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(Circulation Research. 1997;81:672-678.)
© 1997 American Heart Association, Inc.

Articles

Activated RhoA Stimulates c-fos Gene Expression in Myocardial Cells

Tomomi Ueyama, Tsuyoshi Sakoda, Seinosuke Kawashima, Eiji Hiraoka, Ken-ichi Hirata, Hozuka Akita, , Mitsuhiro Yokoyama

From the First Department of Internal Medicine, Kobe (Japan) University School of Medicine.

Correspondence to Mitsuhiro Yokoyama, MD, The First Department of Internal Medicine, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

	Abstract

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Abstract Rho regulates various cell functions, including cell morphology and motility. However, the functional role of Rho on the signaling pathway in myocardial cells (MCs) is unknown. In the present study, we attempted to explore the mode of Rho action for c-fos gene expression in MCs. Expression of the c-fos promoter/enhancer linked to the luciferase reporter gene (c-fos luciferase) was stimulated by the wild type of RhoA and the point-mutated active form of RhoA (RhoA Val14) but not the biologically inactive effector domain mutant of RhoA. Rho GDP dissociation inhibitor inhibited the action of RhoA on c-fos luciferase expression. The deletion analysis revealed that the c-fos serum response element (SRE) and the 12-O-tetradecanoylphorbol-13-acetate response element (TRE) mainly account for c-fos luciferase expression by RhoA Val14. The c-fos SRE mutant, which contains an intact binding site for the serum response factor but lacks the ternary complex factor binding site, was activated by RhoA Val14. The action of RhoA Val14 on c-fos luciferase expression was not inhibited by downregulation of protein kinase C, protein kinase C inhibitors, or tyrosine kinase inhibitors. These results indicate that activated RhoA stimulates c-fos gene expression through the c-fos SRE and TRE and that the signaling pathway from activated RhoA to the c-fos promoter/enhancer is independent of these inhibitor-sensitive pathways in MCs.

Key Words: RhoA • myocardial cell • c-fos promoter/enhancer

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Families of small GTP-binding proteins regulate various cell functions, including cell growth, cell differentiation, gene expression, vesicle transport, cell morphological change, and smooth muscle contraction.^{1 2} Among the many small GTP-binding proteins, the Rho family, consisting of three members (RhoA, RhoB, and RhoC), has been reported to regulate the actomyosin system. Rho also mediates cellular processes, such as stimulus-evoked cell adhesion and motility.^{3 4 5 6} It has been also reported that Rho regulates cytokinesis of the fertilized egg and G₁- to S-phase progression in the cell cycle.^{7 8} Furthermore, overexpression of RhoA in fibroblasts reduces serum dependence for cell growth and is tumorigenic when inoculated into nude mice.⁹ It is suggested that Rho regulates not only cell morphology and motility but also cell growth and tumorigenesis through oncogene-like activity.

The c-fos gene is the most frequently studied member of the cellular immediate-early genes, whose transcription is activated rapidly and transiently within minutes of growth stimulation in a variety of cell types.^{10 11} These immediate-early genes are themselves "third messengers" of intracellular signaling cascades, encoding proteins in transcriptional regulatory complexes. c-fos gene expression is rarely detected in MCs of the normal adult rat or mouse. However, hemodynamic stress and ₁-adrenergic agonists, both of which are known to produce cardiac hypertrophy, rapidly provoke c-fos gene expression in MCs.^{12 13} Thus, the c-fos gene may play an important role in myocardial signal transduction.

