Hisashi Johno

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.

Aberrant, differential and bidirectional regulation of the unfolded protein response towards cell survival by 3'-deoxyadenosine.

The unfolded protein response (UPR) is involved in a diverse range of pathologies triggered by endoplasmic reticulum (ER) stress. Endeavor to seek selective regulators of the UPR is a promising challenge towards therapeutic intervention in ER stress-related disorders. In the present report, we describe aberrant, differential and bidirectional regulation of the UPR by 3′-deoxyadenosine (cordycepin) towards cell survival. 3′-Deoxyadenosine blocked ER stress-induced apoptosis via inhibiting the IRE1–JNK pro-apoptotic pathway. 3′-Deoxyadenosine also inhibited apoptosis through reinforcement of the pro-survival eIF2α signaling without affecting PERK activity. It was associated with depression of GADD34 that dephosphorylates eIF2α, and dephosphorylation of eIF2α by salubrinal mimicked the anti-apoptotic effect of 3′-deoxyadenosine. Unexpectedly, although 3′-deoxyadenosine caused activation of eIF2α, it inhibited downstream pro-apoptotic events including induction of ATF4 and expression of CHOP. Cooperation of adenosine transporter and A3 adenosine receptor, but not A1/A2 receptors, mediated the pluripotent effects of 3′-deoxyadenosine. In mice, ER stress caused activation of JNK, expression of CHOP and induction of apoptosis in renal tubules. The apoptosis was significantly attenuated by administration with 3′-deoxyadenosine, and it was correlated with blunted induction of JNK and CHOP in the kidney. These results disclosed atypical pro-survival regulation of the UPR by 3′-deoxyadenosine, which may be advantageous for the treatment of intractable, ER stress-related disorders.

Anti-inflammatory subtilase cytotoxin up-regulates A20 through the unfolded protein response

We recently reported that subtilase cytotoxin (SubAB) has the potential to attenuate experimental models of inflammatory diseases [3]. Currently, little is known about underlying mechanisms involved in this therapeutic effect. In the present report, we show that SubAB induces A20, the endogenous negative regulator of NF-kappaB, in vitro and in vivo. This stimulatory effect occurred at the transcriptional level, and SubAB induced activation of the A20 promoter. We found that, in the early phase, SubAB triggered activation of NF-kappaB in a dose-dependent manner. Blockade of NF-kappaB abrogated expression of A20 by SubAB. SubAB rapidly triggered the unfolded protein response (UPR), and induction of the UPR by other agents (thapsigargin and A23187) mimicked the stimulatory effects of SubAB, both on NF-kappaB and on A20. The induction of A20 by thapsigargin was correlated with activation of the A20 promoter, which was not observed in the kappaB-mutated A20 promoter. Furthermore, induction of A20 by SubAB was substantially attenuated by treatment with different chemical chaperones. These results elucidated for the first time that the anti-inflammatory SubAB has the potential to induce A20 through the UPR-NF-kappaB-dependent pathway.

Blockade of adipocyte differentiation by cordycepin

BACKGROUND AND PURPOSE Cordyceps militaris has the potential to suppress differentiation of pre‐adipocytes. However, the active entities in the extract and the underlying mechanisms of its action are not known. Hence, we investigated whether and how cordycepin (3′‐deoxyadenosine), a constituent of C. militaris, inhibits adipogenesis.

Induction of CCAAT/enhancer-binding protein-homologous protein by cigarette smoke through the superoxide anion-triggered PERK-eIF2α pathway.

Cigarette smoke triggers apoptosis through oxidative stress- and endoplasmic reticulum (ER) stress-dependent induction of CCAAT/enhancer-binding protein-homologous protein (CHOP) (Tagawa et al., 2008. Free Radic. Biol. Med. 45, 50-59). We investigated roles of individual reactive oxygen/nitrogen species in the transcriptional induction of CHOP by cigarette smoke. Exposure of bronchial epithelial cells to O(2)(-), ONOO(-) or H(2)O(2) induced expression of CHOP, whereas NO alone did not. Induction of CHOP mRNA by cigarette smoke extract (CSE) was attenuated by scavengers for O(2)(-), ONOO(-) or NO, whereas scavenging H(2)O(2) did not affect the induction of CHOP. Like CSE, O(2)(-) and ONOO(-) caused activation of the CHOP gene promoter. Scavengers for O(2)(-), ONOO(-) or NO attenuated CSE-triggered activation of the CHOP gene promoter. CSE, O(2)(-) and ONOO(-) induced phosphorylation of protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) and caused induction of downstream activating transcription factor 4 (ATF4). Scavengers for O(2)(-), ONOO(-) or NO attenuated induction of ATF4 by CSE. Furthermore, dominant-negative inhibition of the PERK-eIF2α pathway exclusively suppressed CSE-triggered induction of CHOP and consequent apoptosis. These results suggest that O(2)(-) and ONOO(-) are selectively involved in CSE-triggered induction of CHOP and that the PERK-eIF2α pathway plays a crucial role in the induction of CHOP and apoptosis downstream of the particular reactive oxygen species.

