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J Am Soc Nephrol 14:1427-1434, 2003
© 2003 American Society of Nephrology

The PI3-Kinase-Akt Pathway Promotes Mesangial Cell Survival and Inhibits Apoptosis In Vitro via NF-{kappa}B and Bad

Haruko Shimamura, Yoshio Terada, Tomokazu Okado, Hiroyuki Tanaka, Seiji Inoshita and Sei Sasaki

The Homeostasis Medicine and Nephrology, Tokyo Medical and Dental University, Tokyo, Japan.

Correspondence to Dr. Yoshio Terada, Homeostasis Medicine and Nephrology, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Phone: 81-3-5803-5214; Fax: 81-3-5803-5215;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. While the serine/threonine protein kinase Akt has attracted attention as a mediator of survival (anti-apoptotic) signal, the regulation and function of the PI3-kinase-Akt pathway in mesangial cells is not well known. To explore the significance of the PI3-kinase-Akt pathway, this study used PI3-kinase inhibitors (Wortmannin and LY294002) and recombinant adenoviruses encoding a dominant-active mutant of Akt (AxCAmyrAkt) and a dominant-negative mutant of Akt (AxCAAkt-AA) in cultured rat mesangial cells. Apoptotic signals were measured by nucleosomal laddering of DNA, caspase 3 assay, and cell death detection ELISA. The PI3 kinase inhibitors and dominant-negative mutant of Akt increased the apoptotic signals in the presence of platelet-derived growth factor (PDGF), while the dominant-active mutant of Akt prevented apoptosis induced by a serum-free medium. In separate experiments, we further investigated downstream signals of Akt in mesangial cells. While PDGF activated NF-{kappa}B and phosphorylated Bad, these reactions were inhibited by overexpression of the dominant-negative mutant of Akt as well as the PI3-kinase inhibitors. These data indicate, firstly, that Akt is phosphorylated by PDGF, and secondly, that the activated Akt prevents apoptotic changes via activation of NF-{kappa}B and phosphorylation of Bad in mesangial cells. This study investigated whether it is Bad phsophorylation or NF-{kappa}B activation that provides the anti-apoptotic effects of Akt, and the data suggested that NF-{kappa}B is probably the principal contributor to the downstream activation of the PI3-kinase-Akt pathway. The findings suggest that the PI3-kinase-Akt pathway acts as a survival signal and plays a key role in the regulation of apoptotic change in mesangial cells principally via NF-{kappa}B. E-mail: yterada.kid@tmd.ac.jp


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Akt (also called protein kinase B), the cellular homologue of the v-akt oncogene (1), is a 60-kD serine/threonine kinase cloned by virtue of its homology to PKA and PKC. The kinase is activated in response to stimulation of tyrosine kinase receptors such as platelet-derived growth factor (PDGF), insulin-like growth factor, and nerve growth factor (2–4). The growth factor receptor stimulation of Akt has been shown to be dependent on phosphatidylinositol 3-kinase (PI3-kinase) activity (5–8).

Growing evidence indicates that Akt is a critical mediator of survival signals that protect cells from apoptosis in multiple cell lines (9,10). These studies show that Akt can exert its anti-apoptotic effects in several different ways. For example, phosphorylation of the pro-apoptotic Bad protein by Akt was found to decrease apoptosis by preventing Bad from binding to an anti-apoptotic protein called Bcl-XL (11,12). Akt was also shown to directly inhibit the activity of a cell death protease caspase-9 (13). Furthermore, Akt is known to promote cell survival by activating nuclear factor-{kappa}B (NF-{kappa}B), which regulates expression of anti-apoptotic genes (14–16). It should be noted, however, these pathways seem cell-specific, and they are still poorly understood in mesangial cells.

Recent experimental evidence suggests that apoptosis plays pathophysiologic roles in glomerulonephritis. While morphologic evidence of mesangial apoptosis was observed in rat Thy1.1 glomerulonephritis (17–19), the regulation and function of the PI3-kinase-Akt pathway in mesangial cells has not been fully demonstrated under in vitro or in vivo conditions. To explore the role of the PI3-kinase-Akt pathway, we investigated the regulation of the Akt pathway in cultured mesangial cells in the present study. We demonstrated that anti-apoptotic cell survival pathways help to maintain a balance between cell survival and apoptosis in mesangial cells. In further experiments with the same cell type, we identified that two signal pathways downstream of Akt, namely activation of NF-{kappa}B and phosphorylation of Bad, and tried to determine which of them contributed as the anti-apoptotic pathway.

