蛋白激酶C-细胞外信号调节激酶12通路介导精氨酸升压素对成年大鼠心肌成纤维细胞的促增殖作用

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用。我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。本研究旨在进一步观察AVP是否对成年大鼠CFs增殖具有促

Acta Physiologica Sinica, June 25, 2008, 60 (3): 333-340

333

Research Paper

Arginine vasopressin stimulates proliferation of adult rat cardiac fibroblastsvia protein kinase C-extracellular signal-regulated kinase 1/2 pathway

HE Yan-Ping, ZHAO Lian-You*, ZHENG Qiang-Sun, LIU Shao-Wei, ZHAO Xiao-Yan, LU Xiao-Long,NIU Xiao-Lin

Department of Cardiology, Tangdu Hospital, the Fourth Military Medical University, Xi’an 710038, China

Abstract: Arginine vasopressin (AVP), a neurohormone and hemodynamic factor implicated in the pathophysiology of hypertensionand congestive heart failure, can also act as a growth-stimulating factor. Our previous work demonstrated that AVP is a mitogen forneonatal rat cardiac fibroblasts (CFs). In the present study, we extended our investigations to adult rat CFs to explore whether AVPcould induce adult rat CF proliferation and, if so, to identify the mechanism involved. Adult rat CFs were isolated, cultured andsubjected to AVP treatment. DNA synthesis and cell cycle distribution were analyzed by [3H]-thymidine incorporation and flowcytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK1/2) activity was measured by in vitro kinase assay using myelin basicprotein (MBP) as a substrate. Protein expressions of total- and phospho-ERK1/2, p27Kip1, cyclins D1, A, E were assessed by Westernblot. The results showed that AVP stimulated DNA synthesis in adult rat CFs, and the effect was abolished by a V1 receptor antagonist,d(CH2)5[Tyr2(Me), Arg8]-vasopressin (0.1 μmol/L), but not by a V2 receptor antagonist, desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (0.1 µmol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator,phorbol 12-myristate 13-acetate (PMA, 30 nmol/L, 5 min), but abolished by depletion of PKC via chronic PMA incubation (2.5 μmol/L, 24 h). In addition, AVP down-regulated protein expression of p27Kip1, increased protein expressions of cyclins D1, A and E, andinduced cell cycle progression from G0/G1 into S stage. Inhibition of ERK1/2 activation by PD98059 (30 μmol/L) abolished the effectof AVP on DNA synthesis, protein expressions of p27Kip1, cyclins D1, A and E as well as cell cycle progression. These results suggestthat AVP is also a growth factor for adult rat CFs. The mitogenic effect of AVP is mediated via V1 receptors and PKC-ERK1/2 pathway.Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27Kip1 and cyclins D1, A and E, which lie downstream ofERK1/2 activation, and induces cell cycle progression in adult rat CFs.

Key words: arginine vasopressin; V1 receptor; extracellular signal-regulated kinase 1/2; protein kinase C; p27Kip1; cyclin; cardiacfibroblast

蛋白激酶C-细胞外信号调节激酶1/2通路介导精氨酸升压素对成年大鼠　心肌成纤维细胞的促增殖作用

何燕萍，赵连友*，郑强荪，刘少伟，赵晓燕，陆晓龙，牛晓琳

第四军医大学唐都医院心血管内科，西安 710038

摘 要：精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子，同时，它还具有直接的生长刺激作用。我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。本研究旨在进一步观察AVP是否对成年大鼠CFs具有促增殖作用，并探计其机制。采用组织块法培养成年大鼠CFs，用[3H]-TdR掺入法和流式细胞仪方法观察AVP作用下CFs的DNA合成和细胞周期分布。根据特异性底物髓磷脂基质蛋白(myelin basic protein,MBP)的磷酸化水平测定细胞外信号调节激酶1/2 (extracellular signal-regulated kinase 1/2, ERK1/2)的活性。用Western blot检测ERK1/2的磷酸化和p27Kip1、细胞周期蛋白D1、A、E的表达。结果显示，AVP (0.1 μmol/L)可促进成年大鼠CFs的DNAReceived 2008-01-15 Accepted 2008-02-25

