Crosstalk between Nrf2 and Notch signaling

https://doi.org/10.1016/j.freeradbiomed.2015.05.017Get rights and content

Highlights

  • Reciprocal Nrf2–Notch transcriptional regulation in the hepatobiliary system.

  • Molecular signaling networks in hepatic regeneration.

  • Biological significance of Nrf2–Notch crosstalk.

Abstract

The transcription factor Nrf2 (nuclear factor, erythroid derived 2, like 2) belongs to the CNC-bZip protein family, forming a transcriptosome with its direct heterodimer partner, sMaf, and co-factors such as CBP/p300. Nrf2 binds to one or more AREs (antioxidant response elements) that are located in the gene regulatory regions of the hundreds of Nrf2 target genes. The AREs are key enhancers that are activated in response to endogenous or exogenous stresses to maintain cellular and tissue homeostasis. Data emanating from gene expression microarray analyses comparing Nrf2-disrupted and wild-type mouse embryonic fibroblasts (MEF) showed that expression of Notch1 and Notch-signaling-related genes were decreased in Nrf2-disrupted cells. This observation triggered our research on Nrf2–Notch crosstalk. A functional ARE has been identified upstream of the Notch1 major transcription start site. Furthermore, an Rbpjκ binding site is conserved on the promoters of Nrf2 among animal species. Notch1 is one of the transmembrane Notch family receptors that drive Notch signaling, together with the Rbpjκ transcription factor. After canonically accepting ligands such as Jags and Deltas, the receptor undergoes cleavage to yield the Notch intracellular domain, which translocates to the nucleus. Recent studies using conditional knockout mice indicate that Notch1 as well as Notch2 plays an important role postnatally in liver development and in maintenance of hepatic function. In this review, we summarize current understanding of the role of reciprocal transcriptional regulation between Nrf2 and Notch in adult liver from studies using Nrf2, Keap1, and Notch1 genetically engineered mice.

Introduction

Molecular pathways that contribute to basic biological functions are often conserved across species. Both the canonical Notch and Nrf2 signaling pathways are evolutionally conserved from C. elegans to higher vertebrate animals [1], [2]. Interestingly, both are transcription factors. Nrf2 and Notch signaling pathways were discovered and described independently. However, the use of conditional knock-out and other genetically engineered mice has begun to unveil both the presence and the functional significance of Nrf2–Notch reciprocal signaling interactions, principally in adult tissues.

In the earliest phase of Notch research, genetic models employed lower species such as Drosophila and C. elegans. Indeed, a gene locus was discovered from the phenotype of a mutant fly with an indentation in the wings. The gene in the locus responsible for this phenotype, which was later called “Notch,” was considered to play a role in cell fate decisions during Drosophila embryogenesis. Moreover, a deletion mutation of this locus resulted in excessive differentiation to neuronal tissue. Subsequent molecular biological analyses revealed that the Notch gene encoded a single-pass transmembrane protein that functioned as a receptor for ligands on the cell surfaces of neighboring cells. This ligand–receptor interaction was verified consequently to enable the fate decisions of the signal-receiving cells to become non-neuronal cells by restraining neuronal differentiation; this process leads to “lateral specification,” which is essential for normal embryonic development [3].

After the concept of Notch signaling had been established in lower animal models, a gene located at the break point on chromosome 9 in the t(7;9)(q34;q34) translocation in a subset of acute T lymphoblastic leukemias in humans was identified as a Notch homolog. It was named translocation-associated Notch homolog 1 (TAN-1) [4]. This gene is currently called NOTCH1, and this discovery revealed that the Notch genes are highly conserved from nematodes up to humans. Leukemia cells harboring the t(7; 9) translocation express a truncated NOTCH1, which does not include a large part of the extracellular domain. This truncated NOTCH1, consequently, is detected in the intracellular space and acts as a constitutively activated transcription factor. Enhanced Notch signaling, such as that transduced by TAN1, has led NOTCH1 to be considered an oncogenic factor.

Nowadays, it is clear that the Notch signaling pathway influences cell fate decisions in animals, such as cell differentiation, survival/apoptosis, and cell cycle in both physiologic and pathologic contexts, while also playing a role in stem cell biology. Many Notch signaling modifier proteins including Rbpjκ have been identified, and the presence of a noncanonical, Rbpjκ-independent pathway has also been described [5], [6]. This review will focus on Rbpjκ-dependent canonical Notch signaling [7].