Recently, it has been reported that RhoA regulates c-fos promoter/enhancer activity through the c-fos SRE in NIH 3T3 cells¹⁴ and that RhoA is involved in G_q and ₁-adrenergic receptor signaling in MCs.¹⁵ Although Rho and Rho GDI are known to be present in the heart,^{16 17} the exact mechanism for signal transduction of Rho has not been elucidated in MCs. Since the c-fos promoter/enhancer region contains major inducible cis-acting elements, ie, SRE, CRE, and TRE, we attempted to explore the signaling pathway by which Rho activates the c-fos promoter/enhancer, including the c-fos SRE, CRE, and TRE in MCs. The present study demonstrates for the first time that the c-fos promoter/enhancer, the c-fos SRE, and the TRE are stimulated by activated RhoA in MCs. Particularly in the c-fos SRE, the SRF binding site rather than the TCF binding site mediates the effect of activated RhoA. Furthermore, the signaling pathway from activated RhoA to the c-fos promoter/enhancer is insensitive to PKC inhibitors or tyrosine kinase inhibitors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Sprague-Dawley rats were purchased from Charles River (Osaka, Japan). The standard culture medium was DMEM/F-12 from GIBCO-BRL. Ro 31-8220, a selective PKC inhibitor, was from Calbiochem-Novabiochem Corp. For luciferase assay, a luciferase assay kit (Toyo Ink Mfg Co, Ltd) was used. Other materials and chemicals were obtained from commercial sources.

Plasmids
The wild type of RhoA, RhoA Val14 (the mutant of Gly to Val at codon 14), which is a point-mutated active form of RhoA,⁶ RhoA Ala37 (the mutant of Thr to Ala at codon 37), which is a biologically inactive effector domain mutant,¹⁸ and Rho GDI constructed into the pcDSR expression plasmid were kindly provided by Prof Y. Takai (Osaka University, Suita, Japan). The c-fos promoter/enhancer linked to the luciferase gene (c-fos luciferase), SRE CAT, CRE CAT, TRE CAT, pTKCAT, and PKCß expression plasmids were generous gifts from Prof K. Kaibuchi (Nara Institute of Science and Technology, Ikoma, Japan).^{19 20 21} The 445-bp fragment of the c-fos promoter/enhancer (positions -404 to +41 in the c-fos gene) was cloned into pSVO-luciferase to construct c-fos luciferase. The deletion fragment (-323 to +41) of the c-fos promoter/enhancer, which contains the c-fos SRE and TRE, was cloned into pSVO-luciferase to construct -323 c-fos luciferase. The deletion fragment (-305 to +41) of the c-fos promoter/enhancer, which contains the c-fos TRE but lacks the c-fos SRE, was cloned into pSVO-luciferase to construct -305 c-fos luciferase. The deletion fragment (-293 to +41) of the c-fos promoter/enhancer, which lacks both the c-fos SRE and TRE, was cloned into pSVO-luciferase to construct -293 c-fos luciferase. The deletion fragment (-323 to +41 without -300 to -294) of the c-fos promoter/enhancer, which contains the c-fos SRE but lacks the c-fos TRE, was cloned into pSVO-luciferase to construct -323 -300/-294 c-fos luciferase. These deletion fragments were made by use of polymerase chain reaction. Nucleotide sequencing confirmed the structure of each deletion fragment. The c-fos SRE, the CRE, and the TRE (5'-CAGGATGTCCATATTAGGACATCTG-3', 5'-GAGC CCGTGACGTTTACAC-3', and 5'-ATGAGTCAGCGCG GATC-3', respectively) were synthesized by a DNA synthesizer. SRE CAT, CRE CAT, and TRE CAT were synthesized by introducing a synthetic c-fos SRE, three synthetic CREs, and three synthetic TREs, respectively, upstream from the IL3 promoter (from positions -50 to +10 in the mouse IL3 gene) fused to the CAT sequence.^{19 20} According to the previous reports,^{22 23 24 25} SRE.L (5'-CTGTATGTCCATATTAGGACATCTG-3'), a derivative of the c-fos SRE that contains the intact binding site for SRF but lacks the TCF binding site, SRE.M (5'-CAGGATGTC CCAATCGGGACATCTG-3'), a derivative of the c-fos SRE that contains the intact binding site for TCF but lacks the SRF binding site, SRE.LM (5'-CTGTATGTCCCAATCGGGACATCTG-3'), a derivative of the c-fos SRE that lacks both TCF and SRF binding sites, and TRE.M (5'-AGGAGTTGGCGCGGATC-3'), a derivative of the TRE that lacks the AP-1 binding site, were synthesized by a DNA synthesizer. SRE.L CAT, SRE.M CAT, SRE.LM CAT, and TRE.M CAT were synthesized by introducing a synthetic SRE.L, a synthetic SRE.M, a synthetic SRE.LM, and three synthetic TRE.Ms, respectively, upstream from the IL3 promoter fused to the CAT sequence. The Pvu II–Pst I fragment (from positions -197 to +18) of the promoter region of the thymidine kinase gene of herpes simplex virus was cloned upstream from the CAT sequence to construct pTKCAT. pcDSR PKCß, the expression plasmid for a constitutively activated mutant of PKC-ß, was constructed by deleting the coding region for amino acids 6 to 159 of PKCß (25% of the V₁ and 90% of the C₁ region) into the pcDSR expression plasmid.²¹ pTKCAT or the RSV promoter linked to the luciferase reporter gene (RSV luciferase) was used as an internal control to standardize the transfection efficiency.

