XBP1U inhibits the XBP1S-mediated upregulation of the iNOS gene expression in mammalian ER stress response

doi:10.1016/j.cellsig.2010.07.006

Cellular Signalling

Volume 22, Issue 12, December 2010, Pages 1818-1828

https://doi.org/10.1016/j.cellsig.2010.07.006 Get rights and content

Abstract

Upregulation of the inducible nitric oxide synthase (iNOS) gene is associated with many pathological conditions such as endoplasmic reticulum (ER) stress, and X-box binding protein 1 (XBP1) is critical in mediating ER-stress responsive genes, including iNOS. Nonetheless, the mechanism by which XBP1 regulates iNOS during ER stress remains unexplored. Here we show that the active/spliced form of XBP1 protein, XBP1S, directly binds to the AABS (A-activator-binding site) in the iNOS promoter in vitro and in living cells. XBP1S exhibits dose-dependent activation of iNOS-specific reporter gene activity and endogenous iNOS expression. XBP1S is elevated whereas the unspliced form of XBP1, XBP1U, reduced in ER stress in HepG2 cells. In addition, XBP1U binds to XBP1S and this complex is associated with the iNOS promoter in response to ER stress. Furthermore, XBP1U acts as a negative mediator and suppresses XBP1S-mediated induction of iNOS. Collectively, we present the first evidence demonstrating the regulation of iNOS gene induction by the interaction between the spliced and unspliced forms of XBP1 in response to ER stress.

Introduction

Eukaryotic cells have evolved sophisticated mechanisms to sense stress in various intracellular compartments and respond appropriately by modulating nuclear gene expression. Within the endoplasmic reticulum (ER), stress is induced by the presence of large amounts of unfolded or misfolded proteins, which lead to signal transduction events in both the cytosol and nucleus. Cells can alleviate this stress by degrading or refolding these improperly folded proteins [1], [2], [3]. ER stress activates a set of signaling pathways, collectively termed the Unfolded Protein Response (UPR). The UPR consists of three molecular branches (inositol requiring enzyme 1, IRE1; PKR-like ER-resistant kinase, PERK and activating transcription factor 6, ATF6), which promote cell survival by reducing misfolded protein levels. Human X-box binding protein 1 (XBP1) is a signaling molecule downstream of IRE1 and ATF6 (in the IRE1-XBP1 pathway of the UPR), and participates in IRE1 α-mediated UPR signal transmission. XBP1 also helps to coordinate IRE1α-dependent XBP1 mRNA splicing [4], [5], [6], [7]. XBP1 exists in two forms; following transcription, XBP1 mRNA can either undergo processing or remain unprocessed to generate spliced (XBP1S) or unspliced (XBP1U) isoforms. In mammalian cells, IRE1 is activated by ER stress and subsequently processes XBP1 mRNA to generate the spliced form of XBP1 protein (XBP1S). XBP1S activates the promoters of many genes, including those coding for enzymes necessary for the degradation of improperly folded ER proteins. XBP1S performs this task by binding to unfolded protein response elements (UPRE) with the consensus sequence GTGNNGT/CAAC [8], [9].

XBP1, which contains a basic region-leucine zipper (bZIP) structure, is an important transcription factor within the cAMP Response Element Binding Protein/Activating Transcription Factor (CREB/ATF) family. XBP1 is ubiquitously expressed in adult tissues. It contains a sequence-specific DNA-binding domain and a transcriptional activation domain, each encoded by a separate open reading frame. In vitro, XBP1-binding sites have been shown to contain a consensus 8-bp core motif that is very similar to CREB binding sites and identical to the ATF6-binding site [10], [11], [12]. Although there is strong evidence that XBP1 plays an important role in regulating the cell's response to ER stress, little is known about the modulation and physiological significance of XBP1 induction. Specifically, efforts to identify and characterize the target genes of XBP1 have been incomplete.

