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Am J Physiol Renal Physiol 295: F1495-F1503, 2008. First published September 17, 2008; doi:10.1152/ajprenal.00602.2007
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Suppression of cytokine responses by indomethacin in podocytes: a mechanism through induction of unfolded protein response

Maro Okamura,1 Yosuke Takano,1 Nobuhiko Hiramatsu,1 Kunihiro Hayakawa,1 Jian Yao,1 Adrienne W. Paton,2 James C. Paton,2 and Masanori Kitamura1

1Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan; and 2School of Molecular and Biomedical Science, University of Adelaide, South Australia, Australia

Submitted 19 December 2007 ; accepted in final form 10 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 [GenBank] reproduced the suppressive effect. Reporter assays showed that indomethacin as well as thapsigargin attenuated activation of NF-{kappa}B by TNF-{alpha}, and it was associated with enhanced degradation of TNF receptor-associated factor 2 (TRAF2) and blunted degradation of I{kappa}Bβ. Subsequent experiments revealed that acute ablation of GRP78 protein by AB5 subtilase cytotoxin caused reinforcement of MCP-1 induction and NF-{kappa}B activation by TNF-{alpha} and that transfection with GRP78 significantly suppressed the cytokine-induced activation of NF-{kappa}B. 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.

monocyte chemoattractant protein 1; nuclear factor-{kappa}B; tumor necrosis factor-{alpha}; unfolded protein response; 78-kDa glucose-regulated protein

INDOMETHACIN AND IBUPROFEN are nonsteroidal anti-inflammatory drugs (NSAIDs) that have been widely used for the relief of pain, fever, and inflammation. The anti-inflammatory potential of NSAIDs is primarily due to their ability to inhibit the cyclooxygenase (COX) enzymes involved in the production of proinflammatory prostaglandins (5). However, several studies demonstrated that certain NSAIDs, such as sodium salicylate, sulindac, ibuprofen, and flurbiprofen, may exert anti-inflammatory effects independently of cyclooxygenase activity and prostaglandin synthesis. Those effects may be mediated by inhibition of certain transcription factors such as activator protein 1 and nuclear factor-{kappa}B (NF-{kappa}B) and/or by alterations in the activity of kinases including I{kappa}B kinase (IKK), mitogen-activated protein kinase, and cyclin-dependent kinase (44). In contrast, indomethacin may activate peroxisome proliferator-activated receptor (PPAR) and thereby exert anti-inflammatory effects without affecting other transcription factors and kinases mentioned above (44).

Previous reports suggested that NSAIDs, especially indomethacin, have the potential to attenuate proteinuria in human nephrotic syndrome (4, 38). Indomethacin also attenuates proteinuria in experimental and human glomerulonephritis, including passive Heymann nephritis and IgA nephropathy (33, 41, 54). In contrast, antiproteinuric effects of other NSAIDs have been less documented. Because podocytes play a crucial role in the maintenance of the integrity of the slit diaphragm that regulates passage of macromolecules in plasma to the urinary space, indomethacin may preserve the function of podocytes under pathological conditions.

Infiltration of leukocytes, especially monocytes/macrophages, plays a decisive role in the development of various proteinuric glomerular diseases (28). Under pathological conditions, activated macrophages secrete inflammatory mediators and stimulate resident cells toward functional alteration. Depletion of macrophages or inhibition of macrophage infiltration attenuates glomerular injury and proteinuria, suggesting a critical role of macrophages in the development of proteinuria (8, 22). Recently, we found that podocytes exposed to macrophages or macrophage-derived cytokines exhibited downregulation of nephrin, the podocyte-specific key regulator for maintaining the structure and function of the slit diaphragm (42, 52). We speculated that, under pathological situations, indomethacin might confer insensitiveness to inflammatory cytokines on podocytes and thereby attenuate proteinuria.

