Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms

Youhua Liu

Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island 02903


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Hepatocyte growth factor (HGF) has been shown to protect renal epithelial cells against apoptosis. To define the mechanism by which HGF inhibits apoptosis, we investigated the effect of HGF on the phosphorylation and expression of the Bcl-2 family proteins. Using a human proximal tubular epithelial cell (HKC) line as a model, we demonstrated that constitutive expression of HGF conveyed marked resistance to apoptotic death induced by serum withdrawal. HGF induced rapid phosphorylation of Akt in HKC cells, which was immediately followed by phosphorylation and resultant inactivation of Bad, a pro-apoptotic member of the Bcl-2 family. Pretreatment of the HKC cells with 10 nM wortmannin completely abolished HGF-induced phosphorylation of Akt and Bad, suggesting that this pathway is dependent on phosphoinositide (PI) 3-kinase. Overexpression of Bad increased apoptotic death in wild-type HKC cells but not in HGF-producing H4 cells. Immunoblotting confirmed that the Bad protein over-expressed in H4 cells was fully phosphorylated at both Ser112 and Ser136 sites. Prolonged incubation of HKC cells with HGF also dramatically induced expression of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. These results suggest that the anti-apoptotic effect of HGF in renal epithelial cells is mediated by dual mechanisms involving two distinct Bcl-2 family proteins. HGF triggers Bad phosphorylation via the PI 3-kinase/Akt pathway, thereby inactivating this pro-apoptotic protein, while simultaneously inducing expression of anti-apoptotic Bcl-xL.

c-met; apoptosis; Bad; Bcl-xL; phosphorylation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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APOPTOTIC CELL DEATH is a genetically controlled, exquisitely orchestrated process that plays a crucial role both in normal biological processes and in diverse disease states (43, 44, 46). Studies indicate that delicate regulation of apoptosis is important for embryonic development and for maintaining normal functions in the tissues of multicellular organisms. Perturbation of the balance between cell survival and apoptosis may lead to either excessive cell death or survival and plays a pivotal role in the pathogenesis of a large number of diseases, ranging from cancer to various autoimmune and degenerative disorders (15, 44). Apoptosis is tightly regulated by diverse factors, including survival signals and extracellular environments (2, 27). Previous studies by us and others demonstrate that hepatocyte growth factor (HGF) is one of these survival factors with potent ability to promote cell survival by inhibiting apoptosis (16, 24, 31, 49).

HGF is a multifunctional protein originally isolated as a potent mitogen for hepatocytes (33, 50). Its functionally pleiotropic nature is demonstrated by the fact that HGF elicits mitogenic, motogenic, and morphogenic action on a wide variety of target cells (33, 50). The receptor for HGF is the product of the c-met protooncogene, which possesses intrinsic tyrosine kinase activity (8). Evidence suggests that the HGF/c-met receptor signaling system plays an essential role in mammalian development, tumorigenesis, and organ regeneration (4, 6, 34, 42). In the kidney, HGF preserves normal renal structure and function and accelerates tubule repair and regeneration in experimental acute renal failure (23, 35). Although mitogenic action of HGF is presumed to mediate these beneficial effects, previous studies in our laboratory demonstrate that HGF is also a survival factor capable of protecting renal epithelial cells from apoptosis (31). This suggests that inhibition of apoptosis may be an additional and novel mechanism accounting for the beneficial effects of HGF to preserve and restore normal organ structure and function after injury.

The notion that HGF is an important modulator of apoptosis is supported by several recent observations in diverse types of cells (16, 24, 31). For example, HGF protects Madin-Darby canine kidney epithelial cells from apoptosis induced by disruption of cell attachment (19). HGF prevents MLP-29 liver progenitor cells from apoptotic death in vitro and inhibits hepatocyte apoptosis in a mouse model of Fas-induced fulminant hepatic failure in vivo (5, 24). Expression of constitutively activated c-met tyrosine kinase in the liver of transgenic mice prevents their hepatocyte from apoptosis and permits immortalization of these cells (3). Recent studies also indicate that HGF protects normal epithelial and various carcinoma cells from apoptosis induced by distinct DNA-damaging agents (16). However, the mechanism by which HGF inhibits apoptosis remains largely unknown, although potential involvement of members of the Bcl-2 gene family, such as BAG-1 and Bcl-xL, has been proposed (5, 16, 24).

