Resistance to TNF-alpha cytotoxicity can be achieved through different signaling pathways in rat mesangial cells

Yan-Lin Guo, Baobin Kang, and John R. Williamson

Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

We reported previously that Ro-318220 blocked expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) induced by tumor necrosis factor-alpha (TNF-alpha ) and subsequently caused apopotosis in mesangial cells (Y.-L. Guo, B. Kang, and J. R. Williamson. J. Biol. Chem. 273: 10362-10366, 1998). These data support our hypothesis that a TNF-alpha -inducible phosphatase may be responsible for preventing sustained activation of c-Jun NH2-terminal protein kinase (JNK) and consequent cell death in these cells (Y.-L. Guo, K. Baysal, B. Kang, L.-J. Yang, and J. R. Williamson. J. Biol. Chem. 273: 4027-4034, 1998). In this study, we investigated the involvement of protein kinase C (PKC) in regulation of MKP-1 expression in mesangial cells together with effects on viability. Although originally characterized as a PKC inhibitor, Ro-318220 inhibited TNF-alpha -induced MKP-1 expression through a mechanism other than blocking the PKC pathway. Furthermore, inhibition of the PKC pathway neither significantly affected TNF-alpha -induced MKP-1 expression nor made cells susceptible to toxic effect of TNF-alpha . Thus PKC activation is not essential for cells to achieve the resistance to TNF-alpha cytotoxicity displayed by normal mesangial cells. However, activation of PKC by phorbol 12-myristate 13-acetate (PMA) dramatically increased cellular resistance to the apoptotic effect of TNF-alpha . Coincidentally, PMA stimulated MKP-1 expression and suppressed JNK activation. Therefore, PMA-induced MKP-1 expression may contribute to the protective effect of PMA. These results provide a mechanistic explanation for previous documentation that PKC activation can rescue some cells from apopotosis.

apoptosis; mitogen-activated protein kinase; mitogen-activated protein kinase phosphatase; c-Jun NH2-terminal protein kinase; tumor necrosis factor-alpha


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

TUMOR NECROSIS FACTOR-alpha (TNF-alpha ) is a polypeptide cytokine that can elicit a wide range of biological responses depending on the cell type and the state of differentiation (3, 39). One of these responses is the induction of apoptosis or programmed cell death in some cells (27). Although certain tumor cells, virus-infected cells, or damaged cells are sensitive to TNF-alpha -induced apopotosis, many normal cells are usually resistant (24, 35). Therefore, TNF-alpha plays an important role in determining cell viability under physiological and pathological conditions.

The current hypothesis to explain how TNF-alpha can selectively kill some cells while not being harmful to others implies the operation of two opposite signaling pathways: a preexisting destructive (apoptotic) pathway and an inducible protective (survival) pathway. Interactions between the two pathways and particularly the balance of their activities will determine whether the cells survive or die (24). Recently, significant progress has been made in clarifying the mechanism of apoptosis induced by TNF-alpha . However, the identities of the protective factors induced by TNF-alpha and their mechanisms of action remain elusive. TNF-alpha activation of extracellular signal-regulated protein kinase (ERK) and nuclear factor-kappa B (NF-kappa B) has been proposed to counteract the cytotoxicity of the apoptotic pathway in certain cells (1, 37, 42) but not in others (10, 20). Some of the putative protective factors so far identified include a manganous superoxide dismutase (41), a zinc finger protein (29), and members of the Bcl-2 family of proteins (11, 19). Given the complexity of TNF-alpha signaling pathways, it is apparent that different protective factors may exert their antiapoptotic effects through different mechanisms and may act at different stages of the apoptotic process in a cell type- and stimulus-dependent manner.

