Arginine vasopressin stimulates mesangial cell proliferation by activating the epidermal growth factor receptor

Paramita M. Ghosh1, Margarita Mikhailova2, Roble Bedolla1, and Jeffrey I. Kreisberg1,3

Departments of 1 Surgery and 2 Pathology, University of Texas Health Science Center at San Antonio, and 3 South Texas Veterans Health Care, San Antonio, Texas 78229


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The potent vasoconstrictor arginine vasopressin (AVP) is also a mitogen for mesangial cells. Treatment with AVP decreased transit time through the cell cycle. AVP-stimulated mesangial cell growth by activating both the Ras mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) cell signaling pathways. Both the selective PI3K inhibitor LY-294002 and the MAPK kinase (MEK) inhibitor PD-98059 inhibited AVP-stimulated mesangial cell proliferation. However, LY-294002 was more potent, indicating an important role for PI3K activation in AVP-stimulated mesangial cell proliferation. AVP appeared to exert its effect on MAPK and PI3K activation, as well as on cell proliferation, by activating the epidermal growth factor receptor (EGF-R). Pretreatment with the tyrphostin-derived EGF-R antagonist AG-1478 inhibited mesangial cell proliferation as well as the activation of extracellular signal-regulated kinase 1/2 (ERK1/2 or p42/p44MAPK), and p70S6 kinase, a downstream effector of PI3K, providing evidence that MAPK and PI3K activation, respectively, occurred downstream of EGF-R activation. Treatment with rapamycin, an inhibitor of the p70S6 kinase activator mTOR, also resulted in growth inhibition, further suggesting the importance of the PI3K signaling pathway in AVP-induced proliferation. AVP treatment appeared to transactivate EGF-R by inducing tyrosine phosphorylation of the Ca2+/protein kinase C (PKC)-dependent nonreceptor tyrosine kinase, Pyk2, leading to Pyk2/c-Src association and c-Src activation. This was followed by association of c-Src with EGF-R and EGF-R activation. These data suggested that AVP-stimulated Pyk2 tyrosine phosphorylation to activate c-Src, thereby leading to EGF-R transactivation.

mitogen-activated protein kinase; phosphatidylinositol 3-kinase; p70S6 kinase; Pyk2; c-Src


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXCESSIVE MESANGIAL CELL PROLIFERATION is a frequent characteristic of many glomerular diseases caused by immunological or nonimmunological types of glomerular cell injury (29, 44). Glomerular disease is also a major cause of end-stage renal disease in humans. The mesangium has been shown to play an important role in maintaining the microcirculation of the glomerulus. During the course of injury, mesangial cells are exposed to a variety of mediators, which are either released locally or are present in the systemic circulation. Among these are platelet-derived growth factor B chain (PDGF-BB), epidermal growth factor (EGF), endothelin-1 (ET-1), angiotensin II (ANG II), interleukin-1 and -6, and arginine vasopressin (AVP), all of which have been shown to be mitogens for mesangial cells in culture (10, 20, 22). It is not surprising, given the importance of mesangial cell proliferation in a variety of glomerular diseases, that a considerable effort has been undertaken to elucidate the mechanisms of glomerular cell proliferation in the hope of designing new specific and effective therapies (49).

Vasoactive agents such as AVP, ANG II, and ET-1 modulate vascular tone and glomerular filtration through their contractile effects on glomerular mesangial cells (20, 22, 42). Additionally, these agents induce hypertrophy and cell growth in both mesangial and smooth muscle cells (22, 25, 54). AVP, rather than ANG II, has been shown to be a potent inducer of mesangial cell proliferation (42). Mesangial cells express the vasopressin V1 receptor, which, when stimulated, induces mobilization of intracellular Ca2+, rather than the V2 receptor, which results in elevations in cAMP levels on stimulation (8, 27). Several lines of evidence indicate the involvement of AVP in proliferative glomerular injury. Chronic blockade of the vasopressin V1 receptor directly inhibits glomerular proliferative injury in salt-loaded spontaneously hypertensive rats (SHR) (36) and in rats showing manifestations of hypercholesterolemia and protein urea (33). In addition, glomeruli of SHR possess a higher number of AVP receptors (V1) than do glomeruli of age-matched Wistar-Kyoto (WKY) rats (36). These results suggest that AVP induces proliferation in glomerular mesangial cells through activation of its V1 receptor. Studies from our laboratory showed that, on binding to its specific V1 receptor, AVP activates a phosphatidylinositol-specific-phospholipase C (PI-PLC) in rat mesangial cells (46). This results in elevated levels of inositol trisphosphate (IP3) and diacylglycerol (DAG), leading to mobilization of intracellular stores of Ca2+ and stimulation of protein kinase C (PKC) (4, 52). Ca2+ mobilization and PKC activation induce increased cell signaling, leading to cell cycle progression (23, 45). These results suggest that AVP-stimulated proliferation may be mediated by Ca2+ mobilization and PKC activation.

In contrast to tyrosine kinase receptors like the PDGF-BB receptor (PDGF-Rbeta ), vasoactive agents such as AVP, ANG II, and endothelin exert their affect by binding G protein-coupled receptors (GPCR) that lack cytoplasmic tyrosine kinase domains. However, like classic growth factors, tyrosine phosphorylation plays a critical role in the growth response elicited by these vasoactive agents (20, 25). Activation of GPCR causes tyrosine phosphorylation of adaptor proteins, such as Shc, Src, or IRS-1, leading to activation of p21Ras and phosphatidylinositol 3-kinase (PI3K). This results in stimulation of the effectors of Ras and PI3K: the mitogen activated protein kinase (MAPK) and p70S6 kinase signaling pathways, respectively (16, 21, 41). The mechanism of adaptor protein activation by the vasopressin V1 receptor is presently unknown.