Cell Culture and Transfection
Single-cell cultures were prepared from neonatal rat hearts as described previously with slight modifications.¹³ MCs were distributed to 60-mm dishes at a density of 6.5x10⁵ cells per dish. The population of nonmyocytes was <10% of the total cell population. The culture medium was DMEM/F-12 supplemented with 5% calf serum. The medium was changed 24 hours after seeding the cells to serum-free medium, which is DMEM/F-12 containing 0.1% bovine serum albumin, ITS (10 µg/mL insulin, 10 µg/mL transferrin, and 10 ng/mL selenious acid), and 30 mmol/L HEPES at pH 7.5. MCs in duplicate dishes were transfected with reporter plasmids by using the modified calcium phosphate precipitation method as described previously.²⁶ The DNA/CaPO₄ prepicitates in each dish (5.0 mL) contained the following if not specifically identified: 5.0 µg of c-fos luciferase, SRE CAT, SRE.L CAT, SRE.M CAT, SRE.LM CAT, CRE CAT, TRE CAT, or TRE.M CAT reporter plasmid; 4.0 µg of pcDSR PKCß, pcDSR RhoA, pcDSR RhoA Val14, pcDSR RhoA Ala37, and/or pcDSR Rho GDI; 4.0 µg of pTKCAT or 0.1 µg of RSV luciferase as an internal control for variations in transfection efficiency; and variable amounts of pcDSR plasmid vector to adjust total DNA. Transfection was carried out using 17 µg of total DNA. Precipitates were removed after 2 hours, and then the cells were maintained in serum-free medium for 48 hours. For the last 24-hour incubation, Ro 31-8220 or herbimycin A was added to inhibit PKC or tyrosine kinase, respectively.

