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Am J Physiol Renal Physiol 294: F534-F541, 2008. First published December 26, 2007; doi:10.1152/ajprenal.00408.2007
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Albumin-stimulated DNA synthesis is mediated by Ca2+/PKC as well as EGF receptor-dependent p44/42 MAPK and NF-{kappa}B signal pathways in renal proximal tubule cells

Yu Jin Lee and Ho Jae Han

Department of Veterinary Physiology, Biotherapy Human Resources Center (BK21), College of Veterinary Medicine, Chonnam National University, Gwangju, Korea

Submitted 3 September 2007 ; accepted in final form 21 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubule cells (PTCs). BSA increased the level of [3H]thymidine incorporation in a dose (≥3 mg/ml)- and time (≥3 h)-dependent manner, intracellular Ca2+ concentration, and the level of protein kinase C (PKC) phosphorylation and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca2+ chelator), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM, intracellular Ca2+ chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or an EGFR inhibitor (AG-1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor-{kappa}B (NF-{kappa}B) and inhibitor of NF-{kappa}B (I{kappa}B) phosphorylation, which was blocked by staurosporine, AG-1478, or PD-98059 (p44/42 MAPK inhibitor). Inhibition of Ca2+, PKC, EGFR, p44/42 MAPK, or NF-{kappa}B signal pathways blocked the BSA-induced incorporation of [3H]thymidine. Consequently, the inhibition of Ca2+, PKC, EGFR, p44/42 MAPKs, or NF-{kappa}B blocked the BSA-induced increases in cyclin D1, cyclin-dependent kinase (CDK)4, cyclin E, or CDK2 and restored the BSA-induced inhibition of p21WAF/Cip1 and p27Kip1 expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca2+/PKC as well as the EGFR-dependent p44/42 MAPK and NF-{kappa}B signal pathways in PTCs.

kidney; cyclins; cyclin-dependent kinases; p21WAF/Cip1; p27Kip1

ALBUMIN is a 69-kDa plasma protein that plays an important role as a binding protein to maintain plasma oncotic pressure and to transport to and from the tissues multiple substances within the bloodstream (28). In the kidney, albumin is filtered by the glomerulus, most of which is effectively reabsorbed by clathrin- and receptor-mediated endocytosis, in which the binding of albumin involves at least a complex of two proteins, megalin and cubilin, in the proximal tubule (14). Albuminuria caused by increased glomerular filtration or tubular injury is a well-known marker of renal disease with direct evidence for its involvement in the progression of chronic kidney disease to end-stage renal failure (21). Under physiological conditions, it has been observed in vitro that albumin inhibits apoptosis and promotes the survival of primary cultures of mouse tubular cells (20). In addition, bovine serum albumin (BSA) has many physiological functions, including increasing Na+-glucose cotransporter activity (18) and stimulating cell growth and L-arginine transport (3) in renal proximal tubule cells (PTCs). These results suggest that normal levels of albumin filtration and absorption are important for health. The exposure of tubule cells to albumin activates a wide array of diverse intracellular signaling pathways. Albumin augments the intracellular Ca2+ concentration ([Ca2+]i) in astrocytes (26) and transactivates epidermal growth factor receptor (EGFR) in renal epithelial cells (30), which mediates the activation of the Ras/ERK and/or phosphatidylinositol 3-kinase (PI3K)/Akt pathways (8). In models of proteinuric nephropathy, albumin causes the generation of reactive oxygen species (25) and the activation of the NF-{kappa}B-dependent proinflammatory genes (37, 42, 43). However, there is little information regarding the effect of BSA on the DNA synthesis of PTCs. Moreover, the signal transduction pathways involved in eliciting these effects are not completely understood.

