Ubiquitin Specific Protease USP48 Destabilizes NF-κB/p65 in Retinal Pigment Epithelium Cells

Abstract

Activation of NF-κB transcription factor is strictly regulated to accurately direct cellular processes including inflammation, immunity, and cell survival. In the retina, the modulation of the NF-κB pathway is essential to prevent excessive inflammatory responses, which plays a pivotal role in many retinal neurodegenerative diseases, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and inherited retinal dystrophies (IRDs). A critical cytokine mediating inflammatory responses in retinal cells is tumor necrosis factor-alpha (TNFα), leading to the activation of several transductional pathways, including NF-κB. However, the multiple factors orchestrating the appropriate regulation of NF-κB in retinal cells still remain unclear. The present study explores how the ubiquitin-specific protease 48 (USP48) downregulation impacts the stability and transcriptional activity of NF-κB/p65 in retinal pigment epithelium (RPE), at both basal conditions and following TNFα stimulation. We described that USP48 downregulation stabilizes p65. Notably, the accumulation of p65 is mainly detectable in the nuclear compartment and it is accompanied by an increased NF-κB transcriptional activity. These results delineate a novel role of USP48 in negatively regulating NF-κB in retinal cells, providing new opportunities for therapeutic intervention in retinal pathologies.

The Deubiquitinating Enzyme USP48 Interacts with the Retinal Degeneration-Associated Proteins UNC119a and ARL3

“…We next assayed the potential effects of silencing USP48 in ciliogenesis. The knockdown of USP48 by siRNA transfection (silencing efficiency previously tested in [ 30 ]), showed similar results to the overexpression experiments: neither ciliary length nor ciliogenesis was clearly affected by USP48 knockdown ( Figure 4 A,B), although the ciliary acetylate α-tubulin increased ( Figure 4 C), suggesting that the lack of functional USP48 resulted in hyper-acetylation of α-tubulin in the cilium.…”

“…USP48 is involved in a wide range of cellular processes such as DNA repair [ 27 , 28 ], cell cycle progression [ 29 ], control of immune response [ 30 , 31 ], regulation of the sonic hedgehog pathway [ 32 ], and tumorigenesis [ 23 , 33 , 34 ]. Data on the function of USP48 beyond cancer and its physiological role in other organs is still very scarce [ 30 , 35 , 36 , 37 ].…”

Ubiquitin-Specific Proteases (USPs) and Metabolic Disorders

“…Among the proinflammatory cytokines, TNF-α provokes NF-κB activation in retinal pigment epithelial cells [ 157 ]. Although USP48 stabilizes NF-κB p65 in HeLa cells via the removal of the K48-linked polyubiquitin chain [ 158 ], gene silencing of Usp48 increases nuclear NF-κB p65 protein in cultured retinal pigment epithelial cells without any stimulation [ 159 ]. After TNF-α stimulation, Usp48 shRNA treatment sustained nuclear NF-κB p65 expression at a high level [ 159 ], and also increased the transcriptional activity of NF-κB in the retinal cells [ 159 ].…”

“…Although USP48 stabilizes NF-κB p65 in HeLa cells via the removal of the K48-linked polyubiquitin chain [ 158 ], gene silencing of Usp48 increases nuclear NF-κB p65 protein in cultured retinal pigment epithelial cells without any stimulation [ 159 ]. After TNF-α stimulation, Usp48 shRNA treatment sustained nuclear NF-κB p65 expression at a high level [ 159 ], and also increased the transcriptional activity of NF-κB in the retinal cells [ 159 ]. Considering that these data show preferential localization of USP48 in the nucleus of retinal cells, nuclear USP48 might mitigate retinal inflammatory responses by promoting the degradation of NF-κB p65 [ 159 ].…”

“…After TNF-α stimulation, Usp48 shRNA treatment sustained nuclear NF-κB p65 expression at a high level [ 159 ], and also increased the transcriptional activity of NF-κB in the retinal cells [ 159 ]. Considering that these data show preferential localization of USP48 in the nucleus of retinal cells, nuclear USP48 might mitigate retinal inflammatory responses by promoting the degradation of NF-κB p65 [ 159 ].…”