In this report we show that AVP stimulates mesangial cell proliferation by activating the EGF receptor (EGF-R). EGF-R activation stimulates mesangial cell growth by activating both the Ras/MAPK and PI3K signaling pathways, which results in activation of their downstream effectors, extracellular signal-regulated kinase (ERK) and p70S6 kinase. Importantly, it appears that GPCR activation of the EGF-R is transduced by the Ca2+/PKC-dependent nonreceptor tyrosine kinase Pyk2, which results in c-Src activation of the EGF-R. Our results attempt to elucidate the signaling steps leading from AVP-induced stimulation of the V1 receptor to cell proliferation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell culture and pharmacological treatments. Rat mesangial cells were isolated and established in culture as previously described (32). The cells were cultured in RPMI 1640 containing 20% fetal bovine serum (FBS) and 1% antimycotic-antibiotic solution (Mediatech-Cellgro). AG 1478, rapamycin, PD-98059, and LY-294002 were obtained from Calbiochem, La Jolla, CA. AVP was from Sigma, St. Louis, MO, and EGF was obtained from GIBCO. Rabbit polyclonal anti-phospho-ERK (Thr202/Tyr204) and anti-phospho-Akt (Ser 473) antibody were from Cell Signaling Technology, Beverly, MA. Monoclonal anti-phosphotyrosine, anti-PDGF-Rbeta , and anti-Pyk2 antibodies were from BD Transduction Laboratories, San Diego, CA. Monoclonal anti-c-Src antibody was from Upstate Biotechnology, Lake Placid, NY. All other antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA.

MTT assay. Cells were plated in triplicate in 20% FBS in 24-well plates at a concentration of 10,000 cells/well in the presence of the agonists to be studied (AVP or EGF, at various concentrations). After 24 h of treatment, 60 µl 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were added per well. This drug is readily taken up by live cells and is absorbed in the mitochondria, where it is converted to a formazan salt (40). Although the original drug is yellow in color, the final product is purple. The formazan crystals thus formed are redissolved in 1 ml DMSO. As this conversion can only take place in the mitochondria of live cells, the extent of formazan formation, as detected by the intensity of the color of the final DMSO solution, gives us a good estimate of the number of cells in the well. The effectiveness of growth inducers in stimulating proliferation can be estimated this way, as cells under highly proliferative conditions exhibit a deep purple color while those under less proliferative conditions exhibit a lighter purple. The intensity of the solution was detected by reading in a spectrophotometer (Beckman).

Flow cytometry. Mesangial cells were grown under desired conditions in the presence of 20% FBS in 100-mm dishes at 500,000 cells/dish. Cells to be processed for flow cytometry were trypsinized, then resuspended in 2 ml cell growth medium, and spun down at 1,000 g for 5 min in a tabletop centrifuge. The medium was aspirated, and the pellet was washed once in PBS. The cells were then resuspended in 500 µl 70% ethanol and incubated 30 min at -20°C. The cells were repelleted and washed twice in 1% BSA/PBS. The cells were suspended in 150 µl PBS, 50 µl of 1 mg/ml RNAse A (Sigma), and 100 µl of 100 µg/ml propidium iodide (Sigma) and incubated overnight at 4°C. Flow cytometry was conducted on FACStar Plus (Becton Dickinson Immunocytometry Systems, San Jose, CA). Cells were illuminated with 200 mW of 488-nm light produced by an argon-ion laser. Fluorescence was read through a 630/22-nm band-pass filter. Data were collected on 20,000 cells as determined by forward and right-angle light scatter and stored as frequency histograms; data used for cell cycle analysis were then analyzed using MODFIT (Verity software, Topsham, ME).

Western blotting. Mesangial cells were grown on 100-mm dishes at 1,000,000 cells/dish and serum starved for 48 h before the experiments. Whole cell extracts were prepared by washing the cells twice in PBS and lysing cells in 250 µl cell lysis buffer (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 10% NP-40, and protease inhibitors: 0.1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml each of phenathroline, leupeptin, aprotinin, and pepstatin A) and phosphatase inhibitors: 20 mM B-glycerol phosphate, 1 mM Na-orthovanadate, and 10 mM NaF. Proteins were quantitated using a BCA assay (Pierce, Rockford IL) and fractionated on SDS-polyacrylamide gels (118:1 acrylamide-bis for p70S6 kinase and 29:1 acrylamide-bis for everything else). Electrophoresis was performed at 45 mA for ~45 min using minivertical electrophoresis cells (Mini-PROTEAN II Electrophoresis Cell, Bio-Rad, Hercules, CA). The gels were electroblotted for 1.5 h at 200 mA using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) onto 0.2-µm polyvinylidene difluoride membrane (Osmonics, Westborough, MA). The blots were stained with primary antibodies at a dilution of 1:500. The staining was detected by enhanced chemiluminescence (Pierce) after incubation with a peroxidase-labeled secondary antibody (Donkey anti-mouse IgG, Chemicon, Temecula, CA, Goat anti-rabbit IgG, Fc specific, Jackson Immunoresearch Laboratories, West Grove, PA).