PTCs have a number of differentiated typical functions of the renal proximal tubules, including a polarized morphology, as well as a distinctive proximal tubule transport and hormone response (9, 17). It was previously reported that the ATP-increased cell proliferation in primary cultured PTCs is consistent with the results obtained from intact renal tissue (23, 24). Therefore, PTCs in hormonally defined, serum-free culture conditions would be a powerful tool for examining the effect of BSA on cell proliferation. Thus this study examined the effect of BSA on DNA synthesis and BSA-related signal cascades in PTCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Male New Zealand White rabbits (1.5–2.0 kg) were purchased from Dae Han Experimental Animal (Chungju, Korea). All procedures for animal management followed the standard operation protocols of Seoul National University. The Institutional Review Board at Chonnam National University approved the research proposal as well as the relevant experimental procedures including animal care. In addition, both authors had the license of Doctor of Veterinary Medicine (DVM) granted by the Ministry of Agriculture and Forestry of the Republic of Korea. Class IV collagenase and soybean trypsin inhibitor were purchased from Life Technologies (GIBCO-BRL, Grand Island, NY). Fatty acid-free BSA, EGTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), AG-1478, bisindolylmaleimide I, Bay 11-7082, and β-actin were obtained from Sigma (St. Louis, MO). PD-98059 and SN50 were acquired from Calbiochem (La Jolla, CA). [3H]thymidine was purchased from NEN (Boston, MA). Phospho-EGFR (Thy 1173), EGFR, cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, p21WAF/Cip1, and p27Kip1 antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-pan-PKC, phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, phospho-NF-{kappa}B, and phospho-inhibitor of NF-{kappa}B (I{kappa}B) antibodies were acquired from Cell Signaling Technology (Hitchin, UK). Goat anti-rabbit IgG was supplied by Jackson Immunoresearch (West Grove, PA). Liquiscint was obtained from National Diagnostics (Parsippany, NJ). All other reagents were of the highest purity commercially available and were used as received.

Cell preparation and culture conditions. The primary rabbit PTC cultures were prepared by the method reported by Chung et al.(9). PTCs were grown in a DMEM-F-12 medium (GIBCO-BRL, Gaithersburg, MD) containing 15 mM HEPES and 20 mM sodium bicarbonate (pH 7.4). Three growth supplements (5 µg/ml insulin, 5 µg/ml transferrin, and 5 x 10–8 M hydrocortisone) were added immediately before the medium was used. The rabbit kidneys were perfused through the renal artery, first with phosphate-buffered saline (PBS) and then with a medium containing 0.5% iron oxide. Renal cortical slices were prepared and homogenized. The homogenate was poured first through a 253-µm-mesh filter and then through an 83-µm-mesh filter. The tubules and glomeruli on the top of the 83-µm filter were transferred to a sterile medium. The glomeruli (containing iron oxide) were removed with a magnetic stir bar. The remaining proximal tubules were incubated briefly in a medium containing collagenase (0.125 mg/ml) and 0.025% soybean trypsin inhibitor. The tubules were then washed by centrifugation, resuspended in a medium containing the three supplements, and transferred into tissue culture dishes. The medium was changed 1 day after plating and every 2 days thereafter. Primary cultured rabbit PTCs were maintained at 37°C in a 5% CO2 humidified environment in a serum-free basal medium that was supplemented with the three growth supplements.

[3H]thymidine incorporation. The medium was changed for the last time when the cells had reached 70–80% confluence. Thymidine incorporation experiments were carried out according to the method described by Gabelman and Emerman (13). The cells were incubated in the medium in the presence or absence of BSA for 12 h and pulsed with 1 µCi of [methyl-3H]thymidine for 1 h at 37°C. The cells were then washed twice with PBS, fixed in 10% trichloroacetic acid (TCA) at room temperature for 15 min, and then washed twice in 5% TCA. The acid-insoluble material was dissolved in 0.2 N NaOH at room temperature, and the level of radioactivity was determined with a liquid scintillation counter (model LS 6500, Beckman Instruments, Fullerton, CA). All experiments were performed in triplicate. The values were converted from absolute counts to a percentage of the control in order to allow comparison between experiments.

