significantly reduced in the PCSK9 siRNA-treated group. The levels of antioxidants, manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1), and oxidative stress-related protein, hypoxia-inducible factor 1α (HIF-1α) were significantly decreased in the PCSK9 siRNA-transfected group. Data in the columns indicate the mean density ratio ± SD, normalized to the level of β-actin in the same sample, and representative gel images are shown (*p < 0.05 between sicon and siPCSK9 on days 0, 3, 5, and 7 of adipogenesis).

Figure 6

Comparison of plasma levels of proprotein convertase subtilisin/kexin type 9 (PCSK9) between Graves’ orbitopathy (GO) patients and non-GO subjects, and correlation analyses between plasma levels of PCSK9, thyrotropin-binding inhibitor immunoglobulin and clinical activity score (CAS). The plasma levels of PCSK9 were measured in GO patients and non-GO subjects using ELISA. The samples were assayed in triplicate. (A) The mean PCSK9 plasma level was significantly higher in the GO patients (n=44, 239.97 ± 48.20 ng/ml) than the healthy subjects (n=26, 190.83 ± 28.77 ng/ml; p < 0.01). A single dot represents the value obtained from a single donor. The results of Spearman’s rank correlation test between plasma levels of PCSK9 (GO patients, n=44) and (B) plasma levels of thyrotropin binding inhibitory immunoglobulin (TBII) (GO patients, n=44) or (C) clinical activity score (CAS) (GO patients, n=44) are shown. The plasma PCSK9 concentrations showed significant associations with the plasma TBII levels (r = 0.576, p < 0.001) and CAS (r = 0.631, p < 0.001).