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.

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.

Direct, continuous monitoring of air pollution by transgenic sensor mice responsive to halogenated and polycyclic aromatic hydrocarbons.

BACKGROUND: The aryl hydrocarbon receptor (AhR, also called the dioxin receptor) plays crucial roles in toxicologic responses of animals to environmental pollutants, especially to halogenated and polycyclic aromatic hydrocarbons. To achieve direct, continuous risk assessment of air pollution using biological systems, we generated transgenic sensor mice that produce secreted alkaline phosphatase (SEAP) under the control of AhR. METHODS: To characterize responses of the mice to AhR agonists, sensor mice were orally administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3MC), benzo[a]pyrene (B[a]P), or beta-naphthoflavone (BNF), and serum levels of SEAP were evaluated. To monitor air pollution caused by cigarette smoke, we placed the mice each day in an experimental smoking room, and evaluated activity of serum SEAP for up to 4 days. Activation of AhR in individual organs was also examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis of SEAP. RESULTS: In response to oral exposure to TCDD, sensor mice exhibited dramatic and sustained activation of AhR. The mice also responded sensitively to 3MC, B[a]P, and BNF. Activation of AhR was dose dependent, and the liver was identified as the main responding organ. After exposure to the smoking environment, sensor mice consistently exhibited transient, reversible activation of AhR. RT-PCR analysis of SEAP revealed that activation of AhR occurred predominantly in the lung. CONCLUSION: We are the first laboratory to demonstrate successfully direct, comprehensive monitoring of air pollution using genetically engineered mammals. The established system would be useful for real risk assessment of halogenated and polycyclic aromatic hydrocarbons in the air, especially in smoking environments.

Real-time detection and continuous monitoring of ER stress in vitro and in vivo by ES-TRAP: evidence for systemic, transient ER stress during endotoxemia.

Activity of secreted alkaline phosphatase (SEAP) produced by transfected cells is rapidly down-regulated by endoplasmic reticulum (ER) stress independent of transcriptional regulation. This phenomenon was observed in a wide range of cell types triggered by various ER stress inducers. The magnitude of the decrease in SEAP was proportional to the extent of ER stress and inversely correlated with the induction of endogenous ER stress markers grp78 and grp94. In contrast to SEAP, activity of secreted luciferase was less susceptible to ER stress. The decrease in SEAP activity by ER stress was caused by abnormal post-translational modification, accelerated degradation and reduced secretion of SEAP protein. In transgenic mice constitutively producing SEAP, systemic induction of ER stress led to reduction in serum SEAP. In these mice, administration with lipopolysaccharide caused rapid, transient decrease in serum SEAP activity, and it was correlated with up-regulation of grp78 in several organs including the spleen, lung, kidney, liver and heart. These results elucidated for the first time a possible involvement of transient, systemic ER stress in endotoxemia and provided evidence for usefulness of ER stress responsive alkaline phosphatase for real-time monitoring of ER stress in vitro and in vivo.

High levels of dioxin-like potential in cigarette smoke evidenced by in vitro and in vivo biosensing.

Cigarette smoke contains low levels of agonists for the aryl hydrocarbon receptor (AhR; also called the dioxin receptor). However, little is understood about the whole potential of cigarette smoke for activating AhR. In this report, we evaluated the total "dioxin-like" activity of cigarette smoke using in vitro and in vivo reporter systems. Cigarette smoke extract (CSE) was prepared from seven cigarette brands (1-20 mg tar content) and subjected to in vitro bioassay based on the xenobiotic-responsive element (XRE) as the sensor and secreted alkaline phosphatase (SEAP) as the reporter. Exposure of reporter cells to CSE triggered activation of XRE in a dose-dependent manner, which was suppressed by functional inhibition of AhR. Direct, brief exposure of the cells to cigarette smoke similarly induced activation of XRE. Using 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) as the standard, the XRE-activating potential (XAP) of individual smoke was evaluated quantitatively. Positive correlation was observed between the tar content and XAP values. The XAP values estimated were extremely high with a range from 18.5 to 51.2 ng 2,3,7,8-TCDD equivalent per cigarette. To further estimate XAP of cigarette smoke in vivo, we generated transgenic reporter mice that secrete SEAP under the control of XRE. After exposure of the mice to smoke, serum levels of SEAP were significantly elevated within 12 hours, peaked at 24 hours, and declined thereafter. These results evidenced for the first time that cigarette smoke has unexpectedly high dioxin-like potential that triggers the AhR-XRE pathway in vitro and in vivo.

