Forkhead Transcription Factor FOXO1 Is Regulated by Both a Stimulatory Thyrotropin Receptor Antibody and Insulin-Like Growth Factor-1 in Orbital Fibroblasts from Patients with Graves' Ophthalmopathy

Abstract

Background: Activation of thyrotropin receptor (TSHR) and/or insulin-like growth factor (IGF-1) receptor (IGF-1R) enhances HA production and adipogenesis in orbital fibroblasts from patients with Graves' ophthalmopathy (GO) and recapitulates the tissue remodeling characteristic of the orbit in GO. A functional relationship between TSHR and IGF-1R has long been postulated, and recently bidirectional crosstalk between the receptors in GO fibroblasts was demonstrated. Because the transcription factor Forkhead box O-1 (FOXO1) was recently shown to be a critical downstream mediator of TSH and IGF-1 effects on thyrocyte proliferation, studies were designed to determine whether FOXO1 might similarly act as a common mediator of M22, a stimulatory TSHR antibody (TSAb), and IGF-1 in GO orbital fibroblasts. Methods: FOXO1 mRNA and protein were measured in orbital tissue specimens derived from normal individuals and patients with GO. In addition, the control of FOXO1 cellular localization was investigated using quantitative Western blotting of fractionated cell lysates from orbital fibroblasts treated with M22 and/or IGF-1 with or without specific TSHR, IGF-1R, or PI3K/AKT1/2 inhibitors. Results: Significantly lower levels of both FOXO1 mRNA and protein were found in GO orbital tissue specimens compared with normal orbital tissues (M = 39%, p = 0.043; M = 46.4%; p = 0.028, respectively). In addition, treatment of GO orbital cultures with M22, IGF-1, or M22 plus IGF-1 increased cytoplasmic FOXO1 compared with control (1.63-fold, p = 0.008; 1.68-fold, p = 0.001; 1.61-fold, p £ 0.001, respectively) and decreased nuclear FOXO1 (M = 28%, p = 0.002; M = 38%, p £ 0.001; M = 35%, p = 0.007, respectively). These effects were inhibited by co-treatment with the respective, but not the opposite, receptor antagonist. AKT inhibition of M22 or IGF-1-treated cultures was found to increase nuclear (1.4-fold, p = 0.026; 1.3-fold, p = 0.001, respectively) and decrease cytoplasmic (24.2%, p = 0.001; 36%, p = 0.004, respectively) FOXO1 localization. Conclusions: These data point to FOXO1 as an important mediator of TSAb and IGF-1 action via their cognate receptors in GO orbital fibroblasts. These findings provide a link between the low FOXO1 protein levels demonstrated in GO orbital tissue and the tissue remodeling characteristic of GO, and suggest novel therapy for GO aimed at increasing nuclear expression of FOXO1 in GO target cells.

Inhibitory Effect of Idelalisib, a Selective Phosphatidylinositol 3-Kinase δ Inhibitor, on Adipogenesis in an In Vitro Model of Graves' Orbitopathy

“…34,35 When FOXO1 is phosphorylated by AKT via the PI3K-AKT cascade, p-FOXO1 is transported from the nucleus to the cytosol, freeing PPARc for activation, while cytosolic FOXO1 is ubiquitinated and degraded. 35,36 In orbital fibroblasts from patients with GO, M22 (a stimulatory TSHR antibody) and IGF-1 increased cytosolic FOXO1 and decreased nuclear FOXO1, indicating that FOXO1 is a downstream mediator of the TSH and IGF1initiated pathogenesis of GO. 36 Moreover, FOXOs act as repressors of hyaluronan production and adipogenesis, as a FOXO enhancer (trifluoperazine hydrochloride) blocks adipogenesis in orbital fibroblasts.…”

“…35,36 In orbital fibroblasts from patients with GO, M22 (a stimulatory TSHR antibody) and IGF-1 increased cytosolic FOXO1 and decreased nuclear FOXO1, indicating that FOXO1 is a downstream mediator of the TSH and IGF1initiated pathogenesis of GO. 36 Moreover, FOXOs act as repressors of hyaluronan production and adipogenesis, as a FOXO enhancer (trifluoperazine hydrochloride) blocks adipogenesis in orbital fibroblasts. 37 In this report, we showed that indirect inhibition of FOXO1 phosphorylation by idelalisib suppresses the action of PPARc, thereby reducing adipogenic differentiation (Fig.…”

New insights into the pathogenesis and nonsurgical management of Graves orbitopathy

“…Taken together these findings suggest a very different mechanism in OAT expansion and underlying orbital fibrosis adipogenesis. Indeed, there is a specific cell-signaling network presenting in fibroblasts from OAT distinct from that in WAT 112 by studies that have highlighted the importance of downstream factors of IGF1-PI3K signalling and revealed that nuclear Forkhead transcriptional factors, FOXOs, serve as convergence points for TSHR and IGF-1R signalling pathways in Graves' orbitopathy 121,122 . Specifically, FOXO1 and FOXO3a served as repressors, which protect orbital fibroblasts from excessive adipogenesis and marked over-production of HA, respectively 121 .…”

MiR-142-3p functions as an oncogene in prostate cancer by targeting FOXO1

“…Forkhead box transcription factor O1 (FOXO1), encoded by the FOXO1 gene in humans, is a member of the Forkhead transcription factor family, and is widely expressed in living organisms, particularly in the liver, spleen, and lung [6,7]. Forkhead box transcription factor O1 has been identified as an important transcriptional effector in the insulin and insulin-like growth factor 1 (IGF-1) signaling pathways [8,9]. In addition, FOXO1 is involved in numerous cellular functions, such as the cell cycle, apoptosis, and metabolism [10].…”