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Angiotensin II and Basic Fibroblast Growth Factor Mitogenic Pathways in Human Fetal Mesangial Cells

Pediatric Research volume 47, pages 614–621 (2000)Cite this article

Abstract

Angiotensin II (Ang II) and basic fibroblast growth factor (bFGF/FGF-2) play relevant roles in renal development. Since the signaling pathways modulating the mitogenic effects of Ang II and bFGF in human fetal mesangial cells (HFMc) are not clearly defined, we carried out experiments to determine whether they would exert their mitogenic effects by modulating the activity of the mitogen-activated protein kinases (MAPK) [extracellular signal-regulated kinase-2 (ERK-2)] and cAMP signaling pathways. In confluent HFMc, bFGF (20 ng/mL) induced a significant 4-fold increase in ERK-2 activity and [3H]-thymidine incorporation (6-fold). In contrast, under similar tissue culture conditions, Ang II (10−6 M) induced a more modest increase in ERK-2 activity (2-fold) and [3H]-thymidine incorporation (35 ± 4%). The mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD098059 (25 μM) almost completely abolished the bFGF-induced proliferation in HFMc but did not significantly affect Ang II proliferative effects. In the presence of the cAMP elevating agent isoproterenol, Ang II and bFGF induced opposite changes in cAMP accumulation and cell growth. Isoproterenol inhibited the basal and bFGF-induced proliferation of HFMc through a MEK-1/2–independent pathway that included the accumulation of cAMP. In contrast, isoproterenol increased Ang II mitogenic effects in correlation with a reduction in cAMP accumulation. We conclude that Ang II and bFGF modulate the proliferation of HFMc through the stimulation of different MEK-1/2–dependent and independent signaling pathways. Activation of MEK-1/2 is required but not sufficient for mitogenesis in HFMc. The accumulation of cAMP in HFMc counteracts the mitogenic effects of bFGF by a MEK-1/2–independent pathway.

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Main

Mesangial cells are specialized smooth muscle cells located inside the glomeruli and in the extraglomerular region in close proximity to the macula densa. They play relevant roles modulating glomerular hemodynamic and immunologic functions (1, 2). The proliferation of human mesangial cells plays an important role during renal development and in the pathogenesis of glomerular renal diseases (1, 2). Several vasoactive agents are known to modulate the growth of human mesangial cells. Among them, Ang II and bFGF/FGF-2 have received considerable attention because of their relevant hemodynamic and growth-promoting effects in the cardiovascular system and kidney (3–7). Unlike bFGF, which induces proliferation of all vascular and renal smooth muscle cell types (4–7), the growth-promoting effects of Ang II are modulated in a tissue-specific manner. For example, Ang II induces hypertrophy in cultured rat aortic smooth muscle cells (3) but stimulates the proliferation of rat type I mesenteric smooth muscle cells (7) and human mesangial (8) or vascular smooth muscle cells (9). Based on these studies, it has been postulated that the growth-promoting effects of Ang II are mediated by other growth factors in a tissue-specific manner (10).

The signal transduction pathways modulating Ang II proliferative effects in HFMc are not clearly defined. It has been suggested that the mitogenic effects of Ang II could be mediated by bFGF or platelet-derived growth factor (PDGF), which are classic activators of the MAPK (6, 11, 12). MAPK, also known as ERK, are serine/threonine kinases that are rapidly activated upon stimulation of a variety of cell surface receptors (13). They function by transmitting signals from the cell surface to the nucleus to regulate the expression of transcription factors such as c-Jun and c-Myc and modulate the process of cell growth and differentiation (14). The binding of growth factors to tyrosine kinase receptors facilitates the exchanges of guanosine diphosphates (GDP) for guanosine triphosphates (GTP)-bound p21ras (15). GTP-bound Ras represents the activated form of Ras, which in turn initiates the activation of a linear cascade of protein kinases defined sequentially as MAP kinase kinase kinase, referred to as MEKK (16), and MAP kinase kinase such as MEK-1 and MEK-2 (17), which ultimately activate MAPK (ERK). At least two independent pathways can lead to MAPK activation in vascular smooth muscle cells. The first one is triggered by activation of tyrosine kinase receptors and includes the protooncogene Ras and Raf and, subsequently, the direct upstream activator of MAPK, MAP kinase kinase or MEK (ERK) (11). The second pathway is stimulated by receptors coupled to heterotrimeric G-proteins and involves protein kinase C (18). In general, whereas receptor tyrosine kinase agonists activate MEK via the protooncogenes c-Ras and c-Raf, the signal through G-protein–coupled receptor agonists is predominately via protein kinase C to either c-Raf or MEK kinase, and these signals may converge again to activate MEK (11, 18). Thus, in a typical fashion, whereas bFGF and PDGF-BB are considered classic activators of the tyrosine kinase receptor pathways, Ang II exerts its growth-promoting effects through calcium/phospholipid-dependent mechanisms via receptors coupled to heterotrimeric G-proteins and phospholipase C and, therefore, represents a typical activator of the second pathway.

In addition to all these upstream regulators of MAPK activity, recent studies have shown that cAMP can also modulate MAPK activity in different cell types (19–24). Moreover, previous studies in vascular smooth muscle cells (25), mesangial cells (26), and renomedullary interstitial embryonic fibroblasts (26) have shown that Ang II or bFGF can modulate the accumulation of cAMP. Furthermore, other soluble factors that increase the accumulation of cAMP, i.e. isoproterenol, usually inhibit the growth of mesangial cells (27, 28). Thus, because the signaling pathways modulating the proliferative effects of Ang II and bFGF have not been examined in HFMc, we carried out this study to determine whether Ang II and bFGF would exert their mitogenic effects by modulating the activity of ERK-1/2 and cAMP in these cells. Our results confirm and extend earlier findings demonstrating that Ang II and bFGF modulate the proliferation of HFMc by stimulating different MEK-1/2 dependent and independent pathways.

