Liver-specific knockout of Nrf1 in the mouse leads to spontaneous development of non- alcoholic steatohepatitis with dyslipidemia, and then its deterioration results in hepatoma, but the underlying mechanism remains elusive to date. A similar pathological model is reconstructed here by using human Nrf1α-specific knockout cell lines. Our evidence has demonstrated that a marked increase of the inflammation marker COX2 definitely occurs in Nrf1α−/− cells. Loss of Nrf1α leads to hyperactivation of Nrf2, which results from substantial decreases in Keap1, PTEN and most of 26S proteasomal subunits in Nrf1α−/− cells. Further investigation of xenograft model mice showed that malignant growth of Nrf1α−/−-derived tumors is almost abolished by silencing of Nrf2, while Nrf1α+/+-tumor is markedly repressed by an inactive mutant (i.e., Nrf2−/−ΔTA), but largely unaffected by a priori constitutive activator (i.e., caNrf2ΔN). Mechanistic studies, combined with transcriptomic sequencing, unraveled a panoramic view of opposing and unifying inter-regulatory cross-talks between Nrf1α and Nrf2 at different layers of the endogenous regulatory networks from multiple signaling towards differential expression profiling of target genes. Collectively, Nrf1α manifests a dominant tumor-suppressive effect by confining Nrf2 oncogenicity. Though as a tumor promoter, Nrf2 can also, in turn, directly activate the transcriptional expression of Nrf1 to form a negative feedback loop. In view of such mutual inter-regulation by between Nrf1α and Nrf2, it should thus be taken severe cautions to interpret the experimental results from loss of Nrf1α, Nrf2 or both.

The full-length Nrf1α is processed into distinct isoforms, which together regulate genes essential for maintaining cellular homeostasis and organ integrity, and liver-specific loss of Nrf1 in mice results in spontaneous hepatoma. Herein, we report that the human constitutive Nrf1α, rather than smaller Nrf1β/γ, expression is attenuated or abolished in the case of low-differentiated high-metastatic hepatocellular carcinomas. Therefore, Nrf1α is of importance in the physio-pathological origin and development, but its specific pathobiological function(s) remains elusive. To address this, TALENs-directed knockout of Nrf1α, but not Nrf1β/γ, is created in the human hepatocellular carcinoma (HepG2) cells. The resulting Nrf1α−/− cells are elongated, with slender spindle-shapes and enlarged gaps between cells observed under scanning electron microscope. When compared with wild-type controls, the invasive and migratory abilities of Nrf1α−/− cells are increased significantly, along with the cell-cycle G2-M arrest and S-phase reduction, as accompanied by suppressed apoptosis. Despite a modest increase in the soft-agar colony formation of Nrf1α−/− cells, its loss-of-function markedly promotes malgrowth of the subcutaneous carcinoma xenograft in nude mice with hepatic metastasis. Together with molecular expression results, we thus suppose requirement of Nrf1α (and major derivates) for gene regulatory mechanisms repressing cancer cell process (e.g. EMT) and malignant behaviour (e.g. migration).

