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Am J Physiol Lung Cell Mol Physiol 290: L1291-L1299, 2006; doi:10.1152/ajplung.00443.2005
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Modulation by bradykinin of angiotensin type 1 receptor-evoked RhoA activation of connective tissue growth factor expression in human lung fibroblasts

Zhenhua Huang, Linda Taylor, Bin Liu, Jun Yu, and Peter Polgar

Department of Biochemistry, Boston University, School of Medicine, Boston, Massachusetts

Submitted 18 October 2005 ; accepted in final form 12 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The mechanisms regulating the opposing physiological actions of bradykinin (BK) and angiotensin II (AngII) are not well understood. Here we investigate signaling interactions between these two effectors. Connective tissue growth factor (CTGF) expression in IMR-90, human lung fibroblasts, is used as the endpoint target. In these cells the BK B2 receptor (BKB2R) is expressed constitutively, while no binding of AngII is detected. An inducible expression system is used to insert AngII receptor 1 (AT1R) and to obtain a signal level in response to AngII at the magnitude of BK. AngII and BK activate G protein-coupled targets, arachidonate release from cellular phospholipid stores, and intracellular phosphatidylinositol turnover equally. Both activate ERK, JNK, and p38 equally. However, AngII activates, whereas BK inactivates, RhoA. AngII induces a rapid (1 h) CTGF mRNA expression. RhoA siRNA and RhoA activation inhibitor, Y-27632, markedly reduce the AngII effect. Simultaneous treatment with BK and AngII attenuates the AT1R action. Additionally, BK in the absence of AngII lowers CTGF mRNA expression below basal levels over a span of 4 h. An AT1R/BKB2R chimera lacking heterotrimeric G protein coupling continues to activate MAP kinases to the same extent as wild-type (WT) AT1R and BKB2R. However, the increase of CTGF mRNA expression by this mutant is low, almost identical with that obtained by the simultaneous treatment of the WT AT1R-expressing cells with BK and AngII. In this context the chimeric receptor displays the characteristics of both receptors. These data demonstrate that, in human lung fibroblasts, BK modulates the action of AngII through the small G protein RhoA, but in a G{alpha}i/G{alpha}q-independent manner.

angiotensin II receptor; bradykinin receptor; RhoA; IMR-90 fibroblasts

THE VASOACTIVE OCTAPEPTIDE, angiotensin II (AngII), is the effector molecule of the renin-angiotensin system. It produces a variety of regulatory actions in the pulmonary, cardiovascular, renal, endocrine, and neural systems through the activation of its AngII receptor 1 (AT1R) (16, 36). Bradykinin (BK) is an active component of the kinin/kininogen system. It is a potent vasoactive nanopeptide that acts through the bradykinin B2 receptor (BKB2R), which has been shown to be involved in many cascading reactions. Both AT1R and BKB2R are members of the seven-transmembrane-spanning heterotrimeric G protein-coupled receptor (GPCR) superfamily and couple to G{alpha}i and G{alpha}q peptides (8, 13, 14, 23) with the generation of cellular inositol trisphosphates (IP) and the release of arachidonate (ARA) from cellular phospholipid stores. AngII has an important role in the fibrotic response to tissue injury in the lung (20), heart (31), and kidney (22). AngII is of particular interest since it plays important roles in human hypertension and fibrosis. BK and AngII are antagonists in a number of physiological processes as recently exemplified in BKB2R knockout mice with respect to cardiac hypertrophy and microvascular deficit (19).

Connective tissue growth factor (CTGF) mRNA expression was chosen as an endpoint target of the actions of BK and AngII in the present study. CTGF is a member of the CAN family (CYR61/CEF10, CTGF/FISP-12, and NOV) (24). This 38-kDa protein is structurally characterized by its cysteine-rich sequence (27) and is expressed in IMR-90 cells (35). High CTGF levels have been demonstrated in many fibrotic human tissues, including lung (30), blood vessels (26), kidney (10), liver (1), and skin (39). Enhanced CTGF expression has been described in the bronchoalveolar lavage from patients with fibrotic lung disease (3).