The Bcl-2 family of proteins are key regulators of apoptosis playing a central role in dictating cell fate in response to diverse stimuli (2). Thus far, at least 15 Bcl-2 family members have been identified in mammalian cells, and several others exist in viruses (2). Although all members share certain structural homology, they are functionally divided. Some members of the family (such as Bcl-2 and Bcl-xL) inhibit apoptosis, whereas others (such as Bax and Bad) promote apoptosis. Hence, the relative levels and ratio of anti-apoptotic proteins to pro-apoptotic proteins are believed to play a critical role in determining whether cells survive or die (2, 12, 47). Recent studies suggest that the biological activity of various members of Bcl-2 family proteins is also regulated by diverse mechanisms, including alterations in expression level or phosphorylation status (13, 36, 52). Based on these findings, we speculated that HGF might inhibit apoptosis in renal epithelial cells by regulating the activity of Bcl-2 family proteins.

In this report, we demonstrated that HGF had marked ability to protect human renal proximal tubular epithelial cells against apoptosis induced by serum withdrawal. We found that this anti-apoptotic effect of HGF appeared to be mediated by phosphorylation of Bad via the phosphoinositide (PI) 3-kinase/Akt pathway. We also demonstrated that HGF could override Bad-induced renal epithelial cell death by promoting Bad phosphorylation at both Ser112 and Ser136 sites. Furthermore, exposure of renal epithelial cells to HGF induced marked elevation of anti-apoptotic protein Bcl-xL. Our data establish a direct link between HGF signaling and the activity of two key members of the Bcl-2 family proteins.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Cell culture and treatment. Human proximal tubular epithelial HKC cells (37) were obtained from Dr. L. Racusen of Johns Hopkins University and were cultured in DMEM-F-12 medium supplemented with 5% FBS. The HGF-producing H4 cell line was established by stable transfection of HKC cells with an expression vector containing human HGF cDNA, as described elsewhere (30). The H4 cells constitutively express 2.3 kb HGF mRNA and secrete biologically active HGF protein into conditioned medium. Determination of HGF protein in the conditioned medium by a specific enzyme-linked immunosorbent assay reveals that H4 cells secrete 1.8 ng HGF · 106 cells-1 · day-1 (38). For investigating the effects of HGF on protein phosphorylation, HKC cells were seeded in standard medium with 5% FBS for 24 h and then were changed to serum-free medium for another 24 h. Human recombinant HGF was added to the culture with fresh serum-free medium at a final concentration of 20 ng/ml. At various time points as indicated, cells were harvested for Western immunoblotting (see Western immunoblot analysis). For some experiments, cells were treated with either various inhibitors at given specified concentrations or vehicle (0.1% DMSO) 0.5 h before HGF stimulation. Wortmannin was obtained from Sigma (St. Louis, MO). PD-98059, myristoylated protein kinase A inhibitor (PKAI), Ro-31-8220, and genistein were purchased from Calbiochem (La Jolla, CA).

Cell survival assay. To determine HGF's effects on survival of renal epithelial cells, cell viability was assessed by counting the number of cells that remain adherent to the cell monolayer and exclude trypan blue. Cells were seeded in 12-well plates and were incubated for a fixed time. Nonadherent cells were removed by two washes with PBS. Adherent cells were harvested by trypsin-EDTA digestion and were stained with 0.04% trypan blue for 5 min. The number of cells excluding trypan blue was determined by counting in a hemacytometer.

Staining of cells with fluorescent dye. Nuclear chromatin morphology was examined by staining with the fluorescent dye H-33342 (1 µg/ml; Calbiochem, San Diego, CA) as described previously (28, 31). Briefly, adherent and detached cells were pooled and stained with H-33342 for 10 min at 37°C. Cells were then washed with PBS one time and were fixed with 3% paraformaldehyde. Cells were cytospun to glass slides, observed, and photographed using fluorescence microscopy. Apoptotic cells with characteristic nuclear fragmentation were counted in at least 10 random fields and were expressed as a percentage of the total cell number (apoptotic index).

TUNEL staining. The detection of apoptotic nuclei using the TdT-mediated dUTP nick end labeling (TUNEL) method was performed using a FragEL DNA fragmentation detection kit according to the protocol specified by the manufacturer (Calbiochem; see Ref. 31). Briefly, adherent and detached cells were harvested and fixed in 3% paraformaldehyde. Cells were cytospun to glass slides and incubated in 0.3% H2O2 in methanol for 5 min to block endogenous peroxidase. Slides were incubated with the labeling reaction mixture containing enzyme and biotin-labeled deoxynucleotides. The slides were then incubated with streptavidin-horseradish peroxidase conjugate, and the apoptotic nuclei were visualized by the addition of diaminobenzidine as substrate and were counterstained with methyl green. Apoptotic cells were counted in at least six random fields and were expressed as a percentage of the total cell number (apoptotic index).

