PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

Tauopathy-associated PERK alleles are functional hypomorphs that increase neuronal vulnerability to ER stress.

Tauopathies are neurodegenerative diseases characterized by tau protein pathology in the nervous system. EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), also known as PERK (protein kinase R-like endoplasmic reticulum kinase), was identified by genome-wide association study as a genetic risk factor in several tauopathies. PERK is a key regulator of the Unfolded Protein Response (UPR), an intracellular signal transduction mechanism that protects cells from endoplasmic reticulum (ER) stress. PERK variants had previously been identified in Wolcott-Rallison Syndrome, a rare autosomal recessive metabolic disorder, and these variants completely abrogated the function of PERK's kinase domain or prevented PERK expression. In contrast, the PERK tauopathy risk variants were distinct from the Wolcott-Rallison variants and introduced missense alterations throughout the PERK protein. The function of PERK tauopathy variants and their effects on neurodegeneration are unknown. Here, we discovered that tauopathy-associated PERK alleles showed reduced signaling activity and increased PERK protein turnover compared to protective PERK alleles. We found that iPSC-derived neurons carrying PERK risk alleles were highly vulnerable to ER stress-induced injury with increased tau pathology. We found that chemical inhibition of PERK in human iPSC-derived neurons also increased neuronal cell death in response to ER stress. Our results indicate that tauopathy-associated PERK alleles are functional hypomorphs during the UPR. We propose that reduced PERK function leads to neurodegeneration by increasing neuronal vulnerability to ER stress-associated damage. In this view, therapies to enhance PERK signaling would benefit at-risk carriers of hypomorphic alleles.

Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells.

Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.

Multiple Mechanisms of Unfolded Protein Response-Induced Cell Death.

Eukaryotic cells fold and assemble membrane and secreted proteins in the endoplasmic reticulum (ER), before delivery to other cellular compartments or the extracellular environment. Correctly folded proteins are released from the ER, and poorly folded proteins are retained until they achieve stable conformations; irreparably misfolded proteins are targeted for degradation. Diverse pathological insults, such as amino acid mutations, hypoxia, or infection, can overwhelm ER protein quality control, leading to misfolded protein buildup, causing ER stress. To cope with ER stress, eukaryotic cells activate the unfolded protein response (UPR) by increasing levels of ER protein-folding enzymes and chaperones, enhancing the degradation of misfolded proteins, and reducing protein translation. In mammalian cells, three ER transmembrane proteins, inositol-requiring enzyme-1 (IRE1; official name ERN1), PKR-like ER kinase (PERK; official name EIF2AK3), and activating transcription factor-6, control the UPR. The UPR signaling triggers a set of prodeath programs when the cells fail to successfully adapt to ER stress or restore homeostasis. ER stress and UPR signaling are implicated in the pathogenesis of diverse diseases, including neurodegeneration, cancer, diabetes, and inflammation. This review discusses the current understanding in both adaptive and apoptotic responses as well as the molecular mechanisms instigating apoptosis via IRE1 and PERK signaling. We also examine how IRE1 and PERK signaling may be differentially used during neurodegeneration arising in retinitis pigmentosa and prion infection.

Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress-induced cell death during the unfolded protein response.

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress-induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop((-)/(-)) cells are partially resistant to ER stress-induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK's down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress-induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a "two-hit" model of ER stress-induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.

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.

The unfolded protein response is a major mechanism by which LRP1 regulates Schwann cell survival after injury.

In peripheral nerve injury, Schwann cells (SCs) must survive to exert a continuing and essential role in successful nerve regeneration. Herein, we show that peripheral nerve injury is associated with activation of endoplasmic reticulum (ER) stress and the adaptive unfolded protein response (UPR). The UPR culminates in expression of C/EBP homology protein (CHOP), a proapoptotic transcription factor in SCs, unless counteracted by LDL receptor-related protein-1 (LRP1), which serves as a major activator of phosphatidylinositol 3-kinase (PI3K). Sciatic nerve crush injury in rats induced expression of the ER chaperone GRP78/BIP, reflecting an early, corrective phase of the UPR. However, when LRP1 signaling was inhibited with receptor-associated protein, PI3K activity was decreased and CHOP protein expression increased, particularly in myelinating SCs. In cultured SCs, the PKR-like ER kinase target eIF2α was phosphorylated and CHOP was induced by (1) inhibiting PI3K, (2) treating the cells with tumor necrosis factor-α (TNF-α), or (3) genetic silencing of LRP1. CHOP gene deletion in SCs decreased cell death in response to TNF-α. Furthermore, the effects of TNF-α on phosphorylated eIF2α, CHOP, and SC death were blocked by adding LRP1 ligands that augment LRP1-dependent cell signaling to PI3K. Collectively, our results support a model in which UPR-activated signaling pathways represent a major challenge to SC survival in nerve injury. LRP1 functions as a potent activator of PI3K in SCs and, by this mechanism, limits SC apoptosis resulting from increased CHOP expression in nerve injury.