In the present study, we used an inducible expression system to establish the presence of the wild-type (WT) AT1R and a mutant AT1R in the normal, diploid IMR-90 fibroblasts. We chose this cell because it does not express the AT1R and thus can be engineered to express the AT1R WT as well as AT1 mutant receptors of choice. With appropriate doxycycline (Dox) treatment, WT AT1R expression, its coupling to heterotrimeric G protein, and its activation of mitogen-activated protein kinases (MAPKs), JNK ERK, and p38 proved to be equivalent to those of the endogenous BKB2R. Utilizing the presence of both BKB2 and AT1 receptors in these cells, we characterized the effect of AngII and BK on CTGF mRNA expression. We found that AngII increases CTGF mRNA rapidly, while BK markedly blunted this action. Furthermore, by generating an AT1R/BKB2R chimera, which binds AngII but failed to interact with G{alpha}i/G{alpha}q, we determined that the activation of MAPKs and the small G protein, RhoA, continued in the absence of this linkage.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [3H]AngII (52.5 Ci/mmol), [3H]BK (78 Ci/mmol), myo-[1, 2-3H]inositol (57.8 Ci/mmol), and [3H]ARA (65.9 Ci/mmol) were obtained from NEN Life Science Products. Analytical-grade Dowex-X8 (AG-1-X8, 100–200 mesh) was obtained from Bio-Rad. RhoA siRNA (sc-29471), control siRNA (sc-37007), and anti-RhoA antibody were purchased form Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, and restriction endonucleases were purchased from New England Biolabs. Y-27632 (a Rho kinase inhibitor), PD-98059 (a specific p42/44 MAPK cascade inhibitor), SB-202190 (a p38 inhibitor), and SP-600125 (a JNK inhibitor) were obtained from Calbiochem. The protease inhibitor cocktail containing pancreatic extract, pronase, thermolysin, chymotrypsin, and papain was obtained from Roche Applied Science (catalog no. 1836153).

Cell culture. IMR-90, human diploid lung fibroblasts (9), at a population doubling level (PDL) of 14 were obtained from the Coriel Institute for Medical Research (Camden, NJ). These are mortal cells, characterized by a limited life span of ~60 doublings (12, 25). Their finite doubling potential makes it particularly difficult to obtain stable transfection. Using an inducible retroviral vector system, we successfully obtained stable and adjustable expression of the AT1R within ~10 PDLs. All experiments were performed with cells at <PDL40. The cells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin.

Construction of the AT1R-BKIC2 chimera. For construction of the AT1R-BKIC2, the second intracellular loop (IC2) of AT1R was substituted with that of BKB2R. To this end, two silent restriction sites AgeI (at position 126) and BstEII (at position 147) were created by site-directed mutagenesis on AT1 cDNA in PCDNA3.1. The corresponding sense and anti-sense oligonucleotides for the IC2 region of BKB2R (5'-CCGGTACCTG GCCATCGTCA AGACCATGTC CATGGGCCGG ATGCGCGGGG and 5'-GTGACCTTGG CCACCAGTAC CCCGCGCATC CGGCCCATGG ACATGGTCTT-3', respectively) were annealed and ligated into the AgeI/BstEII-digested vector cassette. The PmeI-NotI fragments containing the chimeric receptors were subcloned into pCXbsrR.

Retroviral vectors expressing AT1R and AT1R-BKIC2. The tetracycline-inducible expression system used here was as described by Akagi and coworkers (2). To transfect the IMR-90, Bosc23 cells were transfected by using Lipofectamine 2000 with pCL-Ampho plasmid (Imgenex, San Diego, CA) containing retroviral pol, gag, and amphotropic gp70 env genes and constructed vectors [WTAT1R, AT1R-BKIC2, or control empty vector] in the pCXbsr vector. After 48 h, the culture supernatant from the transfected Bosc23 cells containing the virus was filtered, supplemented with 8 µg/ml polybrene, and added to the IMR-90 culture. The cells were selected with 6 µg/ml blasticidin and 400 µg/ml Geneticin (G418) for ~7 and 14 days, respectively. Drug selection was started 2 days after the infection. The selected, drug-resistant populations were used for all experiments.

Effects of guanosine 5’-O-(3-thiotriphosphate) on induced AngII binding in IMR-90 cells. The effect of guanosine 5’-O-(3-thiotriphosphate) (GTP{gamma}S) on AngII binding was determined as described by Seta and coworkers (32) with a few modifications. AT1R- or AT1R-BKIC2-inducible cells were plated onto 20-cm culture dishes and grown in DMEM as described above. After overnight induction with Dox, the cells were washed three times with phosphate-buffered saline (PBS) and scraped into suspension in 5 mM Tris buffer (pH 7.5) containing the protease inhibitor cocktail. After 10 min on ice, the cells were subjected to two freeze/thaw cycles and centrifuged at 21,000 g for 20 min. The pellet containing the membrane fraction was resuspended in 50 mM Tris buffer (pH 7.5) containing 200 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 0.1% bovine serum albumin, and protease inhibitors. Membrane fractions (100 µg of protein) were incubated with 10 nM [3H]AngII at 25°C for 1 h in the presence of varying concentrations of GTP{gamma}S. Free ligand was removed by centrifugation at 10,000 g for 5 min at 4°C. Pellets were washed two times, and the radioactivity remaining in the pellet was determined.

Receptor binding assay. After induction at various Dox concentrations, receptor binding studies were carried out as described previously (28). In brief, 80–100% confluent cell monolayers in 24-well plates (~1.2 x 105 cells/well) were incubated in binding buffer containing specified concentrations of [3H]AngII and [3H]BK in the absence (total binding) or presence (nonspecific binding) of 1 mM AngII and BK for 2 h at 4°C. The cells were washed three times with ice-cold buffer and then solubilized with 0.2% sodium dodecyl sulfate (SDS). Radioactivity was determined in a Packard liquid scintillation counter.