Western immunoblot analysis. The rapid analysis of Akt and Bad phosphorylation was performed using the respective PhosphorPlus antibody kits (New England BioLabs, Beverly, MA). These kits provide phosphospecific Akt and Bad antibodies that detect Akt and Bad only when phosphorylated at specific sites (52). The kits also provide antibodies that detect total Akt and Bad (phosphorylation state-independent) levels. HKC cells and HGF-treated cells were lysed with SDS sample buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.1% bromphenol blue). Samples were heated at 100°C for 10 min before loading and were separated on 10 or 15% SDS-PAGE under nonreducing conditions. The proteins were electrotransferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL) in transfer buffer containing 48 mM Tris · HCl, 39 mM glycine, 0.037% SDS, and 20% methanol at 4°C for 1 h. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% Carnation nonfat milk in 20 mM Tris · HCl, 150 mM NaCl, and 0.1% Tween 20. The membrane was then incubated for 16 h at 4°C with various primary antibodies in blocking buffer containing 5% milk (1:1,000; New England BioLabs) followed by incubation for 1 h at room temperature with a secondary goat anti-rabbit IgG horseradish peroxidase conjugate in blocking buffer. The signals were visualized by the enhanced chemiluminescence system (Amersham).

The effects of HGF on the expression of various apoptosis regulatory proteins were also analyzed by Western blot with their respective antibodies as described above. Briefly, HKC cells were treated with recombinant human HGF for various periods of time as indicated. Cells were lysed in SDS sample buffer and subjected to Western blot. Specific antibodies against Bcl-xL/S, Bax, BAG-1, Bcl-2, p53, Myc, and actin were purchased from Santa Cruz Biochemical (Santa Cruz, CA).

Transfection with Bad expression vector. The Bad eukaryotic expression plasmid (pEBG-mBad), in which mouse Bad is expressed as a glutathione-S-transferase (GST) fusion protein under the control of a strong constitutive mammalian promoter, was purchased from New England BioLabs (PhosphorPlus Bad antibody kit). The eukaryotic expression plasmid vector pcDNA3 (Invitrogen, Carlsbad, CA) was used for vector-alone mock transfection. The HKC cells and HGF-producing H4 cells were transiently transfected with either pEBG-mBad plasmid or pcDNA3 using the calcium phosphate coprecipitation method as previously described (29). After transfection, cell survival as well as Bad protein expression and phosphorylation were determined by the procedures described above.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HGF prevents renal proximal tubular epithelial cells from apoptosis. We previously reported that HGF prevents renal epithelial cells from apoptosis induced by either serum deprivation or treatment with the nephrotoxin cisplatin in an inner medullary collecting duct mIMCD-3 cell line (31). Because the mIMCD-3 cells are originated from distal collecting duct epithelium, the protective role of HGF remains to be established in the proximal tubule, where the greatest injury and regeneration take place in most forms of acute renal injury (22, 45). Therefore, in this study, we used a human proximal tubular epithelial cell line HKC as a model system to test HGF's role in regulating renal epithelial cell apoptosis. As shown in Fig. 1, serum starvation induced significant apoptotic cell death assessed by multiple methods, including fluorescent dye H-33342 staining, TUNEL staining, and morphology. However, constitutive expression of HGF by stable transfection of HKC cells (H4 cells) conveyed marked resistance to apoptotic death induced by serum withdrawal (Fig. 1). The survival rate of HGF-producing H4 cells was more than ninefold greater than that of control HKC cells after serum starvation for 10 days (Fig. 2). Consistently, the percentage of apoptosis positive cells detected by TUNEL staining (apoptotic index) was significantly reduced in HGF-producing H4 cells (Figs. 1 and 2). Similar results were obtained when the cells were stained with fluorescent dye H-33342 (data not shown). To determine the effects of exogenous HGF on renal epithelial cell apoptosis, we incubated the wild-type HKC cells in the serum-free medium in the absence or presence of 20 ng/ml of HGF. Results indicated that exogenous HGF also protected HKC cells from apoptosis induced by serum withdrawal, although at less degree. The number of viable cells in the HGF-treated group was twofold of that in the control group after 8 days of serum starvation (74.1 ± 8.7 vs. 37.0 ± 11.5 × 104/well, P < 0.05, n = 6). Therefore, these data clearly demonstrate that HGF prevents apoptosis of renal epithelial cells derived from both the distal collecting duct (6) and the proximal tubule.