Our results provide insight into the highly organized signaling mechanisms coordinated by the Akt pathway and apoptotic signals in mesangial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The specific PI3-kinase inhibitors Wortmannin and LY294002 were purchased from Sigma Chemical (St. Louis, MO). Human PDGF-BB was obtained from Boehringer Mannheim (Mannheim, Germany). RPMI 1640 medium with 2.05 mM L-glutamine was obtained from Iwaki (Chiba, Japan). Acetylsalicylic acid and dexamethasone were purchased from Sigma Chemical (St. Louis, MO). A rabbit polyclonal antibody against actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Recombinant Adenoviruses
Replication-defective, recombinant adenoviruses encoding a dominant active myristoylated Akt (AxCAmyrAkt) and dominant-negative Akt (AxCAAkt-AA) were prepared as described previously. In brief, rat Akt1 was replaced with the myristoylated form (myrAkt) (20). Both Thr308 and Ser473 of rat Akt1 were replaced by alanine (Akt-AA) (21). The recombinant adenovirus expressing the LacZ gene (AxCALacZ) was prepared as described previously (22). Each adenovirus preparation was titrated by plaque-assay on 293 cells. Viral stocks (1011 plaque-forming units [pfu]/ml) were stored at -80°C and thawed on ice just before use.

Mesangial Cell Culture
Mesangial cell strains from male Sprague-Dawley rats were isolated and characterized as previously reported (23). The cells were maintained in RPMI1640 medium supplemented with 20% fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml of insulin, 5 µg/ml of transferrin, and 5 ng/ml selenite at 37°C in a 5% CO2 incubator. Cells used in experiments were from 5 to10 passages. After an initial incubation in medium plus 20% FCS until approximately 80% confluence, the cells were treated with medium with or without serum, PDGF, LY294002, or Wortmannin for the indicated time and concentration.

Western Blot Analysis
The cell lysates were mixed 1:4 with 5x Laemmli buffer and heated for 5 min at 95°C. Soluble lysates (20 µg) were loaded in each lane and separated by SDS-PAGE using 5% and 20% acrylamide for stacking and resolving gels, respectively. Protein was transferred to nitrocellulose (pore size, 0.45 µm; Schleicher and Schuell, Keene, NH) and probed with polyclonal antibodies against Serine 473-phospho-specific Akt, total-Akt, Serine 136-phospho-specific Bad, total-Bad, and phospho-I{kappa}B-{alpha} (Ser32; New England Biolabs, Beverly, MA). The primary antibodies (diluted 1/1000) and a second antibody consisting of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (diluted 1/2000; New England Biolabs) were used for the detection of phosphospecific-Akt, total-Akt, phospho-I{kappa}B-{alpha} (Ser32). The primary antibodies (diluted 1/500 and 1/1000) and a second antibody consisting of HRP-conjugated goat anti-rabbit IgG (diluted 1/2000; New England Biolabs) were used for the detection of phosphospecific-Bad and total-Bad. The bands were visualized by the ECL detection system with 5 to 10 min exposure after extensive washing of the membranes. Controls for protein loading were identified by actin as the internal standard.

Caspase 3and 9 Assays
A Caspase 3 and 9 Fluorometric Protease Assay Kit (MBL, Tokyo, Japan) was used for measurement of caspase 3 and 9 activities as described previously (24). In brief, cells were plated in six-well dishes and cultured in medium of different conditions. The cells were collected and lysed in lysis buffer at indicated times, and the protein concentration was normalized by the Bradford assay. The lysates were incubated with the same amount of reaction buffer and then incubated with 50 µM DEVD-AFC substrate (caspase 3) or 50 µM LEHD-AFC substrate (caspase 9) for 2 h at 37°C. Fluorescence was monitored with an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

Ladder Assays
Adherent and floating cells were collected and lysed in a medium containing 10 mmol/L Tris (pH 8.0), 100 mmol/L NaCl, and 25 mmol/L ethlenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS), and 1.0 mg/ml proteinase K at 37°C for 4 h. DNA was extracted from the digested cells as described previously (25), divided into 30-µg portions, and subjected to electrophoresis on 1.5% agarose gels.