The number of cells was determined by washing the cells twice with PBS and trypsinizing them from the culture dishes. The cell suspension was mixed with a 0.4% (wt/vol) Trypan blue solution, and the number of live cells was determined with a hemocytometer. Cells that failed to exclude the dye were considered nonviable.

Measurement of [Ca2+]i by confocal microscopy. Changes in [Ca2+]i were monitored by fluo 3-AM, which was initially dissolved in dimethyl sulfoxide and stored at –20°C. PTCs in the 35-mm culture dishes were rinsed twice with bath solution (mM: 140 NaCl, 5 KCl, 1 CaCl2, 0.5 MgCl2, 10 glucose, and 5.5 HEPES, pH 7.4), incubated in bath solution containing 3 µM fluo 3-AM with 5% CO2-95% O2 at 37°C for 40 min, rinsed twice with bath solution, mounted on a perfusion chamber, and scanned every second by confocal microscopy (x400; Fluoview 300, Olympus). The fluorescence was excited at 488 nm, and the emitted light was read at 515 nm. All the analyses of [Ca2+]i were processed at a single-cell level and are expressed as the relative fluorescence intensity (RFI).

Total cell preparations for Western blotting. The medium was then removed, and the cells were washed twice with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in buffer A [mM: 137 NaCl, 8.1 Na2HPO4, 2.7 KCl, 1.5 KH2PO4, 2.5 EDTA, 1 dithiothreitol, and 0.1 PMSF, with 10 µg/ml leupeptin (pH 7.5)]. The resuspended cells were then lysed mechanically on ice by trituration with a 21.1-gauge needle. The cell lysates were initially centrifuged at 1,000 g for 10 min at 4°C. The supernatants were collected as a total cell fraction. The protein was quantified by the Bradford procedure (5).

Western blot analysis. The cell homogenates (30 µg of protein) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. The blots were then washed with H2O, blocked with 5% powder in Tris-buffered saline-Tween 20(10 mM Tris·HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20) for 1 h, and incubated with the primary polyclonal antibody at the dilutions recommended by the supplier. The primary antibodies were detected with goat anti-rabbit IgG conjugated to horseradish peroxidase, and the bands were visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).

Statistical analysis. Results are expressed as means ± SE. The difference between two mean values was analyzed by ANOVA. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of BSA on [3H]thymidine incorporation. To validate the effect of BSA on the incorporation of [3H]thymidine, PTCs were incubated either with different concentrations of BSA (0–20 mg/ml) for 12 h or with 10 mg/ml BSA for various times (0–48 h). As shown in Fig. 1, BSA increased the level of [3H]thymidine incorporation in a time- and dose-dependent manner. BSA, at ≥3 mg/ml, significantly increased the level of [3H]thymidine incorporation after 12-h incubation (Fig. 1A). A significant increase in [3H]thymidine incorporation was observed after 3-h incubation with 10 mg/ml BSA, and the maximum effect was observed at 12 h (Fig. 1B). Therefore, 12-h incubation with 10 mg/ml BSA was used in most experiments.


Figure 1
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  		Fig. 1. Dose (A)- and time (B)-dependent effects of bovine serum albumin (BSA) on [3H]thymidine incorporation. Renal proximal tubule cells (PTCs) were treated with different doses (0–20 mg/ml) of BSA for 12 h (A) or 10 mg/ml of BSA for 0–48 h (B) and pulsed with 1 µCi of [3H]thymidine for 1 h. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control.

 
Effect of BSA on Ca2+/PKC activation. To determine whether the BSA-induced DNA synthesis of PTCs involves an increase in [Ca2+]i, intracellular Ca2+ mobility in response to BSA was examined. As shown in Fig. 2A, BSA induced an increase in [Ca2+]i, which was significantly blocked by EGTA (extracellular Ca2+ chelator, 10–4 M) (Fig. 2B). However, BAPTA-AM (intracellular Ca2+ chelator, 10–6 M) partially blocked the BSA-induced increase in [Ca2+]i (Fig. 2C). In the next steps to determine whether BSA activates PKC signaling, the total protein of PTCs was observed at 5 min after treatment with 10 mg/ml BSA. BSA enhanced the level of PKC phosphorylation in a time-dependent manner (Fig. 2D), which was blocked by pretreatment with EGTA and BAPTA-AM (Fig. 2E). Subsequently, inhibition of Ca2+ with EGTA and BAPTA-AM or inhibition of the PKC pathway with staurosporine and bisindolylmaleimide I (10–7 M) blocked the BSA-induced increase in [3H]thymidine incorporation (Fig. 2, F and G, respectively).