Indomethacin has the ability to induce 70-kDa heat shock protein (HSP70) and to facilitate its recruitment into the nucleus (27, 49). It is known that thermal stress induces HSPs and thereby may attenuate expression of chemokines and adhesion receptors in epithelial and endothelial cells (1, 21). Other investigators also reported that overexpression of HSP70 attenuated expression of NF-{kappa}B-dependent genes in response to proinflammatory stimuli (7, 40). We hypothesized that indomethacin could attenuate responses of podocytes to inflammatory stimuli via induction of the HSP70 family of molecules. The present investigation was initiated to examine this possibility. Using cultured podocytes, we aimed at examining whether indomethacin and ibuprofen attenuate responses of podocytes to inflammatory cytokines, and if so, how the HSP70 family members are involved in the suppressive effect.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells. Murine podocytes (37) were kindly provided by Dr. Karlhans Endlich (University of Heidelberg, Heidelberg, Germany) and cultured as described before (43). For maintenance and/or propagation, podocytes were cultured at 37°C in type I collagen-coated plates using RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS). Expression of nephrin protein was confirmed by Western blot analysis (43, 52). Rat SM43 mesangial cells were established and identified as described before (19). The rat renal tubular epithelial cell line NRK-52E and the porcine renal proximal tubular cell line LLC-PK1 were purchased from American Type Culture Collection (Manassas, VA). These cells were maintained in Dulbecco's modified Eagle's medium/F-12 (GIBCO-BRL, Gaithersburg, MD) supplemented with 5% FBS. Murine podocytes constitutively expressing secreted alkaline phosphatase (SEAP) under the control of the simian virus 40 promoter were established as described before (52) and used for endoplasmic reticulum (ER) stress-responsive alkaline phosphatase (ES-TRAP) assay, as described later.

Pharmacological treatments. Cells were 1) treated with indomethacin (1–500 µM), ibuprofen (250–1,000 µM), aspirin (100–500 µM), sulindac (100–500 µM), tunicamycin (1 µg/ml), thapsigargin (100 nM), A23187 [GenBank] (5 µM), troglitazone (10 µM) or AB5 subtilase cytotoxin (SubAB; 0.1–100 ng/ml) (32); or 2) pretreated with these agents and stimulated by interleukin-1β (IL-1β; human recombinant, 1 ng/ml) or tumor necrosis factor-{alpha} (TNF-{alpha}; human recombinant, 10 ng/ml), and subjected to analyses. Media containing 1% FBS were generally used for experiments. Ibuprofen and sulindac were purchased from Wako (Osaka, Japan), and aspirin was from Cayman Chemical (Ann Arbor, MI). IL-1β and TNF-{alpha} were purchased from Genzyme (Cambridge, MA), and other reagents were obtained from Sigma-Aldrich Japan (Tokyo, Japan).

ES-TRAP assay. Induction of ER stress was evaluated by an ES-TRAP assay (10). It was based on the fact that the activity of SEAP constitutively produced by transfected cells is rapidly downregulated by ER stress (10). The activity of SEAP in culture medium was evaluated by a chemiluminescent method using a Great EscAPe SEAP Detection Kit (BD Biosciences, Palo Alto, CA). The values were normalized by the number of viable cells estimated by formazan assay, as described below.

Formazan assay. The number of viable cells was assessed by a formazan assay using Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan) (52).

Northern blot analysis. Total RNA was extracted by a single-step method, and Northern blot analysis was performed as described previously (18). cDNAs for HSP70 (13), 78-kDa glucose-regulated protein (GRP78; provided by Dr. Kazunori Imaizumi, Nara Institute of Science and Technology, Nara, Japan) (14), CCAAT/enhancer-binding protein-homologous protein (CHOP) (provided by Dr. David Ron, New York University School of Medicine, NY) (50), and monocyte chemoattractant protein 1 (MCP-1) (35) were used to prepare radiolabeled probes. Expression of GAPDH was used as a loading control.

Western blot analysis. Western blot analysis of I{kappa}Bβ, GRP78/94, MCP-1, and TNF receptor-associated factor 2 (TRAF2) was performed using an anti-I{kappa}Bβ antibody (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), anti-KDEL antibody (1:1,000 dilution, Stressgen, Victoria, Canada), anti-MCP-1 antibody (sc-1785, 1:200 dilution, Santa Cruz Biotechnology), and anti-TRAF2 antibody (sc-877, 1:200 dilution, Santa Cruz Biotechnology), as described previously (6, 9). As a loading control, the level of β-actin was assessed using an anti-β-actin antibody (1:30,000 dilution; Sigma-Aldrich Japan). Blots were visualized using the enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). Densitometric analysis was performed using Scion Image (Scion, Frederick, MD).