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Fig. 1.   Hepatocyte growth factor (HGF) protects renal proximal tubular epithelial HKC cells from apoptosis. HGF-producing H4 cells were established by stably transfecting a human renal proximal tubular epithelial cell line (HKC) with an HGF expression vector (pCMV-HGF). Constitutive expression of HGF mRNA and protein in H4 cells was confirmed by Northern blot and enzyme-linked immunosorbent assay, respectively (data not shown). HGF-producing H4 cells (A and C) or HKC cells (B and D) were incubated in serum-free medium for 10 days. A and B: phase-contrast morphology; C and D: TdT-mediated dUTP nick end labeling (TUNEL) staining. Cells (both adherent and detached) were harvested, cytospun to slides, and subjected to staining.



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Fig. 2.   HGF protects renal epithelial cells from apoptotic death induced by serum withdrawal. HKC cells or H4 cells were incubated in serum-free medium for 10 days. A: viable cells (adherent and excluding trypan blue) determined by counting the viable cells per culture well; B: apoptotic index (%) detected by TUNEL staining is shown. ** P < 0.01.

Stimulation of renal epithelial cells with HGF results in Bad phosphorylation. To investigate the mechanism by which HGF inhibits apoptotic cell death, we studied the effects of HGF on the phosphorylation status of the pro-apoptotic protein Bad in human renal epithelial cells. Shown in Fig. 3, stimulation of HKC cells with HGF resulted in Bad phosphorylation at Ser112. Immunoblotting with antibody specific for phospho-Bad revealed a marked increase in phosphorylated Bad protein starting at 30 min, and sustaining until at least 90 min after HGF stimulation (Fig. 3). The abundance of total Bad protein remained relatively constant during this period after HGF treatment (Fig. 3). Densitometric analysis revealed that the ratio of phospho-Bad to total Bad increased by >10-fold at 60 min after HGF stimulation (Fig. 3). These results suggest that HGF activates a signaling pathway leading to phosphorylation of the pro-apoptotic Bad protein and thereby regulates its activity in controlling renal epithelial cell survival or death.



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Fig. 3.   HGF induces Bad phosphorylation in renal epithelial cells. HKC cells were treated with 20 ng/ml HGF for various periods of duration as indicated. Cell lysates were immunoblotted with antibodies either specifically against phospho-Bad or against total Bad protein. A: phospho-Bad; B: total Bad; C: graphic presentation of the ratio of Phospho-Bad to total Bad after HGF stimulation.

Activation of Akt by HGF precedes Bad phosphorylation. Because Bad upstream regulator is recently identified to be Akt, also known as protein kinase (PK) B, a Ser-Thr kinase that plays an essential role in the regulation of cell survival (13, 36, 52), we speculated that Bad phosphorylation by HGF may be mediated through activation of Akt kinase, which is believed to be mediated by phosphorylation of Akt at Ser727 (17, 18). To this end, we studied the effect of HGF on Akt phosphorylation. As demonstrated in Fig. 4, treatment of HKC cells with HGF caused dramatic activation of Akt, as shown by rapid and marked phosphorylation of Akt protein at Ser727 starting as early as 5 min (the earliest time point tested) after HGF stimulation (Fig. 4). Densitometric analysis revealed that the ratio of phospho-Akt to total Akt in HGF-treated cells reached >100-fold of that in the control culture at 30 min after HGF stimulation (Fig. 4). Compared with the kinetics of Bad phosphorylation (Fig. 3), it is clear that Akt phosphorylation and activation by HGF significantly precedes Bad phosphorylation.



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Fig. 4.   Phosphorylation and activation of Akt induced by HGF precedes Bad phosphorylation. HKC cells were treated with 20 ng/ml HGF for various periods of duration as indicated. Cell lysates were immunoblotted with antibodies either specifically against phospho-Akt or against total Akt protein. A: phospho-Akt; B: total Akt; C: graphic presentation of the ratio of Phospho-Akt to total Akt after HGF stimulation.

Of note, the HGF-producing H4 cells also exhibited a remarkable, constitutive Akt phosphorylation when they were cultured in serum-free medium. Densitometric analysis revealed that the ratio of phospho-Akt to total Akt in H4 cells was 28-fold greater than that in the control wild-type HKC cells (Fig. 5). These results suggest that expression of HGF by stable transfection constitutively activates Akt kinase that in turn would phosphorylate Bad protein.


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Fig. 5.   Constitutive phosphorylation of Akt in the HGF-producing H4 cells. HKC cells and the HGF-producing H4 cells were serum-starved for 2 days. Cell lysates were immunoblotted with antibodies either specifically against phospho-Akt or against total Akt protein. A: phospho-Akt; B: total Akt.

