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Annals of Clinical & Laboratory Science 34:277-286 (2004)
© 2004 Association of Clinical Scientists

Review

Angiotensin II Regulation of TGF-ß in Murine Mesangial Cells Involves Both PI3 Kinase and MAP Kinase

Alan Perlman1,3, Loredo M. Lawsin1, Padmaja Kolachana1, Motoyasu Saji1,2, Jack Moore, Jr.1 and Mathew D. Ringel1,2
1 Department of Internal Medicine, Washington Hospital Center, and Medstar Research Institute, Washington, DC; 2 Divisions of Endocrinology and Oncology, Ohio State University, Columbus, OH; and 3 Division of Nephrology and Hypertension, Weil Medical College of Cornell University, New York, NY.

Address correspondence to Alan Perlman, M.D., The Rogosin Institute, Weil Medical College of Cornell University, 505 East 70th Street, New York, NY 10021, USA; tel 212 746 1580; fax 212 746 8439; e-mail perlmaa{at}mail.rockefeller.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Introduction: Chronic activation of the angiotensin II (AngII) type 1 receptor (AT-1) is a central event in the development of chronic kidney disease (CKD), in part through enhanced expression of TGF-ß, and AT-1 receptor blockade inhibits the progression to CKD in a variety of disease states. The AT-1 receptor is a heptahelical Gaq/11-coupled receptor that initiates phospholipase C activity and release of intracellular calcium; recent data suggest that the AT-1 receptor can also activate the epidermal growth factor receptor (EGFR), although the roles of specific EGF-mediated signaling cascades in AT-1 effects on mesangial cell biology are uncertain. We hypothesized that 2 EGFR-activated pathways, PI3 kinase and MAP kinase, are stimulated by the AT-1 receptor and, in part, regulate the effects of AngII on TGF-ß1 levels in mesangial cells. Methods: We examined the effects of AT-1 receptor activation on EGFR, PI3 kinase, and MAP kinase activation in murine mesangial cells. Upon achieving 60–80% confluence, the medium was changed to low-serum for 48 hr and cells were exposed to either the AT-1 receptor blocker, losartan, the EGFR blocker, AG1478, or control medium, and then stimulated with AngII. Similar experiments were performed using LY294002 and U0126, specific inhibitors of PI3 kinase and MEK, respectively. Total cellular protein lysates and RNA were isolated. Activation of the receptors and pathways was evaluated by immunoblotting and levels of TGF-ß mRNA were measured using real-time quantitative RT-PCR. Results: AngII induced autophosphorylation of EGFR (pY1068) and activated Akt and ERK, downstream targets of PI3 kinase and MAP kinase, respectively. AngII-mediated EGFR autophosphorylation was inhibited by losartan and AG1478. AG1478 also inhibited both basal and AngII-mediated activation of Akt and ERK. Finally, AngII-mediated increase in TGF-ß mRNA was inhibited by losartan, AG1478, LY249002, and U0126. Conclusions: Stimulation of the AT-1 receptor in murine mesangial cells results in activation of the EGF receptor with subsequent signaling through PI3 kinase and MAP kinase, thereby regulating TGF-ß mRNA levels. These data suggest that AT-1 receptor signaling pathways through EGFR may serve as a therapeutic target to inhibit the development of CKD.

(received 4 April 2004; accepted 1 May 2004)

Keywords: angiotensin II type 1 receptor, TGF-ß, Akt, ERK, losartan


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic kidney disease (CKD) is an increasingly prevalent condition with current studies suggesting that it affects more than 20 million persons in the USA [1,2]. As its prevalence increases, the development of targeted therapy to retard its progression to end-stage renal disease (ESRD) is urgently needed. To accomplish this goal, it is necessary to clarify the mechanisms that are responsible for the development and progression of CKD.