Luciferase and CAT Assays
The transfected MCs were harvested by scraping. Supernatants were collected after lysis by three cycles of freeze and thaw in 100 µL of lysis buffer including the luciferase assay kit. For luciferase assay, an aliquot of supernatant was added to a buffer containing luciferin in accordance with the luciferase assay kit. Luciferase expression was measured by a luminometer (Bio-Orbit Oy). For the CAT assay, the lysates were incubated in 0.2 mL of a reaction mixture containing 50 nmol/L [¹⁴C]chloramphenicol (0.5 µCi) and 2 mmol/L acetyl coenzyme A in 250 mmol/L Tris-HCl at pH 7.8 for 2 hours at 37°C. CAT expression was assayed by thin-layer chromatography as described.^{19 20 27} The radioactivity was analyzed using a Fujix bioimaging analyzer (BAS2000). Under these conditions, both assays were within the linear range. c-fos luciferase expression in MCs transfected with each expression plasmid and/or treated with each agent was divided by CAT expression in MCs cotransfected with pTKCAT in the same experiment. Similarly, SRE, SRE.L, SRE.M, SRE.LM, CRE, TRE, or TRE.M CAT expression in MCs transfected with pcDSR RhoA Val14 was divided by luciferase expression in MCs cotransfected with RSV luciferase. Luciferase and CAT expressions are expressed relative to the basal condition without stimulation (control) value (1.0). There was no difference in CAT expression among MCs cotransfected with pTKCAT and other expression plasmids. CAT expressions derived from pTKCAT in MCs cotransfected with RhoA expression plasmids or PKCß expression plasmid divided by CAT expressions derived from pTKCAT in MCs transfected with the expression vector (pcDSR) in the same experiment were as follows: pcDSR RhoA, 1.0±0.2; pcDSR RhoA Val14, 1.0±0.0; pcDSR RhoA Ala37, 1.1±0.0; pcDSR Rho GDI, 1.0±0.1; pcDSR RhoA with pcDSR Rho GDI, 0.9±0.2; and pcDSR PKCß, 1.1±0.1 (mean±SEM, n=3, P=NS). Similarly, there was no difference in CAT expression derived from pTKCAT between control and TPA-treated MCs (treated/control ratio, 1.1±0.0 [mean±SEM]; n=3; P=NS). There was no difference in CAT expression derived from pTKCAT between control and Ro 31-8220–treated MCs (treated/control ratio, 1.0±0.1 for 100 nmol/L Ro 31-8220 and 0.9±0.1 for 1 µmol/L Ro 31-8220 (mean±SEM); n=3; P=NS). Similarly, there was no difference in CAT expression derived from pTKCAT between control and herbimycin A–treated MCs (treated/control ratio, 1.0±0.1 for 100 nmol/L herbimycin A and 0.8±0.1 for 1 µmol/L herbimycin A (mean±SEM); n=3; P=NS). Furthermore, there was no difference in RSV luciferase expression between the expression vector (pcDSR) and RhoA Val14 expression plasmids/transfected MCs (RhoA Val14/expression vector [pcDSR] ratio, 1.1±0.1 [mean±SEM]; n=9; P=NS).

Downregulation of PKC by Treatment With TPA in MCs
MCs were transfected with the c-fos luciferase and/or the pcDSR RhoA Val14 by addition of DNA/CaPO₄ solution. After an 8-hour incubation, MCs were then washed with 5 mL of PBS and incubated in 5 mL of serum-free medium with 1 µmol/L TPA for 48 hours at 37°C. The cells were washed with 5 mL of PBS twice and incubated in 5 mL of serum-free medium for 9 hours with or without 1 µmol/L TPA. The luciferase and CAT expressions were then assayed.

Statistical Procedures
All values were expressed as mean±SEM. Statistical evaluation of the data was performed by Student's t test for unpaired observations. When more than two groups were compared, the significance of the difference between group means was analyzed by one-way ANOVA and the Bonferroni test for samples. Values were considered statistically significant at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activated RhoA Stimulates c-fos Promoter/Enhancer in MCs
To examine the effect of RhoA on the c-fos promoter/enhancer, pcDSR RhoA or pcDSR RhoA Val14 was transfected with c-fos luciferase and pTKCAT into MCs. c-fos luciferase expression was activated by the wild type of RhoA and RhoA Val14 in time-dependent (data not shown) and dose-dependent (Fig 1) manners. To confirm that this activation of c-fos luciferase expression is mediated by the action of RhoA, we transfected pcDSR Rho GDI+pcDSR RhoA or pcDSR RhoA Ala37 alone into MCs (Fig 2). Although c-fos luciferase expression was also activated by the wild type of RhoA (3.7±0.1-fold, P<.01 compared with control), Rho GDI inhibited the wild type of RhoA-activated c-fos luciferase expression (1.7±0.3-fold, P<.01 compared with the wild type of RhoA alone). Rho GDI alone did not activate or reduce c-fos luciferase expression (1.3±0.1-fold). Furthermore, RhoA Ala37 did not activate c-fos luciferase expression (1.4±0.2-fold, P<.01 compared with the wild type of RhoA alone). These results indicate that activated RhoA stimulates the c-fos promoter/enhancer in MCs.

