MLN4924, neddylation inhibitor suppresses hypoxia induced retinal angiogenesis by targeting Human Antigen R signaling

Abstract

Purpose

Retinal hypoxia is a key pathological stimulus for neovascularization, leading to abnormal proliferation of blood vessels and vascular endothelial dysfunction leading to vision threatening conditions. The anti-angiogenic potential of MLN4924, a specific inhibitor of neddylation signaling has been evidenced in cancer cells, but remains abstract as therapy for ocular angiogenesis in normal retinal cells. The current work intended to delineate a novel molecular signaling cascade of combating retinal angiogenesis by inhibiting the neddylation-Human Antigen R (HuR) signaling pathway using MLN4924.

Methods

The effect of neddylation inhibition on hypoxia-induced HRMVEC was demonstrated by the real-time PCR, western blotting, immunofluorescence, and functional assays. In silico analysis was used to study the impact of the mutation of the RNA binding protein, HuR neddylation sites on its binding affinity towards VEGF mRNA. RNA immunoprecipitation and Actinomycin D experiments were performed to evidence the effect of neddylation inhibition on HuR binding and stabilization of VEGF mRNA in hypoxic HRMVECs.

Results

Functional angiogenic assays revealed that treatment with MLN4924 could suppress hypoxia-induced angiogenesis by reducing secretory VEGF levels without altering the barrier integrity of HRMVECs. In silico analysis revealed that mutation of two of the HuR neddylation sites decreased its binding affinity to 3′UTR region of VEGF mRNA. Indeed, HuR preferentially bound and stabilized VEGF mRNA upon hypoxia, which was significantly inhibited using MLN4924 in HRMVECs.

Conclusion

Neddylation inhibition could suppress hypoxia-induced angiogenesis through HuR signaling without compromising the barrier integrity of HRMVECs.