Immunoprecipitations. Cells were grown on 100-mm dishes at 500,000 cells/dish and were serum starved for 48 h before the experiment. Five hundred micrograms of protein obtained from whole cell lysates in cell lysis buffer were precleared with 25 µl of 50% protein A-Sepharose beads in 400 µl of lysis buffer containing 1 µg/ml BSA for 1 h. The supernatants were incubated with the appropriate antibody (1-4 µg/sample) overnight. Next, 20 µl of protein A-Sepharose were added for 1 h, and the immunocomplexes were washed three times with lysis buffer. Samples were separated by SDS-PAGE, and proteins were detected by Western blotting.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AVP-stimulated proliferation in rat glomerular mesangial cells. We showed previously that AVP stimulated mesangial cell contraction in a Ca2+-dependent fashion and also induced PKC activation (4, 47). In addition, AVP (1 µM) increased mesangial cell proliferation with a cell doubling time of 34 h compared with 42 h for FCS alone (Fig. 1A). Fluorescent-activated cell sorting (FACS) analysis revealed that AVP stimulated G1 to S-phase transition with ~34% increase of cells in S-phase after 18 h of AVP treatment over and above the effect of FCS alone (Fig. 1B). The results were normalized to the number of cells in S-phase under control conditions. Dose-response experiments showed that 1 µM AVP evoked nearly the same proliferative response in rat mesangial cells as 50 ng/ml EGF (Fig. 2).


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Fig. 1.   A: growth curves demonstrating the effect of 1 µM arginine vasopressin (AVP) with and without the [mitogen-activated protein kinase (MAPK) kinase (MEK)] inhibitor PD-98059 (PD, 100 µM) and the PI3K inhibitor LY-294002 (LY, 25 µM) in the presence of serum. Mesangial cells were plated on 35-mm dishes in the presence of 20% FCS at a concentration of 25,000 cells/dish. At the end of 1, 2, and 3 days, the cells were trypsinized and counted by using a hemocytometer. AVP treatment (1 µM) caused enhanced cell proliferation compared with untreated cells. Estimation of doubling times indicated that AVP-treated cells had a doubling time of ~34 h compared with 42 h for untreated cells. Notice the pronounced inhibition of AVP-stimulated mesangial cell proliferation by LY. The data represent the average of 3 readings. B: change in the number of cells in S-phase of the cell cycle normalized to controls as determined by flow cytometry after 48 h. Typically, under control conditions, 20% of the cells are in S-phase of the cell cycle. Note that AVP-stimulated G1 transition to S-phase that was inhibited by PD-98059 (PD, 100 µM) and LY-294002 (LY, 25 µM). LY inhibited control proliferation, as well. The epidermal growth factor receptor (EGF-R) antagonist AG 1478 (20 µM) as well as rapamycin (100 nM), a potent inhibitor of p70S6 kinase, similarly inhibited mesangial cell growth. The data represent the means of 6 different experiments. P < 0.05: * vs. control; # vs. AVP (ANOVA).



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Fig. 2.   Dose-response of mesangial cell proliferation to AVP (A) and EGF (B) as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (see MATERIALS AND METHODS). These studies were conducted in the presence of 20% FCS. Note that the y-axis of the 2 graphs are different. These graphs represent the average of 3 readings.

AVP-induced mesangial cell proliferation is mediated by the MAPK and PI3K cell signaling pathways. Mitogenesis in mammalian cells takes place by the activation of either of two signaling pathways: the MAPK or the PI3K pathway. Figure 1B indicates that the PI3K pathway plays a greater role in AVP-induced mitogenesis than the MAPK pathway. Inhibition of the PI3K pathway by the selective PI3K inhibitor LY-294002 (25 µM) prevented AVP-stimulated proliferation to a far greater extent (64% decrease in the number of cells in S-phase with respect to AVP treatment alone) than inhibition of the MAPK pathway with the selective inhibitor of MAPK kinase (MEK) PD-98059 (100 µM) (28% decrease in the number of cells in S-phase with respect to AVP treatment alone). The drug concentrations used here were determined by the complete inhibition of ERK phosphorylation with PD-98059 and the complete inhibition of Akt phosphorylation by LY-294002 (not shown). In the absence of serum, these inhibitory effects on ERK and Akt phosphorylation were observed with less than 2 h of incubation in the presence of the drug. However, prolonged incubation times were necessary to induce growth arrest in the presence of serum because of the high doubling times of these cells. To determine the effect of AVP alone on the two signaling pathways in rat mesangial cells, we serum starved the cells for 48 h, followed by AVP treatment for 1-60 min. Treatment with AVP for 15 min stimulated both pathways, as evidenced by increased phosphorylation of ERK 1/2 (p42/p44MAPK) (Fig. 3A) and p70S6 kinase, a downstream effector of PI3K (Fig. 3B). Activation of ERK was determined by immunoblotting with a phospho-specific antibody, whereas p70S6 kinase activation was determined by the appearance of slower migrating forms of the activated protein in SDS-PAGE due to phosphorylation. Phosphorylation of Akt, another downstream effector of PI3K well known for its role in cell survival, was transiently increased by AVP treatment (Fig. 3C). Treatment with 100 nM rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase indispensable for p70S6 kinase activation, inhibited AVP-induced proliferation (49% decrease in the number of cells in S-phase with respect to AVP treatment alone, Fig. 1B), suggesting an important role for the PI3K signaling pathway in AVP-induced mesangial cell proliferation.