Arachidonic acid release. After treatment with Dox, the cells in 24-well plates (~1.2 x 105 cells/well) were labeled with [3H]ARA (0.2 µCi/well) as described previously (28). The labeled cells were washed and incubated with 500 µl of DMEM containing 2 mg/ml bovine serum albumin and incubated with 100 nM BK or AngII for 20 min at 37°C. The medium was removed, and any particular matter was removed by centrifugation at 800 g. The control group was treated identically except for the addition of BK or AngII. Radioactivity in the supernatant was determined in a scintillation counter after addition of 2 ml of Ecolite scintillation fluid.

Phosphoinositide turnover. Dox-treated cells in 12-well plates (~2 x 105 cells/well) were incubated with 1 µCi/ml of myo-[3H]inositol in 1 ml of growth medium for 18 h. The level of inositol phosphate was determined 1 day later as previously described (28). In brief, DMEM containing 20 mM LiCl2 and 20 mM HEPES, pH 7.4, was added to the cultures 10 min before effector stimulation. Cells were then exposed to 100 nM BK or AngII for 30 min at 37°C, and the incubations were terminated by removal of the media and the addition of 0.5 ml of 10 mM ice-cold formic acid. The cells were scraped off the dish and formic acid-soluble material was isolated by centrifugation and neutralized by the addition of 10 ml of 5 mM sodium tetraborate. The total extracted [3H]phosphatidyl-inositol (IP) was bound in a Dowex AG1-X8 formate resin (Bio-Rad) anion exchange column and eluted with 2 M ammonium formate, pH 5.0. Radioactivity was determined in a Packard liquid scintillation counter.

Determinations of CTGF mRNA by real-time PCR. After the exposure of cells expressing either the WT AT1 or AT1-BKIC2 receptors to either BK or AngII and the desired kinase inhibitor, the cellular RNAs were isolated using RNeasy kit (Qiagen). cDNAs were generated using Superscript II (Invitrogen). Gene expression of CTGF was analyzed using an ABI 7700 system (Applied Biosystems). The results were normalized with GAPDH. The primers for real-time PCR for CTGF were: 5'-TGTGTGATGAGCCCAAGGA-3' and 5'-TCAGGGCCAAATGTGTCTTC-3'.

Western blotting of CTGF. After treatment with 10–8 M Dox for 24 h, the AT1R or AT1R-BKIC2 cells were stimulated with 100 nM AngII or BK or both AngII and BK for the indicated times. Cell layers were harvested with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, and complete protease inhibitor cocktail) and centrifuged at 12,000 rpm in a microcentrifuge at 4°C for 20 min. Total protein (50 µg) was fractionated on 12% SDS-PAGE gels and then subjected to Western blot analysis with anti-CTGF antibody. Protein content was determined by the bicinchoninic acid method. beta-Actin was used as a loading control.

MAPK kinase assay. The fibroblasts expressing either WT or mutant AT1R were incubated with or without inhibitors PD-98059, SP-600125, or SB-202190 for 30 min at 37°C and then stimulated with 100 nM AngII or BK for various times up to 10 min. The cells were then washed twice with ice-cold PBS. Cell lysates were prepared by the addition of ice-cold lysis RIPA buffer with complete protease inhibitor cocktail and centrifugation at 12,000 rpm in a microcentrifuge at 4°C for 20 min. The proteins were fractionated on 10% SDS-PAGE gels and Western blots were carried out using antibodies against the phosphorylated or nonphosphorylated ERK1/2, JNK, or p38 MAPK (Cell Signaling Technology).

RhoA activation. The determination of RhoA activation has been described previously by Meacci and coworkers (21). In brief, Dox-treated AT1R- and AT1R-BKIC2 cells in 150 mm x 25 mm plates were incubated with or without 100 nM AngII or BK for 1 and 5 min. The medium was then removed, and the cells were washed twice with ice-cold Tris-buffered saline and scraped in buffer A (50 mM Tris/HCl, pH 7.5, 10 mM NaCl, 1 mM KCl, 2 mM MgCl2) containing 300 mM sucrose and protease inhibitor cocktail. The cells were then lysed (Dounce, 100 strokes). Lysates were centrifuged (10 min, 750 g). Membrane fractions were prepared from the supernatant by centrifugation at 100,000 g for 2 h (21), and the supernatants were saved as soluble fractions (cytosol fractions). Membrane protein (50 µg of protein per lane) was separated by SDS-PAGE. RhoA was detected with monoclonal anti-RhoA antibody (Santa Cruz). As an internal control, beta-actin was determined with monoclonal anti-beta-actin (Sigma). Bound antibody was detected using chemiluminescence reagents (ECL, Amersham).