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.

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-1beta and TNF-alpha 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.

Cigarette smoke as a trigger for the dioxin receptor-mediated signaling pathway.

Dioxins and dioxin-like chemicals cause a wide range of pathologies including carcinogenesis, immune dysfunction, and developmental/reproductive abnormalities. Most of these toxic effects are mediated by aryl hydrocarbon receptor (AhR; also called the dioxin receptor), a ligand-activated transcription factor. Constitutive activation of AhR via genetic manipulation causes development of cancers, inflammation and immune abnormality in mice even without exposure to xenobiotic ligands. Recent investigation disclosed that cigarette smoke contains high levels of agonists for AhR and strongly activates the dioxin signaling pathway. In this review, we describe and discuss possible roles of AhR activation in cigarette smoke-related pathologies, especially focusing on carcinogenesis, inflammation, atherosclerosis, immune dysfunction and teratogenesis.

Novel potential of tunicamycin as an activator of the aryl hydrocarbon receptor -- dioxin responsive element signaling pathway.

Tunicamycin is a well-known inhibitor of protein glycosylation and used as an inducer of endoplasmic reticulum (ER) stress. We found that tunicamycin induced expression of cytochrome P450 1A1 in a dose-dependent manner. Like dioxin, the transcriptional induction was associated with dose-dependent activation of the dioxin responsive element (DRE). This effect was independent of inhibition of protein glycosylation or induction of ER stress. Pharmacological and genetic inhibition of the aryl hydrocarbon receptor (AhR) significantly attenuated activation of DRE by tunicamycin. These results elucidated the novel potential of tunicamycin as an activator of the AhR -- DRE signaling pathway.

Secreted protein-based reporter systems for monitoring inflammatory events: critical interference by endoplasmic reticulum stress.

A number of recent reports have used secreted protein-based reporter assays for monitoring intercellular and intracellular events involved in inflammation. However, we found that these assay systems are critically affected by endoplasmic reticulum (ER) stress. When reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of NF-kappaB were exposed to IL-1beta or TNF-alpha, induction of SEAP activity was markedly reduced under ER stress conditions. Downregulation of SEAP activity was observed regardless of cell types and type of regulatory elements; e.g., when reporter hepatocytes that express SEAP under the control of the dioxin responsive elements were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene, ER stress similarly suppressed the increase in SEAP activity despite its transcriptional upregulation. Activity of constitutively expressed SEAP in various cells was also reduced by ER stress in a magnitude-dependent manner, and it was associated with disturbed subcellular transport of SEAP to the Golgi. Furthermore, interference by ER stress was similarly observed in other reporter assay using secreted luciferase. These results evidenced critical interference by ER stress in secreted protein-based reporter systems. The suppression of reporter responses by ER stress should be considered carefully for experimental design and interpretation of data when secreted protein-based reporter systems are used for investigation.

Profiling of functional phosphodiesterase in mesangial cells using a CRE-SEAP-based reporting system.