METHODS

Materials.

[γ32P]ATP (3000 Ci/mM) (3.3 mM) was purchased from Dupont NEN, Boston, MA. Heat-inactivated FCS and PBS were purchased from GIBCO BRL (Grand Island, NY). 50% Dulbecco's modified Eagle's medium and 50% Ham's F12 were obtained from Biofluid (Rockville, MD). cAMP kits were obtained from Diagnostic Products Corporation (Los Angeles, CA). BCA protein assay kits were obtained from Pierce (Rockford, IL). bFGF was purchased from Bio-source International (Caramillo, CA). ERK-2 polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PD098059 was obtained from Calbiochem (La Jolla, CA). [3H]-Thymidine, 1 mCi/mL, was purchased from ICN Pharmaceutical (Irving, CA). Isoproterenol, 4-(3-butoxy-4-methoxy-benzyl), imidazolidin (cAMP phosphodiesterase inhibitor), myelin basic protein (MBP) were obtained from Sigma Chemical Co. (St. Louis, MO).

HFMc.

HFMc were isolated from two different fetal kidneys as we have previously described in detail (8). These experiments were approved by the Institutional Review Board of the Children's National Medical Center. Briefly, the kidney cortex was minced into 1-mm fragments and passed through graded series of metal sieves. The resulting material containing approximately >95% glomeruli by microscopic examination was incubated with collagenase (type IV, Sigma Chemical Co.) at 37°C. After centrifugation, pelleted glomeruli were resuspended in 50% Dulbecco's modified Eagle's medium, 50% Ham's F12 (Biofluid) containing 4.5 g/L glucose, 20% heat-inactivated FCS (GIBCO), 2 mM glutamate, 100 U/mL penicillin,10 μg/mL streptomycin, and 8 μM insulin (Sigma Chemical Co.) and plated on tissue culture dishes. After approximately 2 or 3 wk of culture, cells with mesangial cell morphology grew from the attached glomeruli. These cells were trypsinized, characterized, and expanded as we have previously described (8). Briefly, HFMc showed a polygonal shape, positive staining for F actin using Bodipy-phallacidin (Molecular Probes, Eugene, OR), lacked the close cell-cell contact typical of endothelial or epithelial cells, and formed ridges composed of multiple layers of cells surrounding regions of monolayers. HFMc did not stain positive with anti-cytokeratin or anti-human von Willebrand's factor antibodies (Dako, Copenhagen, Denmark) but did stain positive with an anti-myosin antibody (Zymed, San Francisco, CA). Finally, HFMc expressed a high number of Ang II type 1 receptors (Bmax of approximately 70 fmol/105 cells) and contracted in response to Ang II when seeded onto the surface of a silicone rubber substratum.

cAMP assays.

The culture medium was aspirated from the HFMc monolayers, and cells were washed three times with PBS. Cell monolayers were overlaid with PBS at room temperature. A cAMP phosphodiesterase inhibitor was added at a final concentration of 10 μM to all incubations to prevent the enzymatic degradation of cellular cAMP. Isoproterenol (10−5 M) with or without bFGF (20 ng/mL) or three doses of isoproterenol (10−9–10−5 M) with 10−6 M Ang II were added to the treatment groups. Cells were incubated for 10 min at 37°C in a CO2 incubator containing 95% air and 5% CO2. After the incubation for 10 min, 1.0 mL/plate aliquot samples were transferred to 12 × 75-mm polypropylene tubes and immediately placed in a 0°C ice bath. The cell monolayers and 1-mL aliquot samples were stored at −20°C for protein determination and cAMP analysis, respectively. Total cellular proteins per plate were determined using the BCA protein assay kit according to the manufacturer's instructions. The extracellular cAMP per well was quantified using Diagnostic Product Corporation kits according to the manufacturer's instructions.

MAPK (ERK-2) assays.

Confluent HFMc were serum-starved overnight and stimulated with different agents (20% FCS, 20 ng/mL bFGF,10−6 M Ang II, 10−5 M isoproterenol with or without bFGF or Ang II). HFMc were treated for 10 min because in preliminary time-course experiments, we found that these growth factors induced a peak activation of ERK-2 activity at approximately 10 min. After incubation, the culture medium was aspirated, and cells were washed with cold PBS and lysed in a buffer containing 20 mM HEPES; pH 7.5, 10 mM EGTA; 40 mM β-glycerophosphate; 1% NP-40; 2.5 mM MgCl2; 1 mM DTT; 2 mM sodium vanadate; 1 mM phenylmethylsulfonyl fluoride; 20 μg/mL aprotinin; and 20 μg/mL leupeptin. Cells were scraped, transferred to ependorf tubes, and centrifuged. The clarified supernatants were recovered, immunoprecipitated with ERK-2 antibody for 1 h at 4°C, and immunocomplexes were recovered using protein G sepharose for 15 min. Pellets were washed three times with PBS supplemented with 1% NP-40 and 2 mM sodium vanadate, once with 0.5 M LiCl in 100 mM, pH 7.5, and once in kinase reaction buffer containing 12.5 mM MOPS, pH 7.5; 12.5 mM β-glycerophosphate; 7.5 mM MgCl2; 0.5 mM EGTA; 0.5 mM sodium fluoride; and 0.5 mM sodium vanadate. The reactions were carried out in 30-mL volume kinase reaction buffer containing 1 μCi of [γ32P]ATP/reaction, 20 μM unlabeled ATP, and 1.5 mg/mL MBP at 30°C for 30 min. The reactions were terminated by the addition of 15 mL 5X Laemmli's buffer before samples were boiled and electrophoresed in 12% PAGE. Phosphorylated MBP was visualized by autoradiography and quantified by scanning densitometer. Experiments were done in the presence or absence of PD098059 (25 μM).

