NFAT signaling induces upregulation of cytokines that promote the pathology of diabetic retinopathy

Abstract

Purpose: The nuclear factor of activated T-cells (NFAT) family of transcription factors consists of five proteins, four of which are tightly regulated by Ca²⁺ through the serine phosphatase calcineurin (CN). Activated calcineurin dephosphorylates a conserved NFAT regulatory domain leading to nuclear translocation of NFAT and increased affinity for its target DNA sites. VEGF signaling is known to stimulate NFAT translocation, and we have previously reported that increased calcium signaling contributes to high glucose-induced VEGF production by retinal Müller cells. We hypothesize that NFAT plays a role in both high glucose-induced VEGF production by Müller cells and VEGF-induced IL-6 production by retinal microvascular endothelial cells (RMEC). The purpose of the present study was to evaluate this hypothesis in vitro using the CN/NFAT inhibitors INCA-6 and FK-506.

Methods: Primary human Müller cells (HMC) were treated with either normal (5mM) or high glucose (25mM) growth medium with and without the NFAT-specific inhibitor INCA-6 (0.5µM and 1µM). Cells were collected 24hrs post treatment and quantitative real-time RT-PCR was performed by co-amplification of VEGF vs. β-actin (endogenous normalization control), using gene-specific TaqMan Gene Expression Assays. Human RMEC were stimulated by VEGF (25ng/ml) with and without the CN inhibitor FK-506 (1µM). 24hrs later, culture medium was collected and analyzed for HMC IL-6 production by ELISA.

Results: Treatment of HMC with high glucose led to a 50% increase in VEGF expression (p<0.005) that was inhibited by INCA-6 treatment at both 0.5µM and 1µM concentrations (p<0.05). Treatment of primary HRMEC with 25ng/ml VEGF resulted in a 2.1-fold increase in secretion of IL-6 (p<0.005). This induction was inhibited by treatment with 1µM FK-506 (p<0.01).

Conclusions: Increased retinal VEGF and IL-6 are hallmarks of diabetic retinopathy (DR). Here we provide evidence that the NFAT family of transcription factors plays a role in the high glucose-induced upregulation of VEGF by HMC and VEGF-induced production of IL-6 by RMEC. Collectively, these findings suggest that NFAT signaling may be a valuable therapeutic target for the treatment of DR, since it promotes both upregulation and bioactivity of retinal VEGF in two critical cell types under disease-relevant conditions.

Keywords: 499 diabetic retinopathy • 748 vascular endothelial growth factor • 490 cytokines/chemokines