The water-soluble Nrf2 (nuclear factor, erythroid 2-like 2, also called Nfe2l2) is accepted as a master regulator of antioxidant responses to cellular stress, and it was also identified as a direct target of the endoplasmic reticulum (ER)-anchored PERK (protein kinase RNA-like endoplasmic reticulum kinase). However, the membrane-bound Nrf1 (nuclear factor, erythroid 2-like 1, also called Nfe2l1) response to ER stress remains elusive. Herein, we report a unity of opposites between these two antioxidant transcription factors, Nrf1 and Nrf2, in coordinating distinct cellular responses to the ER stressor tunicamycin (TU). The TU-inducible transcription of Nrf1 and Nrf2, as well as GCLM (glutamate cysteine ligase modifier subunit) and HO-1 (heme oxygenase 1), was accompanied by activation of ER stress signaling networks. Notably, the unfolded protein response (UPR) mediated by ATF6 (activating transcription factor 6), IRE1 (inositol requiring enzyme 1) and PERK was significantly suppressed by Nrf1α-specific knockout, but hyper-expression of Nrf2 and its target genes GCLM and HO-1 has retained in Nrf1α−/− cells. By contrast, Nrf2−/−ΔTA cells with genomic deletion of its transactivation (TA) domain resulted in significant decreases of GCLM, HO-1 and Nrf1; this was accompanied by partial decreases of IRE1 and ATF6, rather than PERK, but with an increase of ATF4 (activating transcription factor 4). Interestingly, Nrf1 glycosylation and its trans-activity to mediate the transcriptional expression of the 26S proteasomal subunits, were repressed by TU. This inhibitory effect was enhanced by Nrf1α−/− and Nrf2−/−ΔTA, but not by a constitutive activator caNrf2ΔN (that increased abundances of the non-glycosylated and processed Nrf1). Furthermore, caNrf2ΔN also enhanced induction of PERK and IRE1 by TU, but reduced expression of ATF4 and HO-1. Thus, it is inferred that such distinct roles of Nrf1 and Nrf2 are unified to maintain cell homeostasis by a series of coordinated ER-to-nuclear signaling responses to TU. Nrf1α (i.e., a full-length form) acts in a cell-autonomous manner to determine the transcription of most of UPR-target genes, albeit Nrf2 is also partially involved in this process. Consistently, transactivation of ARE (antioxidant response element)-driven BIP (binding immunoglobulin protein)-, PERK- and XBP1 (X-box binding protein 1)-Luc reporter genes was mediated directly by Nrf1 and/or Nrf2. Interestingly, Nrf1α is more potent than Nrf2 at mediating the cytoprotective responses against the cytotoxicity of TU alone or plus tBHQ (tert-butylhydroquinone). This is also further supported by the evidence that the intracellular reactive oxygen species (ROS) levels are increased in Nrf1α−/− cells, but rather are, to our surprise, decreased in Nrf2−/−ΔTA cells.

Our previous work revealed that Nrf1α exerts a tumor-repressing effect because its genomic loss (to yield Nrf1α-/-) results in oncogenic activation of Nrf2 and target genes. Interestingly, β-catenin is concurrently activated by loss of Nrf1α in a way similar to β-catenin-driven liver tumor. However, a presumable relationship between Nrf1 and β-catenin is not yet established. Here, we demonstrate that Nrf1 enhanced ubiquitination of β-catenin for targeting proteasomal degradation. Conversely, knockdown of Nrf1 by its short hairpin RNA (shNrf1) caused accumulation of β-catenin so as to translocate the nucleus, allowing activation of a subset of Wnt/β-catenin signaling responsive genes, which leads to the epithelial-mesenchymal transition (EMT) and related cellular processes. Such silencing of Nrf1 resulted in malgrowth of human hepatocellular carcinoma, along with malignant invasion and metastasis to the lung and liver in xenograft model mice. Further transcriptomic sequencing unraveled significant differences in the expression of both Wnt/β-catenin-dependent and Wnt/β-catenin-independent responsive genes implicated in the cell process, shape, and behavior of the shNrf1-expressing tumor. Notably, we identified that β-catenin is not a target gene of Nrf1, but this CNC-bZIP factor contributes to differential or opposing expression of other critical genes, such as CDH1, Wnt5A, Wnt11A, FZD10, LEF1, TCF4, SMAD4, MMP9, PTEN, PI3K, JUN, and p53, each of which depends on the positioning of distinct cis-regulatory sequences (e.g., ARE and/or AP-1 binding sites) in the gene promoter contexts. In addition, altered expression profiles of some Wnt/β-catenin signaling proteins were context dependent, as accompanied by decreased abundances of Nrf1 in the clinic human hepatomas with distinct differentiation. Together, these results corroborate the rationale that Nrf1 acts as a bona fide dominant tumor repressor, by its intrinsic inhibition of Wnt/β-catenin signaling and relevant independent networks in cancer development and malignant progression.