The underlying pathology of virtually all forms of CKD, irrespective of the underlying etiology, is glomerulosclerosis (GS), characterized by obsolescence of glomeruli and their associated tubulo-interstitial areas. Eventually, viable nephron mass is insufficient to maintain adequate renal function and renal replacement therapy is required. Two decades ago, Hostetter et al [3] proposed that intraglomerular hypertension and glomerular hyper-filtration resulted from renal injury (as in diabetes, hypertension, or chronic allograft nephropathy (CAN)); Brenner and associates [3–5] suggested that such hyperfiltration accelerated the rate of GS, nephron loss, and renal death. Angiotensin II (Ang II) plays a central pathogenic role, as it is a principal mediator of hyperfiltration. Supporting evidence for the role of AngII comes from an overwhelming number of laboratory and clinical studies that have demonstrated the salutary role of AngII interruption or inhibition using pharmacologic agents to arrest or attenuate the progression of CKD [6–8].

More recent data have suggested that chronic exposure to AngII may also result in detrimental effects on the kidney through cellular mechanisms that stimulate mesangial cell proliferation and fibrotic factors, irrespective of hemodynamic alterations. It has been clearly demonstrated that AngII stimulates gene transcription and protein release of transforming growth factor beta (TGF-ß). TGF-ß, a pro-fibrotic molecule, has therefore been implicated in the initiation and promulgation of fibrosis seen in diabetic nephropathy, chronic allograft nephropathy, and in experimental models of immune complex glomerulonephritis [9] and following subtotal nephrectomy [10]. To strengthen the connection of AngII and TGF-ß with renal fibrosis, TGF-ß has been shown to increase the synthesis of extracellular matrix proteins, including fibronectin, biglycan, collagen type 1 [11,12], and collagen type 4 [13]. TGF-ß has also been shown to diminish the degradation of extracellular matrix proteins both by decreasing matrix metalloproteinase 2 levels and by increasing levels of tissue inhibitor of metalloproteinase 2 (TIMP2) [14] and plasminogen activator inhibitor-1 (PAI-1) [15]. PAI-1 inhibits the activity of plasminogen, a proenzyme of the matrix-degrading protein plasmin. AngII treatment of mesangial cells in culture has been demonstrated to induce PAI-1 gene transcription and increase PAI-1 mRNA and protein levels [16].

It has long been recognized that many of the downstream effects of the angiotensin II type 1 (AT-1) receptor occur through protein kinase C activation and intracellular calcium release via G protein signaling. However, Daub et al [17] reported that the EGF-receptor could be activated by G-protein coupled receptors (GPCRs) in rat-1 cells. Further work has demonstrated that the G-protein coupled AT-1 receptor is capable of activating the EGF receptor in NIH3T3 fibroblasts [18], hepatocytes [19], vascular smooth muscle cells through uncertain mechanisms [20–22], and mesangial cells [23–25]. In mesangial cells, both arginine vasopressin [23] and AngII have been shown to activate the EGF receptor, either by direct interactions between the receptors or by release of heparin-binding EGF and autocrine activation of the EGF receptor [23–25].

The EGF receptor initiates signaling through a number of intracellular pathways, principally the MAP kinase and PI3 kinase cascades. While a role for MAP kinase activity has been suggested in AT-1/EGF receptor-mediated effects on mesangial cells [24], the roles of PI3 kinase and its principal downstream effector molecule, Akt (protein kinase B), have not been assessed. The PI3 kinase/Akt pathway is a central signaling cascade whose activation leads to cellular proliferation, anti-apoptotic responses, enhanced metabolism, and regulation of gene transcription [26]. Moreover, Akt is a central regulatory factor for transcription and release of MMPs [27] and TGF-ß [28 29] in non-mesangial cell models, suggesting that this pathway may be important in regulating AngII-mediated effects on key proteins involved in CKD expression by mesangial cells.

