Ceruloplasmin regulating fibrosis in orbital fibroblasts provides a novel therapeutic target for Graves' orbitopathy.

ABSTRACT

PURPOSE: In diagnosing the pathogenesis of Graves' orbitopathy (GO), there is a growing interest in fibrosis generated by orbital fibroblasts (OFs); nevertheless, the involvement of ceruloplasmin (CP) in OFs remains unknown. METHODS: Differentially expressed genes (DEGs) were identified through bioinformatic analysis. OFs were isolated from orbital tissue and identified with immunofluorescent staining. The levels of DEGs were validated in GO tissue samples and TGF-ß-challenged OFs, and CP was selected for the following laboratory investigations. CP overexpression or knockdown was achieved, and cell viability and fibrosis-associated proteins were investigated to assess the cell phenotype and function. Signaling pathways were subsequently investigated to explore the mechanism of CP function in OFs. RESULTS: CP and cathepsin C (CTSC) are two overlapped DEGs in GSE58331 and GSE105149. OFs were isolated and identified through fibrotic biomarkers. CP and CTSC were downregulated in GO tissue samples and TGF-ß-challenged OFs. CP overexpression or knockdown was achieved in OFs by transducing a CP overexpression vector or small interfering RNA against CP (si1-CP or si2-CP) and verified using a qRT-PCR. CP overexpression inhibited cell viability and reduced the levels of α-SMA, vimentin, fibronectin, and collagen I, whereas CP knockdown exerted opposite effects on OFs. CP overexpression inhibited the phosphorylation of Smad3, Erk1/2, p38, JNK, and AKT; conversely, CP knockdown exerted opposite effects on the phosphorylation of factors mentioned above. CONCLUSION: CP was downregulated in GO and suppressed the expression of fibrosis-associated proteins in both GO and normal OFs. CP might serve as a promising therapeutic agent in the treatment regimens for GO.