View larger version (16K): [in this window] [in a new window]   Figure 1. Effect of the wild type of RhoA and RhoA Val14 on c-fos luciferase expression. A line graph shows the effect of the wild type of RhoA () and RhoA Val14 () on c-fos luciferase expression in a dose-dependent manner. Cotransfection of c-fos luciferase with various doses (0.1 to 8.0 µg) of pcDSR RhoA or pcDSR RhoA Val14 into cultured neonatal MCs was carried out. After 48 hours, the cells were harvested. The values are expressed relative to control value (1.0), which is luciferase expression derived from c-fos luciferase in the cells cotransfected without pcDSR RhoA or RhoA Val14, and represent mean±SEM of three to five independent experiments. Luciferase expression was assayed as described in "Materials and Methods." *P<.01 compared with control.

View larger version (27K): [in this window] [in a new window]   Figure 2. Bar graph showing the effect of RhoA and/or Rho GDI on c-fos luciferase expression. Cotransfection of c-fos luciferase with pcDSR RhoA, pcDSR RhoA+pcDSR Rho GDI, pcDSR Rho GDI, pcDSR RhoA Ala37, or pcDSR RhoA Val14 into cultured neonatal MCs was carried out. After 48 hours, the cells were harvested. Luciferase expression was assayed as described in "Materials and Methods." The values are expressed relative to the control value (1.0) and represent mean±SEM of three to five independent experiments. *P<.01 compared with control; **P<.01 compared with the wild type of RhoA alone.

Activated RhoA Stimulates the c-fos Promoter/Enhancer Through the-c-fos SRE and TRE in MCs
To determine the elements responsible for the c-fos promoter/enhancer stimulated by activated RhoA, we performed a deletion analysis of the c-fos promoter/enhancer in MCs. RhoA Val14 activated -323 c-fos luciferase expression as well as the native c-fos luciferase expression (Fig 3). In -305 c-fos luciferase, -293 c-fos luciferase, and -323 -300/-294 c-fos luciferase, the responses to RhoA Val14 were reduced. These results indicated that both the c-fos SRE and TRE are necessary for the stimulation of the c-fos promoter/enhancer by activated RhoA in MCs.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effect of the c-fos SRE and TRE on RhoA Val14–activated c-fos luciferase expression. Cotransfection of c-fos luciferase, -323 c-fos luciferase, -305 c-fos luciferase, -293 c-fos luciferase, or -323 -300/-294 c-fos luciferase with pcDSR RhoA Val14 (4.0 µg) into cultured neonatal MCs was carried out. After 48 hours, the cells were harvested. Luciferase expression was assayed as described in "Materials and Methods." The values are expressed relative to the control values (1.0) of transfected c-fos luciferase and represent mean±SEM of three independent experiments.

Activated RhoA Stimulates the c-fos SRE and the TRE in MCs
To confirm the stimulation of the c-fos promoter/enhancer by activated RhoA, we investigated the individual cis-acting elements, the c-fos SRE, the CRE, and the consensus sequence of TRE. RhoA Val14 activated SRE CAT and TRE CAT expressions (2.7±0.2-fold [P<.01] and 2.1±0.3-fold [P<.05] compared with control, respectively) but not CRE CAT expression (1.1±0.1-fold) (Fig 4A). Furthermore, to confirm that RhoA Val14 actually activates the c-fos SRE and the TRE, we transfected pcDSR RhoA Val14 with SRE.L CAT, SRE.M CAT, SRE.LM CAT, or TRE.M CAT into MCs (Fig 4B). By following the methods of previous studies,^{22 23 24 25} we constructed SRE.L CAT, SRE.M CAT, SRE.LM CAT, and TRE.M CAT. SRE.L CAT and SRE.M CAT have the SRE mutant lacking the TCF binding site and the SRF binding site, respectively, and SRE.LM CAT has the SRE mutant lacking both TCF and SRF binding sites. RhoA Val14 activated SRE.L CAT expression (2.8±0.3-fold, P<.01 compared with control) but not SRE.M CAT and SRE.LM CAT expressions (1.2±0.1-fold and 1.0±0.0-fold, respectively). SRE.L CAT and SRE CAT expressions were almost equally activated by RhoA Val14. Furthermore, RhoA Val14 did not activate TRE.M CAT expression (1.0±0.1-fold). These results indicate that the signaling pathway from activated RhoA to the c-fos SRE is linked to SRF but not TCF and that activated RhoA stimulates the TRE in MCs.

