Protein tyrosine phosphatase 1B as a therapeutic target for Graves’ orbitopathy in an in vitro model

Abstract

Graves’ orbitopathy (GO) is characterised in early stages by orbital fibroblast inflammation, which can be aggravated by oxidative stress and often leads to fibrosis. Protein tyrosine protein 1B (PTP1B) is a regulator of inflammation and a therapeutic target in diabetes. We investigated the role of PTP1B in the GO mechanism using orbital fibroblasts from GO and healthy non-GO subjects. After 24 hours of transfection with PTPN1 siRNA, the fibroblasts were exposed to interleukin (IL)-1β, cigarette smoke extract (CSE), H 2 O 2 , and transforming growth factor (TGF)-β stimulations. Inflammatory cytokines and fibrosis-related proteins were analysed using western blotting and/or enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) release was detected using an oxidant-sensitive fluorescent probe. IL-1β, tumor necrosis factor (TNF)-α, bovine thyroid stimulating hormone (bTSH), high-affinity human stimulatory monoclonal antibody of TSH receptor (M22), and insulin-like growth factor-1 (IGF-1) significantly increased PTP1B protein production in GO and non-GO fibroblasts. PTPN1 silencing significantly blocked IL-1β-induced inflammatory cytokine production, CSE- and H 2 O 2 -induced ROS synthesis, and TGF-β-induced expression of collagen Iα, α-smooth muscle actin (SMA), and fibronectin in GO fibroblasts. Silencing PTPN1 also decreased phosphorylation levels of Akt, p38, and c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER)-stress response proteins in GO cells. PTP1B may be a potential therapeutic target of anti-inflammatory, anti-oxidant and anti-fibrotic treatment of GO.

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

“…Our study with siRNA transfection is limited by the lack of mRNA data, which would have provided additional insights to its effect at transcriptional or translational level. However, like other studies that have employed similar methods ( 53 , 54 ), we believe that we have shown a concrete evidence with western blot and ELISA that PCSK9, regardless of the mechanism with which its level is modified, affects inflammation and adipogenesis in GO. In line with our study, Kumar et al.…”

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“…Our study with siRNA transfection is limited by the lack of mRNA data, which would have provided additional insights to its effect at transcriptional or translational level. However, like other studies that have employed similar methods (53,54), we believe that we have shown a concrete evidence with western blot and ELISA that PCSK9, regardless of the mechanism with which its level is modified, affects inflammation and adipogenesis in GO. In line with our study, Kumar et al have previously reported that an autoantibody against TSH receptor stimulated the phosphoinositide 3-kinase (PI3K)/Akt pathway and induced adipogenesis of orbital preadipocytes in GO (55).…”

Cytokines as Targets of Novel Therapies for Graves’ Ophthalmopathy

“…PTP1B inhibition suppressed IL-1binduced Akt, and JNK phosphorylation, but p38 MAPK phosphorylation was reduced only in GO OF. As the p38 and JNK pathways, and the MAPK pathway, can mediate the transcription and translation of inflammatory cytokines, this study demonstrated that PTP1B mediates inflammatory reactions in GO OF (76).…”

“…Systemic hyperthyroidism and T cell infiltration into orbital tissue leads to ROS production, which can aggravate GO severity by increasing T cell proliferation, adipogenesis, and GAG production in OF (75). Recently, the role of the protein tyrosine phosphatase 1B (PTP1B), encoded by the PTPN1 gene, has been characterized in GO (76). PTP1B is known to be involved in immune cell signaling by regulating cytokines via dephosphorylation of janus kinase (JAK)2, signal transducer and activator of transcription (STAT)5, and tyrosine kinase (TYK)2.…”