Figure 2
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  		Fig. 2. Effect of BSA on Ca2+ influx and PKC activation. A: PTCs were loaded with 3 µM fluo-3 AM in serum-free medium for 40 min and treated with BSA (10 mg/ml). F/F0, relative fluorescence intensity. B and C: cells were pretreated with EGTA (10–4 M) and BAPTA-AM (10–6 M) for 30 min before BSA treatment. A-23187 (10–6 M) was added as a positive control. Similar results were obtained in 5 separate experiments. D and E: cells were incubated with BSA for 0–180 min (D) or pretreated with EGTA or BAPTA-AM for 30 min before BSA treatment for 30 min (E). Total protein was extracted and blotted with antibodies against phospho-pan-PKC and β-actin. Each example shown is a representative of 3 independent experiments. Means ± SE of 3 experiments for each condition determined from densitometry relative to β-actin are shown at bottom. *P < 0.05 vs. control; **P < 0.05 vs. BSA alone. F and G: effect of inhibition of the Ca2+/PKC signal pathways on BSA-induced increase in [3H]thymidine incorporation. The cells were pretreated with EGTA, BAPTA-AM, staurosporine (10–7 M), or bisindolylmaleimide I (10–7 M) for 30 min before BSA treatment for 12 h. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. BSA alone.

 
Effect of BSA on EGFR activation. In experiments to determine whether BSA activates EGFR signaling, BSA increased the phosphorylation of EGFR (Fig. 3A). However, pretreatment with Ca2+ chelators (EGTA and BAPTA-AM) blocked this effect of BSA on EGFR (Fig. 3B). In addition, inhibition of the PKC pathway with staurosporine and bisindolylmaleimide I blocked the BSA-induced increase in EGFR phosphorylation (Fig. 3C). This indicates that the Ca2+/PKC pathways are involved in the BSA-induced increase in EGFR phosphorylation.


Figure 3
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  		Fig. 3. Effect of BSA on epidermal growth factor receptor (EGFR) activation. A: PTCs were incubated with BSA for 0–180 min. B and C: pretreatment with EGTA, BAPTA-AM, staurosporine, or bisindolylmaleimide I for 30 min before BSA treatment for 30 min. Total protein was then extracted and blotted with antibodies to phospho-EGFR and total EGFR. Each example shown is a representative of 4 independent experiments. Means ± SE of 3 experiments for each condition determined from densitometry relative to total EGFR are shown at bottom. *P < 0.05 vs. control; **P < 0.05 vs. BSA alone.

 
Effect of BSA on activation of p44/42 MAPKs and NF-{kappa}B. In experiments to determine whether BSA activates the p44/42 MAPKs, BSA enhanced the level of p44/42 MAPK phosphorylation in a time-dependent manner (Fig. 4A), which was blocked by pretreatment with staurosporine and bisindolylmaleimide I for 30 min before BSA treatment for 30 min (Fig. 4B). In addition to PKC inhibition, inhibition of EGF receptor tyrosine kinase with AG-1478 (10–5 M) or inhibition of p44/42 MAPKs with PD-98059 (10–5 M) attenuated the phosphorylation of p44/42 MAPKs (Fig. 4C). However, AG-1478 and PD-98059 did not block the phosphorylation of PKC (Fig. 4D). Consequently, AG-1478 and PD-98059 also blocked the BSA-induced increase in [3H]thymidine incorporation (Fig. 4E). In an experiment to determine the involvement of NF-{kappa}B and I{kappa}B signal pathways, BSA increased the level of NF-{kappa}B p65 and I{kappa}B phosphorylation in a time-dependent manner (Fig. 5A), which was inhibited by pretreatment with staurosporine, AG-1478, and PD-98059 (Fig. 5B). Finally, inhibition of NF-{kappa}B activation with Bay 11-7082 (I{kappa}B phosphorylation inhibitor, 2 x 10–5 M) or SN50 (NF-{kappa}B nucleus translocation inhibitor, 500 ng/ml) blocked the BSA-induced [3H]thymidine incorporation (Fig. 5C).