Activation of Akt and Bad phosphorylation are dependent on the PI 3-kinase pathway. To investigate the signal pathway involved in Akt activation and Bad phosphorylation by HGF, we studied the effects of specific signal transduction pathway inhibitors on Akt phosphorylation. Because previous studies indicate that PI 3-kinase is essential for activation of Akt (17), we reasoned that the activation of Akt by HGF in renal epithelial cells may be mediated by the PI 3-kinase pathway. To test this hypothesis, we examined whether the PI 3-kinase pathway is absolutely required for HGF-induced Akt activation and Bad phosphorylation in renal epithelial cells. As shown in Fig. 6, preincubation with wortmannin, a specific inhibitor for PI 3-kinase signaling, at a concentration as low as 10 nM completely blocked HGF-induced phosphorylation and activation of Akt in HKC cells. Likewise, inhibition of PI 3-kinase signaling by wortmannin also abolished HGF-induced Bad phosphorylation (Fig. 6C). Of note, blockage of HGF-induced Akt and Bad phosphorylation by wortmannin is specific to the PI 3-kinase pathway, as wortmannin has no effect on HGF-induced Stat-3 phosphorylation even at as high as 100 nM concentration (data not shown). This observation suggests that HGF-induced Akt and Bad phosphorylation is completely dependent on PI 3-kinase signaling.


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Fig. 6.   Phosphorylation of Akt and Bad by HGF is dependent on phosphoinositide (PI) 3-kinase pathway. HKC cells were treated with various signaling transduction inhibitors as indicated 0.5 h before stimulation with 20 ng/ml HGF for 30 min. Final concentrations of the specific inhibitors are as follows: wortmannin, 10 and 100 nM; PD-98059, 10 µM; myristoylated PKAI, 0.3 µM; Ro-31-8220, 50 nM; and genistein, 10 µM. Cell lysates were immunoblotted with antibodies specifically against phospho-Akt, total Akt, phospho-Bad, and total Bad. A: inhibition profile of HGF-induced Akt phosphorylation; B: dose response of Akt phosphorylation to wortmannin; C: dose response of Bad phosphorylation to wortmannin.

To explore whether other signaling pathways also participate in the phosphorylation of Akt and Bad, we studied the effects of other inhibitors specific for diverse signaling pathways on Akt and Bad phosphorylation in renal epithelial HKC cells. As shown in Fig. 6A, pretreatment of HKC cells with the specific MAP kinase inhibitor PD-98059 failed to block HGF-induced Akt and Bad phosphorylation, suggesting that it is independent of MAP kinase pathways. Likewise, HGF-induced Akt and Bad phosphorylation was also independent of PKA and PKC pathways, as myristoylated PKAI and Ro-31-8220, the specific inhibitors for PKA and PKC signaling, respectively, were unable to abolish Akt phosphorylation (Fig. 6A). However, c-met receptor tyrosine phosphorylation appeared to be a prerequisite for HGF-induced Akt and Bad phosphorylation, since blockage of tyrosine phosphorylation with genistein also abrogated Akt and Bad phosphorylation.

Overexpression of Bad promotes apoptosis in renal epithelial cells. To determine whether Bad protein is indeed critical for the regulation of renal epithelial cell survival, we transfected HKC cells with Bad expression vector to produce overexpression of Bad protein in renal epithelial cells. As illustrated in Fig. 7, overexpression of Bad protein in HKC epithelial cells resulted in a remarkable cell death. At the same condition, transfection with empty vector alone did not cause significant apoptosis. The number of viable cells in the HKC cells transfected with Bad expression vector was only 22% of that transfected with empty vector alone after 48 h of incubation after transfection (9.4 ± 2.2 vs. 43.5 ± 2.7 × 104/well, P < 0.01, n = 10). Consistently, fluorescent dye H-33342 staining revealed that more than one-half of the transfected HKC cells underwent apoptosis after transfection with Bad expression vector (data not shown). These results indicate that Bad is a potent apoptosis-promoting protein that plays an essential role in determining renal epithelial cell fate.


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Fig. 7.   Overexpression of Bad induces apoptosis in renal epithelial cells. HKC cells were transfected with either Bad eukaryotic expression plasmid (pEBG-mBad) or vector alone. Bad overexpression markedly promoted renal epithelial cell apoptosis. Photomicrographs were taken at 48 h after transfection. A: HKC cells transfected with pcDNA3; B: HKC cells transfected with pEBG-mBad.