Cell proliferation.

Cell proliferation was measured by [3H]-thymidine incorporation studies and cell counts as previously described (8). For the [3H]-thymidine incorporation studies, cells were serum-starved for 36 h and then treated with each of the following agents: 20% FCS, 20 ng/mL bFGF, 10−6 M Ang II, 10−5 M isoproterenol with or without bFGF or Ang II, and 8-bromo-cAMP (10−3 M) alone. After incubation, 1 μCi/mL [3H]-thymidine was added to each well, and the incubation was continued for an additional 4 h. All incubations were terminated by aspirating the culture medium and doing sequential washes (three times) with cold PBS, followed by the addition of ice-cold 10% trichloride acetic acid (TCA) for 20 min at 4°C to precipitate proteins and nucleic acids and to remove unincorporated [3H]-thymidine. After washing the cells three times with ice-cold water, cells were solubilized with 1 mL of 0.5 M NaOH and 0.1% SDS and incubated at 37°C for 30 min. Upon solubilization, 0.5 mL/well aliquot samples were removed and transferred to scintillation vials, 10 mL of scintillation cocktail was added to each vial, and radioactivity was quantitated by liquid scintillation counter.

Statistical analysis.

Results are expressed as the mean ± SD of values obtained in triplicate from at least three different experiments. Difference between groups was compared by t test, and p values < 0.05 were considered significant. When more than two means were compared, significance was determined by 1-way ANOVA followed by multiple comparisons using the Student-Newman-Keuls test.

RESULTS

Effects of bFGF, Ang II, and isoproterenol on MAPK (ERK-2)activity.

As shown in Figure 1A, control HFMc cells treated with 20% FCS (positive control) showed a significant 8-fold induction of ERK-2 activity (Fig. 1, A and B ). bFGF was also a potent inducer of ERK-2 activity because it caused a 4-fold increase in ERK-2 activation, whereas Ang II and isoproterenol induced an average 2-fold increase in ERK-2 activity, respectively, when compared with control cells (negative control). These findings corroborate previous reports that demonstrated that Ang II activates MAPK in rat mesangial cells (11). The combined treatment of isoproterenol and bFGF did not induce synergistic effects on ERK-2 activity when compared with cells treated with bFGF alone (Fig. 1, A and B ).

Figure 1
figure 1

Effects of bFGF, Ang II, and isoproterenol on ERK-2 activity in HFMc. (A) Confluent HFMc were serum-starved overnight and subjected to one of the following treatments: PBS (negative control), 20% FCS (positive control), bFGF 20 ng/mL, Ang II 10−6 M, or isoproterenol 10−5 M, alone or in combination for 10 min as shown. After incubation, MAPK (ERK-2) assays were done as described in “Methods.” The results shown in (A) represent the mean ± SD values of three different experiments performed in triplicate each time. All groups showed a significant activation of ERK-2 activity when compared with control cells (p < 0.05). ★p < 0.005 when compared with control cells. ★★p < 0.05 when compared with all other groups. (B) and (C) show representative autoradiograms of the phosphorylated MBP extracted from HFMc in one experiment. These cells were treated as shown in (A) in the absence (B) or presence (C) of the MEK inhibitor PD098059 (25 μg/mL).

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[3H]-thymidine incorporation.

Our next objective was to determine whether activation of the MEK-1/2 pathway was required for the mitogenic effects of Ang II or bFGF. We found a positive correlation between the bFGF induction of MEK activation and the stimulation of [3H]-thymidine incorporation. More specifically, bFGF increased [3H]-thymidine incorporation in confluent HFMc by an average of 6-fold in three independent experiments (Fig. 2A), suggesting that bFGF induction of MEK activation is required for its proliferative activity. We then asked whether the proliferative effects of bFGF or Ang II could be impeded by MAPK (MEK) inhibition using PD098059. PD098059 treatment resulted in a significant decrease in bFGF-induced MAPK activation (Fig. 1, B and C ) and subsequent [3H]-thymidine incorporation (Fig. 2B). These findings suggest that the mitogenic effect of bFGF in HFMc requires a MAPK-dependent pathway. In contrast, Ang II induced a modest increase in [3H]-thymidine incorporation (35 ± 4%) in confluent HFMc (Fig. 2A). However, these effects were not significantly affected by PD098059 (Fig. 2B) despite the fact that PD098059 completely blocked Ang II–induced up-regulation of MEK activity (Fig. 1, B and C ). These findings suggest that the mitogenic effects of Ang II in HFMc are mediated through an MEK-independent pathway.

Figure 2
figure 2

Ang II and bFGF-induced [3H]-thymidine incorporation studies in HFMc. For the [3H]-thymidine incorporation studies, confluent HFMc were made quiescent in serum-free medium supplemented with 5 μg/mL insulin for 36 h and then treated with the following agents for 18 h: PBS (control), Ang II 10−6 M, bFGF 20 ng/mL. After 18 h, the cells were pulsed with [3H]-thymidine for an additional 4 h as described in “Methods.” The results are shown in (A) (in the absence of the MEK inhibitor PD098059) and (B) (in the presence of PD098059, 25 μg/mL) and represent the mean ± SD values of three different experiments performed in triplicate each time. ★p < 0.05 when compared with control group. ★★p < 0.005 when compared with all other groups.

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Cell counts.