A sustained activation of c-Jun NH2-terminal protein kinase (JNK) has been shown to be an apoptotic signal in many cells (6, 38). In our previous study (12), we found that rat mesangial cells are normally resistant to TNF-alpha -induced apoptosis. However, they can be made susceptible to the apoptotic effect of TNF-alpha when pretreated with cycloheximide or vanadate. We proposed that a TNF-alpha -induced mitogen-activated protein kinase phosphatase-1 (MKP-1) was responsible for suppressing a prolonged activation of JNK in these cells, thereby protecting them from apopotosis. This hypothesis is supported by our recent finding that blockade of the TNF-alpha -induced expression of MKP-1 by Ro-318220 caused a sustained activation of JNK and resulted in apopotosis (13). A similar conclusion was reached independently of our work by Franklin et al. (9), who demonstrated that conditional expression of MKP-1 protected ultraviolet-induced apoptosis by inhibiting JNK activity in U937 cells. Because Ro-318220 was originally characterized as a protein kinase C (PKC) inhibitor (40), it was of interest to investigate whether it exerted its effect through inhibition of the PKC pathway. PKC activation has been implicated in the regulation of TNF-alpha -initiated cellular processes (17, 23, 31, 32). In several studies, it was found that activation of PKC could protect some cells from apoptosis, but the mechanisms involved are unknown (17, 28). In this study, we extend our investigation of the mechanisms by which Ro-318220 inhibits TNF-alpha -induced MKP-1 and provide some insight into how the PKC pathway may be involved in protecting cells from apoptosis.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Materials. Recombinant TNF-alpha was obtained from Chemicon International (Temecula, CA). Anti-c-Fos antibodies, anti-PKC-delta , -epsilon , and -zeta antibodies, and anti-MKP-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-c-Jun antibodies were from New England Biolabs (Beverly, MA). Anti-PKC-alpha , -beta , -gamma , -lambda , and -iota antibodies were from Transduction Laboratories (Lexington, KY). Ro-318220 (bisindolylmaleimide IX) and GF-109203X (bisindolylmaleimide I) were from LC Laboratories (San Diego, CA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma (St. Louis, MO).

Cell culture. Rat mesangial cells were isolated from male Sprague-Dawley rats under sterile conditions using the sieving technique described previously (21). The cells were maintained in RPMI 1640 medium containing 20% FCS and 0.6 U/ml insulin at 37°C in a humidified incubator (5% CO2 and 95% air). Cells from passages 5-20 were used. After the cells were grown to 80-90% confluence, they were incubated for 16-18 h in insulin-free RPMI 1640 medium containing 2% FCS before the experiments.

Cell viability assay. For cell viability assays, mesangial cells were grown in 12-well plates. They were treated with reagents for the times indicated. Uptake of neutral red dye was used as a measurement of cell viability (12). At the end of the incubations, the medium was removed and the cells were incubated in DMEM containing 2% FCS and 0.001% neutral red for 90 min at 37°C. The uptake of the dye by viable cells was terminated by removing the medium, washing the cells briefly with 1 ml of 4% paraformaldehyde in PBS (pH 7.4), and solubilizing the internalized dye with 1 ml of a solution containing 50% ethanol and 1% glacial acetic acid. The absorbances, which correlate with the amount of live cells, were determined at 540 nm. The cell morphology was also examined under a light microscope periodically during the cell treatment.

Immunocytochemical detection of apoptosis and c-Jun phosphorylation. For apoptosis analysis, cells grown on 25-mm glass coverslips in six-well plates were fixed with 4% paraformaldehyde in PBS after treatment with various reagents as indicated. DNA strand breaks were identified using a TUNEL assay kit (Boehringer Mannheim). Briefly, the fixed cells were treated with terminal deoxyribonucleotidyl transferase, which incorporates fluorescein-tagged nucleotides onto 3'-OH termini of fragmented DNA. After incubation with terminal deoxyribonucleotidyl transferase, the same coverslips were washed briefly with PBS and used for the detection of phospho-c-Jun. Positively stained nuclei were detected with Texas red-conjugated secondary antibodies as described previously (13). Fluorescein-tagged apoptotic nuclei and Texas red-labeled nuclei were visualized using fluorescence microscopy.

Immunocytochemical detection of PKC translocation. Cells grown on 25-mm glass coverslips in six-well plates were fixed with 4% paraformaldehyde in PBS after treatment with PMA or TNF-alpha as indicated. PKC-alpha was immunolocalized using its specific antibodies and visualized with Texas red-conjugated secondary antibodies using fluorescence microscopy following the immunocytochemistry protocol as described (13).

Preparation of whole cell lysate. The cells were treated with reagents for the times indicated, washed twice with ice-cold PBS, and scraped into cell lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM Na3VO4, 50 mM pyrophosphate, 100 mM NaF, 1 mM EGTA, 1.5 mM MgCl2, 1% Triton X-100, 10% glycerol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells were incubated for 10 min on ice and then lysed by sonication (25 pulses, output control 3) using a Branson sonicator and centrifuged at 15,000 g for 15 min. The supernatant was designated whole cell lysate. Protein concentration was determined by the method of Bradford (5) using BSA as standard.