The single Nrf1 gene has capability to be differentially transcripted alongside with alternative mRNA-splicing and subsequent translation through different initiation signals so as to yield distinct lengths of polypeptide isoforms. Amongst them, three of the most representatives are Nrf1α, Nrf1β and Nrf1γ, but the putative specific contribution of each isoform to regulating ARE-driven target genes remains unknown. To address this, we have herein established three cell lines on the base of the Flp-In T-REx system, which are allowed for the tetracycline-inducibly stable expression of Nrf1α, Nrf1β and Nrf1γ. Consequently, the RNA-Sequencing results have demonstrated that a vast majority of differentially expressed genes (i.e. >90% DEGs detected) were dominantly up-regulated by Nrf1α and/or Nrf1β following induction by tetracycline. By contrast, the other DEGs regulated by Nrf1γ were far less than those regulated by Nrf1α/β (i.e. ~11% of Nrf1α and ~7% of Nrf1β). However, further transcriptomic analysis revealed that the tetracycline-induced expression of Nrf1γ significantly increased the percentage of down-regulated genes in total DEGs. These statistical data were further validated by quantitative real-time PCR. The experimental results indicate that distinct Nrf1 isoforms make diverse and even opposing contributions to regulating different subsets of target genes, such as those encoding 26S proteasomal subunits and others involved in various biological processes and functions. Collectively, Nrf1γ acts as a major dominant-negative inhibitor competitively against Nrf1α/β activity, such that a number of DEGs regulated by Nrf1α/β are counteracted by Nrf1γ.

Progressive white matter (WM) impairments caused by subarachnoid hemorrhage (SAH) contribute to cognitive deficits and poor clinical prognoses; however, their pathogenetic mechanisms are poorly understood. We investigated the role of nexilin and oligodendrocyte progenitor cell (OPC)-mediated repair in a mouse model of experimental SAH generated via left endovascular perforation. Nexilin expression was enhanced by the elevated migration of OPCs after SAH. Knocking down nexilin by siRNA reduced OPC migration both in vitro and in vivo and abridged WM repair. In contrast, the protease-activated receptor 1 (PAR1), Ras-proximate-1 (RAP1) and phosphorylated RAP1 (pRAP1) levels in WM were elevated after SAH. The genetic inhibition of PAR1 reduced RAP1 and pRAP1 expression, further enhancing nexilin expression. When delivered at an early stage at a concentration of 25 µg/kg, thrombin receptor antagonist peptide along with PAR1 knockdown rescued the down-regulation of myelin basic protein and improved remyelination at the later stage of SAH. Our results suggest that nexilin is required for OPC migration and remyelination following SAH, as it negatively regulates PAR1/RAP1 signaling, thus providing a promising therapeutic target in WM repair and functional recovery.

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Insufficient remyelination due to impaired oligodendrocyte precursor cell (OPC) differentiation and maturation is strongly associated with irreversible white matter injury (WMI) and neurological deficits. We analyzed whole transcriptome expression to elucidate the potential role and underlying mechanism of action of lipocalin-2 (LCN2) in OPC differentiation and WMI and identified the receptor SCL22A17 and downstream transcription factor early growth response protein 1 (EGR1) as the key signals contributing to LCN2-mediated insufficient OPC remyelination. In LCN-knockdown and OPC EGR1 conditional-knockout mice, we discovered enhanced OPC differentiation in developing and injured white matter (WM); consistent with this, the specific inactivation of LCN2/SCl22A17/EGR1 signaling promoted remyelination and neurological recovery in both atypical, acute WMI due to subarachnoid hemorrhage and typical, chronic WMI due to multiple sclerosis. This potentially represents a novel strategy to enhance differentiation and remyelination in patients with white matter injury.