Small double-stranded RNA-mediated RNA interference. Cells were harvested with 0.25% trypsin, 1 mM EDTA in PBS without Ca2+ and Mg2+. The cells were then plated onto six-well plates at 2 x 104 cells per well in DMEM without antibiotics. After 24 h of incubation when the cultures were 30%-50% confluent, RhoA siRNA or control siRNA were transfected into the cells with Lipofectamine 2000. In brief, we added 80 pmol of siRNA to 250 µl of Opti-MEM medium and 5 µl of Lipofectamine 2000 to a separate 250-µl aliquot of Opti-MEM medium. After 5 min of incubation, the diluted siRNA was combined with the diluted Lipofectamine 2000, mixed gently, and incubated for 20 min at room temperature. We added 500 µl of this complex to one well of a six-well plate. For gene downregulation assays, intact cells were harvested with RIPA buffer for Western blot analysis after incubation at 37°C in a 5% CO2 incubator for 72 h. For assay of CTGF mRNA expression, cells were induced by 1 x 10–8 M Dox for 48 h and were harvested for RNA isolation after 72 h of further incubation.

Statistical analysis. All data are expressed as means ± SE. Significance was established by Student’s t-test. Differences were considered significant at a P value of <0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Inducible receptor expression. Starting at PDL16, stable transfection of the AT1R was achieved by PDL26. In addition to the WT AT1R cDNA, the IMR-90 cells were also transfected with a construct which utilized AT1R as the recipient and BKB2R as the donor of its entire IC2 region (AT1R-BKIC2), as illustrated in Fig. 1A. To characterize the induction of AT1R and AT1R-BKIC2 expression, the cells were incubated with varying concentrations of Dox (10–11–10–6 M) for 24 h. The AT1R expression levels were determined with an [3H]AngII radioligand binding assay. No binding was found in cells transfected with empty vector, in uninfected cells, or AT1R-infected cells not treated with Dox (Fig. 1B). Endogenous binding of BK remained unchanged upon infection with either WT or mutant AT1 receptors. Upon treatment with Dox, inducible receptor expression increased as a function of inducer concentrations ranging from 10–11 to 10–6 M, with AT1R and AT1R-BKIC2 expression increasing from 0 to 20.0 ± 0.9 x 104 and 17.2 ± 0.1 x 104 receptors/cell, respectively.


Figure 1
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  		Fig. 1. Characterizations of angiotensin II receptor 1 (AT1R) and AT1R-BKIC2 expression induced by doxycycline (Dox) (A). Schematic of wild-type (WT) AT1R (black) and bradykinin B2 receptor (BKB2R, gray) and the chimeric AT1R-BKIC2 (black/gray). B: receptor number for endogenous BKB2R and Dox dose-dependent AT1R and AT1R-BKIC2. AT1R-, AT1R-BKIC2-inducible cells, and control vector-transfected cells were incubated with increasing concentrations of Dox, from 10–11 to 10–6 M for 24 h. Their receptor number/cell was compared with receptor number/cell of endogenous BKB2R. *P < 0.01 vs. AT1R without Dox treatment; #P < 0.01 vs. AT1R-BKIC2 without Dox treatment. C: receptor number/cell with time of Dox stimulation. AT1R- and AT1R-BKIC2-inducible and control vector-infected fibroblasts were incubated with 10–6 M Dox for 0–48 h. *P < 0.01 vs. AT1R without Dox treatment; #P < 0.01 vs. AT1R-BKIC2 without Dox treatment. D: decrease in AT1R and AT1R-BKIC2 expression following removal of Dox. The cells were incubated with 10–6 M Dox for 24 h. After removal of Dox, the cells were washed 4 times and then incubated for 0–48 h. *P < 0.01 vs. AT1R without Dox removal; #P < 0.01 vs. AT1R-BKIC2 without Dox removal. The expression level (ligand binding) was determined as described in MATERIALS AND METHODS. Results are means ± SE of 3 independent experiments.

 
A time course of Dox stimulation was performed to determine the time necessary for maximum receptor expression. Cells were incubated with 10–6 M Dox for various times up to 48 h. Expression of both AT1R and AT1R-BKIC2 was initially detected after 3 h. Maximal induction occurred at 24 h after Dox addition (Fig. 1C). To follow the reduction of AT1R expression following withdrawal of the inducer, the inducible cells were incubated for 24 h with 1 x 10–6 M Dox, then washed free of Dox, and thereafter maintained for various times up to 48 h. The AT1 receptor number was determined with the [3H]AngII radio ligand binding assay. The results showed that both AT1R and AT1R-BKIC2 expression remained constant for the first 6 h after Dox removal following which the level of expression decreased by ~48% at 12 h and remained stable up to 48 h (Fig. 1D).

GTP{gamma}S effect on AngII binding to AT1R and AT1R-BKIC2. AngII binding in the presence of increasing GTP{gamma}S concentrations (10–8–10–4 M) was determined to assess heterotrimeric G protein coupling to the AT1 and AT1R-BKIC2 receptors. GTP{gamma}S shifts the GPCR from a high affinity to a low affinity state (32). This shift has been used as an indicator of the interaction of the receptor with its heterotrimeric G protein coupler. As illustrated in Fig. 2, GTP{gamma}S decreased AngII binding to the AT1R in a dose-dependent manner. In contrast, AngII binding to AT1R-BKIC2 was not affected by GTP{gamma}S at any concentration.