The results of the [3H]-thymidine incorporation studies were correlated with the cell counts. When HFMc were seeded at a density of 1.5 × 104 cells/cm2, Ang II (10−6 M) induced a modest but significant increase in cell number (20 ± 4%) when compared with control cells (Ang II–treated cells: 15 856 ± 298 cells/cm2versus control cells: 12 678 ± 350 cells/cm2, mean ± SD values corresponding to two different experiments, p < 0.05). Under similar experimental conditions, bFGF (20 ng/mL) induced an approximately 3-fold increase in cell number (34 678 ± 478 cells/cm2, p < 0.005), and the combined treatment of Ang II and bFGF induced an additional increase in cell number (41 124 ± 289 cells/cm2) when compared with HFMc treated with bFGF alone (p < 0.05). The combined treatment of Ang II and bFGF, however, did not significantly increase the activity of ERK-2 when compared with HFMc treated with bFGF alone (average 4-fold increase induced by either Ang II and bFGF or bFGF alone when compared with control HFMc).

Effects of Ang II and bFGF on cAMP accumulation.

Because previous studies have shown that cAMP elevating agents inhibit the growth of cultured mesangial cells, we studied whether Ang II and bFGF would modulate the accumulation of cAMP in HFMc. These experiments were done in the presence or absence of the cAMP elevating agent isoproterenol. First, we tested the effects of Ang II and bFGF alone and found that only bFGF induced a modest increase in the basal levels of cAMP (36 ± 6%) when compared with control cells (Fig. 3). Moreover, the combined treatment of bFGF and isoproterenol induced a significant 2-fold increase in cAMP accumulation when compared with HFMc treated with isoproterenol alone (Fig. 3). In contrast, Ang II did not induce significant changes in the basal levels of cAMP but attenuated the isoproterenol-induced cAMP accumulation by 40 ± 8% (Fig. 4), corroborating the results of a previous study done in rat mesangial cells, which showed that Ang II attenuated the forskolin-induced accumulation of cAMP by 44% (29).

Figure 3
figure 3

Effects of Ang II, bFGF, and isoproterenol on cAMP accumulation in HFMc. Confluent HFMc were serum-starved overnight before treatment with PBS (control), Ang II 10−6 M, bFGF 20 ng/mL, and isoproterenol 10−5 M in combination with bFGF or alone for 10 min. cAMP assays were done as described in “Methods.” The results represent the mean ± SD values of three different experiments performed in triplicate each time. Basic bFGF but not Ang II induced a modest but significant increase in cAMP accumulation (p < 0.05) when compared with control cells. ★p < 0.005 when compared with control, Ang II, and bFGF groups. ★★p < 0.05 when compared with all other groups.

Full size image
Figure 4
figure 4

Modulation of isoproterenol-induced cAMP accumulation by Ang II in HFMc. Confluent HFMc were serum-starved overnight before treatment with Ang II (10−6 M) or different concentrations of isoproterenol for 10 min. Ang II decreased the accumulation of cAMP induced by isoproterenol. Results represent the mean ± SD values of three different experiments performed in triplicate each time. Differences between isoproterenol and isoproterenol with Ang II–treated cells at 0.1 and 10 μM isoproterenol concentrations were significantly different (p < 0.05).

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Isoproterenol, Ang II, and bFGF effects on mesangial cellgrowth.

To determine whether the changes in cAMP levels were associated with alterations in the growth pattern of HFMc, we studied whether the accumulation of cAMP induced by isoproterenol would modulate the mitogenic effects of Ang II and bFGF and determined whether these changes could be linked to the MEK pathway. HFMc cultured in parallel were treated and examined for both MAPK (ERK-2) activity and DNA synthesis. As shown in Figure 5, isoproterenol (10−5 M) alone decreased the [3H]-thymidine incorporation of HFMc when compared with control cells and blocked the bFGF-stimulated [3H]-thymidine incorporation with similar magnitude compared with PD098059 (data shown in Fig. 2B). However, isoproterenol did not significantly inhibit the bFGF-induced ERK-2 activation (Fig. 1, A and B ), suggesting that the inhibitory effects of isoproterenol should be mediated by an MEK-independent pathway and probably related to the accumulation of cAMP (Fig. 3). In support of this interpretation, 8-bromo-cAMP alone inhibited the growth of HFMc (Fig. 5). In contrast with the inhibitory effects of isoproterenol on bFGF-induced proliferation of HFMc, isoproterenol enhanced Ang II–induced [3H]-thymidine incorporation in HFMc. At the present time, however, we do not know the exact mechanism(s) by which isoproterenol potentiates Ang II mitogenic effects in HFMc.

Figure 5
figure 5

[3H]-Thymidine incorporation studies in HFMc treated with isoproterenol alone or in combination with Ang II or bFGF and Ang II, bFGF, or 8-bromo-cAMP alone. For the [3H]-thymidine incorporation studies, confluent HFMc were made quiescent in serum-free medium supplemented with 5 μg/mL insulin for 36 h and then treated with the following agents for 18 h: PBS (control), isoproterenol (10−5 M), isoproterenol (Iso) (10−5 M) with Ang II (10−6 M), Ang II (10−6 M) alone, bFGF (20 ng/mL) alone, bFGF (20 ng/mL) with isoproterenol (10−5 M), and 8-bromo-cAMP (10−3 M) alone. After 18 h, the cells were pulsed with [3H]-thymidine for an additional 4 h as described in “Methods.” The results represent the mean ± SD values of three different experiments performed in triplicate each time. ★p < 0.05 when compared with control group. ★★p < 0.005 when compared with all other groups.

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DISCUSSION

In the present study, we have shown that the mitogenic effects of Ang II and bFGF in HFMc are predominately mediated by MEK-dependent and independent signaling pathways. In addition, we have demonstrated for the first time that in the presence of isoproterenol, Ang II and bFGF induced opposite changes in cAMP accumulation and cell growth in HFMc. These findings, when interpreted in the context of previous studies (3–11), support the notion that the mitogenic effects of Ang II in HFMc are not predominately mediated by bFGF.