1. Phosphodiesterases (PDEs) are critically implicated in the regulation of mesangial cell function, but profile of functional PDEs in mesangial cells is still unclear. In this study, we investigated roles of individual PDEs in the regulation of mesangial cell behavior by the cAMP pathway. 2. Reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of the cAMP response element (CRE) were exposed to selective PDE inhibitors in the presence or absence of cAMP, and activity of CRE, expression of CRE-regulated protein, mitogenesis and cell survival were examined. 3. Exposure of reporter cells to cAMP-elevating agents resulted in time- and concentration-dependent activation of CRE. Treatment of the cells with any PDE inhibitors alone did not induce CRE activation. Under stimulation with 8-bromo-cAMP or 8-bromo-cGMP, however, inhibitors of PDE2, PDE3, PDE4 and PDE5 enhanced activation of CRE. Inhibition of PDE1 or PDE6 did not affect the CRE activation. 4. Among different combinations tested, only inhibitors of PDE3 and PDE4 cooperatively increased the level of intracellular cAMP, activity of protein kinase A, activation of CRE, and CRE-regulated protein, connexin43. 5. Concomitant inhibition of PDE3 and PDE4 attenuated mitogen-induced activation of extracellular signal-regulated kinases and cell proliferation. Under serum deprivation, combinational inhibition of PDE3 and PDE4 exclusively caused activation of caspase-3 and apoptosis. 6. The present data elucidated that PDE3 and PDE4 play critical roles in the regulation of mesangial cell function. PDE3 and PDE4 were identified as the novel, antiapoptotic machinery that supports survival of mesangial cells.

Transthyretin accelerates vascular Abeta deposition in a mouse model of Alzheimer's disease.

Transthyretin (TTR) binds amyloid-beta (Abeta) and prevents Abeta fibril formation in vitro. It was reported that the lack of neurodegeneration in a transgenic mouse model of Alzheimer's disease (AD) (Tg2576 mouse) was associated with increased TTR level in the hippocampus, and that chronic infusion of anti-TTR antibody into the hippocampus of Tg2576 mice led to increased local Abeta deposits, tau hyperphosphorylation and apoptosis. TTR is, therefore, speculated to prevent Abeta pathology in AD. However, a role for TTR in Abeta deposition is not yet known. To investigate the relationship between TTR and Abeta deposition, we generated a mouse line carrying a null mutation at the endogenous TTR locus and the human mutant amyloid precursor protein cDNA responsible for familial AD (Tg2576/TTR(-/-) mouse) by crossing Tg2576 mice with TTR-deficient mice. We asked whether Abeta deposition was accelerated in Tg2576/TTR(-/-) mice relative to the heterozygous mutant Tg2576 (Tg2576/TTR(+/-)) mice. Contrary to our expectations, the degree of total and vascular Abeta burdens in the aged Tg2576/TTR(-/-) mice was significantly reduced relative to the age-matched Tg2576/TTR(+/-) mice. Our experiments present, for the first time, compelling evidence that TTR does not suppress but rather accelerates vascular Abeta deposition in the mouse model of AD.

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.

Blockade of the dioxin pathway by herbal medicine Formula Bupleuri Minor: identification of active entities for suppression of AhR activation.

Environmental pollutants including dioxins activate the aryl hydrocarbon receptor (AhR) and cause a wide range of pathologies. Development of AhR antagonists will be useful for prevention and treatment of the diseases related to AhR activation. Towards this goal, we aimed at seeking for potential AhR antagonists in herbal medicines using the dioxin responsive element-based sensing via secreted alkaline phosphatase (DRESSA). Through initial rough screening, 4 formulae were selected from 20 herbal medicines and subjected to the second, detailed screening. We found that only Formula bupleuri minor (TJ-9) significantly inhibited activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Among 7 raw herb extracts in TJ-9, Glycyrrhizae Radix and Scutellariae Radix were responsible for the antagonistic effect of TJ-9 against dioxin. Some constituents including Bupleuri Radix and Zingiberis Rhizoma rather activated AhR. Among 12 major constituents of Glycyrrhizae Radix and Scutellariae Radix, we identified that licopyranocoumarin, glycyrrhizic acid and genistein in Glycyrrhizae Radix and baicalein, wogonin and daidzein in Scutellariae Radix had substantial antagonistic effects on AhR. Among these, baicalein most effectively blocked activation of AhR triggered by cigarette smoke, a strong activator of AhR. The antagonistic substances identified here may be useful for prevention from diseases associated with aberrant activation of AhR.

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.

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 O(2)(*), H(2)O(2), or ONOO(-) caused apoptosis, whereas ER stress was induced only by O(2)(*) 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. O(2)(*) 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 O(2)(*) or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) O(2)(*) 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 O(2)(*)-triggered activation of XBP1.