HGF overrides Bad-induced apoptosis by stimulating its phosphorylation. Because Bad protein is a potent pro-apoptotic effector in renal epithelial cells (Fig. 7), we next examined the effects of HGF on the pro-apoptotic action of the overexpressed Bad protein. As demonstrated in Fig. 8, overexpression of Bad by transfection had no significant effect on cell survival in the HGF-producing H4 cells. The number of viable cells was almost identical in the H4 cells transfected with either Bad expression vector or empty vector alone after 48 h of incubation after transfection (79 ± 5.9 vs. 84 ± 8.5 × 104/well, P > 0.05, n = 6). This result is in contrast to that in the HKC cells described above (Fig. 7), suggesting that HGF can abolish the pro-apoptotic effect of the overexpressed Bad. Immunoblotting revealed that transient transfection resulted in overexpression of Bad as a Bad-GST fusion protein with a molecular weight of 49 kDa in these H4 cells (Fig. 8). However, the overexpressed Bad protein was fully phosphorylated at both Ser112 and Ser136 positions in either the presence or absence of serum (Fig. 8). These findings lead us to conclude that HGF can override Bad-induced renal epithelial cell death by triggering its phosphorylation.


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Fig. 8.   Constitutive expression of HGF abolishes Bad-induced renal epithelial cell apoptosis by triggering its phosphorylation at both Ser112 and Ser136 sites. HGF-producing H4 cells were transfected with either Bad eukaryotic expression plasmid (pEBG-mBad) or vector alone (pcDNA3). Transfected cells were grown in standard medium with or without 5% FBS for 48 h. Cell lysates were analyzed by immunoblotting with specific antibodies against Bad, Ser112-phospho-Bad, Ser136-phospho-Bad, and phospho-Akt. Bad overexpression did not significantly affect renal epithelial cell survival and apoptosis. A: H4 cells transfected with pcDNA3; B: H4 cells transfected with pEBG-mBad; C: Western blot with total Bad; D: Western blot with Ser112-phospho-Bad; E: Western blot with Ser136-phospho-Bad; F: Western blot with phospho-Akt.

HGF induces expression of anti-apoptotic protein Bcl-xL. Because the balance of the activity of anti-apoptotic vs. pro-apoptotic proteins is believed to be a key determinant for dictating cell death or survival, we investigated the effects of HGF on the relative abundance of a host of apoptosis regulatory proteins. As shown in Fig. 9, incubation of HKC cells with HGF markedly increased the expression of Bcl-xL, an anti-apoptotic member of Bcl-2 family proteins. The level of Bcl-xL protein started to increase at 24 h and was sustained at least to 48 h after HGF stimulation. Densitometric analysis revealed a 4.5-fold induction of Bcl-xL protein in HKC cells at 36 h after HGF treatment. However, Bcl-xS, the truncated isoform of Bcl-x with a deletion of 63 amino acids exhibiting strong pro-apoptotic activity (2), was not detected in HKC cells either at control culture or after HGF treatment (data not shown). At the same condition, HGF did not significantly affect the abundance of other Bcl-2 family proteins, such as Bcl-2, BAG-1, Bax (Fig. 9), and Bad (data not shown). Detection of other potential apoptosis regulatory proteins such as p53 and Myc also revealed no significant alterations in the expression levels of these proteins after HGF stimulation (Fig. 9).


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Fig. 9.   HGF induces anti-apoptotic protein Bcl-xL expression in renal epithelial cells. HKC cells were treated with 20 ng/ml HGF for various periods of duration as indicated. Cell lysates were immunoblotted with antibodies specifically against various apoptosis regulatory proteins. A: Bcl-xL; B: Bax; C: BAG-1; D: Bcl-2; E: p53; F: Myc; G: actin.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented in this report demonstrate that HGF is a potent survival factor capable of protecting renal proximal tubular epithelial cells from apoptosis. This anti-apoptotic effect of HGF appears to be mediated by dual mechanisms involving two distinct members of Bcl-2 family proteins. As illustrated in Fig. 10, HGF inactivates pro-apoptotic Bad protein through a pathway involving the sequential activation of c-met receptor tyrosine phosphorylation, induction of the PI 3-kinase activity, activation of Akt kinase, and finally Bad phosphorylation. This cascade of events is completed via several posttranslational modifications that occur within a relatively short period of time ranging from minutes to hours (Figs. 3 and 4). In addition to this pathway, HGF also markedly stimulates anti-apoptotic Bcl-xL protein expression. Although the exact mechanism by which HGF upregulates Bcl-xL is unknown, it probably involves de novo gene expression requiring prolonged incubation ranging from hours to days (Fig. 9). The combination and integration of these dual effects of HGF should produce a dramatic shift on the ratio of anti-apoptotic vs. pro-apoptotic proteins in favoring cell survival, thereby having profound effects on preventing renal epithelial cells from apoptosis (Figs. 1 and 10). These observations establish a functional coupling between HGF signaling and the activity of Bcl-2 family proteins, the central components of cell-intrinsic death/survival machinery, leading us to conclude that inactivation of Bad via phosphorylation and induction of Bcl-xL expression likely are the mechanisms by which HGF promotes renal epithelial cell survival.