Influences of Acidic Conditions on Formazan Assay: A Cautionary Note

Formazan assay has been used for several decades to evaluate metabolic activity of eukaryotic and prokaryotic cells. In particular, it has been often applied for quantitative assessment of viable cells under acidic circumstances caused by, e.g., ischemia and hypoxia. However, little attention has been paid to the influence of acidic pH on formazan assays. We found that acidic culture conditions significantly affect outcomes of the assays. Absorbance of tetrazolium–formazan decreased in a pH-dependent manner without affecting cell viability. This nonspecific effect was ascribed to influences of acidic pH on the production of formazan. Replacement of culture media to fresh medium at physiologic pH partially overcame this problem. The influence of acidic culture conditions should be carefully considered when formazan assays are used for the assessment of viable cells under various experimental situations.

Pleiotropic potential of dehydroxymethylepoxyquinomicin for NF-κB suppression via reactive oxygen species and unfolded protein response.

Dehydroxymethylepoxyquinomicin (DHMEQ) is a low-m.w. compound that strongly inhibits NF-κB. Previous reports showed that DHMEQ directly binds to specific cysteine residues of NF-κB subunits and thereby inhibits their nuclear translocation and DNA binding. In this work, we describe novel mechanisms by which DHMEQ suppresses cytokine-triggered activation of NF-κB. We found that sustained exposure of renal tubular cells to DHMEQ blocked TNF-α– and IL-1β–induced TGF-β–activated kinase 1 (TAK1) phosphorylation, a crucial event for NF-κB activation upstream of IκB kinase. This inhibition was mediated by reactive oxygen species (ROS), because of the following: 1) DHMEQ caused generation of ROS; 2) pretreatment with ROS generator inhibited cytokine-induced TAK1 phosphorylation and NF-κB activation; and 3) scavenging of ROS attenuated the suppressive effects of DHMEQ on TAK1 and NF-κB. We also found that DHMEQ caused the unfolded protein response (UPR) through generation of ROS. Alleviation of the UPR by chemical and genetic chaperones partially attenuated the suppressive effect of DHMEQ on NF-κB. The UPR-mediated inhibition of NF-κB occurred downstream of degradation of IκBα and phosphorylation of p65. Subsequent experiments revealed the following: 1) DHMEQ caused selective induction of C/EBPβ through the UPR; 2) overexpression of C/EBPβ suppressed activation of NF-κB; 3) knockdown of C/EBPβ attenuated the inhibitory effect of DHMEQ; and 4) DHMEQ-induced expression of C/EBPβ did not affect TNF-α–triggered degradation of IκBα and phosphorylation of p65. These results suggest that, in addition to its known effect on nuclear translocation of NF-κB, DHMEQ interferes with the cytokine-induced NF-κB signaling via generation of ROS at both upstream and downstream of the IκB kinase–IκB level.

Acidic stress–ER stress axis for blunted activation of NF-κB in mesothelial cells exposed to peritoneal dialysis fluid

BACKGROUND Bacterial peritonitis is a frequent complication in patients on peritoneal dialysis (PD). We previously reported that PD fluid (PDF) suppressed expression of monocyte chemoattractant protein 1 (MCP-1) in mesothelial cells in vitro and in vivo, which was ascribed to the suppression of nuclear factor-κB (NF-κB). To elucidate molecular mechanisms underlying this effect, we tested a role of endoplasmic reticulum (ER) stress. METHODS Mesothelial cells and other cell types were exposed to acidic stress, and induction of the unfolded protein response was examined. Peritoneal induction of ER stress was also tested in mice exposed to acidic and neutralized PDF. Activation of NF-κB and expression of MCP-1 by tumour necrosis factor-α were evaluated in mesothelial cells under acidic and ER stress conditions. Peritoneal expression of MCP-1 and infiltration of monocytes were compared in lipopolysaccharide (LPS)-treated mice between normal and ER stress conditions. RESULTS PDF, but not neutralized PDF, caused ER stress in the peritoneum. In vitro, acidic stress, but not metabolic and osmotic stress, induced ER stress in mesothelial cells and other cell types and suppressed activation of NF-κB and NF-κB-dependent MCP-1 induction. This effect was reproducible by other ER stress inducers, and attenuation of ER stress reversed the suppressive effect of low pH on NF-κB. Like PDF, ER stress inducers suppressed expression of MCP-1 and infiltration of mononuclear cells in the peritoneum of LPS-treated mice. CONCLUSION These results indicate a role for the acidic stress-ER stress pathway in blunted activation of NF-κB, which may cause perturbation of monocyte recruitment by mesothelial cells in PD patients.