Preparation of cytosol and particulate fractions. The cells were treated with 1 µM PMA or 10 ng/ml TNF-alpha for the times indicated. The cells were washed twice with ice-cold PBS and scraped into a buffer containing 25 mM Tris · HCl (pH 7.5), 2 mM EDTA, 1 mM dithiothreitol (DTT), 10% glycerol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF. The cells were incubated for 10 min on ice and then lysed by sonication as described above. The cell lysate was centrifuged for 5 min at 500 g. The pellet containing the cell debris was discarded. The supernatant was centrifuged at 100,000 g for 30 min at 4°C. The supernatant was designated the cytosolic fraction. The pellet was extracted with a buffer containing 25 mM Tris · HCl (pH 7.5), 2 mM EDTA, 1 mM DTT, 10% glycerol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1% Triton X-100 for 30 min on ice. The extract was centrifuged at 100,000 g for 30 min at 4°C. The supernatant, which contained solubilized membranes, was designated the particulate fraction.

Western blot analysis. The protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies according to the manufacturer's instructions with some modifications. The immunoblots were visualized by an enhanced chemiluminescence kit obtained from Amersham Pharmacia Biotech.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Role of PKC in TNF-alpha - and PMA-induced MKP-1 expression. PKC is a protein kinase family that is composed of >10 isoforms. They are classified into three subgroups: conventional PKC isoforms (cPKC) that are activated by Ca2+, phosphatidylserine, and diacylglycerol or PMA; novel PKC isoforms (nPKC) that are activated by diacylglycerol or PMA but not by Ca2+, and atypical PKC isoforms (aPKC) that are insensitive to Ca2+ and PMA but are activated by phosphatidylserine (26). Two approaches were used to investigate whether Ro-318220 inhibited TNF-alpha -induced MKP-1 expression by inhibiting the PKC pathway and to assess the potential involvement of PKC in the expression of MKP-1. First, the cells were treated with PMA for 18 h. This long-term incubation will cause a sustained activation of PMA-sensitive PKC isoforms (cPKC and aPKC) and will eventually result in their degradation, a phenomenon known as PKC downregulation (26). In the second approach, before stimulation with TNF-alpha , the cells were pretreated with GF-109203X, a close structural analog of Ro-318220, which has been shown to be a potent but less specific inhibitor of different PKC isoforms. This treatment acutely inhibits total PKC activity, including aPKC activity (2, 8, 25). As shown in Fig. 1A, whereas Ro-318220 totally blocked TNF-alpha -induced expression of MKP-1, inhibition by GF-109203X or downregulation of PKC by PMA only showed a slight inhibitory effect. In addition to MKP-1, TNF-alpha also induced the expression of c-Fos, which was totally blocked by either Ro-318220 or GF-109203X (Fig. 1B).


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Fig. 1.   Effects of Ro-318220 (Ro), GF-109203X (GF), and protein kinase C (PKC) downregulation on tumor necrosis factor-alpha (TNF-alpha )-induced expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and c-Fos. A: cells were stimulated with 10 ng/ml TNF-alpha for 30 min or were pretreated with 5 µM Ro-318220 or 5 µM GF-109203X for 30 min and then stimulated with 10 ng/ml TNF-alpha for 30 min. For PKC downregulation, cells were incubated with 1 µM phorbol 12-myristate 13-acetate (PMA) for 18 h, medium was changed to fresh starvation medium (RPMI 1640 containing 2% FCS), and then cells were stimulated with TNF-alpha for 30 min. B: cells were treated with TNF-alpha for 30 or 60 min or with combinations of TNF-alpha and Ro-318220 or GF-109203X as described for A. Whole cell lysate was subjected to SDS-PAGE and transferred to nitrocellulose membranes. MKP-1 and c-Fos were identified by Western blot analysis using specific antibodies.

PMA is one of the best-studied PKC activators and has been shown to induce the expression of MKP-1 in some cell types (4, 22). This is also the case in mesangial cells, as shown in Fig. 2. However, in contrast to the expression of MKP-1 induced by TNF-alpha , PMA-induced expression of MKP-1 was totally inhibited by both Ro-318220 and GF-109203X (Fig. 2A). The effect of PMA on the expression of c-Fos was also examined (Fig. 2B). It strongly stimulated c-Fos expression, which was also blocked by both Ro-318220 and GF-109203X. These results indicate that Ro-318220 and GF-109203X were equally effective in inhibiting the effect of PMA, which was most likely mediated by the PKC pathway.


