Maro Okamura

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.

Involvement of Selective Reactive Oxygen Species Upstream of Proapoptotic Branches of Unfolded Protein Response

Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}, H2O2, or ONOO- caused apoptosis, whereas ER stress was induced only by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} or ONOO-. Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO- was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}-triggered activation of XBP1.

Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O(2)(*-) blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O(2)(*-) selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O(2)(*-) may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.

Transcriptional suppression of nephrin in podocytes by macrophages: Roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage‐derived cytokines IL‐1β and TNF‐α markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine‐initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms’ tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol‐3‐kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.

Suppression of NF-κB by Cyclosporin A and Tacrolimus (FK506) via Induction of the C/EBP Family: Implication for Unfolded Protein Response

Immunosuppressive agents cyclosporin A (CsA) and tacrolimus (FK506) inhibit cytokine production by activated lymphocytes through interfering with calcineurin. However, little is known about their effects on the function of nonlymphoid cells. We found that, in renal tubular cells, induction of MCP-1 by inflammatory cytokines was blunted by CsA and FK506. This suppression was correlated with induction of unfolded protein response (UPR) evidenced by endogenous and exogenous indicators. The induction of UPR by these agents was reversible and observed generally in other nonimmune cells. Furthermore, administration with CsA in reporter mice caused rapid, systemic induction of UPR in vivo. In TNF-α-treated cells, suppression of MCP-1 by CsA or FK506 was associated with blunted responses of NF-κB, the crucial regulator of MCP-1. The suppression of NF-κB was reproduced by other inducers of UPR including AB5 subtilase cytotoxin, tunicamycin, thapsigargin, and A23187. CsA and FK506, as well as other UPR inducers, caused up-regulation of C/EBP family members, especially C/EBPβ and CHOP (C/EBP homologous protein), and overexpression of either C/EBPβ or CHOP significantly attenuated TNF-α-triggered NF-κB activation. Furthermore, down-regulation of C/EBPβ by small interfering RNA substantially reversed the suppressive effect of CsA on TNF-α-induced MCP-1 expression. These results suggested that CsA and FK506 confer insensitiveness to TNF-α on resident cells through UPR-dependent induction of the C/EBP family members.

Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice.

Endoplasmic reticulum (ER) stress‐responsive alkaline phosphatase (ES‐TRAP) serves as a sensitive indicator for ER stress. In response to heavy metals including cadmium, nickel and cobalt, hepatocytes and renal tubular cells expressing ES‐TRAP exhibited ER stress and decreased ES‐TRAP activity. In ES‐TRAP transgenic mice, acute exposure to cadmium showed rapid, transient decreases in the activity of serum ES‐TRAP. It was inversely correlated with the induction of endogenous ER stress markers in the liver and kidney. Our result provides first evidence for the acute, reversible induction of ER stress in vivo after exposure to heavy metal.

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 cytokine responses by indomethacin in podocytes: a mechanism through induction of unfolded protein response

We found that, in murine podocytes, expression of monocyte chemoattractant protein 1 (MCP-1) in response to TNF-alpha was suppressed by indomethacin but not by ibuprofen. This anti-inflammatory potential was correlated with induction of 78-kDa glucose-regulated protein (GRP78), a marker of unfolded protein response (UPR). Indomethacin, but not ibuprofen, also triggered expression of CHOP, another endogenous indicator of UPR, as well as repression of endoplasmic reticulum stress-responsive alkaline phosphatase, an exogenous indicator of UPR. Like ibuprofen, other nonsteroidal anti-inflammatory drugs including aspirin and sulindac also did not induce UPR, indicating that the induction of UPR by indomethacin was independent of cyclooxygenase inhibition. The induction of UPR by indomethacin was observed similarly in other cells including mesangial cells and tubular epithelial cells. In tumor necrosis factor (TNF)-alpha-treated cells, suppression of MCP-1 by indomethacin was inversely correlated with induction of UPR, and other inducers of UPR including tunicamycin, thapsigargin, and A23187 reproduced the suppressive effect. Reporter assays showed that indomethacin as well as thapsigargin attenuated activation of NF-kappaB by TNF-alpha, and it was associated with enhanced degradation of TNF receptor-associated factor 2 (TRAF2) and blunted degradation of IkappaBbeta. Subsequent experiments revealed that acute ablation of GRP78 protein by AB5 subtilase cytotoxin caused reinforcement of MCP-1 induction and NF-kappaB activation by TNF-alpha and that transfection with GRP78 significantly suppressed the cytokine-induced activation of NF-kappaB. These results suggested that indomethacin suppressed the response of podocytes to TNF-alpha via UPR and that UPR-triggered induction of GRP78 and degradation of TRAF2 may be responsible, at least in part, for the suppressive effect of indomethacin.

Geranylgeranylacetone, an Inducer of the 70-kDa Heat Shock Protein (HSP70), Elicits Unfolded Protein Response and Coordinates Cellular Fate Independently of HSP70

Geranylgeranylacetone (GGA), an antiulcer agent, has the ability to induce 70-kDa heat shock protein (HSP70) in various cell types and to protect cells from apoptogenic insults. However, little is known about effects of GGA on other HSP families of molecules. We found that, at concentrations ≥100 μM, GGA caused selective expression of 78-kDa glucose-regulated protein (GRP78), an HSP70 family member inducible by endoplasmic reticulum (ER) stress, without affecting the level of HSP70 in various cell types. Induction of ER stress by GGA was also evidenced by expression of another endogenous marker, CCAAT/enhancer-binding protein-homologous protein (CHOP); decreased activity of ER stress-responsive alkaline phosphatase; and unfolded protein response (UPR), including activation of the activating transcription factor 6 (ATF6) pathway and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (IRE1-XBP1) pathway. Incubation of mesangial cells with GGA caused significant apoptosis, which was attenuated by transfection with inhibitors of caspase-12 (i.e., a dominant-negative mutant of caspase-12 and MAGE-3). Dominant-negative suppression of IRE1 or XBP1 significantly attenuated apoptosis without affecting the levels of CHOP and GRP78. Inhibition of c-Jun NH2-terminal kinase, the molecule downstream of IRE1, by 1,9-pyrazoloanthrone (SP600125) did not improve cell survival. Blockade of ATF6 by 4-(2-aminoethyl) benzenesulfonyl fluoride enhanced apoptosis by GGA, and it was correlated with attenuated induction of both GRP78 and CHOP. Overexpression of GRP78 or dominant-negative inhibition of CHOP significantly attenuated GGA-induced apoptosis. These results suggested that GGA triggers both proapoptotic (IRE1-XBP1, ATF6-CHOP) and antiapoptotic (ATF6-GRP78) UPR and thereby coordinates cellular fate even without induction of HSP70.