Suppression of adipocyte differentiation by Cordyceps militaris through activation of the aryl hydrocarbon receptor.

Mycelial extracts have a wide range of biological activities that modulate functions of mammalian cells. In this report, we sought to identify antiadipogenic mycelia with the use of 3T3-L1 cells and found that the extract of Cordyceps militaris exclusively suppressed differentiation of 3T3-L1 preadipocytes into mature adipocytes without affecting cell viability. This inhibitory effect was dose dependent, reversible, and associated with 1) a decrease in lipid accumulation, 2) blunted induction of adipocyte markers including adiponectin, peroxisome proliferator-activated receptor-gamma, and CCAAT/enhancer binding protein-alpha, and 3) sustained expression of a preadipocyte marker, monocyte chemoattractant protein-1. C. militaris also significantly decreased accumulation of lipid and hypertrophy in mature adipocytes and preserved their response to insulin (phosphorylation of Akt) during prolonged culture. Subsequent experiments revealed that C. militaris has the potential to activate the aryl hydrocarbon receptor (AhR). In 3T3-L1 cells, treatment with AhR agonists including benzo[a]pyrene and 3-methylcholanthrene reproduced the antiadipogenic effect of C. militaris. Furthermore, dominant-negative inhibition of AhR abrogated the suppressive effect of C. militaris on adipocyte differentiation. These results suggest that C. militaris has the potential to interfere with adipocyte differentiation through activation of AhR.

Unexpected blockade of adipocyte differentiation by K-7174: implication for endoplasmic reticulum stress.

Preadipocytes constitutively express GATA-2 and GATA-3 that are required to halt the cells at the undifferentiated stage. However, we unexpectedly found that K-7174, a GATA-specific inhibitor, did not induce but rather inhibited differentiation of 3T3-L1 preadipocytes. It was associated with lack of lipid accumulation, blunted expression of adipocyte markers including adiponectin and peroxisome proliferator-activated receptor gamma (PPARgamma), and sustained expression of a preadipocyte marker monocyte chemoattractant protein 1 (MCP-1). Subsequent experiments revealed that K-7174 had the potential to induce endoplasmic reticulum (ER) stress evidenced by induction of GRP78 and CHOP. Other inducers of ER stress completely reproduced the effects of K-7174 including suppression of lipid accumulation, blockade of induction of adiponection and PPARgamma and maintenance of MCP-1 expression. These results indicated a possibility that ER stress suppresses adipocyte differentiation and that GATA inhibitor K-7174 has the potential for interfering with adipogenesis through induction of ER stress.

Recovery and maintenance of nephrin expression in cultured podocytes and identification of HGF as a repressor of nephrin.

Cultured podocytes easily lose expression of nephrin. In this report, we developed optimum media for recovery and maintenance of nephrin gene expression in murine podocytes. Using reporter podocytes, we found that activity of the nephrin gene promoter was enhanced by DMEM/F12 or alpha-MEM compared with RPMI-1640. In any of these basal media, addition of 1,25-dihydroxyvitamin D(3), all-trans-retinoic acid or dexamethasone significantly increased activity of the nephrin promoter. The effects of the supplemental components were synergistic, and the maximum activation was achieved by DMEM/F12 supplemented with three agents. This culture medium was designated as vitamin D(3), retinoic acid and dexamethasone-supplemented DMEM/F12 (VRADD). In reporter podocytes that express nephrin, VRADD induced activation of the nephrin gene promoter up to 60-fold. Even in podocytes that have lost nephrin expression during multiple passages, expression of nephrin mRNA was dramatically recovered by VRADD. However, VRADD caused damage of podocytes in prolonged cultures, which was avoided in the absence of dexamethasone (designated as VRAD). VRAD maintained expression of nephrin for extended periods, which was associated with the differentiated phenotype of podocytes. Using the VRAD-primed podocytes, we revealed that expression of nephrin mRNA as well as nephrin promoter activity was suppressed by a putative dedifferentiation factor of podocytes, hepatocyte growth factor.

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 microM, 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 NH(2)-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.