Shotaro Nakajima

Activation of the Akt-NF-κB Pathway by Subtilase Cytotoxin through the ATF6 Branch of the Unfolded Protein Response

Shiga toxin has the potential to induce expression of inflammation-associated genes, although the underlying mechanisms are not well understood. We examined the effects of subtilase cytotoxin (SubAB), an AB5 toxin produced by some Shiga toxigenic Escherichia coli, on the activation of NF-κB. SubAB is known to be a protease which selectively degrades GRP78/Bip. Treatment of NRK-52E cells with SubAB caused rapid cleavage of GRP78. Following the degradation of GRP78, transient activation of NF-κB was observed with a peak at 6–12 h; the activation subsided within 24 h despite the continuous absence of intact GRP78. The activation of NF-κB was preceded by transient phosphorylation of Akt. Treatment of the cells with a selective inhibitor of Akt1/2 or an inhibitor of PI3K attenuated SubAB-induced NF-κB activation, suggesting that activation of Akt is an event upstream of NF-κB. Degradation of GRP78 caused the unfolded protein response (UPR), and inducers of the UPR mimicked the stimulatory effects of SubAB on Akt and NF-κB. SubAB triggered the three major branches of the UPR including the IRE1-XBP1, PERK, and ATF6 pathways. Dominant-negative inhibition of IRE1α, XBP1, or PERK did not attenuate activation of NF-κB by SubAB. In contrast, genetic and pharmacological inhibition of ATF6 significantly suppressed SubAB-triggered Akt phosphorylation and NF-κB activation. These results suggested that loss of GRP78 by SubAB leads to transient phosphorylation of Akt and consequent activation of NF-κB through the ATF6 branch of the UPR.

mTORC1 serves ER stress-triggered apoptosis via selective activation of the IRE1–JNK pathway

Mammalian target of rapamycin (mTOR) has a key role in the regulation of an array of cellular function. We found that rapamycin, an inhibitor of mTOR complex 1 (mTORC1), attenuated endoplasmic reticulum (ER) stress-induced apoptosis. Among three major branches of the unfolded protein response, rapamycin selectively suppressed the IRE1–JNK signaling without affecting PERK and ATF6 pathways. ER stress rapidly induced activation of mTORC1, which was responsible for induction of the IRE1–JNK pathway and apoptosis. Activation of mTORC1 reduced Akt phosphorylation, which was an event upstream of IRE–JNK signaling and consequent apoptosis. In vivo, administration with rapamycin significantly suppressed renal tubular injury and apoptosis in tunicamycin-treated mice. It was associated with enhanced phosphorylation of Akt and suppression of JNK activity in the kidney. These results disclosed that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the IRE1–JNK pathway.

Bidirectional regulation of NF-κB by reactive oxygen species: A role of unfolded protein response

Nuclear factor-κB (NF-κB) is a transcription factor that plays a crucial role in coordinating innate and adaptive immunity, inflammation, and apoptotic cell death. NF-κB is activated by various inflammatory stimuli including peptide factors and infectious microbes. It is also known as a redox-sensitive transcription factor activated by reactive oxygen species (ROS). Over the past decades, various investigators focused on the role of ROS in the activation of NF-κB by cytokines and lipopolysaccharides. However, recent studies also suggested that ROS have the potential to repress NF-κB activity. Currently, it is not well addressed how ROS regulate activity of NF-κB in a bidirectional fashion. In this paper, we summarize evidence for positive and negative regulation of NF-κB by ROS, possible redox-sensitive targets for NF-κB signaling, and mechanisms underlying biphasic and bidirectional influences of ROS on NF-κB, especially focusing on a role of ROS-mediated induction of endoplasmic reticulum stress.