Figure 2
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  		Fig. 2. Binding properties of AT1R and AT1R-BKIC2 membrane fractions in the presence of guanosine 5’-O-(3-thiotriphosphate) (GTP{gamma}S). Membrane fractions (100 µg of protein) were prepared as described in MATERIALS AND METHODS from either AT1R- or AT1R-BKIC2-expressing cells. The membrane fractions were incubated for 1 h at 25°C in binding buffer containing 10 nM [3H]AngII in the presence of various concentrations of GTP{gamma}S. The ratios of [3H]AngII binding in the presence of GTP{gamma}S to those without GTP{gamma}S were calculated (n = 3). *P < 0.01 vs. AT1R-BKIC2 without GTP{gamma}S addition.

 
G{alpha}q and G{alpha}i coupling. Upon binding with AngII, AT1R activates phospholipase Cbeta via G{alpha}q (33) causing the generation of IPs. AngII also activates phospholipase A2 to generate arachidonic acid through G{alpha}i. BK is known to act in a similar manner upon binding to the BKB2R. As illustrated in Fig. 3A, ARA release increased with increasing number of AT1R per cell in the AT1R-inducible fibroblasts. At 10–8 M Dox, the degree of ARA release activated by 100 nM AngII matched that seen at 100 nM BK. PI turnover also occurred with a similar pattern with somewhat less dependence on receptor number at higher receptor concentrations. The response to BK matched the response to AngII at 10–8 M Dox (Fig. 3B). AngII failed to activate ARA release (Fig. 3A) or PI turnover in cells expressing the AT1R-BKIC2 (Fig. 3B).


Figure 3
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  		Fig. 3. AngII- and BK-induced arachidonate (ARA) release and phosphatidylinositol (PI) turnover. Twenty-four hours before assay, the AT1R- and AT1R-BKIC2-inducible and control vector-transfected cells were incubated with the indicated concentration of Dox. The effects of endogenous BKB2R were also evaluated in the AT1R-inducible cells. A: ARA release. Cells in 24-well plates were labeled with [3H]ARA (0.2 µCi/well) for 16 h, washed, and then stimulated with 100 nM AngII or 100 nM BK for 20 min as described in MATERIALS AND METHODS. The data obtained are means ± SE of 3 separate experiments. B: PI turnover. Cells in 12-well plates were incubated with 1 µCi/ml of myo-[3H]inositol for 18 h, treated with LiCl2, and then exposed to 100 nM AngII or 100 nM BK for 30 min at 37°C as described in MATERIALS AND METHODS. The data obtained are means ± SE of 3 separate experiments. *P < 0.01 vs. AT1R without Dox treatment.

 
CTGF mRNA and protein expression. The expression of CTGF mRNA in the AT1R-inducible cells was tested in response to 100 nM AngII or/and BK. After treatment with 10–8 M Dox for 24 h, the AT1R-inducible cells were exposed to AngII, BK, or both. CTGF mRNA was increased 3.8-fold of the basal value (Fig. 4A) in response to 1 h of exposure to 100 nM AngII. When the cells were exposed to 100 nM BK alone, CTGF mRNA levels did not change but were significantly reduced at 4 h of BK exposure. Simultaneous incubation of the cells with BK and AngII for 1 h resulted in a 50% decrease in CTGF mRNA expression (2.2-fold), compared with incubation with AngII alone. Interestingly, the cells expressing the AT1R-BKIC2 mutant receptor responded to AngII with approximately the same intensity (1.8-fold increase over basal levels) as seen in the simultaneous incubation with AngII and BK of the WT AT1R-infected cells. Simultaneous incubation of these cells with AngII and BK had no further effect on CTGF mRNA levels, with a P value of 0.046 when effects with and without BK addition were compared. Western blot analysis for CTGF was performed to substantiate that changes in CTGF mRNA expression are reflected in CTGF protein production (Fig. 4, B and C). As shown, exposure to AngII for 6 h resulted in 2.0- and 1.5-fold increases in CTGF protein expression in the AT1R and AT1R-BKIC2 cells, respectively. In contrast, corresponding incubation with BK did not change CTGF protein production over control in either the AT1R or AT1R-BKIC2 cells. Simultaneous incubation of the cells with BK and AngII for 6 h resulted in a 0.7-fold decrease in CTGF expression in AT1R cells (Fig. 4B) and a 0.3-fold decrease in CTGF expression in AT1R-BKIC2 cells (Fig. 4C).