View larger version (41K): [in this window] [in a new window]   Figure 4. Effect of RhoA Val14 on SRE, CRE, and TRE CAT expression. A, Bar graphs (top) and autoradiograms of representative transfection experiments (bottom) showing the effect of RhoA Val14 on SRE, CRE, and TRE CAT expression. Cotransfection of SRE, CRE, or TRE CAT with pcDSR RhoA Val14 was carried out. The values are expressed relative to the control value (1.0) and represent mean±SEM of five independent experiments. *P<.05 and **P<.01 compared with control. B, Bar graphs (top) and autoradiograms of representative transfection experiments (bottom) showing the effect of RhoA Val14 on SRE.L, SRE.M, SRE.LM, and TRE.M CAT expression. Cotransfection of SRE.L, SRE.M, SRE.LM, or TRE.M CAT with pcDSR RhoA Val14 was carried out. After 48 hours, the cells were harvested. CAT expression was assayed as described in "Materials and Methods." The values are expressed relative to the control value (1.0) and represent mean±SEM of three independent experiments. CM and AcCM represent chloramphenicol and its acetylated forms, respectively. **P<.01 compared with control.

Effect of PKC or Tyrosine Kinase in the Signaling Pathway From Activated RhoA to the c-fos Promoter/Enhancer in MCs
Since activation of the c-fos SRE and the TRE is known to be linked to both PKC-dependent and PKC-independent pathways,^{19 28 29 30 31 32} we investigated the role of PKC in the action of RhoA in MCs. PKC was downregulated by treatment with 1 µmol/L TPA for 48 hours after transfection of c-fos luciferase with pcDSR RhoA Val14. Without pretreatment with TPA for 48 hours after transfection, RhoA Val14 activated c-fos luciferase expression (4.5±0.3-fold, P<.01 compared with control that was not pretreated with TPA). Addition of TPA without downregulation of PKC also activated c-fos luciferase expression (6.2±0.2-fold, P<.01 compared with control that was not pretreated with TPA) (Fig 5A). c-fos luciferase expression after pretreatment with TPA for 48 hours after transfection did not return completely to the basal level (untreated control). c-fos luciferase expression after pretreatment with TPA was about twice that without pretreatment. Further TPA stimulation after the pretreatment period did not cause any additional activation of c-fos luciferase expression because of downregulated PKC. c-fos luciferase expression activated by RhoA Val14 was not inhibited by pretreatment with TPA (4.6±0.4-fold, P<.01 compared with control that was pretreated with TPA). Furthermore, we investigated the effect of selective PKC inhibitors on the action of RhoA Val14 in MCs (Fig 5B). Ro 31-8220 belongs to a series of bisindolylmaleimides that selectively inhibit PKC, including PKC subtypes , ß, , and .³³ Ro 31-8220 inhibited the action of PKCß on c-fos luciferase expression in a dose-dependent manner. However, Ro 31-8220 did not inhibit the effect of RhoA Val14 on c-fos luciferase expression. In addition, GF 109203X, which inhibited the action of PKCß on c-fos luciferase expression, did not inhibit RhoA Val14–activated c-fos luciferase expression (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 5. Effect of PKC or tyrosine kinase on RhoA Val14–activated c-fos luciferase expression. Cotransfection of c-fos luciferase with pcDSR RhoA Val14 (4.0 µg) into cultured neonatal MCs was carried out. A, PKC was downregulated by treatment with 1 µmol/L TPA for 48 hours after transfection of c-fos luciferase with or without pcDSR RhoA Val14. After treatment with TPA or vehicle for 48 hours after transfection, the cells were subsequently stimulated with TPA (1 µmol/L) or with vehicle for 9 hours. After the stimulation, the cells were harvested. The values are expressed relative to the control values (1.0) treated with or without TPA and represent mean±SEM of three independent experiments. *P<.01 compared with control without pretreatment with TPA; **P<.01 compared with control with pretreatment with TPA. B, For the last 24-hour incubation, Ro 31-8220 or vehicle was added. After the Ro 31-8220 treatment, the cells were harvested. Luciferase expression was assayed as described in "Materials and Methods." The values are expressed relative to the control values (1.0) treated without Ro 31-8220 and represent mean±SEM of three independent experiments. *P<.01 compared with control; **P<.01 compared with PKCß alone; and P<.05 compared with control. C, For the last 24-hour incubation, herbimycin A or vehicle was added. Basic fibroblast growth factor (bFGF, 25 ng/mL) was added for the last 9-hour incubation. After the treatment of herbimycin A, the cells were harvested. Luciferase expression was assayed as described in "Materials and Methods." The values are expressed relative to the control values (1.0) treated without herbimycin A and represent mean±SEM of three independent experiments. *P<.01 compared with control.