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Fig. 3.   Immunoblots displaying an increase in Erk1/2 (A), p70S6 kinase (B), and Akt activation (C). Mesangial cells were grown to confluence in 100-mm dishes, then serum starved for 48 h, at the end of which AVP (1 µM), with and without PD-98059 (PD, 100 µM), was added for 15, 30, or 60 min. The cells were harvested in lysis buffer and separated by PAGE. A: Erk1/2 activation was demonstrated by staining the blots with an antibody to phosphorylated Erk1/2 (Thr202/Tyr204; top 2 lanes). Total Erk1/2 antibody was used to demonstrate equal loading (bottom 2 lanes). Note that AVP stimulated ERK activation and this activation was inhibited by PD-98059 (PD). B: immunoblot demonstrating activation of p70S6 kinase with and without the PI3K inhibitor LY-294002 (LY, 25 µM). Activation was demonstrated by the appearance of slower migrating (phosphorylated) isoforms (arrows). Note that activation of p70S6 kinase occurred by 15 min and this activation was inhibited by LY. C: immunoblot demonstrating transient activation of Akt, a cell survival protein that is also a downstream effector of PI3K. Akt activation was demonstrated by staining the blot with a phospho-specific antibody (Ser 473). These experiments were repeated at least 3 times with similar results.

AVP-induced mesangial cell proliferation requires EGF-R transactivation. GPCRs, like the vasopressin V1 receptor, lack cytoplasmic tyrosine kinase domains (54). In other systems, EGF-R was transactivated in response to GPCR activations, and EGF-R appeared to play a central role in relaying mitogenic signals from GPCRs to the nucleus (12). We therefore determined whether EGF-R was activated in response to AVP treatment as well. Due to its mode of activation, EGF-R activation can be assayed by immunoprecipitation followed by immunoblotting with an anti-phosphotyrosine antibody. As shown in Fig. 4A, mesangial cells displayed EGF-R activation within 15 min of addition of AVP. To determine whether EGF-R activation was necessary for cell proliferation, the cells were treated with the highly selective EGF-R tyrosine kinase inhibitor AG 1478 (20 µM). AG 1478 was found to be 30,000 times more selective for EGF-R than for other receptor tyrosine kinases (IC50 for PDGF-Rbeta or other members of the ErbB family = 100 µM vs. 3 nM for EGF-R) (19). Dose-response studies demonstrated that this dose was necessary to significantly reduce cell proliferation in rat mesangial cells in continuous culture (not shown). AG 1478 completely abolished AVP-induced EGF-R activation (Fig. 4B). On the other hand, AG 1478 did not inhibit the activation of the PDGF-Rbeta (Fig. 4C). Figure 1B shows that 20 µM AG 1478 markedly inhibited AVP-stimulated mesangial cell proliferation in the presence of serum (40% decrease in the number of cells in S-phase with respect to AVP treatment alone), thus supporting a role for EGF-R activation in mesangial cell proliferation. AG 1478 inhibited activation of both ERK and p70S6 kinase (Fig. 4D), indicating that activation of both the MAPK and the PI3K pathways occurred downstream of EGF-R activation.


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Fig. 4.   For these studies, cells were grown to confluence in 100-mm dishes, serum starved for 48 h, at the end of which AVP (1 µM) was added for 15, 30, or 60 min. The cells were harvested in lysis buffer and separated by PAGE. A: immunoblot demonstrating activation of EGF-R by AVP. Activation of the EGF-R was determined by immunoprecipitating (IP) cell extracts with an antibody to the EGF-R, and immunoblotting (IB) with an anti-phosphotyrosine antibody (pY). Note that EGF-R is activated 15 min after AVP treatment and the level of activation continues to increase for 1 h (top). Total EGF-R levels (bottom), determined by immunoblotting the same membrane with an antibody to EGF-R, are unchanged, indicating equal loading of samples. B: immunoblot demonstrating the effect of the tyrphostin AG1478 (20 µM) on EGF-R activation by AVP. RIPA cell extracts were immunoprecipitated with EGF-R antibody, and immunoblots were stained with pY. Notice that AG1478 inhibits EGF-R activation (top). The same immunoblot was stained after stripping for total EGF-R protein to demonstrate equal loading of protein. C: immunoblot demonstrating the effect of AG1478 on [platelet-derived growth factor B chain (PDGF-BB) receptor (PDGF-Rbeta )] activation. AG 1478 failed to inhibit PDGF-induced PDGF-R activation. Cells were serum starved for 48 h and treated with 10 ng/ml PDGF-BB for 1 h. RIPA cell lysates were immunoprecipitated with monoclonal anti-PDGF-Rbeta antibody and immunoblotted with pY. Note that AG1478 failed to inhibit PDGF-Rbeta activation. D: immunoblots demonstrating the effect of AG 1478 on activation of ERK and p70S6 kinase. AVP treatment activated both ERK (top) and p70S6 kinase (bottom), whereas pretreatment with AG1478 (20 µM) inhibited activation of both proteins. Total ERK was stained to demonstrate equal protein loading (middle). All these experiments were repeated at least twice with similar results.