Figure 4
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  		Fig. 4. Effect of BSA on p44/42 MAPK activation. A: PTCs were incubated with BSA for 0–180 min. B: PTCs were pretreated with staurosporine and bisindolylmaleimide I for 30 min before BSA treatment for 30 min, and total protein was then extracted and blotted with antibodies against phospho-p44/42 MAPKs and total p44/42 MAPKs. C and D: cells were pretreated with AG-1478 or PD-98059 for 30 min before BSA treatment for 30 min. Total protein was then extracted and blotted with antibodies against phospho-p44/42 MAPKs and total p44/42 MAPKs (C) or phospho-pan-PKC and β-actin (D). Each example shown is a representative of 3 independent experiments. Means ± SE of 3 experiments for each condition determined from densitometry relative to total p44/42 or β-actin are shown at bottom. E: effect of inhibition of the EGFR or p44/42 MAPK signal pathways on BSA-induced increase in [3H]thymidine incorporation. Cells were pretreated with AG-1478 or PD-98059 for 30 min before BSA treatment for 12 h. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. BSA alone.

 

Figure 5
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  		Fig. 5. Effect of BSA on NF-{kappa}B/inhibitor of NF-{kappa}B (I{kappa}B) phosphorylation. A: PTCs were incubated with BSA for 0–180 min. B: pretreatment with staurosporine, AG-1478, or PD-98059 for 30 min before BSA treatment for 30 min. Total protein was extracted and blotted with antibodies against phospho (p)-NF-{kappa}B, phospho-I{kappa}B, and β-actin. Each example shown is a representative of 3 independent experiments. Means ± SE of 3 experiments for each condition determined from densitometry relative to β-actin are shown at bottom. *,#P < 0.05 vs. control; **,##P < 0.05 vs. BSA alone. C: effect of inhibition of the NF-{kappa}B signal pathway on BSA-induced increase in [3H]thymidine incorporation. Cells were pretreated with Bay 11-7082 (2x10–5 M) or SN50 (500 ng/ml) for 30 min before BSA treatment for 12 h. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. BSA alone.

 
Effect of BSA on cell cycle regulatory protein expression levels. To determine whether BSA-activated Ca2+/PKC, EGFR, p44/42 MAPK, and NF-{kappa}B signaling are involved in cell cycle regulatory protein expression levels, PTCs were treated with these signal pathway inhibitors. The relation between BSA and Ca2+/PKC, EGFR, or p44/42 MAPKs in the expression of cell cycle regulatory proteins (cyclin E/CDK 2 and cyclin D1/CDK 4) and cyclin-dependent protein kinase inhibitors (p21WAF/Cip1 and p27Kip1) was examined. As shown in Fig. 6, A and B, the BSA-induced increase in cyclin E/CDK 2 and cyclin D1/CDK 4 expression levels and the decrease in p21WAF/Cip1 and p27Kip1 expression levels were blocked by the Ca2+ chelators (EGTA or BAPTA-AM), staurosporine, AG-1478, or PD-98059. In addition, the involvement of NF-{kappa}B/I{kappa}B signaling pathway in the effect of BSA on the cell cycle regulatory proteins was examined. Figure 6C shows that Bay 11-7082 and SN50 blocked the BSA-induced increase in cyclin E/CDK 2 and cyclin D1/CDK 4 expression levels and the decrease in p21WAF/Cip1 and p27Kip1 expression levels.