We previously reported that XBP1S can bind to the promoter of the inducible nitric oxide synthase (iNOS) gene, as determined using a human liver cDNA library screen via phage display [20]. Nitric Oxide (NO) is a short-lived biological mediator that plays a key role in many physiological and pathological conditions, including inflammation, neurotransmission, and ER stress. The human iNOS (hiNOS) gene, which regulates NO production, is located on chromosome 17, spans 37 kb, and contains 26 exons. hiNOS is tightly regulated at the level of transcription, and the mechanisms of iNOS induction and suppression are cell, species, and stimulus specific [13], [14]. Moreover, depending on the cell type studied, different regions of the 5′-flanking sequence can mediate cytokine induction of the hiNOS gene. All of these findings suggest that cell and stimulus-specific control of the hiNOS promoter may be important in determining its functional role in different tissues [15], [16]. In the liver, lipopolysaccharide and inflammatory cytokines such as interleukin-1 and tumour necrosis factor-α (TNFα) can induce iNOS expression and provoke prominent NO production. It has been found that the iNOS promoter sequence spans from − 400 bp to the initiation of transcription. Within this region, there exist three conserved motifs for the potential binding of transcription factors. They are as follows: distal binding site for CCAAT/enhancer-binding protein (C/EBP) (− 334 bp to − 326 bp in the human gene); proximal C/EBP-binding site (− 192 bp to − 183 bp) corresponding to the A-activator-binding site (AABS) (“GTGNNGYAA”); and nuclear factor (NF)-κB-binding site (− 115 to − 106 bp). In hepatocytes, iNOS expression is highly regulated by cytokines, endotoxins, and glucocorticoids. The functional binding between AABS and the active form of C/EBPβ (LAP) is essential for liver specific transcriptional regulation of the hiNOS gene [17], [18]. However, the regulatory role of the iNOS gene during conditions of ER stress is unknown. Additionally, it is unknown whether XBP1S, an important activator of the UPR during ERS [6], plays any role in the regulation of the iNOS gene.

In the present study, we analyzed the 1032 bp promoter of the human iNOS gene and constructed a series of iNOS promoter deletion reporter plasmids. We then measured the transcriptional activity of the human iNOS gene promoter and its deletions in HepG2 cells. Our assays revealed that the region of the iNOS promoter from − 286 bp to transcription initiation (including the AABS and NF-κB-binding sites), is required for iNOS gene expression. To study the transcriptional regulation of hiNOS by XBP1 during conditions of ER stress, we determined 1) whether XBP1S can regulate iNOS gene transcription and expression; 2) whether there exists a protein interaction between XBP1U and XBP1S during conditions of ERS; 3) whether XBP1U interferes with XBP1S expression during ERS; and 4) whether XBP1U interferes with XBP1S-mediated regulation of iNOS during ERS. The results we present here indicate that XBP1U can bind to and inhibit XBP1S, thus reversing the XBP1S-mediated upregulation of iNOS expression during ERS.

Section snippets

Construction of plamids

The XBP1S expression plasmid, iNOS reporter plasmid were generated using standard DNA techniques. The human XBP1S, including 1130 bp of open reading frame, was cloned between the BamHI and HindIII (underlined) restriction sites of the pcDNA3.1(−) vector(Oligoengine) using the following primer pair: 5′-GGATCCATGGTGGTGGTGGC AGCCGC-3′, 5′-AAGCTT GACACTAATCAGCTGGGGAAAGAG-3′. pGL2-iNOS, which contains the luciferase reporter gene adjacent to the iNOS 3′-UTR, was generated by cloning the iNOS 3′-UTR

XBP1S binds to the AABS in iNOS promoter in vitro

Using phage display analysis of a human liver cDNA library, we previously discovered that XBP1S binds to the iNOS promoter. In our initial sequence analysis of the human iNOS promoter, we found an AABS at − 192 bp of the hiNOS 5′-flanking region [19], [20]. This site was a 9/9 nucleotide match for the AABS consensus sequence. However, the mere presence of a DNA consensus sequence does not necessarily establish the presence of a functional cis-acting protein–DNA interaction. To determine whether

Discussion

Acknowledgments

This work was supported by the Natural Science Foundation Project of CQ CSTC, No. 2009 BB5062, Education Ministry Study Abroad Staff Foundation, No. 2009-1590, and ChongQing Medical University Key Research Foundation, No. XBZD200803.

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XBP1U inhibits the XBP1S-mediated upregulation of the iNOS gene expression in mammalian ER stress response

Abstract

Introduction

Section snippets

Construction of plamids

XBP1S binds to the AABS in iNOS promoter in vitro

Discussion

Acknowledgments

Cell

J. Biol. Chem.

Cell Metab.

J. Biol. Chem.

J. Biol. Chem.

Cell

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem Biophys Res Commun.

J. Biol. Chem.

Mol. Immunol.

Cell

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods

Nature

Nature

Physiol. Rev.

J. Cell Biol.

J. Biochem.