AVP-induced EGF-R transactivation is mediated by Pyk2 and c-Src. Ca2+/Pyk2 has been implicated in the transactivation of EGF-R by activating c-Src in other systems (14, 18). Because AVP has been shown to induce Ca2+ mobilization and PKC activation (4, 20, 23, 47), we determined whether AVP-induced activation of EGF-R in mesangial cells involved activation of Pyk2 and c-Src. Mesangial cell lysates were immunoprecipitated with a Pyk2 antibody, and immunoblots were analyzed with an antibody to phosphotyrosine. AVP treatment for 30 s to 1 min resulted in Pyk2 activation that was sustained through 60 min, as evidenced by tyrosine phosphorylation (Fig. 5A), with a concomitant transient association of Pyk2 with c-Src determined by immunoprecipitation/immunoblotting experiments (Fig. 5C, top). AVP treatment also resulted in rapid (within 1 min) tyrosine phosphorylation of c-Src (Fig. 5B) with its association with EGF-R (Fig. 5C, middle). EGF-R/c-Src association was determined by immunoprecipitating mesangial cell lysates with an antibody to c-Src and immunoblotting with an antibody to EGF-R. These data suggest that the AVP receptor stimulated Pyk2 tyrosine phosphorylation, resulting in c-Src activation, thereby leading to EGF-R transactivation.


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Fig. 5.   A: immunoblot demonstrating activation of protein tyrosine kinase (Pyk2) by AVP. Mesangial cells were serum starved for 48 h and lysates were collected after AVP treatment and immunoprecipitated (IP) with an antibody to Pyk2. Pyk2 activation was determined by staining immunoblots (IB) with an anti-phosphotyrosine antibody (pY). Pyk2 was activated 1 min after AVP treatment (top), and the activation persisted for 1 h. There was no difference in total Pyk2 expression (bottom), indicating equal loading of proteins. B: immunoblot demonstrating AVP-stimulated tyrosine phosphorylation (activation) of c-Src. Mesangial cell lysates were immunoprecipitated with an antibody to c-Src followed by immunoblotting with pY. Note the sustained increase in tyrosine phosphorylated c-Src after 1 min of AVP treatment (top). Bottom: immunoblots were stained with an antibody to c-Src to demonstrate equal protein loading. C: Pyk2/Src and EGF-R/Src complex formation in response to AVP treatment. Top: mesangial cell lysates were immunoprecipitated with an antibody to c-Src and immunoblots were stained with an antibody to Pyk2. Note that Pyk2/c-Src complex formation occurred within 1 min of AVP treatment. Middle: immunoblot is the same as in top after stripping and staining with an EGF-R antibody to demonstrate cSrc/EGF-R complex formation. Note the transient increase in c-Src/EGF-R association after AVP treatment. Immunoblotting with a total c-Src antibody reveals equal protein loading (bottom). This experiment was repeated twice with similar results.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several groups of investigators have reported that AVP is a strong mitogen for mesangial cells (22, 23, 25, 42). Earlier studies showed that AVP stimulates MAPK activity in mesangial cells (1) and is a very potent inducer of the immediate early genes c-fos, c-jun, and Egr-1 (42). In this report, we extend these observations and show that AVP treatment of mesangial cells stimulates cell cycle progression and proliferation by inducing PI3K and MAPK activation through a novel mechanism involving the activation of EGF-R (a scheme is shown in Fig. 6). Although AVP was shown to stimulate p70S6 kinase activation in rat cardiomyocytes (53), this is the first time that AVP has been shown to stimulate p70S6 kinase in mesangial cells.


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Fig. 6.   Proposed pathways activated by AVP treatment in mesangial cells. Phospholipase C (PLC); protein kinase C (PKC); mammalian target of rapamycin (mTor).

The role of p42/p44MAPK (ERK2/1) in the transduction of proliferative signals to the nucleus is well known (39, 43). ERK is activated by receptor tyrosine kinases (e.g., growth factors) and by stimulation of GPCRs (e.g., V1) (34). Binding of ligand transduced proliferative or differentiation signals to the nucleus resulting in activation of cyclin-dependent kinases and cell cycle progression (24). The signaling pathways leading from activated growth factor receptors to ERKs showed that the small GTPase Ras played a prominent role (15, 31). Because full activation of ERK requires both threonine and tyrosine phosphorylation, it suggested that integration of multiple signaling pathways was required for the activation of the kinase (28). In accordance with previously published data, our results indicate that AVP-induced mesangial cell growth was dependent on ERK phosphorylation, because inhibition of ERK activation by PD-98059 inhibited AVP-induced proliferation. However, our data also show, for the first time in mesangial cells, that the PI3K pathway plays an important role in AVP-induced proliferation.

PI3K signaling through p70S6 kinase acts synergistically with Ras/MAPK to stimulate the G1 to S-phase transition of the cell cycle (7, 30). PI3K is composed of two subunits (regulatory p85 and catalytic p110) that appear to possess both lipid kinase and protein kinase activities (9, 13). PI3K activation occurs after binding of p85, through its SH2 domain, to the cytoplasmic region of receptor tyrosine kinases, which recruit p110 to the plasma membrane where the lipid substrates are localized. Also, GTP-bound Ras can bind to and activate PI3K (38). p70S6 kinase is a prominent downstream effector of PI3K (26). Cell proliferation requires the coordinate activation of p70S6 kinase. This kinase participates in the translation of mRNAs that encode many of the components of the translational apparatus, including ribosomal proteins and elongation factors. Thus activation of p70S6 kinase is a prerequisite for protein synthesis (17). p70S6 kinase is activated in vivo by phosphorylation, mediated, in part, by PI3K and by a phosphatidylinositol kinase-related kinase, mTOR (2, 17, 26). Rapamycin inhibits mTOR and prevents it from activating p70S6 kinase by forming a stable complex with FK506 binding protein, which binds mTOR (5). Results presented in this report show that both rapamycin and LY-294002, which inhibit PI3K directly, potently inhibited control and AVP-stimulated mesangial cell proliferation, indicating the importance of p70S6 kinase activation in mesangial cell proliferation.