Figure 4
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  		Fig. 4. Time-dependent expression of connective tissue growth factor (CTGF) mRNA and protein levels in response to AngII or BK. A: CTGF mRNA expression. After treatment with 10–8 M Dox for 24 h, the AT1R and AT1R-BKIC2 cells were incubated with 100 nM AngII and/or 100 nM BK for 0–4 h at 37°C. Total RNAs were then isolated from the cells using an RNeasy kit. cDNAs were produced from 10 µg of RNA using SuperScript II reverse transcriptase. The expression of CTGF mRNA was determined by real-time PCR as described in MATERIALS AND METHODS. Results are means ± SE of 3 independent experiments. *P < 0.05 vs. AT1R with only Dox treatment. #P <0.05. B and C: CTGF protein expression. After treatment with 10–8 M Dox for 24 h, AT1R- or AT1R-BKIC2-expressing cells were stimulated with 100 nM AngII or BK or both AngII and BK for the indicated time. Then, the cell layer was harvested, and 50 µg of total protein were resolved by SDS-PAGE. CTGF was detected with anti-CTGF antibody as described in MATERIALS AND METHODS. *P < 0.05 vs. control. The results shown are representative of 3 separate experiments.

 
MAPKs. MAPKs ERK, JNK, and p38 have been shown to be linked to CTGF production (33, 34). We investigated the role of these three MAPKs in the signal transduction responses to AngII and BK in the human lung fibroblasts by Western blot assays for their phosphorylated (Tyr/Thr) forms using specific antiphospho-kinase antibodies. All three phosphorylated kinases increased to a maximum at 2 min and then decreased gradually within 10 min after treatment of the cells with either 100 nM AngII or BK (Fig. 5A). Both the time requirement to reach the maximum responses as well as the degrees of phosphorylation of all three MAPKs proved to be very similar in response to AngII or BK. Importantly, the AT1R-BKIC2, lacking functional coupling to heterotrimeric G protein, activated all three MAPKs as WT AT1R (Fig. 5B). PD-98059, an activated ERK inhibitor; SB-202190, a p38 MAPK inhibitor; and SP-600125, a JNK inhibitor blocked ERK, p38, and JNK activation by AngII or BK (Fig. 5C).


Figure 5
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  		Fig. 5. Time course of AngII and BK-induced ERK1/2, p38, JNK1/2 activation and inhibition. After treatment with 10–8 M Dox for 24 h, the AT1R (A)- and AT1R-BKIC2 (B)-expressing cells were stimulated with 100 nM AngII or BK for indicated time intervals. (C). The AT1R-inducible cells were treated with 30 µM PD-98059 (an inhibitor of ERK), 10 µM SB-202190 (a p38 MAPK inhibitor), and 50 µM SP-600125 (a JNK inhibitor), respectively, for 1 h. The cells were stimulated with 100 nM AngII or BK for 5 min and harvested in radioimmunoprecipitation assay buffer at the indicated times. Aliquots (50 µg) of cell lysates were subjected to Western blotting using antibodies as described in MATERIALS AND METHODS. *P < 0.05 vs. phosphorylated (P)-ERK1/2 in untreated inducible IMR-90. #P < 0.05 vs. P-p38 in untreated inducible IMR-90. {ddagger}P < 0.05 vs. P-JNK1/2 in untreated inducible IMR-90.

 
Effects of inhibition of MAPKs on CTGF mRNA expression. To determine whether any of these MAPKs were involved in the AngII-dependent induction of CTGF mRNA expression, we selectively blocked the activation of ERK, p38, and JNK and then measured the relative CTGF mRNA levels in response to AngII. After treatment with 10–8 M Dox for 24 h, the AT1R-inducible cells were incubated with inhibitors of MAPKs for 1 h and then stimulated with 100 nM AngII for 1 h. All three inhibitors decreased basal and AngII-stimulated CTGF mRNA expression (Fig. 6A). However, a more detailed inspection of the results with the use of the inhibitor effect ratio (7) illustrated that these MAPK inhibitors reduce only basal CTGF mRNA expression (Fig. 6B). No effect was seen in cells exposed to either AngII or BK. On the other hand, Y-27632, which inhibits RhoA activation, depressed both AngII-stimulated and basal CTGF mRNA expression (Fig. 6B). This inhibitor also proved to affect the time dependency of BK-induced (4 h) downregulation of CTGF mRNA expression.


Figure 6
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  		Fig. 6. Effect of inhibitors of MAPKs on CTGF mRNA expression. A: after treatment with 10–8 M Dox for 24 h, the AT1R-inducible cells were incubated with the inhibitors of MAPKs for 1 h and then stimulated with 100 nM AngII for 1 h or BK for 4 h. Total RNA was then isolated from the cells using the RNeasy kit. cDNAs were made from 10 µg of RNA with SuperScript II reverse transcriptase. The expression of CTGF mRNA was measured by real-time PCR. For each time point, RNA was prepared from 3 separate cultures and each sample was run in triplicate. B: values represent CTGF mRNA expression in response to AngII in the presence or absence of specific inhibitors (inhibitor effect ratio). These values were obtained as described in MATERIALS AND METHODS. *P < 0.05 vs. control. SP, SP-600125; SB, SB-202190; PD, PD-98059.

