Possible targets for nonimmunosuppressive therapy of Graves' orbitopathy.
BACKGROUND
Graves' orbitopathy (GO) is caused by expansion of the orbital contents by excess adipogenesis and overproduction of hyaluronan (HA). Immunosuppressive and antiinflammatory treatments of GO are not always effective and can have side effects, whereas targeting GO-associated tissue remodeling might be a more logical therapeutic strategy. Previously we reported that signaling cascades through IGF1 receptor and thyrotropin receptor within orbital preadipocytes/fibroblasts drove adipogenesis and HA production. Our current study combined the stimulation of IGF1 receptor and thyrotropin receptor increase of HA accumulation, which we hypothesize is by activation of phosphatidylinositol 3-kinase (PI3K)-1A/PI3K1B, respectively. The central aim of this study was to investigate whether PI3K/mammalian target of rapamycin complex 1 (mTORC1) inhibitors affected adipogenesis and/or HA production within orbital preadipocyte/fibroblasts.


METHODS
Human orbital preadipocytes were treated with/without inhibitors, LY294002 (PI3K1A/mTORC1), AS-605240 (PI3K1B), or PI103 (PI3K1A/mTORC1) in serum-free medium for 24 hours or cultured in adipogenic medium for 15 days. Quantitative PCR was used to measure hyaluronan synthases (HAS2) transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA.


RESULTS
Unlike AS-605240, both LY294002 (10 μM) and PI-103 (5 μM) significantly decreased HAS2 transcripts/HA accumulation and adipogenesis. Because PI-103 and LY294002 are dual PI3K/mTOR inhibitors, we investigated the inhibition of mTORC1 (rapamycin 100 nM), which significantly decreased adipogenesis but had no effect on HAS2 transcripts/HA, implicating PI3K-1A in the latter.


CONCLUSIONS
The combined inhibition of PI3K1A and mTORC1 signaling in vitro decreased both HA accumulation and adipogenesis. Because PI3K and mTOR inhibitors are clinically used to treat other conditions, they have the potential to be repositioned to be used as an alternative nonimmunosuppressive therapy of GO.
Institute of Molecular and Experimental Medicine (L.Z., F.G.-J., M.S.D., C.M.D., M.L.), Department of Medical Genetics (A.R.T.), Institute of Cancer and Genetics, School of Medicine, Cardiff University, and Department of Ophthalmology (C.L., D.M.), Cardiff and Vale University Health Board, Heath Park, Cardiff CF14 4XN, United Kingdom.
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Zhang
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Grennan-Jones
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Draman
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Lane
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Morris
C M
CM
Dayan
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Tee
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Ludgate
D050156:Adipogenesis / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D055808:Drug Discovery; D005786:Gene Expression Regulation / Q000187:drug effects; D049970:Graves Ophthalmopathy / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D006820:Hyaluronic Acid / Q000235:genetics / Q000378:metabolism; D058990:Molecular Targeted Therapy; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D017526:Receptor, IGF Type 1 / Q000819:agonists / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D045162:Thiazolidinediones / Q000494:pharmacology
C505144:5-quinoxalin-6-ylmethylenethiazolidine-2,4-dione; D002867:Chromones; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D011810:Quinoxalines; D011989:Receptors, Thyrotropin; D045162:Thiazolidinediones; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D006820:Hyaluronic Acid; D017526:Receptor, IGF Type 1