Thus, in the present study, we evaluated a continuous cultured model of murine mesangial cells for AngII activation of PI3 kinase and for subsequent regulation of TGF-ß mRNA levels. We demonstrate that AngII-mediated transactivation of the EGF receptor results in activation of both MAP kinase and PI3 kinase in mesangial cells, and that both pathways are involved in the regulation of TGF-ßmRNA levels. In these cells, pharmacologic blockade of both MAP kinase and PI3 kinase pathways results in the inhibition of TGF-ß mRNA expression. Thus, in this study, we define a novel role for PI3 kinase signaling in AT-1-mediated cellular effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.  A mouse mesangial cell line (ATCC CRL-1927) was obtained from American Type Culture Collection (Manassas, VA). DMEM (glucose-, glutamine-, and phenol red-free) and Coon’s modified F-12 media were obtained from Sigma Chemical Co (St. Louis, MO). Fetal bovine serum (FCS), TRIzol reagent, glutamine, and the reagents and nitrocellulose membranes for immunoblotting were obtained from Invitrogen Corp (Carlsbad, CA). AngII, AG1478, and okaidic acid were purchased from Calbiochem-Novabiochem (San Diego, CA). LY294002, U0126, PD98059, rapamycin, and antibodies to total Akt, phospho-Akt (ser 473), total p44/42 MAP kinase (ERK), phospho-ERK, and phospho-EGF receptor (pY1068) were obtained from Cell Signaling Technology (Beverly, MA). EGF receptor antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Leupeptin, pepstatin, and 4-amidino-phenylmethane-sulfonyl fluoride (APMSF) were from Roche Molecular Biochemicals (Indianapolis, IN). The BCA protein assay kit and SuperSignal West Pico chemiluminescence substrate were from Pierce Biotechnology (Rockford, IL). RNeasy mini kit was purchased from Qiagen, Inc, (Valencia, CA). Losartan was a generous gift from Dupont-Merck Pharmaceuticals (Wilmington, DE). Other chemicals were purchased from Sigma Chemical Co unless otherwise specified.

Preparation of cellular protein extracts.  To determine the effects of AngII upon the EGF receptor and its downstream effectors, mesangial cells were propagated in 10 cm dishes in DMEM/ F-12 (3:1) medium containing 2 mM glutamine and 5% FCS in a humidified 5% carbon dioxide incubator. Upon 60–80% confluence, the cells were washed with Dulbecco’s phosphate buffered saline (PBS) and cultivated in 10 ml of experimental medium containing no glucose, no glutamine, and 0.2% FCS. After 24 hr, the medium was replaced with 3.6 ml of pre-warmed fresh experimental medium 1.5 to 2 hr before the experiments, and the EGF receptor blocker, 250 nM AG1478, or the AT-1 receptor blocker, 10 µM losartan, was added to experimental medium 1 hr prior to the addition of 1 µM AngII. To stop reactions, cells were washed with ice-cold DPBS and immediately lysed with the addition of RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 5 mM sodium vanadate, 1 µg/ml leupeptin, 20 µM APMSF, 0.1% SDS, 1% IPGAL CA-630, 1 µg/ml pepstatin, and 0.3 µM okadaic acid). The cells were then scraped and homogenized with a #20–21 gauge needle and 3 ml syringe. The cells were centrifuged at 12,000 x g for 10 min at 4°C. The supernatant was transferred into fresh tubes and stored at –80°C. The protein concentrations of cell lysates were measured using a micro BCA protein assay reagent kit (Pierce Chemical Co). Initial dose- and time-response curves were obtained for all blocking and stimulation experiments (data not shown). In all experiments, the blocking reagents were dissolved in 0.1% DMSO as recommended by the manufacturer. The control experiments therefore contain 0.1% DMSO without the blocking reagents.