 
RhoA and the regulation of CTGF mRNA expression. The inhibitor results suggested participation of RhoA in the regulation of CTGF mRNA expression by AngII and BK in the human fibroblasts. We have previously proposed that the small G protein, RhoA, may be involved in AT1R signaling (38). Watts and Spiteri (35) more recently demonstrated that RhoA is involved in transforming growth factor-beta regulation of CTGF expression in these fibroblasts. Generally, RhoA is located in the cytosol in its inactive state (6, 40). Upon activation, RhoA becomes membrane bound. Thus the role of RhoA in AngII-activated CTGF mRNA expression was explored by following RhoA membrane translocation, a hallmark of RhoA activation. As shown (Fig. 7A), AngII induced an increase in membrane translocation of RhoA as rapidly as 1 min, while translocation continued to increase for up to 5 min. In contrast, BK dissociated membrane binding of RhoA as seen at after 1-min and 5-min periods of incubation. The increase in RhoA translocation to the membrane in response to AngII also occurred to a lesser degree in the cells containing the AT1R-BKIC2 mutant receptor (Fig. 7B). The Western blot analysis also showed that the amount of RhoA detectable in the cytosol did not change after AngII or BK stimulation in inducible IMR-90 (Fig. 7C). These results indicated that upon activation only a small portion of inactive RhoA translocates from the cytosol to membrane. This result was in line with the report of Aoki et al. (4).


Figure 7
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  		Fig. 7. AngII- and BK-induced translocation of RhoA. After treatment with 10–8 M Dox for 24 h, AT1R- and AT1R-BKIC2-inducible IMR-90 were stimulated with 100 nM AngII or BK for the indicated time. Membrane fractions and cytosol fractions were isolated as described in MATERIALS AND METHODS. An equal amount (50 µg) of protein was loaded onto each lane. Immunoblot analysis was performed using specific anti-RhoA antibody. beta-Actin was used as an internal control. A: membrane factions in AT1R. B: membrane factions in AT1R-BKIC2 *P < 0.05 vs. samples treated without AngII and BK. C: cytosol fractions in AT1R and AT1R-BKIC2. The result shown is representative of 3 independent experiments.

 
siRNA inhibition of RhoA activation of CTGF mRNA expression. The AT1R-inducible cells were transiently transfected with control siRNA or with siRNA directed against RhoA. Suppression of RhoA expression in response to the specific siRNA was confirmed by Western blotting (Fig. 8A). Consequently, RhoA activation by AngII was markedly decreased following RhoA siRNA treatment but not in cells treated with the control siRNA. As shown in Fig. 8B, the RhoA siRNA markedly reduced AngII increased CTGF mRNA levels at 1 h of stimulation from an initial value of approximately fourfold to a value of 1.5-fold of basal. The basal CTGF mRNA levels were also reduced by the RhoA siRNA. Control siRNA had no effect on these parameters.


Figure 8
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  		Fig. 8. CTGF mRNA expression in siRNA-transfected AT1R-inducible cells. AT1R-inducible cells were transfected with RhoA siRNA as described under MATERIALS AND METHODS. A: for gene downregulation assay, cells were harvested for Western blot after incubation at 37°C in a 5% CO2 incubator for 72 h after siRNA transfection. B: to determine CTGF mRNA expression, cells were induced with 10–8 M Dox at 48 h after siRNA transfection. Twenty-four hours after incubation with Dox, the AT1R-inducible cells were stimulated with either 100 nM AngII or BK or with both AngII and BK for 1 h. Total RNA was isolated. cDNA was made from 10 µg RNA and then diluted to 100 ng/µl for real-time PCR. For each time point, samples were prepared from 3 separate cultures, and each sample was run in triplicate. *P <0.05 vs. AT1R treated with only Dox. #P < 0.05 vs. control RNA interference (RNAi) in AT1R treated with only Dox. {ddagger}P < 0.05 vs. RhoA RNAi in AT1R treated with only Dox.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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CTGF mRNA expression has been followed as a target with which to analyze opposing signaling actions by AngII and BK. The IMR-90 cells express CTGF at a fairly high level (17, 29). These cells are untransformed and largely retain their in vivo phenotype. Because they are characterized by a finite lifespan of ~60 PDLs, stable transfection of the AT1R cDNA is not possible. The use of the Dox-inducible retroviral vector permitted the controllable expression of AT1R at an early PDL value of 26. Once transfection of the AT1R cDNA was accomplished, known heterotrimeric G protein-coupled signaling molecules were targeted to obtain an AT1R expression level (receptor/cell number) comparable to levels resulting from the signaling actions of BKB2R.

We observed that AngII rapidly increased CTGF mRNA to a level 3.8-fold above basal after 1 h of incubation of the AT1R-inducible cells with this effector. However, CTGF mRNA levels in cells exposed to BK did not change after 1 h and were markedly reduced by 4 h of exposure. Perhaps more importantly, the rapid (1 h) AngII-evoked CTGF mRNA expression was attenuated by ~50% when the cells were treated at the same time with BK. Determinations of CTGF protein levels generally agreed with the results obtained for the CTGF mRNA expression. AngII increased CTGF protein production in both the AT1R and AT1R-BKIC2 cells.