PI3K activation is important not only in cell proliferation but also in cell survival. A second PI3K effector, Akt, also known as protein kinase B, is also affected by growth factors and plays a role in cell survival (6). Activation of Akt and p70S6 kinase by a similar range of mitogens and phosphatase inhibitors implied a close connection between the signaling pathways to the two kinases. Earlier studies suggested that p70S6 kinase activation was mediated by Akt; however, it now appears that p70S6 kinase can be activated independently of Akt (reviewed in 17, 11, 35). Our results do not allow us to conclude whether p70S6 kinase activation is dependent on Akt or not; rather, it demonstrates the importance of the PI3K signaling pathway and p70S6 kinase activation in AVP-stimulated mesangial cell proliferation.

Although AVP-induced MAPK activation has been reported (1), the mechanism by which AVP activates MAPK and PI3K signaling cascades leading to cell growth was not previously known. GPCR transactivation of receptor tyrosine kinases was shown to be necessary for transmission of mitogenic and other signals to the nucleus (12). Data presented in this paper indicated for the first time that the EGF-R was potently activated by AVP stimulation. In addition, this activation was inhibited by the EGF-R kinase inhibitor AG 1478. As further proof of the involvement of EGF-R in AVP-induced cell signaling, AG 1478 inhibited both AVP-induced cell proliferation as well as MAPK and PI3K activation. In this paper, we suggest that AVP-stimulated EGF-R activation is induced by intracellular signals. There is presently no evidence of EGF-R ligand (EGF or TGFalpha ) precursor production stimulated by AVP, as has been shown in other models (37, 55). Future studies will reveal the feasibility of such pathways of AVP-induced EGF-R transactivation.

We have previously shown that AVP induces Ca2+ mobilization and PKC activation in mesangial cells (4, 47). We therefore tried to determine whether EGF-R activation by AVP takes place in a Ca2+/PKC-dependent manner. Eguchi et al. (18) showed that the Ca2+/PKC-sensitive, nonreceptor tyrosine kinase Pyk2 was activated after ANG II treatment in vascular smooth muscle cells. We have shown here that AVP also induced Pyk2 activation in mesangial cells, suggesting that AVP-induced EGF-R activation is mediated by Pyk2. Both Pyk2 and EGF-R formed complexes with c-Src, suggesting a role for c-Src in Pyk2-mediated EGF-R transactivation by AVP. Further studies using EGF-R mutants lacking c-Src binding domains will reveal the importance of c-Src binding in EGF-R activation. Our results suggest that EGF-R serves as a scaffold for preactivated c-Src and for downstream adaptors that lead to MAPK and PI3K activation in mesangial cells treated with AVP. This is in accordance with the mechanism of MAPK activation by ANG II in smooth muscle cells suggested by Eguchi et al. (19).

In conclusion, in this report we have attempted to elucidate the mechanism by which AVP induces mesangial cell proliferation. We have previously shown that AVP binding to mesangial cells results in activation of PKC and elevation in intracellular levels of Ca2+ (4, 47). We now show that within 30 s of AVP treatment, the protein tyrosine kinase Pyk2 is tyrosine phosphorylated (activated), resulting in its association with and activation of c-Src by 1-5 min. This results in Src/EGF-R association with activation of EGF-R within 15 min of AVP treatment, leading to MAPK and p70S6 kinase activation and cell proliferation (a scheme is presented in Fig. 6). These studies may be important for the future development of therapies for treatment of glomeruloproliferative diseases.


    ACKNOWLEDGEMENTS

We to thank Dr. C. Alex McMahan for the enormous help with the statistical analysis and Charles A. Thomas for help with FACS analysis.


    FOOTNOTES

This work was supported by a Merit Review from the Department of Veterans Affairs (J. I. Kreisberg), University of Texas Health Science Center at San Antonio Institutional Research Grant (P. M. Ghosh), and National Heart, Lung, and Blood Institute Training Grant 2T32-HL-07446-16 (P. M. Ghosh). J. I. Kreisberg is a Career Scientist with the Department of Veterans Affairs.

Address for reprint requests and other correspondence: J. I. Kreisberg, Dept. of Surgery, Univ. of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229 (E-mail: kreisberg{at}uthscsa.edu).

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.

Received 1 August 2000; accepted in final form 26 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aharonovitz, O, Aboulafia-Etzion S, Leor J, Battler A, and Granot Y. Stimulation of 42/44 kDa mitogen-activated protein kinases by arginine vasopressin in rat cardiomyocytes. Biochim Biophys Acta 1401: 105-111, 1998[ISI][Medline].

2.   Alessi, DR, Kozlowski MT, Weng QP, Morrice N, and Avruch J. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol 8: 69-81, 1998[ISI][Medline].

3.   Ausiello, DA, Kreisberg JI, and Roy C. Contraction of cultured rat glomerular cells of apparent mesangial origin after stimulation with angiotensin II and arginine vasopressin. J Clin Invest 65: 754-760, 1980[ISI][Medline].