Next, we investigated the role of tyrosine kinase on the action of RhoA in MCs. Herbimycin A, which is a tyrosine kinase inhibitor, did not inhibit RhoA Val14–activated c-fos luciferase expression (Fig 5C). Genistein (10 µmol/L for the last 24-hour incubation) also did not inhibit RhoA Val14–activated c-fos luciferase expression (data not shown). These results suggest that the tyrosine kinase inhibitor–sensitive pathway is not involved in the signaling pathway from activated RhoA to the c-fos promoter/enhancer in MCs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We demonstrate in the present study the role of Rho in the signaling pathway of c-fos gene expression in MCs by analyzing the c-fos promoter/enhancer region. Activated RhoA stimulated the c-fos promoter/enhancer in MCs. The deletion mutant of the c-fos promoter/enhancer, which lacks the c-fos SRE and/or TRE, decreased its response to activated RhoA in MCs. Furthermore, we showed that the individual c-fos SRE and consensus sequence of TRE were stimulated by activated RhoA in MCs.

In the present study, the deletion mutant of the c-fos promoter/enhancer, which lacks both the c-fos SRE and TRE, did not completely abolish luciferase expression by RhoA Val14. In addition, in our assay system, this deletion mutant also did not abolish luciferase expression by TPA. These results are consistent with the previous report demonstrating that the deletion mutant of the c-fos promoter/enhancer, which lacks both the c-fos SRE and TRE, did not abolish CAT expression by epidermal growth factor or TPA.³⁴ Our results may indicate that both the c-fos SRE and TRE are necessary but not sufficient for the stimulation of the c-fos promoter/enhancer by activated RhoA in MCs. Other RhoA-mediating sites in the c-fos promoter/enhancer may exist.

The c-fos SRE is a regulatory sequence required for serum-induced and growth factor–induced c-fos transcriptional activation. This response is mediated by TCF and/or SRF, which are known to bind the SRE both in vitro and in vivo.³⁵ We have also shown that RhoA Val14 activates SRE.L CAT expression but not SRE.M CAT and SRE.LM CAT expressions and that SRE.L CAT expression activated by RhoA Val14 is almost equal to SRE CAT expression. Our results indicate that the signaling pathway from activated RhoA to the SRE may link SRF, but not TCF. These results are consistent with the previous report demonstrating that in NIH 3T3 cells two kinds of signaling pathways converge at the SRE: one links TCF activity to MAP kinase activation, and the other links SRF activity to the Rho family–induced signals.¹⁴

The TRE is defined as an AP-1 binding site. AP-1 activity appears to be regulated by the following signaling pathways: the TPA-inducible PKC pathway,³⁶ PKC-independent pathways, including cell surface tyrosine kinase and Ras proteins,^{19 31} and another pathway that is independent of PKC and PKA.³² Recently, it has been reported that in Saccharomyces cerevisiae PKC1 is a downstream target of RHO1.³⁷ Therefore, to investigate whether the signaling pathway from activated RhoA to the c-fos promoter/enhancer is mediated by PKC, we carried out the experiment of PKC downregulation by TPA and that using selective PKC inhibitors. We demonstrated that neither PKC downregulation by TPA nor selective PKC inhibitors (such as Ro 31-8220 and GF 109203X) inhibited RhoA Val14–activated c-fos luciferase expression in MCs. The conventional PKC subfamily (PKC and PKCß1), novel PKC subfamily (PKC, PKC, and PKC), and atypical PKC subfamily (PKC) are present in rat cultured neonatal MCs.³⁸ GF 109203X has been shown to act as a competitive inhibitor of ATP for the conventional PKC subfamily (PKC, PKCß, and PKC).³⁹ Ro 31-8220 is an inhibitor of not only the conventional PKC subfamily (PKC, PKCß, and PKC) but also PKC.³³ PKC is neither activated by phorbol esters nor downregulated by TPA treatment.⁴⁰ Therefore, our study has demonstrated that at least phorbol ester–sensitive or bisindolylmaleimide (Ro 31-8220 and GF 109203X)–sensitive PKC is not involved in the downstream effector of RhoA on the activation of the c-fos promoter/enhancer in MCs.