Immunoblotting.  Twenty µg of total protein lysate was suspended in reduced SDS sample buffer and boiled for 5 min. Protein lysates were subjected to SDS-PAGE (6% or 4–20%), and separated proteins were transferred to nitrocellulose membranes (0.45 µm pore size, Invitrogen) by electrophoretic blotting (Invitrogen). Nonspecific binding was prevented by blocking the membrane with Tris buffered saline (TBS)-T (0.1% Tween 20 in 20 mM Tris-HCl, pH 7.6, and 137 mM NaCl) containing 5% nonfat dry milk overnight at 4°C. Immunoblotting was performed as follows: Membranes were washed 4 times (15 min/wash) with TBS-T and incubated with the primary antibody in TBS-T buffer containing 5% nonfat dry milk for 2 hr at room temperature (RT). After washing 4 times (15 min/wash) with TBS-T, membranes were then incubated for 1 hr with the secondary antibody conjugated with peroxidase in TBS-T containing 5% nonfat dry milk at RT. After washing with TBS-T 4 times (15 min/wash), immunodetection was performed with the SuperSignal West Pico staining kit (Pierce). The stained membranes were exposed to CL-Xposure films (Pierce) using a series of exposure times to obtain the optimal image and were developed with a standard X-ray film developer.

RNA isolation and real-time quantitative RT-PCR.  Mesangial cells were cultured in growth medium in 10 cm dishes. Upon 60–80% confluence, cells were washed with PBS and further cultivated in 10 ml of experimental medium. After 24 hr, the medium was replaced with 10 ml of fresh experimental medium and AngII was added. In blocking experiments, 10 nM losartan, 250 nM AG1478, 20 µM LY249002, or 2 µM U0126, was added 1 hr before the addition of AngII. After 24, 48, and 72 hr, the medium was aspirated and RNA was isolated with RNeasy mini kit (Qiagen). The RNA concentration was measured by spectrophotometry at 260 nm.

Intron-spanning TGF-ß1 oligonucleotide primers and internal Taqman probes were designed using Primer Express (Applied Biosystems, Foster City, CA). The sequences are shown below and were confirmed to be sequence-specific by BLAST search. Probes were fluorescently labeled with FAM.

Forward primer : 5'-ACT GGA GTT GTA CGG CAG TGG-3'

Reverse primer : 5'-GCA GTG AGC GCT GAA TCG A-3'

Probe : 5'-FAM-TGA ACC AAG GAG ACG GAA TAC AGG GCT-TAMRA –3'

Two hundred ng of total RNA for each sample was reverse-transcribed in a 20 µl reaction using 0.75 U/µl Moloney murine leukemia virus reverse transcriptase and reverse transcriptase buffer containing 5.5 mM MgCl2, 500 µM each dNTPs, 2.5 µM random hexamers, and 0.4 U/µl RNase inhibitor. Quantitative PCR was performed in 96-sample plates. cDNA equivalent of 50 ng total RNA (5 µl of RT reaction mixture) per 25 µl tube containing TaqMan PCR Universal Master Mix (Applied Biosystems), 100 nM probe, and 200 nM of each TGF-ß1 primer were used. As a control for RNA integrity and for assay normalization, 18S ribosomal RNA was amplified with a TaqMan ribosomal RNA control reagents kit (Applied Biosystems) using cDNA equivalent of 0.25 ng total RNA, 40 nM 18S probe, and 20 nM 18S primers in 25 µl tube.

PCR was performed in the following manner: Following an initial 10 min denaturation at 95°C, samples were subjected to 40 cycles of a 2-step amplification protocol that included 15 sec of denaturation at 95°C and 60 sec of an annealing-elongation step, using the standard protocol of the manufacturer. TGF-ß1 and 18S were amplified from all samples in duplicate in 3 separate reactions. Interassay variability was <5%. Negative controls were included for the entire RT-PCR and for the PCR alone in each reaction.

Normalized results for TGF-ß1 were calculated using the mean threshold cycle (C{tau} Akt) of all reactions for each sample and the mean threshold cycle of 18S (C{tau}18S) amplification for each sample by calculating 2–(C{tau} Akt – C{tau} 18S) as recommended by the manufacturer (Applied Biosystems). Several samples were run by electrophoreses through agarose gels and all showed a single unique band at the expected size location for each amplicon.