Transient transfection and reporter assays. Using GeneJuice Transfection Reagent (Novagen), podocytes were transiently transfected with pNF-{kappa}B-Luc (Panomics, Fremont, CA) or pPPRE(A)-Luc (provided by Dr. Shunji Ishihara, Shimane University School of Medicine, Shimane, Japan) (36) that introduces a luciferase gene under the control of the {kappa}B site or the peroxisome proliferator-responsive element (PPRE), respectively. The cells were then pretreated with indomethacin, thapsigargin, or troglitazone and stimulated with TNF-{alpha}. In some experiments, cells were transiently transfected with pNF-{kappa}B-Luc together with pcDNA3.1-GRP78 (provided by Dr. Richard C. Austin, Henderson Research Center, Ontario, Canada) (50) or pCI-neo-ORP150 (provided by Dr. Satoshi Ogawa, Kanazawa University, Kanazawa, Japan) (29) encoding GRP78 or 150-kDa oxygen-regulated protein (ORP150), respectively. After stimulation by TNF-{alpha} for 6–9 h, cells were subjected to luciferase assay using the Luciferase Assay System (Promega, Madison, WI).

Statistical analysis. Assays were performed in quadruplicate. Data are expressed as means ± SE. Statistical analysis was performed using a nonparametric Mann-Whitney U-test to compare data in different groups. A P value <0.05 was considered to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Induction of unfolded protein response by indomethacin, but not by other NSAIDs, in podocytes. Previous reports showed that NSAIDs, especially indomethacin, have the potential to induce HSPs (12, 27) which may suppress expression of inflammation-associated genes in epithelial and endothelial cells (1, 7, 21, 40). The renoprotective effect of indomethacin in glomerulonephritis (33, 41, 54) may be ascribed to induction of HSPs. To examine this possibility, podocytes were treated with serial concentrations of indomethacin or ibuprofen for 6 h, and expression of HSP70 and GRP78, another member of the HSP70 family, was examined by Northern blot analysis. We found that, in cultured podocytes, expression of HSP70 was not affected by either indomethacin or ibuprofen even at the highest concentrations (Fig. 1A, top row). However, GRP78, an endogenous indicator of unfolded protein response (UPR) (23), was substantially upregulated by indomethacin, but not by ibuprofen (Fig. 1A, second row). Similarly, expression of CHOP, another marker of UPR, was also induced by indomethacin, but not by ibuprofen (Fig. 1A, third row). The induction of UPR by indomethacin was observed at low concentrations (≥50 µM) (Fig. 1B). Time-lapse experiments revealed that induction of GRP78 and CHOP was observed within 3 h and lasted for 24 h (Fig. 1C, second and third rows). Of note, the basal level of HSP70 was not increased but rather decreased by indomethacin after 12–24 h (Fig. 1C, top row).


Figure 1
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  		Fig. 1. Induction of unfolded protein response (UPR) by indomethacin, but not by ibuprofen in podocytes. A–C: murine podocytes were treated with ibuprofen (250–1,000 µM), indomethacin (1–500 µM), or tunicamycin (an inducer of UPR; 1 µg/ml) for 6 h (A and B) or with 100 µM indomethacin for 3–24 h (C), and expression of HSP70, GRP78, and CHOP was examined by Northern blot analysis. Expression of GAPDH is shown at the bottom as a loading control. D: podocytes constitutively expressing SEAP were treated with indomethacin (100–1,000 µM) for 24 h, and activity of SEAP in culture media was evaluated by chemiluminescent assay. Activity of SEAP was normalized by the number of viable cells estimated by formazan assay. Assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant differences (P < 0.05). E: NRK-52E, SM43, and LLC-PK1 cells were treated with indomethacin (50–500 µM) for 6 h, and expression of GRP78 and CHOP was examined. F: podocytes were treated with aspirin (100–500 µM), sulindac (100–500 µM), or 200 µM indomethacin for 6 h, and expression of GRP78 and CHOP was examined.