The mitogenic pathways modulating the growth-promoting effects of Ang II in HFMc are not completely understood. A previous study done in rat mesangial cells (11, 30) suggested that the different potency and duration of activation of the MAP kinase cascade induced by PDGF-BB and Ang II may explain why PDGF-BB is a potent mitogen for rat mesangial cells, whereas Ang II only induces hypertrophy in these cells. In the present study, we have extended the results of this study to HFMc and demonstrated that bFGF, a more potent activator of MAPK than Ang II, is also a more powerful mitogenic agent than Ang II. Moreover, we have shown that the mitogenic effects of bFGF on HFMc are predominately mediated through an MEK-dependent pathway, because the treatment of HFMc with bFGF in the presence of PD098059, an inhibitor of MEK activity, almost completely abolished the bFGF-induced MAPK activation and subsequent [3H]-thymidine incorporation in these cells. In contrast, the mitogenic effects of Ang II persisted in the presence of PD098059, indicating that they are not restricted to the MEK pathway. These findings are supported by a previous study done in vascular smooth muscle cells from hypertensive rats, which demonstrated that the full Ang II–induced mitogenic response in these cells required both MAPK in conjunction with phospholipase D or other signaling components (18, 31). In addition, recent studies done in cultured vascular smooth muscle cells have shown that Ang II can stimulate the Jak/STAT (32) and the c-Jun NH2 terminal protein kinase pathways (33). Thus, it is possible that other MEK-independent pathways may play a relevant role in this process.

In previous studies (8, 34), we have found that Ang II has more significant mitogenic effects in subconfluent or rapidly proliferating HFMc when compared with confluent mesangial cells. The reduced mitogenic response of Ang II in confluent HFMc is associated with the down-regulation of Ang II receptors in confluent HFMc (8). In the present study, we have confirmed that Ang II, unlike bFGF, is a very modest mitogenic growth factor in confluent HFMc. We tested the effects of both growth factors in HFMc seeded at high density because cultured confluent mesangial cells are most likely to represent the physiologic conditions of the glomerular mesangial cells in vivo. Thus, on the basis of these results, it is possible to speculate that bFGF and the MEK pathway may have the most relevant in vivo mitogenic role in glomerular mesangial cells. However, because bFGF lacks a signal peptide for secretion and is normally not released into the circulation, mesangial cells should not be exposed to bFGF under normal conditions. During renal development or in the presence of glomerular injury, bFGF will be released from the extracellular matrix, basement membranes, or injured cells (35). Thus, both bFGF and Ang II may have a synergistic proliferative effect during renal development and/or glomerular injury.

Another interesting finding of this study is that isoproterenol inhibited the bFGF-stimulated [3H]-thymidine incorporation with similar potency compared with the MEK inhibitor PD098059. To the best of our knowledge, this is the first study demonstrating that isoproterenol can inhibit bFGF-induced mitogenic effects in human mesangial cells. Isoproterenol is a β-adrenergic agonist that binds to receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) (22, 36). The binding of isoproterenol to its receptors promotes the exchange of GDP for GTP by the G-protein, causing the activation of the Gαs subunit of the G-protein (36). Additionally, the nucleotide exchange also facilitates the dissociation of the Gαs subunit from the G-protein, generating two protein molecules: Gαs and Gβγ subunits. The Gαs activates adenylyl cyclase (36), which further activates cAMP-dependent protein kinase (PKA) (22), resulting in pleiotropic effects in cell growth. Additionally, the Gαs subunit may negatively regulate MAPK activity, whereas the Gβγ subunits stimulate MAPK activity (22). Thus, the interplay between the stimulatory Gαs and inhibitory Gβγ subunits ultimately determines MAPK activity. In this study, we found that isoproterenol inhibited the mitogenic effects of bFGF in HFMc without reducing ERK-2 activity. Taken together, these findings suggest that the growth inhibitory effects of isoproterenol in HFMc may be mediated by intracellular targets distinct from the MEK-1/2 pathway. In support of these findings, previous studies have demonstrated that cAMP-induced growth-related effects in vascular smooth muscle cells (37) and fibroblasts (38) are not mediated by the MAPK pathway. For example, the activation of cAMP modulates the growth of vascular smooth muscle cells (39) and fibroblasts (40) by affecting the activity of the cell cycle inhibitor p27Kip1 and cyclin D1. Thus, the target of isoproterenol inhibition of mesangial cell growth may lie downstream of MEK or it may be related to the accumulation of cAMP. The last one is the most likely mechanism because other agents that stimulate adenyl cyclase activity, i.e. PGE2 and PGI2, also diminish the rate of [3H]-thymidine incorporation into mesangial cells (41, 42). The synergistic effect of isoproterenol and bFGF on cAMP accumulation was observed in the presence of phosphodiesterase inhibitors, suggesting that these changes are probably due to the stimulation of adenyl cyclase rather than to inhibition of the degradation of the cyclic nucleotide. cAMP may convey its physiologic effects by phosphorylating amino acid residues in the free catalytic subunits of cAMP-dependent protein kinase or by transporting the protein kinase-cAMP complex to the nucleus where it can bind to DNA and alter the transcription of genes (43, 44). However, in the present study, we did not exclude the possibility that other mechanisms could also be involved. For example, isoproterenol has been shown to decrease the adhesion of cultured rat mesangial cells (27, 28) and to stimulate the expression of 5′-nucleotidase activity in rat mesangial cells (42), and both factors may contribute to the inhibition of mesangial cell growth.