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.

Selective abrogation of BiP/GRP78 blunts activation of NF-κB through the ATF6 branch of the UPR: involvement of C/EBPß and mTOR-dependent dephosphorylation of Akt.

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/EBPß. Overexpression of C/EBPß suppressed TNF-α-induced NF-κB activation, and knockdown of C/EBPß 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/EBPß. 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/EBPß and mTOR-dependent dephosphorylation of Akt.

Impairment of MCP-1 expression in mesothelial cells exposed to peritoneal dialysis fluid by osmotic stress and acidic stress.

BACKGROUND: Bacterial peritonitis is one of the most frequent complications in patients on peritoneal dialysis. In the present study, we investigated effects of peritoneal dialysis fluid (PDF) on mesothelial cell recruitment of macrophages, focusing especially on unphysiological properties of PDF. METHODS: Human and murine mesothelial cells were exposed to PDF or individual properties of PDF (low pH, high glucose concentration, hyperosmolality, high lactate concentration) in vitro and in vivo, treated with inflammatory stimuli, and subjected to analyses of monocyte chemoattractant protein-1 (MCP-1), nuclear factor-κB (NF-κB), mitogen-activated protein (MAP) kinases, and MAP kinase phosphatase-1 (MKP-1). RESULTS: We found that intraperitoneal administration of PDF suppressed expression of MCP-1 and infiltration of mononuclear cells in the peritoneum of mice following injection with lipopolysaccharide. Among the unphysiological properties of PDF, low pH and hyperosmolality caused blunted induction of MCP-1 in cytokine-stimulated mesothelial cells. The attenuated response was ascribed to suppression of NF-κB by low pH and inhibition of p38 MAP kinase by hyperosmolality. Furthermore, the attenuated phosphorylation of p38 MAP kinase by osmotic stress was associated with induction of MKP-1. CONCLUSION: These results suggest a possibility that mesothelial cells exposed to PDF exhibit attenuated MCP-1 expression and consequent impairment of macrophage recruitment through dual mechanisms, that is, inhibition of NF-κB by acidic stress and blunted activation of p38 MAP kinase by osmotic stress. In patients on peritoneal dialysis, blunted expression of chemokines may lead to perturbation of bacterial clearance by macrophages in the peritoneal cavity.

Selective deletion of adipocytes, but not preadipocytes, by TNF-alpha through C/EBP- and PPARgamma-mediated suppression of NF-kappaB.

Tumor necrosis factor-alpha (TNF-alpha) is a key regulator of adipose tissue mass, but mechanisms underlying this effect have not been fully elucidated. We found that exposure to TNF-alpha caused a significant decrease in the number of adipocytes, but not preadipocytes. Subsequent experiments revealed that TNF-alpha selectively deleted adipocytes through induction of apoptosis. Following exposure to TNF-alpha, rapid activation of nuclear factor-kappaB (NF-kappaB) was observed only in preadipocytes, but not in adipocytes. Inhibition of NF-kappaB rendered preadipocytes susceptible to TNF-alpha-induced apoptosis, suggesting that different activity of NF-kappaB is the determinant for the distinct apoptotic responses. During adipocyte differentiation, expression and activity of peroxisome proliferator-activated receptor-gamma (PPARgamma) were upregulated. Treatment of preadipocytes with a PPARgamma agonist attenuated NF-kappaB activation and rendered the cells vulnerable to TNF-alpha-induced apoptosis. Conversely, treatment of adipocytes with a PPARgamma antagonist enhanced NF-kappaB activation and conferred resistance to TNF-alpha-induced apoptosis, suggesting involvement of PPARgamma in the suppression of NF-kappaB in adipocytes. We also found that, following differentiation, expression and activity of CCAAT/enhancer binding protein (C/EBP), especially C/EBPalpha and C/EBPbeta, were upregulated in adipocytes. Overexpression of individual C/EBPs significantly inhibited activation of NF-kappaB in preadipocytes. Furthermore, transfection with siRNA for C/EBPalpha or C/EBPbeta enhanced activity of NF-kappaB in adipocytes, suggesting that C/EBP is also involved in the repression of NF-kappaB in adipocytes. These results suggested novel mechanisms by which TNF-alpha selectively deletes adipocytes in the adipose tissue. The C/EBP- and PPARgamma-mediated suppression of NF-kappaB may contribute to TNF-alpha-related loss of adipose tissue mass under certain pathological situations, such as cachexia.