Cell Death ELISA
Histone-associated DNA fragments were quantitated by ELISA (Boerhinger, Mannheim, Germany). All cells from each well were collected by trypsinization and pipetting, pelleted (800 rpm, 5 min), lysed, and subjected to the capture ELISA according to the manufacturer’s protocol. Cytosolic proteins were collected using cell lysis buffer according to the manufacturer’s protocol. After a 30-min incubation of the cells with cell lysis buffer, the samples were centrifuged for 10 min (15,000 rpm), the nuclei were formed into pellets, the cytoplasmic fraction became supernatant, and the supernatants were collected for the ELISA assay. Each experiment was carried out in triplicate and repeated independently at least five times.

Transient Transfection and Luciferase Assay
Rat mesangial cells were transfected with NF-{kappa}B-luciferase-plasmid (NF-{kappa}B element x 5 and luciferase fusion plasmid) from Stratagene (La Jolla, CA), and the lysate was used for promoter assay. Data are representative of at least five experiments performed in duplicate. Plasmid DNA (10 µg) were transfected by electroporation method. For experiments performed in exponentially growing cells, luciferase activity was measured 48 h after transfection. Normalization was achieved by cotransfecting 1.0 µg of pCH110, a {beta}-galactosidase reporter construct, as an internal control for the transfection efficiency, as previously reported (26).

Statistical Analyses
The results were given as means ± SEM. The differences were tested using two-way analysis of variance followed by the Scheffe test for multiple comparisons. Two groups were compared by the unpaired t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PDGF on Akt Phosphorylation in Cultured Rat Mesangial Cells
To explore the significance of the Akt pathway, we first examined the regulation of the PI3-kinase-Akt pathway in cultured rat mesangial cells. In preliminary experiments, we performed time course of PDGF-induced Akt phosphorylation from 0 to 60 min, the phosphorylation peaked at 15 min. Thus, mesangial cells were exposed to PDGF (5 to 50 ng/ml) for 15 min. Immunoblot analyses for phosphorylation of Akt were performed using phospho-Akt-specific antibody. As shown in Figures 1A and 1B, PDGF treatment increased the Akt phosphorylation dose-dependently, while treatment with the PI3-kinase inhibitor LY294002 (5 µM and 30 µM) reduced it. Similar findings were observed when the cells were pretreated with Wortmannin (20 nM and 50 nM; Figure 1C). These results suggested that Akt is a downstream mediator of PI3-kinase activation in mesangial cells.



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  		Figure 1. Effect of platelet-derived growth factor (PDGF) on phosphorylation of Akt in cultured rat mesangial cells. (A) PDGF dose-dependently stimulates phosphorylation of Akt in rat mesangial cells. Mesangial cells were incubated with PDGF for 15 min. (B) Inhibition of PDGF-induced phosphorylation of Akt by LY294002 in rat mesangial cells. Mesangial cells were pre-incubated with or without LY294002 for 30 min and then treated with PDGF for 15 min. (C) Inhibition of PDGF-induced phosphorylation of Akt by Wortmannin in rat mesangial cells. Mesangial cells were pre-incubated with or without Wortmannin for 30 min and then treated with PDGF for 15 min. Upper gel, immunoblots using an antibody specific to the phosphorylated form of Akt; lower gel, immunoblots with an antibody to total Akt.