In most tissues, including liver, pituitary, proximal tubular cells, and embryonic renomedullary interstitial cells, Ang II inhibits the production of cAMP through interactions of the Ang II type 1 receptor with the guanyl nucleotide-binding protein Gαi, which is known to have an inhibitory effect on adenyl cyclase (22, 44). In other systems, i.e. fetal fibroblasts, distal tubular epithelial cells, and bovine adrenal cells, Ang II stimulates the accumulation of cAMP by different mechanisms that include the modulation of adenyl cyclase, prostaglandin secretion, or protein kinase C stimulation, depending on each cell type (44). Moreover, recent studies suggest that Ang II increases the accumulation of hormone-stimulated cAMP in cultured vascular smooth muscle cells (25) and in cultured preglomerular microvascular smooth muscle cells from normotensive and genetically hypertensive rats (45). In contrast with these findings, Ang II inhibits the release of cAMP induced by isoproterenol in isolated perfused kidneys from genetically hypertensive rats (46). Thus, the effects of Ang II on cAMP seem to be specifically modulated in each cell type. In the present study, we have found that Ang II did not affect the basal levels of cAMP in HFMc but significantly reduced the isoproterenol-induced accumulation of cAMP by approximately 40%, which is consistent with a 44% Ang II–mediated inhibition of forskolin-stimulated cAMP accumulation reported in rat mesangial cells (29).

The clinical relevance of the Ang II inhibitory effect on cAMP production in HFMc may not be limited to cell proliferation. Ang II directly stimulates both collagen and fibronectin synthesis in cultured HFMc independently of the cAMP pathway (34, 47). However, the synthesis of collagen in arterial smooth muscle cells subjected to stretching is inhibited whenever the intracellular levels of cAMP are raised (48), and agents that stimulate cAMP production (i.e. forskolin) induce a significant increase in fibronectin synthesis in mesangial cells (49). Thus, Ang II could potentially modulate the synthesis of collagen and fibronectin in HFMc through a cAMP-dependent mechanism. In addition, these findings may be relevant for the process of renin synthesis in juxtaglomerular cells because the synthesis of renin is increased after the activation of the cAMP–responsive elements in the renin gene and inhibited by Ang II (50). We found that bFGF, in contrast with Ang II, increases the production of cAMP. This finding is supported by the work of Kunz et al. (51), which showed that bFGF enhances the cAMP-induced transcriptional activation of inducible nitric oxide synthase in rat mesangial cells. Of interest, bFGF induces systemic hypotensive effects that may be mediated by the nitric oxide pathway (52). Thus, although bFGF and Ang II may have synergistic mitogenic effects in cultured human mesangial cells, both growth factors may induce opposite hemodynamic changes and, therefore, may modulate the in vivo behavior of mesangial cells in a different manner.

In conclusion, we have shown that bFGF is a potent mitogen that utilizes an MEK-dependent pathway to elicit its growth-promoting effects in cultured confluent HFMc. On the other hand, under similar tissue culture conditions, we have found that Ang II is a modest mitogen that induces proliferation of HFMc predominately by signaling through an MEK-independent pathway. Moreover, in the presence of isoproterenol, both growth factors induce opposite changes in cAMP accumulation and mesangial cell growth. The bFGF mitogenic effects can be almost completely abolished by MEK inhibitors or by isoproterenol treatment. More studies will be required to determine how bFGF, Ang II, and isoproterenol modulate the growth of glomerular mesangial cells in vivo.

Abbreviations

HFMc:

human fetal mesangial cells

bFGF:

basic fibroblast growth factor

Ang II:

angiotensin II

MAPK:

mitogen-activated protein kinases

ERK:

extracellular signal-regulated kinases

MEK-1 and -2:

MAP kinase kinase 1 and 2

References

  1. Mene P, Simonson MS, Dunn MJ 1989 Physiology of the mesangial cell. Physiol Rev 69: 1347–1424.

    CAS  PubMed  Google Scholar 

  2. Eddy AA 1994 Immune mechanisms of glomerular injury. In: Holliday MA, Barratt TM, Avner ED (eds) Pediatric Nephrology. Williams & Wilkins, Baltimore, MD, 672–696.

    Google Scholar 

  3. Morishita R, Gibbons GH, Ellison KE, Lee W, Zhang L, Yu H, Kaneda Y, Ogihara T, Dzau VJ 1994 Evidence for direct local effect of angiotensin in vascular hypertrophy. J Clin Invest 94: 978–984.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Lindner V, Reidy MA 1991 Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc Natl Acad Sci USA 88: 3739–3743.

    CAS  PubMed  Google Scholar 

  5. Damon DH, Lange DL, Hattler BG 1997 In vitro and in vivo vascular actions of basic fibroblast growth factor (bFGF) in normotensive and spontaneously hypertensive rats. J Cardiovasc Pharmacol 30: 278–284.

    CAS  PubMed  Google Scholar 

  6. Francki A, Uciechowski P, Floege J, von der Ohe J, Resch K, Radeke HH 1995 Autocrine growth regulation of human glomerular mesangial cells is primarily mediated by basic fibroblast growth factor. Am J Pathol 147: 1372–1382.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Su EJ, Lombardi DM, Wiener J, Daemen MJ, Reidy MA, Schwartz SM 1998 Mitogenic effect of angiotensin II on rat carotid arteries and type II or III mesenteric microvessels but not type I mesenteric microvessels is mediated by endogenous basic fibroblast growth factor. Circ Res 82: 321–327.

    CAS  PubMed  Google Scholar 

  8. Ray PE, Aguilera G, Kopp JB, Horikoshi S, Klotman PE 1991 Angiotensin II receptor-mediated proliferation of cultured human fetal mesangial cells. Kidney Int 40: 764–771.

    CAS  PubMed  Google Scholar 

  9. Campbell-Boswell M, Robertson AL Jr 1981 Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro. Exp Mol Pathol 35: 265–276.

    CAS  PubMed  Google Scholar 

  10. Itoh H, Mukoyama M, Pratt RE, Gibbons GH, Dzau VJ 1993 Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II. J Clin Invest 91: 2268–2274.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Huwiler A, Stabel S, Fabbro D, Pfeilschifter J 1995 Platelet-derived growth factor and angiotensin II stimulate the mitogen-activated protein kinase cascade in renal mesangial cells: comparison of hypertrophic and hyperplastic agonists. Biochem J 305: 777–784.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Silver BJ, Jaffer FE, Abboud HE 1989 Platelet-derived growth factor synthesis in mesangial cells: induction by multiple peptide mitogens. Proc Natl Acad Sci USA 86: 1056–1060.