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.

The oxidative stress: endoplasmic reticulum stress axis in cadmium toxicity.

Cadmium preferentially accumulates in the kidney, the major target for cadmium-related toxicity. Several underlying mechanisms are postulated, and reactive oxygen species (ROS) have been considered as crucial mediators for tissue injuries. In addition to oxidative stress, we recently disclosed that endoplasmic reticulum (ER) stress also plays a critical role. Cadmium causes ER stress in vitro and in vivo and mediates induction of apoptosis in target tissues. In this article, we describe a role for ER stress and involvement of particular branches of the unfolded protein response (UPR) in cadmium-triggered tissue injury, especially nephrotoxicity. We also discuss relationship between oxidative stress and ER stress, and involvement of selective ROS in the induction of pro-apoptotic branches of the UPR.

Downregulation of gap junction expression and function by endoplasmic reticulum stress.

Gap junctional intercellular communication (GJIC) plays a critical role in the control of multiple cell behavior as well as in the maintenance of tissue and organ homeostasis. However, mechanisms involved in the regulation of gap junctions (GJs) have not been fully understood. Given endoplasmic reticulum (ER) stress and dysfunction of GJs coexist in several pathological situations, we asked whether GJs could be regulated by ER stress. Incubation of mesangial cells with ER stress-inducing agents (thapsigargin, tunicamycin, and AB(5) subtilase cytotoxin) resulted in a decrease in connexin 43 (Cx43) expression at both protein and mRNA levels. This was accompanied by a loss of GJIC, as evidenced by the reduced numbers of dye-coupled cells after single cell microinjection or scrape loading dye transfer. Further studies demonstrated that ER stress significantly inhibited the promoter activity of the Cx43 gene, reduced [(35)S]-methionine incorporation into Cx43 protein and accelerated degradation of Cx43. ER stress also decreased the Cx43 protein levels in several different cell types, including human umbilical vein endothelial cells, mouse-derived renin-secreting cells and human hepatoma cells. Furthermore, induction of ER stress by hypoxic chemicals thenoyltrifluoroacetone and cobalt chloride was found to be associated with a reduction in Cx43. Our findings thus reveal a close link between ER stress and GJs. ER stress may represent a novel mechanism underlying the altered GJs in a variety of pathological situations.

Activation of the Akt-NF-kappaB 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 AB(5) toxin produced by some Shiga toxigenic Escherichia coli, on the activation of NF-kappaB. 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-kappaB 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-kappaB 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-kappaB activation, suggesting that activation of Akt is an event upstream of NF-kappaB. Degradation of GRP78 caused the unfolded protein response (UPR), and inducers of the UPR mimicked the stimulatory effects of SubAB on Akt and NF-kappaB. SubAB triggered the three major branches of the UPR including the IRE1-XBP1, PERK, and ATF6 pathways. Dominant-negative inhibition of IRE1alpha, XBP1, or PERK did not attenuate activation of NF-kappaB by SubAB. In contrast, genetic and pharmacological inhibition of ATF6 significantly suppressed SubAB-triggered Akt phosphorylation and NF-kappaB activation. These results suggested that loss of GRP78 by SubAB leads to transient phosphorylation of Akt and consequent activation of NF-kappaB through the ATF6 branch of the UPR.