 
The PI3-Kinase Inhibitors and the Dominant-Negative Mutant of Akt Promoted Apoptosis in PDGF-Treated Mesangial Cells
To investigate the functional roles of the PI3-kinase-Akt pathway, we examined how treatment with PI3-kinase inhibitors (50 nM Wortmannin and 30 µM LY294002) and adenovirus-mediated gene transfer of the dominant-negative mutant of Akt (AxCAAkt-AA) and AxCALacZ (control) influenced the apoptotic phenomena in mesangial cells. Apoptotic signals were measured by nucleosomal laddering of DNA, caspase 3 assay, and cell death detection ELISA. As shown in Figure 2A, the PI3–kinase inhibitors and the dominant-negative mutant of Akt induced nucleosomal laddering of cells in the presence of PDGF. Transfection of AxCAAkt-AA and the PI3–kinase inhibitors also induced caspase 3 activity (Figure 2B). Cell death ELISA examination showed that transfection of AxCAAkt-AA and the PI3-kinase inhibitors significantly increased the apoptotic signals (Figure 2C). According to these results, the inhibition of the PI3-kinase-Akt pathway promoted apoptotic changes in the mesangial cells.



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  		Figure 2. Induction of apoptosis by PI3-kinase inhibitors and dominant-negative Akt in rat mesangial cells. Mesangial cells were initially transfected with AxCAAkt-AA or AxCALacZ for 48 h, incubated with or without PI3-K inhibitors (wortmannin, LY294002) at 30 min before, and then exposed to PDGF for 12 h. (A) Extracted DNA from adherent and floating cells was subjected to electrophoresis on 1.5% agarose gels. (B) Cell lysate was used for caspase 3 assay (n = 5, mean ± SEM, *P < 0.05 versus PDGF only, #P < 0.05 AxCALacZ versus AxCAAkt-AA). (C) Cell lysate from adherent and floating cells was subjected to cell death ELISA assay (n = 5, mean ± SEM, *P < 0.05 versus PDGF only; n = 5, mean ± SEM, #P < 0.05 AxCALacZ versus AxCAAkt-AA).

 
The Dominant-Active Form of Akt Prevented the Induction of Apoptosis by Serum Deprivation in Mesangial Cells
To further examine the functional roles of Akt, we employed adenovirus-mediated gene transfer of the dominant-active mutant of Akt (AxCAmyrAkt) in mesangial cells. Preliminary, we found that apoptotic phenomena such as DNA laddering, increment of caspase 3 activity, and cell death in ELISA assay could be observed in mesangial cells when the cells were incubated in a serum-free condition for 48 h. We then transfected mesangial cells with adenovirus encoding AxCAmyrAkt and AxCALacZ (control) and measured the apoptotic signals. As shown in Figure 3A, the dominant-active mutant of Akt reduced nucleosomal laddering of cells caused by serum withdrawal. Transfection of AxCAmyrAkt also significantly reduced caspase 3 activity (Figure 3B). Cell death ELISA examination showed that the apoptotic signals were significantly inhibited by the transfection of AxCAmyrAkt (Figure 3C). These results demonstrated that the stimulation of the Akt pathway inhibited apoptotic changes induced by serum deprivation in mesangial cells.



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  		Figure 3. Inhibition of apoptosis by dominant-active Akt in rat mesangial cells. Mesangial cells were transfected with AxCAmyrAkt or AxCALacZ for 48 h and incubated by serum-free medium for 48 h. (A) Extracted DNA from adherent and floating cells was subjected to electrophoresis on 1.5% agarose gels. (B) Cell lysate was used for caspase 3 assay (n = 5, mean ± SEM, *P < 0.05). (C) Cell lysate from adherent and floating cells was subjected to cell death ELISA assay (n = 5, mean ± SEM, *P < 0.05).

 
Signal Pathways Downstream of Akt in Cultured Rat Mesangial Cells
Several downstream signals of Akt, such as NF-{kappa}B, Bad, and caspase 9, have been reported in different cell lines. To examine the signal pathway downstream of Akt in mesangial cells, we began by examining the activation of NF-{kappa}B by transfection of NF-{kappa}B-luciferase –plasmid (NF-{kappa}B element x 5 and luciferase fusion plasmid) and promoter assay. As shown in Figure 4A, the NF-{kappa}B luciferase activity stimulated by PDGF was reduced to 35% by a PI3-kinase inhibitor, LY 294002. This stimulation was also reduced to 31% by Wortmannin. In separate experiments using mesangial cells that had been initially transfected with AxCAAkt-AA or AxCALacZ, the NF-{kappa}B luciferase activity was reduced to 48% in the AxCAAkt-AA-transfected cells. Noting that NF-{kappa}B is held in the cytoplasm as an inactive complex with inhibitor I{kappa}B and remains in an inactivate state without I{kappa}B phoshorylation, we used Western bolt analysis further evaluate the phoshorylation of I{kappa}B with phospho-I{kappa}B-specific antibodies. PDGF stimulated the phosphorylation of I{kappa}B, and this phosphorylation was inhibited by LY294002, Wortmannin, and transfection of AxCAAkt-AA (Figure 4B). These results suggested that NF-{kappa}B is one of the downstream mediators of the PI3-kinase-Akt pathway in mesangial cells.