Blockade of Smad signaling by 3′-deoxyadenosine: a mechanism for its anti-fibrotic potential

Cordyceps militaris has been used in Eastern countries for the treatment of various diseases including chronic kidney diseases. However, there are no reports that identified its active entities and molecular mechanisms underlying its therapeutic effectiveness. 3′-Deoxyadenosine is a major nucleoside derivative isolated from C. militaris. Some reports suggested that both C. militaris and 3′-deoxyadenosine have anti-inflammatory and anti-fibrotic effects. In the present report, we investigated whether and how 3′-deoxyadenosine interferes with fibrogenic processes in the kidney. For this purpose, we examined effects of 3′-deoxyadenosine on the expression of collagens triggered by transforming growth factor-β (TGF-β1) and bone morphogenetic protein-4 (BMP-4), especially focusing on the regulation of Smad signaling in vitro and in vivo. We found that 3′-deoxyadenosine suppressed expression of collagens induced by TGF-β1 and BMP-4 dose dependently. This suppression occurred at the transcriptional level and was correlated with blunted activation of the CAGA box and the BMP-responsive element. The suppressive effect on the TGF-β/BMP signaling was mediated mainly by adenosine transporter and partially by the A3 adenosine receptor, but not A1/A2 adenosine receptors. 3′-Deoxyadenosine reduced levels of both phosphorylated and total Smad proteins (Smad1, 2 and 3) dose dependently. It was mainly ascribed to transcriptional suppression, but not to enhanced protein degradation and eIF2α-mediated translational suppression. Consistent with the in vitro results, in vivo administration with 3′-deoxyadenosine reduced the levels of phosphorylated and total Smad proteins, as well as the levels of Smad mRNAs, in the kidney subjected to unilateral ureteral obstruction. It was associated with blunted induction of type I collagen and α-smooth muscle actin, a decrease in the number of interstitial myofibroblasts and reduced fibrotic area. These results suggest that 3′-deoxyadenosine interferes with the TGF-β and BMP signaling via downregulation of Smads, which may underlie the anti-fibrotic effect of this agent. 3′-Deoxyadenosine may be useful for therapeutic intervention in various TGF-β-related fibrotic disorders.

Unfolded Protein Response Causes a Phenotypic Shift of Inflamed Glomerular Cells toward Redifferentiation through Dual Blockade of Akt and Smad Signaling Pathways

During recovery from acute glomerulonephritis, cell proliferation, matrix expansion, and expression of the dedifferentiation marker α-smooth muscle actin (α-SMA) subside spontaneously. However, the molecular mechanisms underlying this recovery process remain elusive. In mesangioproliferative glomerulonephritis, the unfolded protein response (UPR) is induced in activated, dedifferentiated mesangial cells. We investigated the role of the UPR in mesangial cell deactivation and redifferentiation and found that, during experimental glomerulonephritis in rats, reinforcement of the UPR significantly attenuated mesangial cell proliferation, matrix expansion, and expression of α-SMA. Consistent with this in vivo result, induction of the UPR suppressed cell proliferation and transcriptional expression of type IV collagen (ColIV) and α-SMA in activated mesangial cells. The UPR reduced phosphorylation of Akt in vitro and in vivo, and it was responsible for attenuation of cell proliferation. The UPR also preferentially depressed levels of total and phosphorylated Smads without affecting transcriptional levels, and it was responsible for suppression of ColIV and α-SMA. Translational suppression via the eIF2α pathway, but not proteasome-mediated protein degradation, was responsible for the down-regulation of Smads. These results suggest the novel potential of the UPR to facilitate a phenotypic shift of activated glomerular cells toward deactivation and redifferentiation. The UPR may serve as endogenous machinery that supports recovery of glomeruli from acute inflammation.