 
UPR is triggered by ER stress caused by accumulation of unfolded/misfolded proteins in the ER. Induction of ER stress in indomethacin-treated podocytes was further confirmed by the ES-TRAP assay (10) using podocytes constitutively expressing SEAP (designated as ES-TRAP podocytes) (52). ES-TRAP podocytes were treated with indomethacin (100–1,000 µM) for 24 h, and the activity of SEAP in culture media was evaluated by chemiluminescent assay. The values were normalized by the number of viable cells estimated by formazan assay. As shown in Fig. 1D, the activity of SEAP was reduced by indomethacin dose dependently. Of note, cell viability was not affected significantly by indomethacin at concentrations of 100–500 µM. Indomethacin at 1,000 µM modestly reduced (24%) the number of viable cells.

To examine whether the induction of UPR by indomethacin is a phenomenon specific to podocytes, responses of other renal cells were tested. NRK-52E rat tubular epithelial cells, SM43 rat mesangial cells, and LLC-PK1 porcine proximal tubular cells were treated with serial concentrations of indomethacin, and expression of endogenous markers for UPR was examined. Northern blot analysis revealed that expression of CHOP was markedly induced in all cell types. Similarly, expression GRP78 was also upregulated modestly in NRK-52E cells and LLC-PK1 cells (Fig. 1E). To further confirm that the induction of UPR is specific to indomethacin and independent of COX inhibition, we also tested effects of other NSAIDs, aspirin and sulindac. As shown in Fig. 1F, like ibuprofen, neither aspirin nor sulindac induced expression of GRP78 and CHOP.

Inhibition of TNF-{alpha}-induced MCP-1 expression by indomethacin and other UPR inducers, but not by ibuprofen. Inflammatory cytokines, especially TNF-{alpha}, trigger glomerular cells to express MCP-1 and thereby cause accumulation of macrophages in glomeruli (15, 30). TNF-{alpha} also causes activation and dedifferentiation of podocytes (42) and may therefore induce proteinuria. Using MCP-1 as an indicator of inflammatory responses, we examined whether induction of MCP-1 by cytokines is influenced by indomethacin in podocytes. Treatment of podocytes with TNF-{alpha} for 6 h markedly induced expression of MCP-1. When the podocytes were pretreated with indomethacin, the induction was attenuated (Fig. 2A, top row). It was associated with substantial induction of GRP78, a marker of UPR (Fig. 2A, middle row). Similarly, thapsigargin, an inducer of UPR, markedly induced expression of GRP78 and abrogated induction of MCP-1 by TNF-{alpha}. In contrast, pretreatment with ibuprofen did not induce GRP78 and did not attenuate TNF-{alpha}-induced MCP-1 expression (Fig. 2A). These results indicated that induction of UPR is correlated with the suppression of MCP-1 in TNF-{alpha}-exposed podocytes. Interestingly, expression of MCP-1 by IL-1β was modestly suppressed by ibuprofen and thapsigargin, and markedly by indomethacin. In contrast to TNF-{alpha}-exposed cells, inverse correlation of MCP-1 and GRP78 was not evident in IL-1β-exposed cells (Fig. 2B). Consistent with the results of Northern blot analysis, indomethacin markedly downregulated the level of MCP-1 protein in TNF-{alpha}- and IL-1β-treated cells, and a suppressive effect of ibuprofen was observed only in IL-1β-treated cells (Fig. 2C).


Figure 2
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  		Fig. 2. Inhibition of TNF-{alpha}-induced MCP-1 expression by indomethacin and other UPR inducers, but not by ibuprofen. A and B: podocytes were pretreated with 500 µM ibuprofen, 100 µM indomethacin, or 100 nM thapsigargin for 6 h and treated with 10 ng/ml TNF-{alpha} (A) or 1 ng/ml IL-1β (B) for 6 h, and expression of MCP-1 and GRP78 was examined by Northern blot analysis. C: podocytes were pretreated with ibuprofen or indomethacin for 6 h, treated with TNF-{alpha} or IL-1β for 16 h, and subjected to Western blot analysis of MCP-1. D: podocytes were pretreated with 1 µg/ml tunicamycin, 100 nM thapsigargin, 5 µM A23187 [GenBank] , or 100 µM indomethacin for 6 h, treated with TNF-{alpha} for 6 h, and subjected to Northern blot analysis.