Statistical Analyses.  The presented experiments are representative of immunoblots performed on protein extracted from at least 3 independent experiments. Quantitative RT-PCR was performed in duplicate on 3 occasions. Statistical analyses by ANOVA were performed using StatView (Abacus Concept, Berkeley, CA). When ANOVA exhibited significance, groups were compared using a post hoc Fisher PDSL test. Significance was set at p <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGF receptor is phosphorylated by AngII.  AngII transactivates the EGF receptor via the AngII receptor in vascular smooth muscle cells [17–19]. Therefore, we examined the levels of EGF receptor expression and activation at basal conditions and following AngII stimulation by Western blotting using antibodies that recognize total EGF receptor and phosphorylated EGF-R at tyrosine 1068, a site that is required for binding to GRB2 and GAB1, leading to MAP kinase and PI3 kinase, respectively, and that correlates with EGF receptor activity [30, 31]. Minimal autophosphorylation of EGF receptor at tyrosine 1068 was detected in basal conditions, in the absence of AngII. AngII incubation increased the level of phosphorylated EGF-R at 1 min (data not shown), with maximal stimulation of the EGF receptor between 5 and 15 min (Figs. 1AGo,1BGo).



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  		Fig. 1. Losartan inhibits AngII-induced phosphorylation of EGFR. Mesangial cells cultured in medium containing 0.2% serum for 1 day were exposed to either AG 1478 250 nM, Losartan 10 µM, or additional experimental medium containing 0.1% DMSO (for control) 1 hr prior to the addition of AngII (1 µM). At times 0, 5, and 15 min, the reactions were terminated, cellular proteins were collected, and immunoblots were performed using specific antibodies against autophosphorylated (pY1068) and total EGF receptor. Panel A: The level of phosphorylated EGFR was increased by Ang I and this effect was inhibited by both the EGFR blocker (AG 1478) and the AT-1 blocker (losartan). Panel B: After scanning, the bands were quantified and the ratios of pTyr1068 EGFR/tEGFR were calculated. Results are shown relative to the appropriate control lane for each experiment (0 min). Results are typical of experiments performed on 3 separate occasions.

 
Losartan inhibits AngII-induced phosphorylation of the EGFR.  To test the hypothesis that trans-activation of EGF-R occurs following stimulation of the AT-1 receptor, mesangial cells were exposed to the angiotensin receptor blocker, losartan, or the EGF receptor blocker, AG1478, 1 hr prior to addition of AngII into the medium. AG1478 inhibited EGF receptor phosphorylation under both basal and stimulated conditions. Under identical conditions, losartan also inhibited EGF receptor phosphorylation to levels comparable to those seen in the presence of AG1478. (Figs. 1AGo,1BGo) These findings give further support for transactivation from the AT-1 receptor to the EGF receptor.

Losartan and AG1478 inhibit AngII-induced Akt and ERK phosphorylation.  We examined whether AngII stimulation resulted in activation of PI3 kinase and MAP kinase pathways by immunoblotting for phosphorylation (activation) of Akt and ERK, respectively. Phosphorylation of both ERK and Akt was demonstrated after 15 min of AngII stimulation (Fig. 2Go). Treatment with AG1478 completely inhibited the phosphorylation of Akt and ERK basally and following stimulation by AngII (Fig. 2Go), indicating that EGF receptor inhibition dramatically inhibits Akt activation basally, and blocks the ability of AngII to activate Akt. Treatment with losartan showed similar effects (data not shown), confirming that the effect was mediated by the AT-1 receptor.



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  		Fig. 2. AngII-induced phosphorylation of Akt and ERK is EGF receptor-dependent. Panel A: After mesangial cells were cultured in the absence of serum for 1 day, 100 nM AngII was added to the medium. The EGF receptor inhibitor, AG1478 (250 nM), or 0.1% DMSO alone (control), was added 30 min before the addition of AngII. After 15 min, cellular protein was collected and total and phosphorylated (activated) Akt and ERK levels were measured by immunoblotting. AG1478 treatment completely eliminated phosphorylation of Akt and ERK in both basal and AngII-stimulated cells. Panel B: Similar experiments demonstrate that LY294002 and PD98059 (specific MEK inhibitor) block the ability of angiotensin II to activate Akt or ERK, demonstrating that angiotensin II-mediated activation of Akt and Erk occur via PI3 kinase and MEK, respectively.