4.   Bonventre, JV, Skorecki KL, Kreisberg JI, and Cheung JY. Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 251: F94-F102, 1986[ISI][Medline].

5.   Brown, EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, and Schreiber SL. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369: 756-758, 1994[ISI][Medline].

6.   Burgering, BMT, and Coffer P. Protein kinase B (c-Akt) in phosphatidylinositol 3-kinase signal transduction. Nature 376: 599-602, 1995[ISI][Medline].

7.   Cantley, LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, and Soltoff S. Oncogenes and signal transduction. Cell 64: 281-302, 1991[ISI][Medline].

8.   Carmichael, MC, and Kumar R. Molecular biology of vasopressin receptors. Semin Nephrol 14: 341-348, 1994[ISI][Medline].

9.   Carpenter, CL, Auger K, Duckworth B, Hou WM, Schaffhausen B, and Cantley LC. A tightly regulated serine/threonine protein kinase regulates phosphatidylinositol 3-kinase activity. Mol Cell Biol 13: 1657-1665, 1993[Abstract].

10.   Choudhury, GG, Karamitsos C, Hernandez J, Gentilini A, Bardgette J, and Abboud HE. PI-3-kinase and MAPK regulate mesangial cell proliferation and migration in response to PDGF. Am J Physiol Renal Physiol 273: F931-F938, 1997[ISI][Medline].

11.   Conus, NM, Hemmings BA, and Pearson RB. Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k. J Biol Chem 273: 4776-4782, 1998[Abstract/Free Full Text].

12.   Daub, H, Wallasch C, Lankenau A, Herrlich A, and Ullrich A. Signal characteristics of G protein-transactivated EGF receptor. EMBO J 16: 7032-7044, 1997[Abstract/Free Full Text].

13.   Dhang, R, Hiles I, and Pahayatou G. Phosphatidylinositol 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity. EMBO J 13: 522-533, 1994[Abstract].

14.   Dikic, I, Tokiwa G, Lev S, Courtneidge SA, and Schlessinger J. A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature 383: 547-550, 1996[ISI][Medline].

15.   Downward, J. Cell cycle: routine role for Ras. Curr Biol 7: R258-R260, 1997[ISI][Medline].

16.   Duff, JL, Marrero MB, Paxton WG, Schieffer B, Bernstein KE, and Berk BC. Angiotensin II signal transduction and the mitogen-activated protein kinase pathway. Cardiovasc Res 30: 511-517, 1995[ISI][Medline].

17.   Dufner, A, and Thomas G. Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res 253: 100-109, 1999[ISI][Medline].

18.   Eguchi, S, Iwasaki H, Inagami T, Numaguchi K, Yamakawa T, Motley ED, Owada KM, Marumo F, and Hirata Y. Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells. Hypertension 33: 201-206, 1999[Abstract/Free Full Text].

19.   Eguchi, S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, and Inagami T. Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 273: 8890-8896, 1998[Abstract/Free Full Text].

20.   Force, T, Kyriakis JM, Avruch J, and Bonventre JV. Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells. J Biol Chem 266: 6650-6656, 1991[Abstract/Free Full Text].

21.   Foschi, M, Chari S, Dunn MJ, and Sorokin A. A biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase. EMBO J 16: 6439-6451, 1997[Abstract/Free Full Text].

22.   Ganz, MB, Pekar SK, Perfetto MC, and Sterzel RB. Arginine vasopressin promotes growth of rat glomerular mesangial cells in culture. Am J Physiol Renal Fluid Electrolyte Physiol 255: F898-F906, 1988[Abstract/Free Full Text].

23.   Ganz, MB, Perfetto MC, and Boron WF. Effects of mitogens and other agents on rat mesangial cell proliferation, pH, and Ca2+. Am J Physiol Renal Fluid Electrolyte Physiol 259: F269-F278, 1990[Abstract/Free Full Text].

24.   Grana, X, and Reddy EP. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin dependent kinase inhibitors (CKIs). Oncogene 11: 211-219, 1995[ISI][Medline].

25.   Granot, Y, Erikson E, Freidman H, Van Putten V, Williams B, Schrier RW, and Maller JL. Direct evidence for tyrosine and threonine phosphorylation and activation of mitogen-activated protein kinase by vasopressin in cultured rat vascular smooth muscle cells. J Biol Chem 268: 564-569, 1993.

26.   Grewe, M, Gansauge F, and Schmid RM. Regulation of cell growth and cyclin D1 expression by the constitutively active FRAP-p70S6 kinase pathway in human pancreatic cancer cells. Cancer Res 59: 3581-3587, 1999[Abstract/Free Full Text].

27.   Jard, S, Lombard J, Marie J, and Devilliers G. Vasopressin receptors from cultured mesangial cells resemble V1a type. Am J Physiol Renal Fluid Electrolyte Physiol 253: F41-F49, 1987[Abstract/Free Full Text].

28.   Johnson, GL, and Vaillancourt RR. Sequential protein kinase reactions controlling cell growth and differentiation. Curr Opin Cell Biol 6: 230-238, 1994[ISI][Medline].

29.   Johnson, RJ. The glomerular response to injury: progression or resolution? Kidney Int 45: 1769-1782, 1994[ISI][Medline].

30.   Klippel, A, Escobedo MA, and Wachowicz MS. Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation. Mol Cell Biol 18: 5699-5711, 1998[Abstract/Free Full Text].

