TNFα and SOCS3 regulate IRS-1 to increase retinal endothelial cell apoptosis

Abstract

Rates of diabetes are reaching epidemic levels. The key problem in both type 1 and type 2 diabetes is dysfunctional insulin signaling, either due to lack of production or due to impaired insulin sensitivity. A key feature of diabetic retinopathy in animal models is degenerate capillary formation. The goal of this present study was to investigate a potential mechanism for retinal endothelial cell apoptosis in response to hyperglycemia. The hypothesis was that hyperglycemia-induced TNFα leads to retinal endothelial cell apoptosis through inhibition of insulin signaling. To test the hypothesis, primary human retinal endothelial cells were grown in normal glucose (5 mM) or high glucose (25 mM) and treated with exogenous TNFα, TNFα siRNA or suppressor of cytokine signaling 3 (SOCS3) siRNA. Cell lysates were processed for Western blotting and ELISA analyses to verify TNFα and SOCS3 knockdown, as well as key pro- and anti-apoptotic factors, IRS-1, and Akt. Data indicate that high glucose culturing conditions significantly increase TNFα and SOCS3 protein levels. Knockdown of TNFα and SOCS3 significantly increases anti-apoptotic proteins, while decreasing pro-apoptotic proteins. Knockdown of TNFα leads to decreased phosphorylation of IRS-1Ser307, which would promote normal insulin signaling. Knockdown of SOCS3 increased total IRS-1 levels, as well as decreased IRTyr960, both of which would inhibit retinal endothelial cell apoptosis through increased insulin signaling. Taken together, our findings suggest that increased TNFα inhibits insulin signaling in 2 ways: 1) increased phosphorylation of IRS-1Ser307, 2) increased SOCS3 levels to decrease total IRS-1 and increase IRTyr960, both of which block normal insulin signal transduction. Resolution of the hyperglycemia-induced TNFα levels in retinal endothelial cells may prevent apoptosis through disinhibition of insulin receptor signaling.

SOCS3 is an endogenous inhibitor of pathologic angiogenesis

“…1,5 It also regulates endothelial cell (EC) apoptosis. 4 However, the role of SOCS3 in regulating angiogenesis in vivo is unknown. If SOCS3 were angiostatic, as has been hypothesized, then its suppression would increase neovascularization in pathologic conditions (see Figure 1A).…”

“…1,2 SOCS3 is transiently induced by inflammatory mediators such as lipopolysaccharide, 3 IL-6, 3 and TNF␣. 4 SOCS3 inhibits cytoplasmic effectors such as the JAK/STAT kinases and deactivates tyrosine kinase receptor signaling, including the IGF-1 receptor. 1,5 It also regulates endothelial cell (EC) apoptosis.…”

miR15a regulates NLRP3 inflammasome proteins in the retinal vasculature

“…miR15a decreased tumor necrosis factor alpha (TNFα) levels, which can help to restore normal insulin signaling (Jiang et al, 2012, 2018). In addition to TNFα, other inflammatory cytokines, such as IL-1β, are also upregulated in diabetic retinopathy and indicated in diabetic complications (Liu et al, 2012).…”

TLR4 regulates insulin-resistant proteins to increase apoptosis in the mouse retina

“…We have previously reported that β-adrenergic receptors can regulate TLR4 in the diabetic retina, as well as retinal endothelial cells (REC) an Müller cells [7]. Additionally, we have reported that a β-adrenergic receptor agonist can reduce functional, neuronal, and vascular phenotypes associated with retinopathy with rodents [8], as well as restore normal insulin signal transduction in cells grown in high glucose [9, 10]. …”