To elucidate the underlying mechanisms involved in the opposing regulation of CTGF expression in the human lung fibroblasts by AngII and BK, we first investigated the action of the MAPKs ERK, JNK, and p38. These kinases have been reported to participate in the regulation of CTGF mRNA expression by other effectors (15, 18). However, as illustrated here, all three MAPKs (JNK, ERK, and p38 MAP) were activated to the same extent in the human lung fibroblasts by both AngII and BK (Fig. 5A), suggesting that MAPKs don’t participate in the direct induction by AngII of CTGF mRNA expression in these cells. In confirmation of these results, specific MAPK inhibitors for ERK, p38, and JNK decreased the stimulation by AngII but also reduced basal expression of CTGF mRNA. The results in terms of inhibitor effect ratios showed that all three kinases decreased basal CTGF mRNA expression but did not participate in the AngII-evoked CTGF mRNA expression (Fig. 6B).

Activation of RhoA by AngII has been previously reported to participate in the induction of CTGF mRNA expression (15). Experiments here with the inhibitor of RhoA activation, Y-27632, supported a role for this small G protein in the AngII-evoked increase of CTGF mRNA expression in the human lung fibroblasts. Western blot analysis showed that AngII increased membrane translocation of RhoA, whereas BK dissociated the membrane binding of RhoA (Fig. 7, A and B). The inhibitor effect ratio substantiated that RhoA participates in the rapid AngII-evoked increase in CTGF mRNA expression (Fig. 6B) and that BK decreased CTGF mRNA expression over a longer, 4-h span. The involvement of RhoA in the effects of AngII and BK on CTGF mRNA expression was further confirmed with the use of RhoA siRNA. It is interesting to note that both BKB2R and AT1R increase CTGF mRNA expression in the Rat-1 fibroblasts (37) via ERK and JNK pathways (5). These observations combined with the present results illustrate that cell type and species source are crucial determinants in the agonist and antagonist actions of these two receptors.

Traditionally, the signaling pathways of the GPCR have been portrayed as entirely dependent on coupling to heterotrimeric G protein (11). We demonstrated previously that BKB2 receptor mutations involving the COOH terminus result in receptors that signal outside this traditional coupling (37). Here we show that exchanging the IC2 of the AT1R with the corresponding IC2 of the BKB2R results in a receptor whose binding to AngII is unaffected by GTP{gamma}S (Fig. 2), suggesting that this mutant receptor is not heterotrimeric G protein coupled. Additionally, this chimera failed to release ARA or cause PI turnover (Fig. 3, A and B), another indication of lack of G{alpha}i/G{alpha}q coupling, although the mutant receptor continued to signal fully in response to AngII through MAPK cascades (Fig. 5B). However, the activation of RhoA was considerably less (Fig. 7B) than in WT AT1R. With respect to CTGF mRNA expression, the cells expressing the AT1R-BKIC2 mutant receptor responded to AngII, alone, with approximately the same intensity as cells containing WT AT1R that were treated with AngII and BK simultaneously. The cells transfected with the IC2 chimera express CTGF mRNA in response to AngII in a very similar manner to cells expressing WT AT1R treated with RhoA siRNA and exposed to AngII (Fig. 8B). In summary, these results suggest that the hybrid receptor assumed signaling characteristics of both receptors and that the incorporation of the IC2 from the BKB2R enabled the AT1R to express certain characteristics of the BKB2R. More specific insights into the mechanism of RhoA activation by AT1R and its inactivation by the BKB2R remain to be determined. Thus far our studies suggest that activation of AT1R and BKB2R utilize different, receptor-specific signaling motifs. Combining the present results and past findings (35), we should like to note that the regions coupling to G protein differ in AT1R and BKB2R. These observations demonstrate that even closely related GPCR such as the AT1 and BKB2 do not employ the same regions for similar actions. Therefore, care must be taken when applying results obtained with one GPCR to another GPCR.

We have identified a defining point in the opposing AngII- and BK-regulated signaling. Our results demonstrate that BK attenuates the rapid AngII-dependent change in CTGF mRNA expression. The present results and the future investigations based on these results should provide information that ultimately could ameliorate pathologic malfunctions brought forth by AngII via AT1R.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported in part by National Institutes of Health Grants HL-25776 and AG-00115.


   ACKNOWLEDGMENTS
 
We thank Dr. Hidesaburo Hanafusa (Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan) for providing the inducible retroviral expression systems. We also thank Dr. Kathrin H. Kirsch (Department of Biochemistry Boston University School of Medicine) for experimental advice and Dr. Herbert Kagan for help in preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Polgar, Dept. of Biochemistry, Boston Univ. School of Medicine, Boston, MA 02118 (e-mail: peterp{at}bu.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.


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