Figure 6
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  		Fig. 6. Effect of BSA on cell cycle regulatory protein expression levels. PTCs were pretreated with EGTA/BAPTA-AM (A), staurosporine, AG-1478, or PD-98059 (B), or Bay 11–7082 or SN50 (C) for 30 min before BSA treatment for 12 h. Total protein was extracted and blotted with antibodies against cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, p21WAF/Cip1, and p27Kip1. Each example shown is representative of 4 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrated that BSA stimulates the DNA synthesis of PTCs mediated by the Ca2+/PKC, p44/42 MAPK, NF-{kappa}B, and EGFR signal pathways. BSA might play an important role in maintaining proximal tubular integrity and functions. Our hypothesis was that BSA treatment would stimulate DNA synthesis and cell proliferation in PTCs. This was based on previous studies that showed that BSA increased the Na+-glucose cotransporter (18) and stimulated cell growth, L-arginine transport, and metabolism to polyamines (3). Interestingly, low concentrations of albumin can act as a survival factor that is able to inhibit apoptosis of macrophages (20), whereas at high concentrations it can actually be toxic to cells (11). This apparent discrepancy may indicate that albumin could have a dual effect on PTCs (7). Although it is known that albuminuria is caused by increased glomerular filtration or tubular injury, a recent study demonstrated that nephritic levels of albumin are normally filtered across the glomerular capillary wall and albuminuria can result if there is inhibition of the retrieval process (31). Accordingly, micropuncture studies revealed that albumin concentrations range from 20 to 30 µg/ml in the proximal tubules of rodents. The induction of nephrosis causes up to a 100-fold increase in the albumin concentration prevailing in the proximal tubule (22, 27). Therefore, a range of protein concentrations comparable to those used in other studies were chosen. These concentrations were higher than those measured in the proximal tubular fluid of rats with experimental nephrosis by micropuncture studies, but the in vivo tubular changes manifest only after prolonged exposure of proximal tubule to filter proteins (41, 43, 44).

Albumin itself may act as a signaling molecule and play an important role in the activation of many intracellular signaling cascades in PTCs (6). In this study, BSA increased [Ca2+]i, PKC, p44/42 MAPKs, and the level of NF-{kappa}B phosphorylation. In addition, the inhibition of these molecules blocked BSA-induced increase of [3H]thymidine incorporation. Previous studies reported that albumin augments [Ca2+]i and DNA synthesis in astrocytes (26). These results showed that the phosphorylation of p44/42 MAPKs, NF-{kappa}B, and I{kappa}B by BSA was inhibited by the PKC inhibitors or PD-98059. Previously, NF-{kappa}B dimers, which are activated in experimental renal disease, were shown to remain in the cytoplasm in an inactive form bound to I{kappa}B. On stimulation, I{kappa}B is phosphorylated, ubiquitinylated, and ultimately degraded through proteolytic cleavage by the proteasome system (16). This is consistent with a previous report showing that the binding of glycated albumin to MonoMac 6 cells leads to the activation of p44/42 and p38 MAPKs with the subsequent translocation of NF-{kappa}B to the nucleus (1). In addition, the p44/42 MAPKs-dependent activation of NF-{kappa}B occurs in macrophage RAW cells after NADPH oxidase stimulation (10). Hence, these results suggest that BSA-induced DNA synthesis is mediated by Ca2+, PKC, p44/42 MAPK, and NF-{kappa}B signal pathways.