 
To investigate involvement of UPR in the suppression of the response of podocytes to TNF-{alpha}, other inducers of UPR, tunicamycin and A23187 [GenBank] , were tested. Consistent with the results by indomethacin and thapsigargin, pretreatment with tunicamycin or A23187 [GenBank] induced GRP78 and suppressed MCP-1 (Fig. 2D). Again, close, inverse correlation was observed between the level of MCP-1 and the level of UPR. To further conform that UPR is involved in the suppression of MCP-1 expression, we used SubAB that specifically degrades GRP78 protein (32). GRP78 serves as a master regulator for the UPR network by binding to the transducers of ER stress; protein kinase-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) (2). Removal of GRP78 causes activation of these transducers and selective induction of UPR (32). Podocytes were pretreated with SubAB for 6 h and treated with TNF-{alpha} for an additional 6 h. As shown in Fig. 3A, pretreatment with SubAB suppressed induction of MCP-1, and it was correlated with induction of endogenous markers for UPR, GRP78 and CHOP. Dose-response experiments revealed that the suppressive effect of SubAB was observed at concentrations of ≥1–10 ng/ml, and inverse correlation was observed between the level of MCP-1 and the levels of GRP78 and CHOP (Fig. 3B).


Figure 3
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  		Fig. 3. Inhibition of TNF-{alpha}-induced MCP-1 expression by AB5 subtilase cytotoxin (SubAB)-induced UPR. Podocytes were pretreated with 100 ng/ml SubAB (A) or serial concentrations (0.1–100 ng/ml) of SubAB (B) for 6 h, treated with 10 ng/ml TNF-{alpha} for 6 h, and subjected to Northern blot analysis of MCP-1, GRP78, and CHOP.

 
NF-{kappa}B, but not PPAR-{gamma}, as a target of UPR-mediated suppression of MCP-1. In general, induction of MCP-1 by TNF-{alpha} is regulated by NF-{kappa}B (47). We investigated whether activation of NF-{kappa}B by TNF-{alpha} is attenuated by UPR triggered by indomethacin and other agents. For this purpose, podocytes were transiently transfected with pNF-{kappa}B-Luc, pretreated with indomethacin or thapsigargin and stimulated by TNF-{alpha}. The reporter assay revealed that induction of UPR by either indomethacin or thapsigargin significantly attenuated activation of NF-{kappa}B by TNF-{alpha} (Fig. 4A).


Figure 4
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  		Fig. 4. Suppression of TNF-{alpha}-induced NF-{kappa}B activation by indomethacin and thapsigargin. A: podocytes were transiently transfected with pNF-{kappa}B-Luc, pretreated with 200 µM indomethacin or 100 nM thapsigargin for 6 h, and treated with (+) or without (–) 10 ng/ml TNF-{alpha} for 9 h. Activity of luciferase was evaluated by chemiluminescent assay and normalized by the number of viable cells estimated by formazan assay. Assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant differences (P < 0.05). B and C: podocytes were pretreated with 200 µM indomethacin or 100 nM thapsigargin for 6 h and stimulated with 10 ng/ml TNF-{alpha} for 30 min or 24 h. Protein level of I{kappa}Bβ was evaluated by Western blot analysis. The level of β-actin is shown at the bottom as a loading control. Densitometric analysis of individual bands normalized by the levels of β-actin is shown (C). D: podocytes were pretreated with ibuprofen, indomethacin, or thapsigargin for 16 h and subjected to Western blot analysis of TRAF2.

 
Under unstimulated conditions, NF-{kappa}B locates in the cytoplasm as complexes with I{kappa}B. When cells are stimulated, IKK phosphorylates I{kappa}B and causes its rapid degradation via the proteasome pathway. It allows for translocation of NF-{kappa}B into the nucleus and its binding to the {kappa}B sites. To confirm involvement of NF-{kappa}B as the target of UPR-mediated inhibition of MCP-1, the level of I{kappa}B protein was evaluated. Western blot analysis revealed that, following stimulation with TNF-{alpha}, I{kappa}Bβ was rapidly degraded within 30 min, and the reduced level lasted for at least 24 h. However, induction of UPR by indomethacin or thapsigargin substantially inhibited the degradation of I{kappa}Bβ at both 30 min and 24 h (Fig. 4, B and C), confirming the result of the reporter assay.

