Sp1 is a substrate of Keap1 and regulates the activity of CRL4AWDR23 ubiquitin ligase toward Nrf2

Abstract

Nuclear factor erythroid 2–related factor 2 (Nrf2) is a critical transcription factor that orchestrates cellular responses to oxidative stress. Because the dysregulation of Nrf2 has been implicated in many diseases, precise regulation of its protein level is crucial for maintaining homeostasis. Kelch-like-ECH-associated protein 1 (Keap1) and WD40 repeat protein 23 (WDR23) directly regulate Nrf2 levels via similar but distinct proteasome-dependent pathways. WDR23 forms a part of the WDR23-Cullin 4A-RING ubiquitin ligase complex (CRL4A WDR23 ), whereas Keap1 serves as a substrate adaptor for the Cullin 3–containing ubiquitin ligase complex. However, the mechanisms underlying crosstalk between these Keap1 and WDR23 pathways for the regulation of Nrf2 levels have not been investigated. Here, we showed that knockdown (KD) of Keap1 upregulated the expression of Cullin4A ( CUL4A ) in a specificity protein 1 (Sp1)–dependent manner. We also revealed that Sp1 interacted with Keap1, leading to ubiquitination of Sp1. Increases in Sp1 by Keap1 KD triggered Sp1 binding to the fourth Sp1 binding site (Sp1_M4) within the −230/+50 region of the CUL4A gene. We also demonstrated that the overexpression and KD of Sp1 reduced and increased Nrf2 protein levels, respectively. These effects were abrogated by the WDR23 KD, suggesting that Sp1 also regulates Nrf2 levels via the ubiquitin ligase complex CRL4A WDR23 . In conclusion, we discovered Sp1 as a novel substrate of Keap1 and provided evidence that Sp1 regulates the expression of CUL4A . We revealed a novel role for Sp1 in mediating crosstalk between two independent regulators of Nrf2 protein levels.

Sp1-Mediated Prdx6 Upregulation Leads to Clasmatodendrosis by Increasing Its aiPLA2 Activity in the CA1 Astrocytes in Chronic Epilepsy Rats

“…Seizures increase Sp1 activity, which is one of the upstream regulators for clasmatodendrosis [ 13 , 36 ]. Furthermore, Sp1 overexpression reduces Nrf2 protein level [ 37 ], and CDDO-Me decreases Sp1 expression [ 38 ]. Therefore, it is likely that Sp1 activation would lead to Prdx6 upregulation in CA1 astrocytes within the epileptic hippocampus.…”

“…However, NAC and MMA reduced Prdx6 and Sp1 levels in CA1 astrocytes without inducing Nrf2 upregulation. Sp1 is a transcription factor to regulate expressions of Nrf2 and HSP25, which are involved in clasmatodendrosis [ 13 , 20 , 22 , 37 ]. Furthermore, Sp1 inhibition abolishes curcumin-induced Prdx6 upregulation following cerebral ischemia [ 61 ].…”

Redox signaling at the crossroads of human health and disease

“… 61 The N ‐terminal Neh2 domain is involved in the Keap1‐Nrf2 interaction, 62 as well as Nrf2 stability and ubiquitination. 63 The Neh4 and Neh5 domains are transcription activation domains that interact with the CREB binding protein (CBP) to promote Nrf2 transcription, 64 leading to enhanced expression of Nrf2‐targeted ARE genes. It has been reported that retinoid X receptor alpha (RXR‐α) suppresses the function of Nrf2 by interacting with the Neh7 domain (also known as the RXR‐α interaction domain).…”

“…There are seven functional domains (Neh1‐7) that regulate the stability and transcriptional activity of Nrf2 61 . The N ‐terminal Neh2 domain is involved in the Keap1‐Nrf2 interaction, 62 as well as Nrf2 stability and ubiquitination 63 . The Neh4 and Neh5 domains are transcription activation domains that interact with the CREB binding protein (CBP) to promote Nrf2 transcription, 64 leading to enhanced expression of Nrf2‐targeted ARE genes.…”

The Antioxidant Transcription Factor Nrf2 in Cardiac Ischemia–Reperfusion Injury

“…Transcription mediated by Nrf2 is activated by several transcription factors, which expands the types of stressors that induce Nrf2-target genes. These factors include the aryl hydrocarbon receptor (AhR) [ 44 , 45 ], peroxisome proliferator-activated receptor (PPAR)γ [ 46 , 47 ], nuclear factor-κB (NF-κB) [ 48 , 49 ], specificity protein 1 (Sp-1) [ 50 ], p53 [ 51 ], c-Jun, c-Myc [ 52 , 53 ], and breast cancer 1 (BRCA1) [ 54 ]. As mentioned earlier, Nrf2 also enhances its own transcription [ 32 ].…”