Albumin also activates EGFR in renal epithelial cells (30). While most EGFR is expressed on the basolateral surfaces in kidney, 10% of the EGFR expressed on the luminal membrane of tubular cells might function as a scaffold to facilitate transmembrane signaling in response to albumin (19). Therefore, this study examined how the EGFR might be involved in BSA-induced signaling in cultured PTCs, and whether the EGFR could be linked to BSA-induced cell proliferation. In this study, BSA increased the level of EGFR phosphorylation that had been blocked by Ca2+/PKC inhibitors. The inhibition of EGFR by AG-1478 significantly blocked the BSA-induced phosphorylation of p44/42 MAPKs or the NF-{kappa}B/I{kappa}B signal pathways, as well as the BSA-induced increase in [3H]thymidine incorporation. These results are supported by previous observations that, in some cases, EGFR transactivation is Ca2+- and PKC dependent (4, 12, 15, 36). It has been suggested the kinases of the Src family play an essential role upstream of the EGFR. Moreover, the EGFR has been shown to function as an important component of the signaling pathways linking G protein-coupled receptor activation and a variety of physical stimuli to the activation of the Ras/ERK and/or PI3K/Akt pathways (8). Therefore, the disruption of EGFR signaling has been targeted as a potential therapeutic maneuver in animal models of polycystic kidney disease (38) or renal cell cancer (2). Overall, these results suggest that the EGFR in response to BSA might contribute to the activation of the p44/42 MAPK and NF-{kappa}B/I{kappa}B signal pathways.

These signal molecules can regulate cell cycle regulatory protein expression levels. Positive regulators include the cyclins and their catalytic partners, the CDKs, which are essential for the progression of cells through each phase of the cell cycle and various cell cycle checkpoints (32, 33). Negative regulators include cyclin kinase inhibitors (CKIs), which inhibit the cell cycle at multiple checkpoints by inactivating the cyclin-CDK complexes (34). p21WAF/Cip1 and p27Kip1, members of the CIP/KIP family of CKIs, are important in regulating the cyclin-CDK complexes, which are active in the G1-S phase transition (29, 39), and cyclin E/CDK2 can be activated by the phosphorylation of p27Kip1 to induce its degradation (40). In addition, the regulation of cyclin D1 expression is mediated by the Ras/ERK signaling pathway (35). In this study, inhibition of Ca2+, PKC, EGFR, p44/42 MAPK, or NF-{kappa}B pathway blocked the BSA-induced expression of cyclins and CDKs and restored the BSA-induced inhibition of p21WAF/Cip1 and p27Kip1 expression (Fig. 6). These results showed that the decrease in p21WAF/Cip1 and p27Kip1 level is associated with an increase in proliferation in response to mitogenic stimuli and demonstrated that BSA promotes the cell cycle to faster cycling and cell proliferation. Therefore, BSA can stimulate DNA synthesis and proliferation in PTCs that are dependent on the Ca2+, PKC, EGFR, p44/42 MAPK, and NF-{kappa}B pathways. To our knowledge, this is the first report showing that BSA inhibits p21WAF/Cip1 and p27Kip1 expression and activates proliferation in PTCs. Figure 7 shows a hypothetical model of the signaling mechanisms involved in mediating the BSA-induced increase in PTC proliferation. However, whether this correlates with physiological/pathological modulation of PTC function deserves further study.


Figure 7
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  		Fig. 7. Hypothesized model for the signal pathways involved in BSA-induced cell proliferation. BSA augments intracellular Ca2+ influx and PKC phosphorylation. In turn, PKC activates EGFR phosphorylation and subsequent activation of p44/42 MAPKs. Subsequently, p44/42 MAPK activations induce phosphorylation of NF-{kappa}B and I{kappa}B ubiquitination. Finally, these molecules increase cell cycle regulatory protein expression levels.

 
In summary, BSA increased DNA synthesis of primary cultured PTCs through the Ca2+/PKC-mediated activation of the EGFR and subsequent activation of p44/42 MAPK and NF-{kappa}B signaling pathways.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant (code no. 20070401034006) from BioGreen 21 Program, Rural Development Administration, Republic of Korea. The authors acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development through the Brain Korea 21 project.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Han, Dept. of Veterinary Physiology, Coll. of Veterinary Medicine, Chonnam National Univ., Gwangju 500-757, Korea (e-mail: hjhan{at}chonnam.ac.kr)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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Y. J. Lee, H. N. Suh, and H. J. Han
Effect of BSA-induced ER stress on SGLT protein expression levels and {alpha}-MG uptake in renal proximal tubule cells
Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1405 - F1416.
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