ER Stress Depresses NF- B Activation in Mesangial Cells through Preferential Induction of C/EBP

Modest induction of endoplasmic reticulum (ER) stress confers resistance to inflammation in glomeruli. Recently, we found that ER stress leads to mesangial insensitivity to cytokine-induced activation of NFB, but the underlying mechanisms are incompletely understood. ER stress can trigger expression of CCAAT/enhancer-binding proteins (C/EBPs), which interact with transcription factors including NFB. Here, we investigated a role for C/EBPs in the ER stress–induced resistance to cytokines. Mesangial cells preferentially induced C/EBP after exposure to thapsigargin or tunicamycin; induction of C/EBP was modest and transient, and expression of C/EBP was absent. The induction of C/EBP correlated with accumulation of C/EBP protein and enhanced transcriptional activity of C/EBP. Overexpression of C/EBP markedly suppressed TNF–induced activation of NFB, independent of its transacting potential. Knockdown of C/EBP by small interfering RNA reversed the suppressive effect of ER stress on NFB. In vivo, preconditioning of mice with ER stress induced renal C/EBP and suppressed NFB– dependent gene expression in response to LPS. Using dominant negative mutants and null mutants for individual branches of the unfolded protein response, we identified the RNA-dependent protein kinase– like ER kinase (PERK) and the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) pathways as the unfolded protein response responsible for ER stress–induced C/EBP . These results suggest that ER stress blunts cytokine-triggered activation of NFB, in part through PERKand IRE1-mediated preferential induction of C/EBP . J Am Soc Nephrol 21: 73–81, 2010. doi: 10.1681/ASN.2009040432 Endoplasmic reticulum (ER) stress is defined as accumulation of unfolded or misfolded proteins in the ER, which triggers an adaptive program called the unfolded protein response (UPR). 1,2 Three major transmembrane transducers for sensing ER stress have been identified in the ER. Those are RNA-dependent protein kinase–like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring ER-to-nucleus signal kinase 1 (IRE1). Activation of PERK leads to phosphorylation of eukaryotic translation initiation factor 2 (eIF2 ), which causes general inhibition of protein synthesis and induction of a transcription factor ATF4 that binds to the amino acid response element. In response to ER stress, p90ATF6 transits to the Golgi, where it is cleaved by the proteases S1P and S2P, yielding a free cytoplasmic domain p50ATF6, which functions as an active transcription factor. Similarly, activated IRE1 catalyzes removal of a small intron from X-box– binding proReceived April 23, 2009. Accepted September 18, 2009. Published online ahead of print. Publication date available at www.jasn. org. Correspondence: Dr. Masanori Kitamura, Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi 409-3898, Japan. Phone: 81-55-273-8054; Fax: 81-55-273-8054; E-mail: masanori@yamanashi.ac. jp Copyright 2010 by the American Society of Nephrology BASIC RESEARCH www.jasn. org J Am Soc Nephrol 21: 73–81, 2010 ISSN : 1046-6673/2101-73 73 tein 1 (XBP1) mRNA. This splicing event creates a translational frameshift in XBP1 mRNA to produce an active transcription factor. p50ATF6 and XBP1 subsequently bind to the ER stress response element (ERSE) and the UPR element (UPRE), leading to expression of target genes including ER chaperones and genes involved in ER stress–associated degradation. Activation of IRE1 also allows for interaction of IRE1 with TNF receptor–associated factor 2 (TRAF2), leading to recruitment and activation of apoptosis signal–regulating kinase 1 (ASK1) and downstream c-Jun N-terminal kinase (JNK). 1,2 ER stress is observed under various pathologic situations, including inflammation.3 Previous studies suggested that ER stress has the potential to activate NFB and thereby contributes to the development of inflammation, as reviewed by Zhang and Kaufman4; however, in the kidney, previous reports showed that UPR was induced in glomeruli of rats with passive Heymann nephritis and that ER stress preconditioning attenuated proteinuria in this experimental model.5,6 Inagi et al.7 also reported that, in the anti-Thy1 model of mesangioproliferative glomerulonephritis in rats, UPR was induced in nephritic glomeruli, especially in podocytes and mesangial cells. They also showed that preconditioning with subnephritogenic doses of ER stress inducers ameliorated the pathologic manifestations of the disease; however, mechanisms underlying the anti-inflammatory potential of ER stress and UPR have not been elucidated. Recently, we reported that preceding ER stress causes insensitivity of mesangial cells to cytokine-induced activation of NFB via upregulation of A20 and downregulation of TRAF28; however, our data indicated that the roles of A20 and TRAF2 are partial and that other major targets of ER stress may also be present. To uncover additional mechanisms involved, we focused this investigation on a role of CCAAT/enhancerbinding protein (C/EBP) in the ER stress–induced cytokine insensitivity in mesangial cells. C/EBP is a family of transcription factors that are required for development, physiologic function, and responses to injury in various tissues.9 A previous report showed that some ER stress inducers triggered expression and production of C/EBP in human hepatoma cells.10 It is also known that C/EBP family members can interact with various transcription factors, including NFB,11 which may modulate function of NFB. We therefore investigated a role of C/EBP in the ER stress–induced suppression of NFB in mesangial cells. In this report, we demonstrate that ER stress depresses cytokine-induced activation of NFB through preferential induction of C/EBP . We also elucidate that particular UPRs, especially the PERKand IRE1-initiated pathways, are involved in this process.