It has been reported that Rho regulates the signal transduction pathway of tyrosine kinase in Swiss 3T3 cells.⁴¹ Therefore, we further investigated whether the signaling pathway from activated RhoA to the c-fos promoter/enhancer is mediated by tyrosine kinase. Our results have indicated that the signaling pathway from activated RhoA to the c-fos promoter/enhancer is insensitive to tyrosine kinase inhibitors (herbimycin A and genistein) in MCs. In COS-7 cells, It has been recently reported that activated RhoA forms a complex with PKN, which is a serine-threonine protein kinase, and activates it.^{42 43} Although the functional role of PKN is not clear in MCs, there is a possibility that RhoA activates c-fos gene expression through the PKN pathway. Furthermore, Rho family GTPases Rac and Cdc42 are shown to regulate the activity of the c-Jun amino-terminal kinase through p21-activated kinase 1 without affecting PKC, PKA, or "classical" MAP kinase in COS-7 cells and NIH 3T3 cells.^{44 45 46} We have shown here that the PKC inhibitor–or tyrosine kinase inhibitor–sensitive pathway does not work on the downstream of activated RhoA. However, it is suggested that Rho works on the downstream of PKC in platelets and lymphocytes.^{4 5} Therefore, in MCs RhoA may act as the downstream effector molecule of PKC, as in platelets and lymphocytes, or operate in quite different pathways, including PKC and PKN as its downstream molecules. Further experiments are required to examine the mechanisms.

In summary, activated RhoA stimulated the c-fos promoter/enhancer in MCs in the transient transfection assay. This stimulation by activated RhoA was mediated by the c-fos SRE and TRE. At the c-fos SRE, the signaling pathway of activated RhoA linked to the SRF binding site. In addition, the signaling pathway from activated RhoA to the c-fos promoter/enhancer was insensitive to PKC inhibitors and tyrosine kinase inhibitors. The potentially important role of Rho in myocardial signal transduction and myocardial hypertrophy should be explored further.

	Selected Abbreviations and Acronyms

 

AP-1 = activator protein-1 CAT = chloramphenicol acetyltransferase CRE = Ca2+/cAMP response element GDI = GDP dissociation inhibitor GF 109203X = 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide IL3 = interleukin 3 MC = myocardial cell PKA, PKC, PKN = protein kinase A, C, and N pTKCAT = thymidine kinase CAT Ro 31-8220 = 3-[1-3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide methane sulfonate RSV = Rous sarcoma virus SRE = serum response element SRF = serum response factor TCF = ternary complex factor TPA = 12-O-tetradecanoylphorbol 13-acetate TRE = TPA response element

	Acknowledgments

 
This study was supported by grants-in-aid for scientific research (Nos. 0627410 and 08670794) from the Ministry of Education, Science, and Culture (1996), a grant from the Japan Cardiovascular Research Foundation, a grant from the Study Group of Molecular Cardiology, and a grant from the Kanae Foundation of Research for New Medicine. We greatly appreciate the supply of plasmids from Prof Y. Takai (Osaka University, Suita, Japan) and Prof K. Kaibuchi (Nara Institute of Science and Technology, Ikoma, Japan). We are grateful to Seiko Tsutsui for her skillful technical assistance.

Received June 11, 1996; accepted August 15, 1997.

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