    CAS  PubMed  Google Scholar 

  13. Ray LB, Sturgill TW 1987 Rapid stimulation by insulin of a serine/threonine kinase in 3T3–L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. Proc Natl Acad Sci USA 84: 1502–1506.

    CAS  PubMed  Google Scholar 

  14. Marshall CJ 1995 Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80: 179–185.

    CAS  PubMed  Google Scholar 

  15. McCormick F 1993 Signal transduction. Nature 363: 15–16.

    CAS  PubMed  Google Scholar 

  16. Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, Johnson GL 1993 A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science 260: 315–319.

    CAS  PubMed  Google Scholar 

  17. Zheng CF, Guan KL 1993 Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2. J Biol Chem 268: 11435–11439.

    CAS  PubMed  Google Scholar 

  18. Schieffer B, Bernstein KE, Marrero MB 1996 The role of tyrosine phosphorylation in angiotensin II mediated intracellular signaling and cell growth. J Mol Med 74: 85–91.

    CAS  PubMed  Google Scholar 

  19. Sevetson BR, Kong X, Lawrence JC Jr 1993 Increasing cAMP attenuates activation of mitogen-activated protein kinase. Proc Natl Acad Sci USA 90: 10305–10309.

    CAS  PubMed  Google Scholar 

  20. Haneda M, Araki S, Sugimoto T, Togawa M, Koya D, Kikkawa R 1996 Differential inhibition of mesangial MAP kinase cascade by cyclic nucleotides. Kidney Int 50: 384–391.

    CAS  PubMed  Google Scholar 

  21. Bokemeyer D, Sorokin A, Dunn MJ 1996 Multiple intracellular MAP kinase signaling cascades. Kidney Int 49: 1187–1198.

    CAS  PubMed  Google Scholar 

  22. Crespo P, Cachero TG, Xu N, Gutkind JS 1995 Dual effect of beta-adrenergic receptors on mitogen-activated protein kinase. J Biol Chem 270: 25259–25265.

    CAS  PubMed  Google Scholar 

  23. Wu J, Dent P, Jelinek T, Wolfman A, Weber MJ, Sturgill TW 1993 Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3′,5′-monophosphate. Science 262: 1065–1069.

    CAS  PubMed  Google Scholar 

  24. Yamada T, Terada Y, Homma MK, Nonoguchi H, Sasaki S, Yuasa Y, Tomita K, Marumo F 1995 AVP inhibits EGF-stimulated MAP kinase cascade in Madin-Darby canine kidney cells. Kidney Int 48: 745–752.

    CAS  PubMed  Google Scholar 

  25. Kubalak SW, Webb JG 1993 Angiotensin II enhancement of hormone-stimulated cAMP formation in cultured vascular smooth muscle cells. Am J Physiol 264: H86–H96.

    CAS  PubMed  Google Scholar 

  26. Maric C, Aldred GP, Harris PJ, Alcorn D 1998 Angiotensin II inhibits growth of cultured embryonic renomedullary interstitial cells through the AT2 receptor. Kidney Int 53: 92–99.

    CAS  PubMed  Google Scholar 

  27. Kreisberg JI, Venkatachalam MA 1986 Vasoactive agents affect mesangial cell adhesion. Am J Physiol 251: C505–C511.

    CAS  PubMed  Google Scholar 

  28. Kreisberg JI, Venkatachalam MA, Patel PY 1984 Cyclic AMP-associated shape change in mesangial cells and its reversal by prostaglandin E2. Kidney Int 25: 874–879.

    CAS  PubMed  Google Scholar 

  29. Zhou J, Ernsberger P, Douglas JG 1993 A novel angiotensin receptor subtype in rat mesangium. Hypertension 21: 1035–1038.

    CAS  PubMed  Google Scholar 

  30. Huwiler A, Fabbro D, Pfeilschifter J 1995 Platelet-derived growth factor stimulates de novo synthesis of mitogen-activated protein kinase in renal mesangial cells. Eur J Biochem 227: 209–213.

    CAS  PubMed  Google Scholar 

  31. Wilkie N, Morton C, Ng LL, Boarder MR 1996 Stimulated mitogen-activated protein kinase is necessary but not sufficient for the mitogenic response to angiotensin II. J Biol Chem 271: 32447–32453.

    CAS  PubMed  Google Scholar 

  32. Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk BC, Delafontaine P, Bernstein KE 1995 Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature 375: 247–250.

    CAS  PubMed  Google Scholar 

  33. Kudoh S, Komuro I, Mizuno T, Yamazaki T, Zou Y, Shiojima I, Takekoshi N, Yazaki Y 1997 Angiotensin II stimulates c-Jun NH2-terminal kinase in cultured cardiac myocytes of neonatal rats. Circ Res 80: 139–146.

    CAS  PubMed  Google Scholar 

  34. Ray PE, Bruggeman LA, Horikoshi S, Aguilera G, Klotman PE 1994 Angiotensin II stimulates human fetal mesangial cell proliferation and fibronectin biosynthesis by binding to AT1 receptors. Kidney Int 45: 177–184.

    CAS  PubMed  Google Scholar 

  35. Ray PE, Bruggeman LA, Weeks BS, Kopp JB, Bryant JL, Owens JW, Notkins AL, Klotman PE 1994 bFGF and its low affinity receptors in the pathogenesis of HIV- associated nephropathy in transgenic mice. Kidney Int 46: 759–772.