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Fig. 2.   Expression of MKP-1 and c-Fos induced by PMA and effects of Ro-318220 and GF-109203X. Cells were stimulated with 1 µM PMA or pretreated with 5 µM Ro-318220 or GF-109203X for 30 min, followed by 1 µM PMA, for times indicated. Whole cell lysate was subjected to SDS-PAGE and transferred to nitrocellulose membranes. MKP-1 (A) and c-Fos (B) were identified by Western blot analysis using specific antibodies.

The effectiveness of PKC downregulation by PMA was confirmed by Western blot analysis. Only PKC-alpha (conventional), -delta and -epsilon (novel), and -zeta (atypical) were detected in the whole cell lysate using PKC isoform-specific antibodies (Fig. 3), consistent with the results reported by other investigators in mesangial cells (16). After incubation for 18 h with PMA, the content of PMA-insensitive PKC-zeta in the whole cell lysate was only slightly affected, as expected (Fig. 3). It should be pointed out that the anti-PKC-zeta antibody cross-reacted with three bands in the region between the 66- and 116-kDa molecular markers (Fig. 3). A nearly identical pattern was seen in mesangial cells using the same antibody by Rzymkiewicz et al. (30). The band in the middle was identified as PKC-zeta based on the fact that its expression was blocked by an antisense oligonucleotide to PKC-zeta . The lower band was not affected by the same treatment; therefore, it may represent a nonspecific binding protein (30). The higher band was identified as a PMA-sensitive PKC isoform that cross-reacted with anti-PKC-zeta antibodies as noted in a number of publications (7, 34). Similarly, in mesangial cells, this band was sensitive to PMA downregulation (Fig. 3) and translocated to the membrane fraction upon PMA stimulation (see Fig. 5). As shown in Fig. 3, PKC-alpha and -delta were essentially below the detection level, and the amounts of PKC-epsilon were greatly diminished after long-term incubation of the cells with PMA (Fig. 3). Therefore, PMA downregulation of PKC isoforms would significantly but not completely diminish PKC activity, because of the insensitivity of PKC-zeta to PMA downregulation. However, the inhibition of total PKC activity by GF-109203X was expected to be more effective. Many studies suggested that GF-109203X is a stronger but less selective PKC inhibitor than Ro-318220 as discussed by Beltman et al. (2). The selectivity of Ro-318220 and GF-109203X was tested in an alpha T3-a gonadotroph-derived cell line that expressed PKC-alpha , -epsilon , and -zeta . In vivo, Ro-318220 was shown to selectively inhibit PKC-alpha and -epsilon , whereas GF-109203X was an equally effective inhibitor of PKC-alpha , -epsilon , and -zeta , with IC50 values in the nanomolar range (18). In an in vitro study, GF-109203X inhibited all PKC isoforms tested with an order of potency of alpha  > beta 1 > epsilon  > delta  > zeta  (25). Thus, under our experimental conditions, the total activity of PKC, including PKC-zeta , should be significantly inhibited by GF-109203X.


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Fig. 3.   Downregulation of PKC by PMA. Cells were treated with (+) or without (-) PMA for 18 h. PKC isoforms in whole cell lysate were identified by Western blot analysis using specific antibodies.

Effects of Ro-318220, GF-109203X, and PKC downregulation on the viability of cells treated with TNF-alpha . To investigate the possibility of a correlation between expression level of MKP-1 and the protection effect against apoptosis induced by TNF-alpha , the cell viability was tested under experimental conditions described in Fig. 1. When TNF-alpha -induced MKP-1 expression was blocked by Ro-318220 (Fig. 1A), the effect of TNF-alpha on cell viability was dramatic (Fig. 4A). However, the same concentration of GF-109203X showed a much weaker effect on TNF-alpha -induced cell death (Fig. 4A), which correlated with it being ineffective in blocking the expression of MKP-1 (Fig. 1A). Similarly, downregulation of PKC with PMA (18 h) showed no significant effects on cell viability when the cells were stimulated with TNF-alpha (Fig. 4B). The overall pattern of cell viability to TNF-alpha and Ro-318220 treatment (Fig. 4B) was very similar to that observed when the cells were not pretreated with PMA (Fig. 2A). Thus the cellular resistance to TNF-alpha toxicity displayed by normal mesangial cells seems to correlate with the level of expression of MKP-1 but not with activity of PKC.