A recent report showed that, in certain cancer cells, ER stress downregulated the level of TRAF2 selectively (11). TRAF2 is an essential component for TNF-{alpha}-induced NF-{kappa}B signaling (48), and the suppression of NF-{kappa}B by indomethacin-induced UPR may be ascribed to depletion of TRAF2. To examine this possibility, podocytes were treated with ibuprofen, indomethacin, or thapsigargin for 16 h, and the level of TRAF2 was examined by Western blot analysis. As shown in Fig. 4D, the level of TRAF2 was depressed in indomethacin- or thapsigargin-treated cells, but not in ibuprofen-treated cells.

Previous reports showed that indomethacin may activate PPAR-{gamma} (24, 44) and thereby exert anti-inflammatory effects via inhibition of NF-{kappa}B (3, 34). We speculated that activation of PPAR-{gamma} might be involved in the suppression of NF-{kappa}B by indomethacin-induced UPR. To examine this possibility, an effect of indomethacin on the activity of PPAR-{gamma} was tested. Podocytes were transiently transfected with pPPRE(A)-Luc and treated with indomethacin or the PPAR-{gamma} agonist troglitazone. The reporter assay showed that troglitazone significantly induced activation of PPRE. In contrast, indomethacin did not activate PPAR-{gamma} in podocytes (Fig. 5A). Furthermore, activation of NF-{kappa}B by TNF-{alpha} was not affected by the pretreatment with troglitazone (Fig. 5B). These results excluded a possibility that PPAR-{gamma} is involved in the suppression of NF-{kappa}B by UPR triggered by indomethacin.


Figure 5
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  		Fig. 5. Lack of involvement of peroxisome proliferator-activated receptor (PPAR)-{gamma} in indomethacin-induced suppression of NF-{kappa}B. A: podocytes were transiently transfected with pPPRE(A)-Luc, treated with 200 µM indomethacin or 10 µM troglitazone for 9 h, and subjected to chemiluminescent assay. Activity of luciferase was normalized by the number of viable cells estimated by formazan assay. Assays were performed in quadruplicate, and data are expressed as means ± SE. NS, not statistically significant. *Statistically significant difference (P < 0.05). B: podocytes were transiently transfected with pNF-{kappa}B-Luc, pretreated with or without 10 µM troglitazone for 6 h, stimulated with or without 10 ng/ml TNF-{alpha} for 9 h, and subjected to chemiluminescent assay. Normalized activity of NF-{kappa}B is shown.

 
Suppression of TNF-{alpha}-induced MCP-1 expression and NF-{kappa}B activation by GRP78. As demonstrated, indomethacin induced GRP78, and close, inverse correlation was observed between the level of MCP-1 and the level of GRP78 in TNF-{alpha}-stimulated podocytes. We examined whether induction of GRP78 per se is involved in the suppression of NF-{kappa}B and NF-{kappa}B-mediated MCP-1 expression by indomethacin. For this purpose, SubAB that specifically degrades GRP78 protein was used. Following the treatment with SubAB, the reduction of GRP78 triggers UPR and consequently upregulates synthesis of GRP78 in the late phase. However, in the early phase, treatment with SubAB abrogates GRP78 protein before its secondary induction. As shown in Fig. 6A, treatment of podocytes with SubAB abolished GRP78 protein without reduction of GRP94. The abrogation of GRP78 protein was observed within 1 h (data not shown). To examine a role of GRP78 in the UPR-induced insensitiveness of podocytes to TNF-{alpha}, cells were pretreated with SubAB for 1 h and stimulated by TNF-{alpha} for 6 h. In contrast to the cells pretreated with SubAB for 6 h (Fig. 3A), the lack of GRP78 rather enhanced induction of MCP-1 by TNF-{alpha} (Fig. 6B). Furthermore, reporter assays showed that activation of NF-{kappa}B occurred in the deprivation of GRP78 and that NF-{kappa}B activation by TNF-{alpha} was not inhibited but was rather enhanced (Fig. 6C).