    CAS  PubMed  Google Scholar 

  36. Lefkowitz RJ, Caron MG 1988 Adrenergic receptors. J Biol Chem 263: 4993–4996.

    CAS  PubMed  Google Scholar 

  37. Takahashi T, Kawahara Y, Okuda M, Yokoyama M 1996 Increasing cAMP antagonizes hypertrophic response to angiotensin II without affecting Ras and MAP kinase activation in vascular smooth muscle cells. FEBS Lett 397: 89–92.

    CAS  PubMed  Google Scholar 

  38. McKenzie FR, Pouyssegur J 1996 cAMP-mediated growth inhibition in fibroblasts is not mediated via mitogen-activated protein (MAP) kinase (ERK) inhibition. J Biol Chem 271: 13476–13483.

    CAS  PubMed  Google Scholar 

  39. Fukumoto S, Koyama H, Hosoi M, Yamakawa K, Tanaka S, Morii H, Nishizawa Y 1999 Distinct role of cAMP and cGMP in the cell cycle control of vascular smooth muscle cells: cGMP delays cell cycle transition through suppression of cyclin D1 and cyclin-dependent kinase 4 activation. Circ Res 26: 985–991.

    Google Scholar 

  40. L'Allemain G, Lavoie JN, Baldin V, Pouyssegur J 1997 Cyclin D1 expression is a major target of the cAMP-induced inhibition of cell cycle entry in fibroblast. Oncogene 14: 1981–1990.

    CAS  PubMed  Google Scholar 

  41. Mene P, Dunn MJ 1990 Prostaglandins and rat glomerular mesangial cell proliferation. Kidney Int 37: 1256–1262.

    CAS  PubMed  Google Scholar 

  42. Savic V, Blanchard A, Vlahovic P, Stefanovic V, Ardaillou N, Ardaillou R 1991 Cyclic adenosine monophosphate-stimulating agents induce ecto-5′-nucleotidase activity and inhibit DNA synthesis in rat cultured mesangial cells. Arch Biochem Biophys 290: 202–206.

    CAS  PubMed  Google Scholar 

  43. Muraguchi A, Miyazaki K, Kehrl JH, Fauci AS 1984 Inhibition of human B cell activation by diterpine forskolin: interference with B cell growth factor-induced G1 to S transition of the B cell cycle. J Immunol 133: 1283–1287.

    CAS  PubMed  Google Scholar 

  44. Johnson MC, Aguilera G 1991 Angiotensin-II receptor subtypes and coupling to signaling systems in cultured fetal fibroblasts. Endocrinology 129: 1266–1274.

    CAS  PubMed  Google Scholar 

  45. Mokkapatti R, Vyas SJ, Romero GG, Mi Z, Inoue T, Dubey RK, Gillespie DG, Stout AK, Jackson EK 1998 Modulation by angiotensin II of isoproterenol-induced cAMP production in preglomerular microvascular smooth muscle cells from normotensive and genetically hypertensive rats. J Pharmacol Exp Ther 287: 223–231.

    CAS  PubMed  Google Scholar 

  46. Vyas SJ, Mi Z, Jackson EK 1996 The inhibitory effect of angiotensin II on stimulus-induced release of cAMP is augmented in the genetically hypertensive rat kidney. J Pharmacol Exp Ther 279: 114–119.

    CAS  PubMed  Google Scholar 

  47. Wolf G, Haberstroh U, Neilson EG 1992 Angiotensin II stimulates the proliferation and biosynthesis of type I collagen in cultured murine mesangial cells. Am J Pathol 140: 95–107.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Kollros PR, Bates SR, Mathews MB, Horwitz AL, Glagov S 1987 Cyclic AMP inhibits increased collagen production by cyclically stretched smooth muscle cells. Lab Invest 56: 410–417.

    CAS  PubMed  Google Scholar 

  49. Kreisberg JI, Kreisberg SH 1995 High glucose activates protein kinase C and stimulates fibronectin gene expression by enhancing a cAMP response element. Kidney Int Suppl 51: S3–S11.

    CAS  PubMed  Google Scholar 

  50. Horiuchi M, Pratt RE, Nakamura N, Dzau VJ 1993 Distinct nuclear proteins competing for an overlapping sequence of cyclic adenosine monophosphate and negative regulatory elements regulate tissue-specific mouse renin gene expression. J Clin Invest 92: 1805–1811.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Kunz D, Walker G, Eberhardt W, Messmer UK, Huwiler A, Pfeilschifter J 1997 Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1 beta- and cAMP-induced nitric oxide synthase expression in rat renal mesangial cells. J Clin Invest 100: 2800–2809.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Cuevas P, Carceller F, Ortega S, Zazo M, Nieto I, Gimenez-Gallego G 1991 Hypotensive activity of fibroblast growth factor. Science 254: 1208–1210.

    CAS  PubMed  Google Scholar 

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Affiliations

  1. Center for Molecular Physiology Research, Children's Research Institute, Children's National Medical Center, Washington, 20010, DC

    Ernest B Izevbigie & Patricio E Ray

  2. Oral and Pharyngeal Cancer Branch Program [J.S.G.], NIDR, National Institutes of Health, Bethesda, 20892, Maryland, U.S.A.

    J Silvio Gutkind

Authors
  1. Ernest B Izevbigie
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Supported by USPHS awards R0–1DK 4919 and RO-1HL 55605, “FADI” from Buenos Aires, Argentina, and NIDDK training grant # DK49419-S1 (E.B.I.).

Presented in part at the American Pediatric Society–Society for Pediatric Research Annual Meeting, San Francisco, May 1–4, 1999.

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Izevbigie, E., Gutkind, J. & Ray, P. Angiotensin II and Basic Fibroblast Growth Factor Mitogenic Pathways in Human Fetal Mesangial Cells. Pediatr Res 47, 614–621 (2000). https://doi.org/10.1203/00006450-200005000-00010

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