 
AngII stimulation of TGF-ß1 mRNA levels is mediated by PI3 kinase and MAP kinase via the EGF-receptor.  Mouse mesangial cells constitutively expressed TGF-ß1 mRNA. AngII treatment increased the TGF-ß expression level in a dose-dependent fashion after 48 hr (Fig. 3Go). Losartan and Ag1478 both inhibited AngII-mediated TGF-ß1 mRNA levels (p <0.005 and <0.001, respectively). Because PI3K/Akt and ERK pathways were stimulated by AngII, we examined the effects of pharmacological inhibitors for both pathways. The PI3 kinase inhibitor, LY249002, decreased AngII-induced TGF-ß1 expression to control level (p <0.001 vs AngII-stimulated mesangial cells). The ERK kinase inhibitor, U0126, also decreased Ang II-induced TGF-ß expression, although to lesser extent than LY 249002 (p <0.005 vs AngII-stimulated mesangial cells). These results demonstrate that the AngII effects on TGF-ß1 mRNA levels are, in part, dependent on ERF receptor activation, and on the activation of both PI3 kinase and MAP kinase.



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  		Fig. 3. AngII up-regulation of TGF-ß mRNA levels is dependent on PI3 kinase and MAP kinase and occurs via the EGF receptor. MES cells were seeded in growth medium. At 24 hr before the experiments, medium was changed to experimental medium. At day 0, fresh experimental medium was added to the culture and LY294002 (20 µM, LY), U0126 (10 µM, U), losartan (10 mM, Los), or AG 1478 (250 nM, AG) was added. After 1 hr, control medium or AngII (1 µM, Ang II) with 0.1% DMSO was added. Cells were harvested 48 hr later and RNA was isolated. TGF-ß1 mRNA levels were calculated using the following formula, in order to standarize all experiments: 100 x ([experiment medium]-[control medium])/([angiotensin II, 1 µ M]-[control medium]). *p <0.005, ** p <0.001 comparing AngII (1 µM) alone vs AngII (1 µM + blocker).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chronic kidney disease is a leading cause of morbidity and mortality in the United States. While initial gains in clinical outcome clearly occur with blood pressure control and other measures, recent data suggest a unique renoprotective effect for ACE inhibitors and angiotensin II receptor blockers [6–8]. These clinical data corroborate laboratory data that demonstrate a key role for angiotensin II receptor activation in the pathogenesis of GS in models of hypertensive, diabetic, and post-transplantation nephropathies. Experimentally, much research has focused on TGF-ß as the cytokine responsible for the fibrosis common to these forms of CKD, and it has been reported that angiotensin II enhances TGF-ß gene transcription and release.

Recent data have suggested that G-protein-coupled receptors activate classical growth factor receptors through intracellular cross-talk as well as induction of extracellular ligand activation [17–25]. With novel therapies designed to block growth factor signaling in clinical trials for other diseases, such as monoclonal antibodies and small molecule inhibitors [32], we deemed it important to characterize the potential roles these pathways might play in mesangial cell biology. In particular, we wished to determine if these pathways might be involved in the development and progression of GS and progressive CKD.

We initially demonstrated that AngII induced the phosphorylation (activation) of Akt and ERK, downstream effectors of PI3 kinase and MAP kinase pathways, respectively. While MAP kinase can clearly be activated by G-protein-coupled receptors via cross-talk [32], activation of both pathways suggested that growth factor receptor stimulation might be involved. Because transactivation of EGFR has been demonstrated in various cell systems, we focused on this pathway of activation [17–25].

