Graphs are means? SD from three independent experiments. of Sp1 reduced and increased Nrf2 protein levels, respectively. These effects were abrogated by the WDR23 KD, suggesting that Sp1 also regulates Nrf2 levels the ubiquitin ligase complex CRL4AWDR23. In conclusion, we discovered Sp1 as a novel substrate of Keap1 and provided evidence that Sp1 regulates the expression of ubiquitination, including Holliday junction resolvase GEN-1 (14), p21 (15), stem-loop binding protein (16), and Nrf2 (11). The transient activation of Nrf2 in normal cells is beneficial for cytoprotection and the prevention of pathological conditions; however, its consecutive activation in cancer cells is responsible for chemoresistance and is associated with a poor prognosis (17). Therefore, the precise regulation of Nrf2 levels is crucial. A somatic mutation in highly conserved Kelch or the intervening region domain of the Keap1 protein that results in the constitutive activation of Nrf2 often occurs in cancer cells (18, 19). Therefore, the second layer of Nrf2 regulation is important for preventing carcinogenesis and chemoresistance. We previously reported that the knockdown (KD) of WDR23 was sufficient to increase the level and transactivity of Nrf2, whereas its overexpression only affected Nrf2 under Keap1 KD (12). These findings indicate that WDR23 regulates Nrf2 under basal conditions, whereas the further induction Stachyose tetrahydrate of WDR23 activity toward Nrf2 requires the inhibition of Keap1. Therefore, WDR23 plays a major role in the regulation of Nrf2 in cancer cells bearing mutation. However, the molecular mechanisms underlying crosstalk between these two independent and parallel regulators of Nrf2, particularly that by which WDR23 senses the function of Keap1, have not yet been elucidated. Specificity protein 1 (Sp1) is a ubiquitously expressed nuclear transcription factor belonging to the Stachyose tetrahydrate C2H2-type zinc-finger protein family. Sp1 regulates gene expression proteinCprotein interactions, such as with vascular endothelial growth factor receptor-2 (20), or acts in concert with other transcription factors, including Stat1 (21), nuclear factor-B (22), and EGR-1 (23), in the absence of the TATA box. It binds to the sequence known as the GC box (GGGCGG or CCGCCC) in the Stachyose tetrahydrate promoters of numerous genes with high affinity (24, 25). Sp1 was initially regarded as a coordinator of the constitutive expression of housekeeping genes; however, recent studies showed that Sp1 responded to physiological and pathological stimuli (26, 27). Previous findings clearly demonstrated that Sp1 protein levels and transcriptional activity were induced by oxidative stress (28, 29, 30). For example, we found that high glucose-induced oxidative stress increased nuclear Sp1 levels, which inhibited the expression of (27). Increases in the level and activity of Sp1 have been widely proven to be responsible for oxidative stress-related carcinogenesis, including proliferation, the cell cycle, invasion, metastasis, angiogenesis, and the inhibition of apoptosis in hepatocellular carcinoma (31). Stachyose tetrahydrate Although Sp1 plays a role in the oxidative stress response pathway, the underlying molecular mechanisms have not yet been elucidated in detail. We herein demonstrated the role of Sp1 as a mediator of Keap1CWDR23 crosstalk for the regulation of Nrf2. The results obtained herein revealed that Keap1 directly regulates Sp1. The stabilization of Sp1 during the KD of Keap1 resulted in the transcriptional activation of Cullin4A (but not (Fig.?1, mRNA levels, whereas those of remained unchanged (Fig.?1, was also increased by the H2O2 treatment (Fig.?1, were measured by RT-PCR. in pGL3 or its 5-endo deletion construct was co-transfected with pRL-null as an internal control plasmid into control and Keap1 KD Hep3B cells. Luciferase activity was assessed 48?h after transfection using the Dual-Luciferase Reporter Assay system relative to the promoter-less construct pGL3-Basic. Results are shown Stachyose tetrahydrate as a ratio relative to the activity of promoter??1920/+50 in control cells. All values are means? SD from three independent experiments. N.S. not significant, ?the indicated cells. CUL4A, Cullin4A; DDB1, DNA damage-binding protein 1; KD, knockdown; Keap1, Kelch-like ECH-associated protein 1; RBX1, Ring-box 1; shRNA, short hairpin RNA; tBHQ, tert-butylhydroquinone; WDR23, WD40 repeat protein?23. To elucidate the mechanisms underlying the regulation of expression by Keap1, we investigated promoter activity under Keap1 KD conditions using a luciferase reporter assay. Luciferase reporter plasmids (pGL3-basic) containing??1920/+50?bp of the genomic region were constructed and transfected into Hep3B cells. The KD of Keap1 induced a 2.5-fold increase in promoter activity Rock2 (Fig.?1by Keap1 is present within this segment. The role of Sp1 on the Keap1 KD-mediated induction of CUL4A To elucidate.