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Fig. 4.   Effects of Ro-318220, GF-109203X, and PKC downregulation on viability of cells stimulated with TNF-alpha . A: cells were incubated with 10 ng/ml TNF-alpha for 3 h (T) or 10 µM Ro-318220 (R) or 10 µM GF-109203X (G) for 3.5 h. Other cells were pretreated with 10 µM Ro-318220 or GF-109203X for 30 min and then stimulated with 10 ng/ml TNF-alpha for 3 h (RT, GT respectively). B: for PKC downregulation, cells were incubated with 1 µM PMA for 18 h, medium was changed to fresh starvation medium, and cells were stimulated with TNF-alpha for 3 h (PT), with Ro-318220 for 3.5 h (PR), or with Ro-318220 for 30 min followed by TNF-alpha for 3 h (PRT). Cells treated with PMA only were used as control (PC). Cell viability was determined by neutral red assay method. Results are means ± SE of 3 experiments performed in triplicate.

Activation of PKC by TNF-alpha and PMA. It has been reported that TNF-alpha was able to activate PKC in some cells but not in others (32). Our results thus far favor the notion that PKC activation may not be required for the protective effect against apoptosis elicited by TNF-alpha . We next examined whether TNF-alpha could activate PKC in mesangial cells. It is known that, upon activation by various stimuli, PKC translocates from the cytosol to the cell membrane. Redistribution of PKC, therefore, has been regarded as an indication of its activation (15). As shown in Fig. 5, PMA caused a significant increase of PKC-alpha , -delta , and -epsilon in the membrane fractions (Fig. 5A), with a concurrent decrease in the cytosolic fraction (Fig. 5B), whereas the content of PKC-zeta was not changed in either the cytosolic or membrane fractions. On the other hand, TNF-alpha caused only a slight increase of PKC-epsilon in the membrane fraction (Fig. 5A). Therefore, PKC activation stimulated by TNF-alpha was negligible compared with that stimulated by PMA in mesangial cells. The redistribution of PKC was further confirmed by immunolocalization using anti-PKC-alpha antibodies as an example. PKC-alpha is mainly in cytoplasm in the resting cells. PMA caused a clear redistribution of PKC-alpha within the cells, whereas TNF-alpha had no apparent effect (data not shown). This is consistent with results obtained from Western blot analysis (Fig. 5).


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Fig. 5.   Translocation of PKC isoforms induced by PMA and TNF-alpha . Cells were treated with 10 ng/ml TNF-alpha or 1 µM PMA for 15 min. CON, control. Cell lysates were fractionated to particulate (A) and cytosolic (B) fractions and subjected to SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes. PKC isoforms were identified by Western blot analysis using specific antibodies.

Pretreatment of mesangial cells with PMA increases resistance to cell death caused by TNF-alpha and cycloheximide. It was reported that activation of PKC could protect some cells from apoptosis induced by stimuli such as radiation (14) and ceramide (17, 28). Therefore, we examined whether activation of PKC could rescue mesangial cells from TNF-alpha -induced apoptosis. As shown in Fig. 6, incubation of mesangial cells with TNF-alpha in the presence of cycloheximide caused a significant loss of cell viability (Fig. 6). Interestingly, this effect could be largely reversed by pretreatment of the cells with PMA for 2 h before addition of TNF-alpha and cycloheximide (Fig. 6). The PMA-induced cellular resistance to cell death was PKC dependent. This was shown by the fact that the PMA-induced protective effect was abolished when PMA was added together with either GF-109203X or Ro-318220 (Fig. 6). The morphology of the cells was examined under a microscope. At the end of 3 h of incubation with TNF-alpha and cycloheximide, ~70% of the cells were undergoing apoptosis, as judged by their characteristic morphology (Fig. 7B) and by TUNEL analysis (Fig. 8B). Pretreatment of the cells with PMA for 2 h dramatically decreased the incidence of cell death caused by subsequent treatment with TNF-alpha and cycloheximide (Figs. 7C and 8C). When GF-109203X was added together with PMA, the protective effect elicited by PMA was totally abolished (Fig. 7D), indicating that the antiapoptotic effect elicited by PMA was PKC dependent. Therefore, it seems that, although PKC activation contributes little to the TNF-alpha -mediated cellular resistance to TNF-alpha toxicity, PKC activation by PMA can dramatically potentiate the ability of the cells to counteract the apoptotic effect of TNF-alpha .