AngII activation of EGFR was confirmed in experiments that showed that AngII incubation resulted in EGFR phosphorylation and that this activity was blocked by pretreatment with losartan. Autophosphorylation of the receptor at 1068 corresponds tightly with EGF receptor activity [30,31]. Additional functional support for this relationship was demonstrated by the ability of the EGF receptor blocker to inhibit both basal and AngII-stimulated activation of Akt and ERK at low doses. The exact mechanism of the transactivation is uncertain. PKC inhibition with staurosporine abrogated, in part, AngII-mediated EGF phosphorylation (data not shown). Other workers have demonstrated autocrine production of heparin binding-EGF by mesangial cells in a primary cultured system [24]. Thus, the mechanism of cross-talk between the 2 receptors is complex and may be somewhat cell system-specific.

Because a role for the PI3 kinase pathway in AngII-mediated cellular effects in mesangial cells has not been reported, we were interested in determining the relative roles of MAP kinase or PI3 kinase. Defining the critical pathways is particularly important since small molecule inhibitors of each pathway are in clinical trials. Distinct from prior studies in mesangial cells, we demonstrate that AngII-activated MAP kinase and PI3 kinase both regulate AngII-induced TGF-ß increased mRNA levels. The PI3 kinase pathway is a critical regulator of cell growth and cell motility, MMP production, protein synthesis, and a variety of other cellular processes [26,27]. Of particular interest in mesangial cells, regulation of TGF-ß gene transcription by Akt, a critical downstream regulator of the PI3 kinase pathway, has recently been shown to occur through regulation of forkhead transcription factors and SMAD 3 [28,29]. These pathways also may be involved in the regulation of TGF-ß mRNA levels by Akt in mesangial cells.

Stimulants other than AngII are likely to be involved in the basal activation of the EGF receptor, since mild activation was detected in the absence of serum or other growth factors. These data suggest that additional autocrine or paracrine mechanisms are involved and that they are also inhibited by the EGF receptor blocker. The particular stimuli for the paracrine/autocrine activation remain to be defined, but may be important in the regulation of TGF-ß release in mesangial cells.

Like other workers, we have evaluated a murine mesangial cell model. Distinct from these studies, we have utilized a continuous cultured model of SV40-transformed cells. Disadvantages of this model include the transformed nature of the cells. However, there are advantages to using this model rather than primary cultured murine mesangial cells, the most significant of which is the ability to study receptor tyrosine kinase pathways in the absence of high serum concentrations. This is particularly important for the PI3 kinase pathway, which is highly regulated by serum. This is emphasized by the requirement for PI3 kinase activation of 5% ser um concentration to demonstrate AngII-induced TGF-ß1 protein release in murine mesangial cells, while the mRNA levels are regulated in the absence of serum (data not shown).

If the present findings are confirmed in other cell lines and in vivo, the potential clinical implications of these results are significant. It has been shown in vivo that rat glomeruli stimulated by EGF have decreased single nephron plasma flow and glomerular filtration rate [33]. In addition, increased EGF receptor staining has been reported in patients with IgA mesangial proliferative lesions [34]. Thus, it may be possible that blockers of EGF or its downstream pathways can be used to improve renal hemodynamics, reduce the release of profibrotic factors, such as TGF-ß and PAI, and thereby reduce fibrosis in patients with renal disease. Additional work in a variety of models is needed to determine the clinical potential of EGF receptor inhibition in renal disease.

In conclusion, AngII, through stimulation of the AT-1 receptor, leads to transactivation of the EGF receptor, which then stimulates the MAP kinase and PI3 kinase pathways in murine mesangial cells (Fig. 4Go). Inhibition of this pathway reduces basal and AngII-stimulated activation of these pathways and decreases TGF-ß mRNA levels. Both the MAP kinase and PI3 kinase pathways regulate AngII-induction of TGF-ß mRNA levels. These data suggest that the angiotensin-EGFR-PI3K/ MAPK-TGF-ß pathway is important in the pathogenesis and/or progression of glomerulosclerosis.



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  		Fig. 4. Potential model of AngII-induced TGF-ß release in mesangial cells. AngII binds with the type I receptor and transactivates the EGF receptor through uncertain mechanisms. Once the EGF receptor is activated, both PI3 kinase/Akt kinase and MAP kinase pathways are activated, ultimately leading to increased TGF-ß1 mRNA levels.

 


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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