METTL3 overexpression and ATF6 silencing-induced Inhibition in HAS2 expression relieves PDGF-BB stimulation-induced HA production and the proliferation of human orbital fibroblasts in graves' ophthalmopathy

Background: Graves' ophthalmopathy (GO), a common extrathyroidal manifestation of Graves' disease, is characterized by inflammation and tissue remodeling in the orbit. Hyaluronan synthase 2 (HAS2) plays a pivotal role in hyaluronan (HA) production, which is implicated in the pathophysiology of GO. Understanding the regulatory mechanisms of HAS2 could provide novel therapeutic targets for managing this debilitating condition.

Methods: The study treated human orbital fibroblasts with platelet-derived growth factor BB (PDGF-BB) to mimic an in vitro GO model. Methyltransferase like 3 (METTL3) inhibitor, STM2457, was used to analyze METTL3-induced effects on PDGF-BB-induced HA production and proliferation in human orbital fibroblasts. Quantitative Real-Time PCR (qRT-PCR) and western blotting assays were performed to assess mRNA and protein expressions, respectively. HA concentration was measured using a colorimetric assay. Cell viability was analyzed using a cell counting kit-8 (CCK-8) assay. Cell proliferation was evaluated using a 5-Ethynyl-2'-deoxyuridine (EdU) assay. The interactions between HAS2 and METTL3, YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), and activating transcription factor 6 (ATF6) were investigated using RNA immunoprecipitation assay, m6A methylated RNA immunoprecipitation assay, dual-luciferase reporter assay, and/or chromatin immunoprecipitation assay.

Results: HAS2 and ATF6 expression levels were upregulated in PDGF-BB-induced human orbital fibroblasts, whereas METTL3 expression was downregulated. Knockdown of HAS2 reduced PDGF-BB-induced HA production and proliferation in human orbital fibroblasts. METTL3 was found to destabilize HAS2 mRNA through m6A modification and promote its degradation in a YTHDF1-dependent manner. Treatment with STM2457 enhanced PDGF-BB-induced effects. In addition, METTL3 overexpression attenuated PDGF-BB-induced HA production and proliferation of human orbital fibroblasts by regulating HAS2. The results also showed that ATF6 transcriptionally activated HAS2, and HAS2 overexpression counteracted the effects of ATF6 knockdown on HA production and proliferation of PDGF-BB-stimulated human orbital fibroblasts.

Conclusion: METTL3 overexpression and ATF6 silencing led to an inhibition in HAS2 expression, which in turn mitigated PDGF-BB-induced hyaluronan production and the proliferation of human orbital fibroblasts in GO. These findings offer a new avenue for the development of targeted therapies.

HAS2; ATF6; Graves’ ophthalmopathy; METTL3; YTHDF1.