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Fig. 6.   Pretreatment of cells with PMA increased viability of mesangial cells. C, control; T, TNF-alpha for 3 h; Ch, cycloheximide for 3.5 h; ChT, cycloheximide for 30 min followed by TNF-alpha for 3 h; PChT, PMA for 2 h and then, after change to fresh starvation medium, cycloheximide for 30 min followed by TNF-alpha for 3 h; PGChT and PRChT, treatment same as for PChT, except GF-109203X (PGChT) or Ro-318220 (PRChT) was added with PMA. Concentrations of reagents were 10 ng/ml TNF-alpha , 5 µg/ml cycloheximide, 1 µM PMA, 10 µM Ro-318220, and 10 µM GF-109203X. Cell viability was determined by neutral red assay method. Results are mean ± SE of 3 experiments performed in triplicate.


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Fig. 7.   Pretreatment of cells with PMA increased resistance to cell death caused by subsequent treatment with TNF-alpha and cycloheximide. A: control. B: cells pretreated with 5 µg/ml cycloheximide for 30 min followed by 10 ng/ml TNF-alpha for 3 h. C: cells treated with 1 µM PMA for 2 h and, after change to fresh starvation medium, cycloheximide for 30 min followed by TNF-alpha for 3 h. D: cells treated as for C, except 10 µM GF-109203X was added with PMA. Cell morphology was examined under a phase-contrast light microscope.


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Fig. 8.   Pretreatment with PMA protected cell from apoptosis and attenuated c-Jun phosphorylation caused by subsequent treatment with TNF-alpha and cycloheximide. A and a: control. B and b: cells were pretreated with 5 µg/ml cycloheximide for 30 min followed by 10 ng/ml TNF-alpha for 3 h. C and c: cells were treated with 1 µM PMA for 2 h, medium was changed to fresh starvation medium, and cells were treated with cycloheximide for 30 min followed by TNF-alpha for 3 h. Apoptosis and c-Jun phosphorylation were examined in same cells. Apoptotic cells were identified by TUNEL analysis (A, B, C). Phospho-c-Jun was detected by anti-phospho-c-Jun antibodies and visualized with Texas red-conjugated secondary antibodies (a, b, c) by fluorescence microscopy.

Pretreatment of mesangial cells with PMA attenuates JNK activation induced by TNF-alpha and cycloheximide. Our results indicate that cellular resistance to TNF-alpha toxicity seems to correlate with the expression level of MKP-1. If PMA-induced MKP-1 could dephosphorylate and inactivate JNK activation, one would expect that pretreatment of the cell with PMA should be able to suppress JNK activation induced by TNF-alpha and cycloheximide. As illustrated in Fig. 8, the cells undergoing apoptosis induced by TNF-alpha and cycloheximide (Fig. 8B) also show heavy phosphorylation of c-Jun, indicating strong activation of JNK in these cells (Fig. 8b). Pretreatment of the cells with PMA protected the cells from apoptosis (Fig. 8C) and also greatly attenuated c-Jun phosphorylation (Fig. 8c). These results provide a strong correlation between apopotosis and activities of JNK and MKP-1 and support the notion that PMA-induced MKP-1 may contribute to the protective effect of PMA by attenuation of a sustained JNK activation.


    DISCUSSION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Ro-318220 and GF-109203X are two structurally related analogs of bisindolylmaleimide that have been widely used as ATP-competitive inhibitors of PKC (25, 36, 40). In a recent study, however, it was found that, unlike GF-109203X, Ro-318220 has some distinct cellular effects that are independent of its ability to inhibit PKC (2). In accordance with this finding, the present study shows that Ro-318220 inhibited TNF-alpha -induced MKP-1 in rat mesangial cells through a mechanism other than inhibition of PKC. This was demonstrated by two independent approaches, i.e., by acute inhibition of PKC with GF-109203X and by downregulation of PKC with PMA (Fig. 1). More importantly, our results also indicate that the induction of MKP-1 by TNF-alpha and PMA acted through different mechanisms, i.e., PKC activation was essential for PMA-induced MKP-1 expression, but it contributed only marginally to TNF-alpha -induced MKP-1 expression.