Selective Abrogation of BiP/GRP78 Blunts Activation of NF-κB through the ATF6 Branch of the UPR: Involvement of C/EBPβ and mTOR-Dependent Dephosphorylation of Akt

ABSTRACT Subtilase cytotoxin (SubAB) that selectively cleaves BiP/GRP78 triggers the unfolded protein response (UPR) and protects mice from endotoxic lethality and collagen arthritis. We found that pretreatment of cells with SubAB suppressed tumor necrosis alpha (TNF-α)-induced activation of NF-κB and NF-κB-dependent chemokine expression. To elucidate underlying mechanisms, the involvement of C/EBP and Akt, putative regulators of NF-κB, was investigated. Among members of the C/EBP family, SubAB preferentially induced C/EBPβ. Overexpression of C/EBPβ suppressed TNF-α-induced NF-κB activation, and knockdown of C/EBPβ attenuated the suppressive effect of SubAB on NF-κB. We identified that the ATF6 branch of the UPR plays a crucial role in the induction of C/EBPβ. In addition to this effect, SubAB depressed basal and TNF-α-induced phosphorylation of Akt via the UPR. It was mediated by the induction of ATF6 and consequent activation of mTOR that dephosphorylated Akt. Inhibition of Akt attenuated activation of NF-κB by TNF-α, suggesting that the mTOR-Akt pathway is another target for SubAB-initiated, UPR-mediated NF-κB suppression. These results elucidated that SubAB blunts activation of NF-κB through ATF6-dependent mechanisms, i.e., preferential induction of C/EBPβ and mTOR-dependent dephosphorylation of Akt.

Acquisition of Anergy to Proinflammatory Cytokines in Nonimmune Cells through Endoplasmic Reticulum Stress Response: A Mechanism for Subsidence of Inflammation

Acute endoplasmic reticulum (ER) stress causes induction of inflammatory molecules via activation of NF-κB. However, we found that, under ER stress conditions, renal mesangial cells acquire anergy to proinflammatory stimuli. Priming of the cells with ER stress inducers (tunicamycin, thapsigargin, A23187, and AB5 subtilase cytotoxin) caused blunted induction of MCP-1 in response to TNF-α, IL-1β, macrophage-derived factors, or bystander macrophages. The magnitude of suppression was closely correlated with the level of GRP78, an endogenous indicator of ER stress. The suppression of MCP-1 under ER stress conditions was reversible and observed in general regardless of cell types or triggers of ER stress. The decrease in the level of MCP-1 mRNA was ascribed to transcriptional suppression via unexpected inhibition of NF-κB, but not to accelerated mRNA degradation. Subsequent experiments revealed that TNFR-associated factor 2, an essential component for TNF-α signaling, was down-regulated by ER stress. We also found that, under ER stress conditions, expression of NF-κB suppressor A20 was induced. Overexpression of A20 resulted in suppression of cytokine-triggered NF-κB activation and knockdown of A20 by RNA interference significantly attenuated induction of anergy by ER stress. In contrast, other ER stress-inducible/-related molecules that may suppress NF-κB (e.g., GRP78, NO, reactive oxygen species, and IκB) were not involved in the inhibitory effects of ER stress. These results elucidated ER stress-dependent mechanisms by which nonimmune cells acquire anergy to inflammatory stimuli under pathological situations. This self-defense machinery may play a role in halting progression of acute inflammation and in its spontaneous subsidence.

ER Stress Depresses NF-κB Activation in Mesangial Cells through Preferential Induction of C/EBPβ

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 NF-kappaB, 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 NF-kappaB. Here, we investigated a role for C/EBPs in the ER stress-induced resistance to cytokines. Mesangial cells preferentially induced C/EBPbeta after exposure to thapsigargin or tunicamycin; induction of C/EBPdelta was modest and transient, and expression of C/EBPalpha was absent. The induction of C/EBPbeta correlated with accumulation of C/EBPbeta protein and enhanced transcriptional activity of C/EBP. Overexpression of C/EBPbeta markedly suppressed TNF-alpha-induced activation of NF-kappaB, independent of its transacting potential. Knockdown of C/EBPbeta by small interfering RNA reversed the suppressive effect of ER stress on NF-kappaB. In vivo, preconditioning of mice with ER stress induced renal C/EBPbeta and suppressed NF-kappaB-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/EBPbeta. These results suggest that ER stress blunts cytokine-triggered activation of NF-kappaB, in part through PERK- and IRE1-mediated preferential induction of C/EBPbeta.