Suppression of NF-kappaB 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-alpha-treated cells, suppression of MCP-1 by CsA or FK506 was associated with blunted responses of NF-kappaB, the crucial regulator of MCP-1. The suppression of NF-kappaB was reproduced by other inducers of UPR including AB(5) 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/EBPbeta and CHOP (C/EBP homologous protein), and overexpression of either C/EBPbeta or CHOP significantly attenuated TNF-alpha-triggered NF-kappaB activation. Furthermore, down-regulation of C/EBPbeta by small interfering RNA substantially reversed the suppressive effect of CsA on TNF-alpha-induced MCP-1 expression. These results suggested that CsA and FK506 confer insensitiveness to TNF-alpha on resident cells through UPR-dependent induction of the C/EBP family members.

Dual suppression of adipogenesis by cigarette smoke through activation of the aryl hydrocarbon receptor and induction of endoplasmic reticulum stress.

Cigarette smoking decreases body weight, whereas molecular mechanisms underlying this phenomenon have not been elucidated. In this report, we investigated regulation of adipogenesis by cigarette smoke and involvement of aryl hydrocarbon receptor (AhR) and endoplasmic reticulum (ER) stress. We found that cigarette smoke extract (CSE) inhibited differentiation of preadipocytes into adipocytes dose dependently. It was associated with a decrease in lipid accumulation, blunted expression of adipocyte markers (adiponectin, PPAR-gamma, and C/EBPalpha), and sustained expression of a preadipocyte marker MCP-1. CSE markedly induced activation of AhR, and AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene and 3-methylcholanthrene) reproduced the inhibitory effect of CSE on adipocyte differentiation. Furthermore, knockout of the AhR gene or blockade of AhR by a dominant-negative mutant attenuated the suppressive effects of CSE on adipocyte differentiation. We also found that CSE induced ER stress in preadipocytes, and ER stress inducers (thapsigargin, tunicamycin, and A23187) reproduced the suppressive effect of CSE on the differentiation of preadipocytes. Interestingly, AhR agonists did not cause ER stress, and ER stress inducers did not activate AhR. These results suggested that cigarette smoke has the potential to inhibit adipocyte differentiation via dual, independent mechanisms, i.e., through activation of the AhR pathway and induction of the unfolded protein response.

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-kappaB. 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-alpha, IL-1beta, 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-kappaB, but not to accelerated mRNA degradation. Subsequent experiments revealed that TNFR-associated factor 2, an essential component for TNF-alpha signaling, was down-regulated by ER stress. We also found that, under ER stress conditions, expression of NF-kappaB suppressor A20 was induced. Overexpression of A20 resulted in suppression of cytokine-triggered NF-kappaB 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-kappaB (e.g., GRP78, NO, reactive oxygen species, and IkappaB) 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.

Blockade of the aryl hydrocarbon receptor pathway triggered by dioxin, polycyclic aromatic hydrocarbons and cigarette smoke by Phellinus linteus.

Environmental pollutants including halogenated and polycyclic aromatic hydrocarbons activate the aryl hydrocarbon receptor (AhR) and thereby cause a wide range of pathological changes. Development of AhR antagonists will be useful for prevention and treatment of diseases related to AhR activation. Towards this end, we aimed in the present study at seeking for potential inhibitors of the AhR pathway in mycelial extracts using the dioxin responsive element-based sensing via secreted alkaline phosphatase (DRESSA). Through the screening of 13 mycelia, extracts prepared from Phellinus linteus, Cordyceps militaris and Hericium erinaceum inhibited activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene or 3-methylcholanthrene. Subsequent studies revealed that only Phellinus linteus suppressed activation of AhR and AhR-dependent gene expression triggered by all of these agonists. Cigarette smoke is known to contain a number of halogenated and polycyclic aromatic hydrocarbons. We found that Phellinus linteus has the potential to block activation of AhR and AhR-dependent gene expression triggered by cigarette smoke. Furthermore, the inhibitory effect of Phellinus linteus on the AhR pathway was independent of; 1) depression of AhR or AhR nuclear translocator, and 2) induction of AhR repressor. We conclude that Phellinus linteus contains potent inhibitor(s) of AhR activation and may be useful for prevention of pathologies associated with aberrant activation of AhR.

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.