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
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ABSTRACT |
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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
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INTRODUCTION |
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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-R), 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.
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MATERIALS AND METHODS |
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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-R, 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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RESULTS |
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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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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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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-R 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-R
(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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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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DISCUSSION |
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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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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 TGF) 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.
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ACKNOWLEDGEMENTS |
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We to thank Dr. C. Alex McMahan for the enormous help with the statistical analysis and Charles A. Thomas for help with FACS analysis.
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FOOTNOTES |
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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.
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