The mammalian target of rapamycin contributes to synovial fibroblast pathogenicity in rheumatoid arthritis

Abstract

ObjectivesThe mammalian target of Rapamycin (mTOR) is a metabolic master regulator of both innate and adaptive immunity; however, its exact role in stromal cell biology is unknown. In this study we explored the role of the mTOR pathway on Rheumatoid Arthritis synovial fibroblast (RASF) metabolism and activation and determined if crosstalk with the Hippo-YAP pathway mediates their effects.MethodsPrimary RA synovial fibroblasts (RASF) were cultured with TNFα alone or in combination with the mTOR inhibitor Rapamycin or YAP inhibitor Verteporfin. Chemokine production, matrix metalloproteinase (MMP) production, and adhesion marker expression were quantified by real-time PCR, ELISA, and/or Flow Cytometry. Invasion assays were performed using Transwell invasion chambers, while wound repair assays were used to assess RASF migration. Cellular bioenergetics was assessed using the Seahorse XFe96 Analyzer. Key metabolic genes (GLUT-1, HK2, G6PD) were measured using real-time PCR. Reanalysis of RNA-Seq analysis was performed on RA (n = 151) and healthy control (HC) (n = 28) synovial tissue biopsies to detect differential gene and pathway expression. The expression of YAP was measured by Western Blot.ResultsTranscriptomic analysis of healthy donor and RA synovial tissue revealed dysregulated expression of several key components of the mTOR pathway in RA. Moreover, the expression of phospho-ribosomal protein S6 (pS6), the major downstream target of mTOR is specifically increased in RA synovial fibroblasts compared to healthy tissue. In the presence of TNFα, RASF display heightened phosphorylation of S6 and are responsive to mTOR inhibition via Rapamycin. Rapamycin effectively alters RASF cellular bioenergetics by inhibiting glycolysis and the expression of rate limiting glycolytic enzymes. Furthermore, we demonstrate a key role for mTOR signaling in uniquely mediating RASF migratory and invasive mechanisms, which are significantly abrogated in the presence of Rapamycin. Finally, we report a significant upregulation in several genes involved in the Hippo-YAP pathway in RA synovial tissue, which are predicted to converge with the mTOR pathway. We demonstrate crosstalk between the mTOR and YAP pathways in mediating RASF invasive mechanism whereby Rapamycin significantly abrogates YAP expression and YAP inhibition significantly inhibits RASF invasiveness.ConclusionmTOR drives pathogenic mechanisms in RASF an effect which is in part mediated via crosstalk with the Hippo-YAP pathway.

Recent developments in the synovial fibroblast pathobiology field in rheumatoid arthritis

“…The role of the crosstalk between mTOR and the Hippo-YAP pathways in mediating RASF invasive mechanism was demonstrated. Here, Rapamycin abrogated YAP expression, and YAP inhibition inhibited RASF invasiveness [27] showing that multiple pathways contribute to the invasion of RASF into cartilage. Another study showed that PU.1, a central regulator of the pro-fibrotic system, led to hyper-activation and an inflammatory status of not only RASF but also of macrophages, whereas FMS-like tyrosine kinase 3 (FLT3) showed opposite effects [28].…”

“…mTOR (mammalian target of Rapamycin), a metabolic regulator of innate and adaptive immunity, was evaluated regarding RASF activation and invasive properties [27]. RASF after TNF stimulation showed higher phosphorylation of S6 (a downstream target of mTOR) and were responsive to mTOR inhibition via Rapamycin.…”

The role of YAP/TAZ on joint and arthritis

“…74,80,160 Still, the mechanisms involved in RA-FLS phenotype alteration remain to be elucidated. Recent literature has reported increased expression of YAP/TAZ in synovium in both antigen-induced arthritis (AIA) mice and RA patients, 79,160,161 and enriched epigenetic modifications of the YAP/TAZ were found in RA-FLS compared to OA-FLS. 78,80 These results suggest that YAP/TAZ might function as an important pathogenic regulator in RA-FLS.…”

“…The typical features of RA are synovial inflammation and the concomitant destruction of adjacent cartilage and bone, and evidence is accumulating that FLSs play a pivotal role in this process 34,76,158,159 . FLSs are one of the primary pathological cell types within the RA joint, which acquire an aggressive phenotype and promote inflammation when RA progresses 74,80,160 . Still, the mechanisms involved in RA‐FLS phenotype alteration remain to be elucidated.…”

The Hippo-YAP Signaling Pathway in Osteoarthritis and Rheumatoid Arthritis

“…118 Researchers have also shown that rapamycin regulates genes in the Hippo-YAP pathway, which are predicted to converge with the mTOR pathway, in RA synovial tissue. 119 These activities ultimately alter rheumatoid arthritis synovial fibroblast metabolism and inhibit glycolysis and the expression of rate-limiting glycolytic enzymes in human synovial fibroblasts. 119 Bottini et al 72,120 reported that drugs aimed at decreasing the expression of YAP and nuclear YAP in FLS could inhibit the migration, invasion and inflammatory activity of FLS and ameliorate arthritis severity.…”