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  		Figure 4. Effect of PDGF on nuclear factor-{kappa}B (NF-{kappa}B) luciferase activity and phosphorylation of I{kappa}B in cultured rat mesangial cells. (A) Mesangial cells were initially transfected with NF-{kappa}B luciferase plasmid, and then transfected AxCAAkt-AA or AxCALacZ for 48 h, or incubated with or without PI3-kinase inhibitors (Wortmannin, LY294002) for 30 min. The cells were exposed to PDGF for 6 h and harvested for luciferase assay. (n = 5, mean ± SEM, *P < 0.05 versus PDGF only; #P < 0.05 AxCALacZ versus AxCAAkt-AA). (B) Mesangial cells were incubated with PDGF for 30 min. Phosphorylation of I{kappa}B in cultured rat mesangial cells was detected by immunoblots using an antibody specific to the phosphorylated form of I{kappa}B. Inhibition of PDGF-induced phosphorylation of I{kappa}B by LY294002 and Wortmannin in rat mesangial cells. Mesangial cells were pre-incubated with or without LY294002 or Wortmannin for 30 min and then treated with PDGF for 30 min. Mesangial cells were transfected with AxCAAkt-AA or AxCALacZ for 48 h and then treated with PDGF for 30 min. Upper gel, immunoblots using an antibody specific to the phosphorylated form of I{kappa}B; lower gel, immunoblots with an antibody to actin.

 
We next examined whether Bad is associated as a downstream mediator of the PI3-kinase-Akt pathway in cultured rat mesangial cells. Mesangial cells were exposed to PDGF for 20 min. Immunoblot analyses for Bad phosphorylation were performed using phospho-Bad-specific antibody (Ser136). As shown in Figure 5, PDGF treatment increased Bad phosphorylation and LY294002 (30 µM) inhibited this effect. We performed the same experiments using Wortmannin (50 nM) and obtained similar results. Adenovirus-mediated gene transfer of the dominant-negative mutant of Akt (AxCAAkt-AA) to mesangial cells also reduced the phosphorylation of Bad. These results suggest that Bad is a downstream mediator of the PI3-kinase activation in mesangial cells. In further experiments, we examined whether inhibition of Akt phosphorylaion by LY294002, Wortmannin, and the dominant-negative mutant of Akt (AxCAAkt-AA) altered the activity of caspase 9. As shown in Figure 6, caspase 9 activity was higher in the condition without serum. Caspase 9 activity was increased in mesangial cells cultured with PDGF alone than it was in cells cultured with FCS. We used this pro-apoptotic condition in the following experiments. In the experiments using transfected AxCAAkt-AA and PI3–kinase inhibitors, caspase 9 activity was not significantly changed from the activity observed in the presence of PDGF alone at neither 12 h nor 24 h (Figure 6 shows at 12 h). Thus, the Akt pathway seemed to play only a minor role in caspase 9 activity under our experimental condition. Judging from these data, there are at least two signaling pathways (via NF-{kappa}B and Bad) downstream of the PI3-kinase-Akt pathway in mesangial cells.



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  		Figure 5. Effect of PDGF on phosphorylation of Bad in cultured rat mesangial cells. Mesangial cells were pre-incubated with or without LY294002 or Wortmannin for 30 min and then treated with PDGF for 20 min. Mesangial cells were transfected with AxCAAkt-AA or AxCALacZ for 48 h, and then treated with PDGF for 20 min. Upper gel, immunoblots using an antibody specific to the phosphorylated form of Bad; lower gel, immunoblots with an antibody to total Bad.

