Reversal of Pathological Features of Graves' Orbitopathy by Activation of Forkhead Transcription Factors, FOXOs.
BACKGROUND
Graves' orbitopathy (GO) is a disfiguring/distressing, inflammatory autoimmune condition. This intractable problem is caused by expansion of the orbital contents around the eye by excessive fat generation (adipogenesis) and overproduction of extracellular matrix components, especially hyaluronan (HA) from preadipocytes/fibroblasts (PFs). Current immunosuppressive/antiinflammatory treatments are largely ineffective and have unpleasant side effects, and a better therapeutic strategy through understanding GO-associated pathological features is needed.


OBJECTIVE
Previously we identified depot-specific HA synthase 2 regulation (HAS2; major source of HA), which facilitates orbit-specific HA accumulation during adipogenesis, and targeting phosphatidylinositol-3-kinase/mechanistic target of rapamycin-complex-1 pathways blocked both pathological features. The current study revealed low expression levels of Forkhead box O (FOXOs; critical downstream effectors of phosphatidylinositol-3-kinase) in orbital PFs through adipogenesis compared with sc levels. We aimed to dissect the role of FOXOs in GO pathogenesis to identify nonimmunosuppressive targets for GO treatment.


METHODS
Human orbital and sc primary PFs were treated with small interfering RNA/chemical inhibitor (AS1842856) of FOXOs or FOXO enhancer trifluoperazine hydrochloride (TFP; Food and Drug Administration approved drug), in serum-free medium for 24 hours, or TFP treatment in adipogenic medium for 15 days.


METHODS
Quantitative PCR was used to measure HAS2 transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA.


RESULTS
Substantially increased or decreased HAS2/HA production was observed by inhibiting (small interfering RNA or chemical inhibitor) or enhancing (TFP) FOXO expression, respectively. TFP treatment is also sufficient to counteract thyrotropin receptor-activated HAS2/HA production and block adipogenesis in orbital PFs.


CONCLUSIONS
FOXOs play a crucial repressor role in the regulation of HAS2/HA production and adipogenesis in orbital PFs. Our data reveal for the first time that resetting GO-associated pathological features through drug-targeted activation of FOXOs could provide a feasible nonimmunosuppressive therapeutic strategy for GO.
School of Medicine (L.Z., F.R., A.R.T., C.M.D., M.L.), Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom; and Department of Endocrinology (Q.H.J.), First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032, Shaanxi, China.
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Zhang
School of Medicine (L.Z., F.R., A.R.T., C.M.D., M.L.), Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom; and Department of Endocrinology (Q.H.J.), First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032, Shaanxi, China.
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Ji
School of Medicine (L.Z., F.R., A.R.T., C.M.D., M.L.), Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom; and Department of Endocrinology (Q.H.J.), First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032, Shaanxi, China.
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Ruge
School of Medicine (L.Z., F.R., A.R.T., C.M.D., M.L.), Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom; and Department of Endocrinology (Q.H.J.), First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032, Shaanxi, China.
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Lane
School of Medicine (L.Z., F.R., A.R.T., C.M.D., M.L.), Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom; and Department of Endocrinology (Q.H.J.), First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032, Shaanxi, China.
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Morris
School of Medicine (L.Z., F.R., A.R.T., C.M.D., M.L.), Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom; and Department of Endocrinology (Q.H.J.), First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032, Shaanxi, China.
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Tee
School of Medicine (L.Z., F.R., A.R.T., C.M.D., M.L.), Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom; and Department of Endocrinology (Q.H.J.), First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032, Shaanxi, China.
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Dayan
School of Medicine (L.Z., F.R., A.R.T., C.M.D., M.L.), Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom; and Department of Endocrinology (Q.H.J.), First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032, Shaanxi, China.
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Ludgate
D050156:Adipogenesis / Q000187:drug effects / Q000235:genetics; D000328:Adult; D005260:Female; D051858:Forkhead Transcription Factors / Q000235:genetics*; D014453:Glucuronosyltransferase / Q000235:genetics; D049970:Graves Ophthalmopathy / Q000235:genetics* / Q000473:pathology*; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000378:metabolism; D066298:In Vitro Techniques; D008875:Middle Aged; D058539:Phosphatidylinositol 3-Kinase / Q000235:genetics; D015363:Quinolones / Q000627:therapeutic use; D034741:RNA, Small Interfering / Q000037:antagonists & inhibitors; D011989:Receptors, Thyrotropin / Q000187:drug effects; D050151:Subcutaneous Fat / Q000378:metabolism; D015533:Transcriptional Activation; D014268:Trifluoperazine / Q000494:pharmacology
C553926:5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid; D051858:Forkhead Transcription Factors; D015363:Quinolones; D034741:RNA, Small Interfering; D011989:Receptors, Thyrotropin; D014268:Trifluoperazine; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; C101754:HAS2 protein, human; D000076002:Hyaluronan Synthases; D058539:Phosphatidylinositol 3-Kinase