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Fig. 10.   Schematic model showing that HGF protects renal epithelial cells against apoptosis by dual mechanisms. HGF inactivates pro-apoptotic Bad protein through a cascade of events involving the sequential activation of c-met, PI 3-kinase, Akt, and finally Bad phosphorylation, which leads to its cytosolic sequestration by association with 14-3-3 proteins. These are completed via posttranslational modification within short periods of time ranging from minutes to hours. At the same time, HGF markedly induces anti-apoptotic Bcl-xL protein expression probably through de novo gene expression. Increased Bcl-xL protein blocks the activation of Apaf-1 and caspase. Integration of these dual effects of HGF should produce a dramatic shift on the ratio of anti-apoptotic vs. pro-apoptotic proteins in favoring renal epithelial cell survival. P, phosphorylated.

The pro-death activity of Bad is believed to be dependent on its phosphorylated status (39, 48). In the absence of phosphorylation, Bad may suppress cell survival by heterodimerizing with Bcl-xL or Bcl-2, the pro-survival member of the Bcl-2 family, and such heterodimer formation causes cell death either directly or indirectly, by inducing Bax homodimer formation (20, 48). The phosphorylation of Bad results in its cytosolic sequestration and inactivation by its association with 14-3-3 proteins, thereby preventing its binding to Bcl-xL or Bcl-2 at intracellular membrane sites (51). Although the role of Bad in regulating cell death may not be ubiquitous, members of the Bcl-2 family, including Bad, likely play important roles in mediating HGF-induced cell survival in diverse types of cells. In fact, overexpression of Bad alone can induce marked renal epithelial cell apoptosis (Fig. 7), and HGF is capable of overriding this increased apoptotic cell death apparently via a mechanism that involves Bad phosphorylation (Fig. 8). These results suggest that HGF-induced Bad phosphorylation is necessary and may be sufficient for renal epithelial cell survival.

The signaling pathway leading to Bad phosphorylation by HGF is evidently involved in activation of Akt, which in turn results from c-met receptor-mediated activation of PI 3-kinase. Akt is a survival-promoting Ser-Thr protein kinase whose activity is regulated by a variety of growth factors in a PI 3-kinase-dependent manner (17, 18). Activation of Akt is previously shown to deliver survival signals leading to inhibition of apoptosis in several types of cells, including primary culture of cerebellar neurons, Rat-1, and COS-7 cells (14, 25). Kinetic studies of Akt and Bad phosphorylation suggest that Akt is probably the upstream signal activating Bad phosphorylation upon HGF stimulation in renal epithelial cells (Figs. 3 and 4). This view is further supported by recent observations that Bad phosphorylation is mediated by Akt kinase in interleukin (IL)-3-stimulated lymphoid progenitor cells (36) and in insulin-like growth factor (IGF) I-stimulated cerebellar neurons (13).

The phosphorylation of Bad triggered by HGF occurs at both Ser112 and Ser136 sites of Bad protein in renal epithelial cells (Fig. 8). This differs somewhat from a recent observation that IGF-I and its downstream kinase Akt primarily trigger Bad phosphorylation at Ser136, but not at Ser112, for suppressing Bad-mediated cell death in cerebellar granule cells (13). The explanation for this discrepancy remains uncertain. It may represent a potential difference in the signaling pathway leading to Bad phosphorylation triggered by HGF vs. IGF-I. Alternatively, it could simply be due to different cell types that were examined. Consistent with our results, IL-3 prevents cell death by triggering Bad phosphorylation at both Ser112 and Ser136 in hematopoietic cells (36, 51). It is also suggested that phosphorylation of Bad at both sites may be achieved through simultaneous activation of different survival pathways from a combination of factors (13). The fact that HGF alone triggers concomitant Bad phosphorylation at both Ser112 and Ser136 likely suggests that HGF might be more effective in blocking Bad-induced apoptosis than other cytokines that induce phosphorylation at only a single site.