Suppression of nephrin expression by TNF-α via interfering with the cAMP-retinoic acid receptor pathway

Nephrin, a crucial component of the slit diaphragm, is downregulated in proteinuric glomerular diseases including glomerulonephritis. We previously reported that 1) expression of nephrin in cultured podocytes is reinforced by retinoic acid (RA) and 1,25-dihydroxyvitamin D(3), 2) these effects are mediated by retinoic acid receptor (RAR) and vitamin D receptor (VDR), and 3) basal and inducible expression of nephrin is downregulated by TNF-alpha. In the present investigation, we identified that TNF-alpha selectively represses activity of RAR but not VDR. To elucidate mechanisms underlying this observation, we tested involvement of downstream targets for TNF-alpha: nuclear factor-kappaB (NF-kappaB), mitogen-activated protein (MAP) kinases, phosphatidylinositol 3-kinase (PI3K)-Akt, and cAMP-protein kinase A (PKA). TNF-alpha caused activation of NF-kappaB, MAP kinases, and PI3K-Akt in podocytes, whereas blockade of these molecules did not affect inhibition of RAR by TNF-alpha. In contrast, TNF-alpha depressed activity of cAMP-PKA, and blockade of PKA inhibited basal and RA-induced activation of RAR. Furthermore, activity of RAR was significantly upregulated by cAMP, and the suppressive effect of TNF-alpha on RAR was reversed by cAMP-elevating agents. These results suggest that 1) expression of nephrin in podocytes is regulated by the cAMP-RAR pathway and 2) suppression of nephrin by TNF-alpha is caused, at least in part, through selective inhibition of this pathway.

Inhibition of NF-κB by MG132 through ER stress-mediated induction of LAP and LIP

Proteasome inhibitor MG132 blocks activation of NF‐κB by preventing degradation of IκB. In this report, we propose an alternative mechanism by which MG132 inhibits cytokine‐triggered NF‐κB activation. We found that MG132 induced endoplasmic reticulum (ER) stress, and attenuation of ER stress blunted the suppressive effect of MG132 on NF‐κB. Through ER stress, MG132 up‐regulated C/EBPβ mRNA transiently and caused sustained accumulation of its translational products liver activating protein (LAP) and liver‐enriched inhibitory protein (LIP), both of which were identified as suppressors of NF‐κB. Our results disclosed a novel mechanism underlying inhibition of NF‐κB by MG132.

Selective, potent blockade of the IRE1 and ATF6 pathways by 4‐phenylbutyric acid analogues

4‐Phenylbutyric acid (4‐PBA) is a chemical chaperone that eliminates the accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, its chaperoning ability is often weak and unable to attenuate the unfolded protein response (UPR) in vitro or in vivo. To develop more potent chemical chaperones, we synthesized six analogues of 4‐PBA and evaluated their pharmacological actions on the UPR.

Purinergic control of AMPK activation by ATP released through connexin 43 hemichannels - pivotal roles in hemichannel-mediated cell injury.

ABSTRACT Connexin hemichannels regulate many cell functions. However, the molecular mechanisms involved remain elusive. Hemichannel opening causes loss of ATP, we therefore speculated a potential role for AMPK in the biological actions of hemichannels. Activation of hemichannels by removal of extracellular Ca2+ led to an efflux of ATP and a weak activation of AMPK. Unexpectedly, dysfunction of hemichannels markedly potentiated AMPK activation, which was reproduced by promotion of extracellular ATP degradation or inhibition of P2 purinoceptors but counteracted by exogenous ATP. Further analysis revealed that ATP induced a purinoceptor-dependent activation of Akt and mTOR. Suppression of Akt or mTOR augmented AMPK activation, whereas activation of Akt by transfection of cells with myristoylated Akt, a constitutively active form of Akt, abolished AMPK activation. In a pathological model of hemichannel opening triggered by Cd2+, disclosure of hemichannels similarly enhanced AMPK activity, which protected cells from Cd2+-induced cell injury through suppression of mTOR. In summary, our data point to a channel-mediated mechanism for the regulation of AMPK through a purinergic signaling pathway. Furthermore, we define AMPK as a pivotal molecule that underlies the regulatory effects of hemichannels on cell survival.