Notch encodes single-pass transmembrane receptors: there are 4 Notch genes (Notch1Notch4) and 5 specific ligand genes (Jag1, Jag2, Delta-like <Dll> 1, Dll3, Dll4). The ligand genes also encode single-pass transmembrane proteins in mammals. As summarized in Fig. 1, Notch protein products synthesized de novo first undergo intramolecular cleavage to form heterodimers, composed of extracellular and transmembrane subunits localized to the plasma membrane. Once receptor–ligand interactions occur, the Notch molecules in the target cells are processed by two successive proteolytic cleavages. The first cleavage begins extracellularly, close to the transmembrane domain, and is mediated by metalloproteases of the ADAM family [8]. The cleaved extracellular domain of Notch is trans-endocytosed by the ligand-presenting neighboring cell. The second cleavage proceeds within the transmembrane domain and is mediated by γ-secretase, which is a multiple protein complex consisting of Presenilin, Nicastrin, Aph1a (Anterior pharynx defective 1 homolog), and Psenen (presenilin enhancer 2 homolog) proteins. The cleavage by γ-secretase permits translocation of the Notch intracellular domain (NICD) fragment into the nucleus, where it binds to Rbpjκ, a direct cis-element binding protein, through the RAM (Rbp-associated molecule) domain and ankyrin repeats. Further, NICD associates with Mastermind-like proteins (MAMLs) through ankyrin repeats and recruits transcriptional activators such as p300, finally converting the Rbpjκ complex from a transcriptional repressor into an activator. Then target genes begin to be expressed. Hes (hairy and enhancer of split) [9], [10] encodes a basic helix loop helix inhibitory transcription factor that leads to self-renewal of target cells by inhibiting differentiation. Hes is one of the best characterized of the Notch target genes. The cell cycle promoter CyclinD1, the proliferation-related gene c-Myc, the anti-apoptotic gene Bcl2, the gene for Notch-regulated ankyrin repeat protein (Nrarp), Deltex1, and the pre–T-cell receptor gene (Ptcra) have also been identified as Notch target genes [11]. Thus, mechanistically, Notch transduces the signal received on the plasma membrane of the target cell into the nucleus to contribute to gene expression through collaboration with the specific transcription factor Rbpjκ. Responses are dependent upon cellular context.

The Notch pathway in mammals can exert pleiotropic effects in each tissue that expresses Notch. Thus, Notch-signaling networks regulate a wide range of events in embryonic and postnatal development, including proliferation, apoptosis, border formation, and cell fate decisions. Four major events are regulated through Notch. (i) Prevention of differentiation: Notch maintains stem cells and/or transit amplifying cells in an undifferentiated state in the intestinal crypt by inhibiting expression of differentiation-promoting genes [12], [13]. (ii) Binary cell fate decisions: In the lymphoid system Notch specifies the T-cell lineage at the expense of the B-cell lineage from bipotent early thymocyte progenitors [14]. Equipotent precursor cells provide two alternative cell fates depending on whether an uncommitted progenitor cell receives a strong Notch signal or not [15]. Hepatocholangiogenesis in the liver may be included in this context. (iii) Induction of differentiation: This phenomenon follows in a different or an opposite context from (i). For example, Notch induces terminal differentiation of transit-amplifying cells in the skin. During thymocyte differentiation Notch1 promotes differentiation of pro-T cells into pre-T cells [14]. (iv) Tumorigenesis: Constitutive overexpression of Notch within hematopoietic bone marrow cells or in T-cell progenitors results in T-cell leukemias. Hence, Notch functions as an oncogene in this case [16]. However, Notch seems to function as a tumor suppressor in the skin, since the loss of Notch signaling causes the development of basal cell carcinoma-like tumors [17].

Thus, although both the canonical Notch pathway and the Notch–Rbpjκ transcriptional machinery are conserved, the resulting biological responses are rich in variety, sometimes even bringing about opposite outcomes in different tissues of mammals. Such diversity in responses is a peculiarity of Notch signaling. Elucidation of Notch signaling crosstalk with other signaling pathways might shed light on these Notch-mediated effects and provide better understanding of their underlying mechanisms.

Nrf2 is a member of the CNC family of transcription factors, which includes Nrf1, 2, and 3, Bach1 and 2, and NF-E2p45 [2]. Expression of Nrf2 target genes is highly and rapidly inducible following exposure of cells to endogenous and exogenous oxidants and electrophilic stresses [18], [19]. Control of this expression is mediated through one or more cis-elements located on the 5’ flanking gene regulatory regions as enhancers, or sometimes suppressors, for gene expression. Initially, this cis-element was called the “electrophile-responsive element” (EpRE) because the first compounds demonstrated to activate this element were electrophiles or compounds easily oxidized to electrophiles [20]. Follow-up studies by others using phenolic antioxidants provided the nomenclature “antioxidant responsive element” (ARE) [21], which has largely superseded the use of EpRE, despite a poor reflection of the chemistry of pathway activation. The CNC family transcription factors possess a basic leucine zipper domain for direct DNA binding, principally to the ARE. CNC transcription factors, including Nrf2, are conserved among species, as happens with the Notch family. Nrf2 heterodimerizes with small Maf protein (sMaf) to elicit the most potent gene expression mediated through the ARE transcriptosome. Global gene expression analyses in Nrf2 null mouse embryonic fibroblasts or in Nrf2 null mice have revealed that ARE-containing genes regulated by Nrf2 include a wider array of genes, which have important biological functions for cell and organism survival. Furthermore, these studies have confirmed that Nrf2 can contribute to the basal and/or induced or repressed expression of its target genes, depending on the particular gene and its cellular context [18].