The involvement of PKC in the regulation of TNF-alpha -mediated processes has been documented in a number of cells (3, 17, 28, 31, 33). However, its role in the regulation of TNF-alpha toxicity remains largely unknown. Activation of PKC by TNF-alpha seems to be cell type dependent. For instance, among human leukemic cell lines, TNF-alpha activated PKC in Jurkat cells, U937 cells, and K562 cells but not in CCD18 cells (32). In mesangial cells, TNF-alpha only induced a slight translocation of PKC-epsilon . Although it is unclear to exactly what level the activity of PKC was changed upon stimulation with TNF-alpha , this uncertainty does not detract from the major conclusions to be drawn from our data; i.e., activation of PKC is not essential for TNF-alpha -induced expression of MKP-1 and for the cellular resistance to apoptosis exhibited by mesangial cells under normal conditions.

The resistance to apoptosis induced by TNF-alpha and cycloheximide seems to correlate with the expression level of MKP-1 induced by both TNF-alpha and PMA. Inhibition of PKC neither significantly affected TNF-alpha -induced expression of MKP-1 nor made the cells susceptible to the toxic effect of TNF-alpha . However, the cells underwent apoptosis when MKP-1 expression was blocked under our experimental conditions (Figs. 1 and 4) (12, 13). Another interesting observation is that, in addition to MKP-1, TNF-alpha also induced c-Fos expression, both effects being blocked by Ro-318220 (Fig. 1). Thus an important question to ask is whether TNF-alpha -induced c-Fos also plays a role in protecting the cells from apoptosis. This question is addressed by the observations that GF-109203X selectively inhibited TNF-alpha -induced c-Fos but not MKP-1 expression (Fig. 1) and that the cells did not become susceptible to TNF-alpha -induced apoptosis under these conditions (Fig. 4). It is likely that expression of MKP-1 but not c-Fos is responsible for the antiapoptotic effect. These results further support our hypothesis that MKP-1 may act as a protective factor against TNF-alpha -induced apoptosis by suppressing JNK activation (12, 13). Recently, a similar conclusion was reached independently of our work by Franklin et al. (9), who demonstrated conditional expression of MKP-1-protected ultraviolet light-induced apoptosis by inhibiting JNK activity in U937 cells. On the basis of this assumption, one would expect that other agents that are able to induce MKP-1 expression should show a similar protective effect. This speculation is supported by the results from PMA experiments. PMA induced MKP-1 expression within the time period from 30 to 120 min (Fig. 2) and also elicited resistance to apoptosis caused by TNF-alpha and cycloheximide (120 min; Figs. 6 and 7), with a concurrent attenuation of JNK activity (Fig. 8). Unlike TNF-alpha -induced MKP-1, the effects of PMA were dependent on PKC activation. Thus the involvement of the PKC pathway in the mediation of resistance to apoptosis seems to be stimulus dependent in rat mesangial cells.

In several studies, it was reported that activation of PKC could protect the cells from apoptosis. For example, in leukemia cells, the PKC activator dioctanylglycerol and PMA blocked TNF-alpha - and ceramide-induced apoptosis (17, 28). Lotem et al. (23) reported that activation of PKC could rescue myeloid cells from apoptosis induced by withdrawal of growth factors. It is known that PMA activates several signaling pathways in addition to PKC. These include ERK and NF-kappa B, both of which have been implicated in protecting cells from apoptosis (37, 42). It is likely that multiple signaling pathways are involved in mediating the antiapoptotic effect elicited by PMA. The present study provides a possible mechanistic explanation for how activation of PKC elicits resistance to apoptosis. Specifically, induction of MKP-1 by PMA may play a role in protecting cells from apoptosis by attenuating JNK activation. The expression of MKP-1 induced by TNF-alpha (12, 13) and PMA (present study) or by molecular biology techniques (9) showed similar effects against apoptosis. It is likely, therefore, that MKP-1 may act as a common protective molecule in the cells in which the JNK pathway is involved in the mediation of apoptosis.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-15120 and DK-48493. Y.-L. Guo is a recipient of NIDDK Training Grant DK-07314.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: J. R. Williamson, Dept. of Biochemistry and Biophysics, University of Pennsylvania, 601 Goddard Labs, 37th and Hamilton Walk, Philadelphia, PA 19104.

Received 11 June 1998; accepted in final form 2 November 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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