Figure 6
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  		Fig. 6. Suppression of TNF-{alpha}-induced MCP-1 expression and NF-{kappa}B activation by 78-kDa glucose-regulated protein (GRP78). A: podocytes were treated with or without 100 ng/ml SubAB for 4 h. Protein levels of GRP78 and GRP94 were examined by Western blot analysis. B: podocytes were pretreated with or without 100 ng/ml SubAB for 1 h and stimulated by 10 ng/ml TNF-{alpha} for 6 h. Expression of MCP-1, GRP78, and CHOP was examined by Northern blot analysis. C: podocytes were transiently transfected with pNF-{kappa}B-Luc, pretreated with or without 100 ng/ml SubAB for 1 h, stimulated with or without 10 ng/ml TNF-{alpha} for 6 h, and subjected to chemiluminescent assay. Normalized activity of NF-{kappa}B is shown. D and E: podocytes were transiently transfected with pNF-{kappa}B-Luc together with pcDNA3.1 (Vector) or pcDNA3.1-GRP78 (GRP78) (D), or pCI-neo (Vector) or pCI-neo-ORP150 (ORP150) (E), and stimulated with or without TNF-{alpha} for 9 h. Assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant difference (P < 0.05).

 
To further examine the role of GRP78 in the suppression of NF-{kappa}B, a transfection study was performed. Podocytes were transiently transfected with pNF-{kappa}B-Luc together with GRP78 and stimulated by TNF-{alpha}. Consistent with the result of the SubAB-based loss-of-function experiment, overexpression of GRP78 significantly suppressed activation of NF-{kappa}B by TNF-{alpha} (Fig. 4D). In contrast, overexpression of ORP150, another ER chaperone that attenuates ER stress (53), did not affect TNF-{alpha}-triggered NF-{kappa}B activation (Fig. 6E). These results implied a possibility that the suppressive effect of GRP78 may be not via attenuation of ER stress but via a more specialized, GRP78-dependent mechanism.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present report, we describe that UPR inhibits cellular responses to TNF-{alpha}, i.e., activation of NF-{kappa}B and consequent expression of MCP-1 in podocytes. In contrast to our current results, several previous reports showed that ER stress and consequent UPR trigger activation of NF-{kappa}B (16). The reason for the discrepancy is currently unclear, but one possibility is that, in the early phase, ER stress induces rapid, modest activation of NF-{kappa}B, whereas in the later phase, consequent UPR may induce insensitiveness to subsequent stimuli. Indeed, as demonstrated in this report, in the early phase, UPR triggered by SubAB (pretreatment for 1 h) caused transient induction of MCP-1 and NF-{kappa}B activation and rather enhanced TNF-{alpha}-induced MCP-1 expression (Fig. 6, B and C). In contrast, in the later phase, UPR triggered by SubAB (pretreatment for 6 h) blunted induction of MCP-1 by TNF-{alpha} (Fig. 3, A and B). It is worthwhile to note that the inhibition of cellular responses to TNF-{alpha} by UPR is not specific to podocytes but is a more general phenomenon observed in other cell types, including mesangial cells and tubular epithelial cells (our unpublished data).

Currently, molecular mechanisms involved in the induction of tolerance by UPR are not fully elucidated. However, we have indicated a possibility that the anti-inflammatory potential of UPR may be mediated, at least in part, by the induction of GRP78. GRP78 is a multifunctional molecule composed of the ATPase domain, the peptide-binding domain, and a COOH-terminal domain with unknown function (17). Principally, GRP78 functions as a master regulator for the UPR network by binding to PERK, IRE1, and ATF6 to inhibit their activation (2). GRP78 also binds to unfolded proteins through the peptide-binding domain, promotes proper protein folding, and prevents aggregation of unfolded proteins (20). As described, several previous reports showed that, in the early phase, ER stress triggers activation of NF-{kappa}B. If so, it may not be surprising that GRP78, an important reliever of ER stress, has the potential to attenuate activation of NF-{kappa}B. In addition to these intracellular functions, GRP78 may have anti-inflammatory effects when it is secreted into extracellular space (31). In podocytes, GRP78 might be secreted under ER stress conditions and could attenuate activation of NF-{kappa}B.