HGF elicits multiple, diverse biological actions in a variety of target cells, including renal epithelial cells (10, 30). Because a single HGF receptor, c-met, has been identified, these diverse functions are believed to be mediated through different postreceptor signaling transduction pathways. The mitogenic action of HGF is presumably mediated through the c-met receptor-activated Ras-MAP kinase cascade (1, 21), whereas the motogenic action (scattering effect) is thought to be dependent on PI 3-kinase and Rac activation (40, 41). Recent studies indicate that the Stat-3-mediated pathway may be essential for the tubulogenic action of HGF, as blockage of the association of Stat-3 to c-met receptor inhibits tubule formation in vitro without affecting either HGF-induced cell scattering or growth (7). The results in this report suggest that activation of Akt and Bad by HGF does not appear to be in the pathways leading to the activation of MAP kinase (Fig. 6). Rather, it is largely dependent on activation of PI 3-kinase, which activates downstream Akt kinase and finally Bad phosphorylation. The finding that HGF inhibits apoptosis in a PI 3-kinase-dependent fashion is consistent with the notion that a signaling pathway from PI 3-kinase plays an essential role in mediating growth factor-dependent cell survival (13, 17, 36).

Induction of anti-apoptotic Bcl-xL protein expression may represent another mechanism by which HGF inhibits apoptosis. This notion is also consistent with a recent observation that IGF-I promotes cell survival by a new signaling pathway independent of PI 3-kinase/Akt in fibroblasts overexpressing the IGF-I receptor (26). The biological significance of HGF-induced Bcl-xL in renal epithelial cells is not fully characterized; however, it is reasonable to assume that the integration of the inactivation of pro-apoptotic Bad coupled with the induction of anti-apoptotic Bcl-xL should have dramatic effects on promoting cell survival in response to more severe death challenge. Furthermore, induction of Bcl-xL by HGF may also be important in long-term pro-survival signaling, since kinetic studies revealed that HGF-induced Akt phosphorylation was only sustained for ~6 h (data not shown).

It should be noted that, in addition to the pathways described here, HGF may promote cell survival by other mechanisms as well. In this regard, it is reported that HGF receptor, c-met, can physically associate with BAG-1 protein, an anti-apoptotic member of the Bcl-2 family, and thereby promotes MLP-29 liver progenitor cell survival (5). Recent studies also demonstrate that HGF restores an adriamycin-induced decrease in Bcl-xL protein in MDA-MB-453 human breast carcinoma cells, although it fails to upregulate Bcl-xL expression under normal conditions (16). Furthermore, Akt has been recently shown to directly phosphorylate cell death protease pro-caspase-9 in the prostate epithelial cell line 267 (11) and a fork head transcription factor in 293T cells (9), suggesting that inactivation of caspase or/and transcription factor via Akt-induced phosphorylation may be additional mechanisms by which HGF promotes cell survival.

Evidence suggests that cell survival mechanisms may be downregulated in acute and chronic diseases leading to increased cell death and organ failure. If so, then protection from apoptosis by HGF might represent a new therapeutic strategy to preserve organ function. For example, although cell necrosis is classically considered to be the major form of renal tubular epithelial cell death in acute renal failure, emerging evidence suggests that a significant portion of cells die of apoptosis after injury (27). Rapid activation of HGF in vivo (32) suggests that HGF may act as a survival factor tending to lessen initial injury by inhibition of apoptosis in addition to promoting cell proliferation and tubule repair. Consistent with this view, administrations of exogenous HGF in animals preserves normal nephron structure and functions and significantly ameliorates the initial decline in kidney function in experimental acute renal failure (23, 35). Likewise, specific blocking of HGF signaling in vivo by a neutralizing antibody in rats with remnant kidneys after <FR><NU>5</NU><DE>6</DE></FR> nephrectomy significantly promotes renal cell apoptosis and expedites the onset and progression of renal disease (38).


    ACKNOWLEDGEMENTS

I am grateful to Dr. L. Dworkin for invaluable discussions during the course of these studies and critical review of this manuscript. I also thank L. Lin for excellent technical assistance and Dr. L. Racusen for providing HKC cells.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant R01 DK-54922 and by the Distinguished Young Investigator Grant from the National Natural Science Foundation of China (no. 39825508). Y. Liu is a recipient of the Independent Scientist Award from NIDDK (K02 DK-02611).

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 and other correspondence: Y. Liu, Department of Pathology, University of Pittsburgh School of Medicine, S-428 Biomedical Science Tower, Pittsburgh, PA 15261.

Received 21 January 1999; accepted in final form 26 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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