 


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  		Figure 6. Induction of caspase 9 by PI3-kinase inhibitors and dominant-negative Akt in rat mesangial cells. Mesangial cells were transfected with either AxCAAkt-AA or AxCALacZ for 48 h, incubated with or without PI3-kinase inhibitors (Wortmannin, LY294002) for 30 min, and then immediately exposed to PDGF for 12 h. All cell lanes were treated with FCS-free medium except the lane of FCS(+). Cell lysate was used for caspase 9 assay (n = 6, mean ± SEM, *P < 0.05).

 
Next, we tried to assess the relative contributions of Bad and NF-{kappa}B signaling downstream of the PI3-kinase-Akt pathway to determine which plays a principal role. Our first step was to examine the effects of the NF-{kappa}B inhibitors (acetylsalicylic acid [ASA] and dexamethasone [Dex]). As shown in Figure 7, PDGF stimulated the phosphorylation of I{kappa}B, and this phosphorylation was dose-dependently inhibited by ASA and Dex. We next examined the apoptotic signal. As shown in Figure 7C, the transfection of AxCAmyrAkt significantly reduced caspase 3 activity, but this attenuating effect was abolished in the cells treated with ASA and Dex. These results demonstrated that NF-{kappa}B activation was essential for the prevention of apoptotic change in the Akt pathway.



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  		Figure 7. Inhibition of PDGF-induced phosphorylation of Akt by NF-{kappa}B inhibitors in rat mesangial cells. (A) Mesangial cells were incubated with PDGF (20 µg/ml) in the presence or absence of acetylsalicylic acid (ASA) for 30 min. (B) Mesangial cells were incubated with PDGF (20 µg/ml) and in the presence or absence of dexamethasone (Dex) for 30 min. (C) Mesangial cells were transfected with AxCAmyrAkt or AxCALacZ for 48 h, incubated in serum-free medium for 48 h, and then incubated with or without NF-{kappa}B inhibitors (ASA, 50 µM; Dex, 0.5 µM). Cell lysate was used for caspase 3 assay (n = 5, mean ± SEM, *P < 0.05).

 
We also examined the time course of Bad and I{kappa}B phosphorylation. In the assessment of Bad phosphorylation, the Bad stayed phosphorylated for 3 h after the PDGF stimulation (Figure 8A). The I{kappa}B phosphorylation appeared at 30 min after PDGF stimulation and lasted for 12 h (Figure 8B). As shown in Figure 8C, the Akt phosphorylation appeared at 10 min after PDGF stimulation and Akt stayed phosphorylated for 12 h. Since there was no way to directly inhibit Bad phoshorylation, we could not make any comparison by inhibiting the pathway. However, the longstanding activation of NF-{kappa}B up to the appearance of the apopototic changes and the effect of NF-{kappa}B in abolishing the anti-apoptotic effect of Akt, both suggest that NF-{kappa}B could be a dominant pathway in rat mesangial cells.



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  		Figure 8. Time courses of phosphorylation of Bad, I{kappa}B, and Akt. Rat mesangial cells were incubated with PDGF (20 µg/ml) for the indicated periods. (A) Upper gel, immunoblots using an antibody specific to the phosphorylated form of Bad; lower gel, immunoblots with an antibody to total Bad. (B) Upper gel, immunoblots using an antibody specific to the phosphorylated form of I{kappa}B; lower gel, immunoblots with an antibody to actin. (C) Upper gel, immunoblots using an antibody specific to the phosphorylated form of Akt; lower gel, immunoblots with an antibody to total Akt.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that Akt is phosphorylated by PDGF and that activated Akt prevents apoptotic changes in rat mesangial cells. In addition, we characterized the downstream signals of the PI3-kinase-Akt pathway. Our in vitro data suggest that this pathway acts as a survival signal and plays a key role in the regulation of apoptotic change in mesangial cells.