Keap1 was strategically discovered using the yeast two-hybrid system based on the predicted existence of a negative regulator acting through the Neh2 domain of Nrf2 [22]. Keap1 possesses bric à brac, tram track and broad complex (BTB) domains and β-propeller structures carrying 6-blades contained in the Kelch domain. Keap1, principally localized in the cytoplasm, serves as a scaffold for the degrasome complex for Nrf2 by coupling with the Cul3–Ubiquitin system [23]. Keap1 null mice died postnatally due to malnutrition caused by constriction of the esophagus and cardia of the stomach due to hyperkeratinization. However, the Keap1 null mice were rescued completely by deleting the Nrf2 gene [24]. Furthermore, the relative expression of almost all Nrf2 target genes, such as Nqo1, Gclc, and Gclm, which were initially defined by comparisons between Nrf2 null and wild-type mice, has a generally inverse relation to the relative expression of the same genes when Keap1 null and wild-type mice are compared, due to the constitutive expression of Nrf2 in the Keap1 null mice. Thus, the specificity of Keap1–Nrf2 molecular interaction was elucidated in vivo. ChIP-seq and other approaches have greatly subsequently expanded the list of Nrf2 target genes [25].

The essence of the Nrf2–ARE signaling system lies in the sequestration of Nrf2 away from the nucleus in the basal state and the translocation and accumulation in the nucleus as a rapid response to stressors, including ROS, in the induced state [18]. Mechanistically, in the basal state, the majority of Nrf2 synthesized de novo is undergoing proteasomal degradation. When phosphorylation occurs on the Neh6 domain of Nrf2, the Cul1–β-Trcp system can modify it, either in the cytoplasm or in the nucleus [26]; however, the Keap1–Cul3 system marks Nrf2 synthesized de novo through polyubiquitination, leading to rapid proteasomal degradation. In the nucleus, the exportin complex transports Nrf2 to the cytoplasm. In this situation, basal expression of target genes is maintained, whereas, in the induced state, modification of the Keap1–Cul3 complex through interactions of reactive cysteines with electrophiles or ROS leads to conformational changes disrupting the degrasome function of the complex. This outcome allows newly synthesized Nrf2 access to the nucleus and results in altered expression of target genes [27] (Fig. 2).

Section snippets

Nrf2 and Notch1 expression in MEF

Differential gene expression analyses using microarrays, and more recently ChIP-Seq and RNA-Seq technologies, have been the most effective ways to provide insights into the downstream targets of signaling pathways. Many microarray analyses have been performed in various tissues of wild-type and Nrf2 null mice to define cytoprotective pathways and to characterize the role of the Keap1–Nrf2–ARE pathway in the pharmacodynamic action of several classes of anticarcinogens [28]. One limitation of

Biological significance of Nrf2–Notch crosstalk

In general, Nrf2 is widely expressed in tissues and various cell types. However, careful analysis of the data by the Kan group [30] indicates that Nrf2 mRNA expression patterns are not held at a constant level. Their in situ hybridization analyses clearly showed differences in expression levels among tissues and cells types. Hence the conclusion “The various cells can express the Nrf2 gene opportunely when it is required.” This variable expression pattern makes Nrf2–Notch crosstalk much more

Conclusions

Nrf2–ARE signaling and Notch signaling can be regulated by reciprocal transcriptional machinery, at least postnatally. Namely, Notch1 is an Nrf2 target gene and Nrf2 is a Notch–Rbpjκ target gene. Nrf2–Notch crosstalk influences the expression of defense systems against endogenous and exogenous stressors, leading to cytoprotection, and enhances maintenance of cellular homeostasis and tissue organization through actions on cell proliferation kinetics and cell fate determinations of stem cell

Acknowledgments

This work is supported by National Institutes of Health Grants CA094076 and CA197222 to TWK. DVC is supported by a Marie Curie IOF (PIOF-GA-2012–329442) within the 7th European Community Framework Programme.

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