31.   Kolch, W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 351: 289-305, 2000[ISI][Medline].

32.   Kreisberg, JI, and Wilson PW. Renal cell culture. J Electron Microsc (Tokyo) 9: 238-263, 1988.

33.   Kurihara, I, Saito T, Obara K, Shoji Y, Hirai M, Soma J, Sato H, Imai Y, and Abe K. Effect of a nonpeptide vasopressin V1 antagonist (OPC-21268) on experimental accelerated focal glomerulosclerosis. Nephron 73: 629-636, 1996[ISI][Medline].

34.   Lopez-Ilasaca, M. Signaling from G-protein-coupled receptors to mitogen-activated protein (MAP)-kinase cascades. Biochem Pharmacol 56: 269-277, 1998[ISI][Medline].

35.   Nave, BT, Ouwens M, Withers DJ, Alessi DR, and Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 344: 427-431, 1999[ISI][Medline].

36.   Otsuka, F, Ogura T, Yamauchi T, Oishi T, Hashimoto M, Mimura Y, and Makino H. Effects of OPC-21268, a vasopressin V1-receptor antagonist, on expression of growth factors from glomeruli in spontaneously hypertensive rats. Regul Pept 72: 87-95, 1997[ISI][Medline].

37.   Prenzel, N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, and Ullrich A. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 402: 884-888, 1999[ISI][Medline].

38.   Rodriguez-Viciana, P, Warne PH, Dhand R, Vanhaesebroeck B, Gout I, Fry MJ, Waterfield MD, and Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370: 527-532, 1994[ISI][Medline].

39.   Roovers, K, and Assoian RK. Integrating the MAP kinase signal into the G1 phase cell cycle machinery. Bioessays 22: 818-826, 2000[ISI][Medline].

40.   Sargent, JM, and Taylor CG. Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukemia. Br J Cancer 60: 206-210, 1989[ISI][Medline].

41.   Saward, L, and Zahradka P. Angiotensin II activates phosphatidylinositol 3-kinase in vascular smooth muscle cells. Circ Res 81: 249-257, 1997[Abstract/Free Full Text].

42.   Schulze-Lohoff, E, Kohler M, Fees H, Reindl N, and Sterzel RB. Divergent effects of arginine vasopressin and angiotensin II on proliferation and expression of the immediate early genes c-fos, c-jun and Egr-1 in cultured rat glomerular mesangial cells. J Hypertens 11: 127-134, 1993[ISI][Medline].

43.   Seger, R, and Krebs EG. The MAPK signaling cascade. FASEB J 9: 726-735, 1995[Abstract/Free Full Text].

44.   Shankland, SJ, Hugo C, and Coats J. Changes in cell-cycle protein expression during experimental mesangial proliferative glomerulonephritis. Kidney Int 50: 1230-1239, 1996[ISI][Medline].

45.   Sjoholm, A, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. Rapid Ca2+ influx and diacylglycerol synthesis in growth hormone-mediated islet beta-cell mitogenesis. J Biol Chem 275: 21033-21040, 2000[Abstract/Free Full Text].

46.   Troyer, DA, Gonzalez OF, Douglas JG, and Kreisberg JI. Phorbol ester inhibits arginine vasopressin activation of phospholipase C and promotes contraction of, and prostaglandin production by, cultured mesangial cells. Biochem J 251: 907-912, 1988[ISI][Medline].

47.   Troyer, DA, Gonzalez OF, Padilla RM, and Kreisberg JI. Vasopressin and phorbol ester-stimulated phosphatidylcholine metabolism in mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 262: F185-F191, 1992[Abstract/Free Full Text].

48.   Troyer, DA, Kreisberg JI, Schwertz DW, and Venkatachalam MA. Effects of vasopressin on phosphoinositides and prostaglandin production in cultured mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 249: F139-F147, 1985[ISI][Medline].

49.   Tsuboi, Y, Shankland S, Grande JP, Walker HJ, Johnson RJ, and Dousa TP. Suppression of mesangial proliferative glomerulonephritis development in rats by inhibitors of cAMP phosphodiesterase isozymes types III and IV. J Clin Invest 98: 262-270, 1996[Abstract/Free Full Text].

50.   Ullrich, A, and Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell 61: 203-212, 1990[ISI][Medline].

51.   Van der Geer, P, Hunter T, and Lindberg RA. Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 10: 251-337, 1994[ISI].

52.   Venkatachalam, MA, and Kreisberg JI. Agonist-induced isotonic contraction of cultured mesangial cells after multiple passage. Am J Physiol Cell Physiol 249: C48-C55, 1985[Abstract/Free Full Text].

53.   Xu, YJ, Ouk Kim S, Liao DF, Katz S, and Pelech SL. Stimulation of 90- and 70-kDa ribosomal protein S6 kinases by arginine vasopressin and lysophosphatidic acid in rat cardiomyocytes. Biochem Pharmacol 59: 1163-1171, 2000[ISI][Medline].

54.   Zachary, I, Woll PJ, and Rozengurt E. A role for neuropeptides in the control of cell proliferation. Dev Biol 124: 295-308, 1987[ISI][Medline].

55.   Zwick, E, Hackel PO, Prenzel N, and Ullrich A. The EGF receptor as central transducer of heterologous signaling systems. Trends Pharmacol Sci 20: 408-412, 1999[ISI][Medline].


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