In this study, we demonstrated that the PI3-kinase-Akt pathway is activated by PDGF in mesangial cells. PDGF is known to be a potent mediator in the proliferation of mesangial cells, and the MAP kinase and the JAK-STAT pathway have been reported to be involved in this PDGF-mediated signaling cascade (27,28). PDGF drives the mesangial cell proliferation in many progressive renal diseases (6). A recent investigation showed that Akt promotes mesangial cell proliferation via inhibition of P27kip1 transcription in mesangial cells (29). PDGF is also to be a principal survival factor that inhibits apoptosis and promotes proliferation in several cell types. In this report, we demonstrated that the PI3-kinase-Akt pathway acts as a survival (anti-apoptotic) signal and that PDGF activates that pathway in mesangial cells. While PDGF has been reported to mediate the proliferation that deeply involved in the mesangial proliferation of Thy 1.1 nephritis and IgA nephritis (17–19), our data suggest that PDGF serves a separate function as a survival factor acting through the PI3-kinase-Akt pathway in glomerulonephritis.

Several targets of Akt survival signal have been identified in different cell types. Among them, the NF-{kappa}B protein has been shown to promote expression of anti-apoptotic genes. Recent reports demonstrated a direct association between Akt and phosphorylation of I{kappa}B in fibroblasts by identifying NF-{kappa}B as a target of Akt in anti-apoptotic PDGF signaling (14,15,30,31). In contrast, other investigators working with human vascular smooth muscle cells and skin fibroblasts concluded that Akt phosphorylation and NF-{kappa}B activation share no association (32). Thus, it remains controversial whether NF-{kappa}B is indeed involved in the PI3-kinase-Akt. Our data identified that NF-{kappa}B as a downstream mediator of the PI3-kinase-Akt pathway in mesangial cells.

We also demonstrated that Bad is phosphorylated by Akt in mesangial cells. Bad is a proapoptotic protein form of the Bcl-2 family members. Phosphorylation of Bad promotes cell survival in many cell types, and several reports have suggested that Bad is a downstream signal of the Akt pathway. In neurons, growth factor activation of the PI3-kinase-Akt pathway stimulates the phosphorylation of Bad, thereby suppressing apoptosis and promoting cell survival. Phosphorylation of Bad at Ser-136 by activated Akt results in its dissociation from Bcl-XL and association with the adapter protein 14-3-3 (12). The free Bcl-XL released from sequestration by Bad promotes cell survival. Our data indicate that part of the anti-apoptotic action of the PI3-kinase-Akt pathway is elicited via phosphorylation of Bad in mesangial cells.

A previous report suggested that the phosphorylation of Akt directly suppresses caspase 9 activity and inhibits apoptosis in human umbilical endothelial cells (14). When we attempted to examine whether the inhibition of Akt phosphorylaion would alter the activity of caspase 9, no significant difference was observed under our experimental condition. Taken together, these results imply that NF-{kappa}B and Bad are both involved in the signal pathway downstream of the PI3-kinase-Akt-pathway in mesangial cells.

The remaining question is which of these, NF-{kappa}B or Bad, acts as the principal signaler downstream of the PI3-kinase-Akt pathway. As we mentioned above, the Akt-induced activation of NF-{kappa}B and phosphorylation of Bad both seem to be cell-specific events. For two reasons, our data suggested that NF-{kappa}B contributes more as a downstream signaler in rat mesangial cells: NF-{kappa}B was still activated by the time the apopototic changes were observed, and inhibition of NF-{kappa}B activation reduced the anti-apoptotic effect of dominant-active Akt.

In summary, we investigated the regulation and function of the PI3-kinase-Akt pathway and the downstream signal of Akt in cultured rat mesangial cells. Our findings indicate that Akt is phosphorylated by PDGF and that the activated Akt prevents apoptotic changes partly via the downstream signaling of NF-{kappa}B and Bad. In our data characterizing the relative contributions of NF-{kappa}B and Bad to this pathway, the former seemed to contribute more. These results suggest that the PI3-kinase-Akt pathway acts as a survival signal and plays a key role in the regulation of apoptotic change in mesangial cells.


   Acknowledgments
 
We are grateful to Dr. Wataru Ogawa and Dr. Masato Kasuga for providing the recombinant adenoviruses encoding a dominant active myristoylated Akt (AxCAmyrAkt) and dominant-negative Akt (AxCAAkt-AA).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication February 24, 2002. Accepted for publication February 15, 2003.




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