In some cell types, however, GRP78 may also be involved in the activation of NF-{kappa}B. For example, a recent report provided evidence that TNF-{alpha} induced association of GRP78 with IKK and that knockdown of GRP78 abolished phosphorylation of RelA by TNF-{alpha} (39). Another report also showed that, in human prostate cancer cells, binding of activated forms of the proteinase inhibitor 2-macroglobulin to cell surface-associated GRP78 induced NF-{kappa}B activation, and silencing of GRP78 by RNAi suppressed this process (26). The role of GRP78 in the regulation of NF-{kappa}B is complicated and, possibly, dependent on cellular contexts.

In podocytes, UPR suppressed activation of NF-{kappa}B triggered by TNF-{alpha}. However, in IL-1β-stimulated cells, an inverse correlation between the level of UPR and suppression of MCP-1 was not evident. It suggests a possibility that UPR interferes with the TNF-{alpha} signaling upstream of the common pathway to NF-{kappa}B activation by TNF-{alpha} and IL-1β, i.e., upstream of IKK activation (48). In the TNF-{alpha} signaling, TNF receptor 1 (TNFR1), TNFR1-associated death domain (TRADD), receptor-interacting protein (RIP), TRAF2, and IKK complex are essential for NF-{kappa}B activation (48). In contrast, the proximal signaling event involved in IL-1β-triggered activation of NF-{kappa}B is different from that of TNF-{alpha}. In particular, in IL-1 signaling, TRAF2 is not required for activation of NF-{kappa}B. Recently, Hu et al. (11) reported that, in thapsigargin- or tunicamycin-treated MCF-7 cells and L929 cells, TNFR1, TRADD, RIP, and IKK proteins were maintained at the same levels as those in untreated control, whereas the level of TRAF2 protein was decreased significantly. Consistent with this report, we demonstrated that the level of TRAF2 protein was lower in indomethacin- and thapsigargin-treated cells, but not in ibuprofen-treated cells. The downregulation of TRAF2 by UPR may explain the different responses of MCP-1 to UPR in TNF-{alpha}- and IL-1β-stimulated podocytes.

The molecular mechanisms involved in the induction of UPR by indomethacin are currently unclear. However, the fact that other COX inhibitors did not induce UPR in podocytes even at high concentrations suggested that inhibition of COX is not causative of the induction of UPR by indomethacin. Previous reports showed that certain NSAIDs increased intracellular calcium levels and thereby caused UPR, including induction of GRP78 (45, 46). The increase in the intracellular calcium level may be caused by direct permeabilization of the cellular membrane by NSAIDs (45). The similar mechanism could underlie the induction of UPR by indomethacin. A putative, theoretical model for anti-inflammatory action of indomethacin in podocytes is illustrated in Fig. 7.


Figure 7
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  		Fig. 7. Hypothetical model for anti-inflammatory action of indomethacin in podocytes. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit inflammatory responses of cells via inhibition of cyclooxygenase (COX) and consequent suppression of proinflammatory prostaglandins (PGs). In addition to this known mechanism, indomethacin, but not other NSAIDs, inhibits TNF-{alpha}-triggered activation of NF-{kappa}B via induction of UPR and consequent downregulation of TNF receptor-associated factor 2 (TRAF2) and upregulation of GRP78.

 
Under ER stress, distinct death signals as well as survival signals are transduced (16). UPR induced by indomethacin may have not only the light side (anti-inflammatory) but also the dark side (proapoptotic) that triggers cellular damage. It is known that NSAIDs including indomethacin can cause nephrotoxicity (25). Several mechanisms have been postulated to explain the phenomenon, but our current result indicates a possibility that ER stress triggered by indomethacin might also contribute to its nephrotoxic effect.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (nos. 16390243, 17651026, and 19651024) to M. Kitamura.


   ACKNOWLEDGMENTS
 
We appreciate Dr. Karlhans Endlich (University of Heidelberg) for providing with murine podocytes. We also thank Dr. Kazunori Imaizumi (Nara Institute of Science and Technology), Dr. David Ron (New York University School of Medicine), Dr. Richard C. Austin (Henderson Research Center), Dr. Satoshi Ogawa (Kanazawa University), and Dr. Shunji Ishihara (Shimane University School of Medicine) for providing with plasmids.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Kitamura, Dept. of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, Univ. of Yamanashi, Shimokato 1110, Chuo, Yamanashi 409-3898, Japan (e-mail: masanori{at}yamanashi.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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