This work was supported by the Natural Science Foundation of Shaanxi Province, China (No. 2004C2-21). *Corresponding author. Tel: +86-29-84777656; Fax: +86-29-84777656; E-mail: LianY_Zhao@

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用。我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。本研究旨在进一步观察AVP是否对成年大鼠CFs增殖具有促

334Acta Physiologica Sinica, June 25, 2008, 60 (3): 333-340

合成，该作用可被V1受体拮抗剂d(CH2)5[Tyr2(Me), Arg8]-vasopressin (0.1 μmol/L)阻断，而不受V2受体拮抗剂desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (0.1 μmol/L)的影响。AVP可激活ERK1/2，用蛋白激酶C (protein kinase C, PKC)激动剂佛波酯(phorbol 12-myristate 13-acetate, PMA, 30 nmol/L, 5 min)急性刺激可模拟该作用，而PMA持续慢性作用(2.5 μmol/L, 24 h)耗竭PKC后则抑制AVP对ERK1/2的激活。AVP可抑制p27Kip1的蛋白表达，升高细胞周期蛋白D1、A和E的表达，同时促进细胞周期由G0/G1期进入S期。ERK1/2抑制剂PD98059 (30 μmol/L)阻断AVP对DNA合成、p27Kip1、细胞周期蛋白D1、A和E蛋白表达的作用，并抑制细胞周期进程。以上结果表明，AVP可促进成年大鼠CFs增殖，该作用由V1受体和PKC-ERK1/2通路介导。AVP可通过ERK1/2调控p27Kip1、细胞周期蛋白D1、A和E的表达，从而促进成年大鼠CFs的细胞周期进程。关键词：精氨酸升压素；V1受体；细胞外信号调节激酶1/2；蛋白激酶C；p27Kip1；细胞周期蛋白；心肌成纤维细胞中图分类号：Q25

Adverse cardiac remodeling is characterized by intersti-tial fibrosis, cardiac myocyte death or hypertrophy andproliferation of cardiac fibroblasts (CFs)[1-3]. Despite itsadaptive nature, cardiac hypertrophy is independentlyassociated with increased risks in cardiovascular morbidityand mortality. It has been critically implicated in thepathophysiology of chronic congestive heart failure, whichfeatures a worsening neurohormonal profile. As a neurohor-mone and hemodynamic factor, elevated plasma argininevasopressin (AVP) has been documented in both hypertensionand congestive heart failure[4,5]. Sustained and chronic acti-vation of AVP system, as triggered by congestive heart failure,leads to progressive cardiac remodeling[6]. AVP can also actas a potent growth-stimulating factor for a variety of celltypes, including mesangial cells[7], hepatocytes[8], osteo-blast-like cells[9], and intestinal epithelial cells[10]. CFs accountfor up to two-thirds of the total cells in the normal heart andare responsible for maintaining its structural integrity throughcontrolled proliferation and extracellular matrix production.Our previous work showed that AVP induces pro-fibroticresponse in neonatal rat CFs[11]. Importantly, AVP also in-duces neonatal rat CF proliferation via the V1 receptor[12].However, the mechanism for this mitogenic effect is lessthan well-defined. In the present study, we extended ourinvestigations to adult rat CFs to explore whether AVP couldinduce adult rat CF proliferation and, if so, to provide fur-ther insight into the underlying mechanism.

Gibco) containing 10% fetal bovine serum (FBS; Hyclone),100 U/mL penicillin and 100 μg/mL streptomycin, and in-cubated in a humidified atmosphere of 5% CO2 at 37 ºC.Immunocytochemical examinations showed that all cul-tured cells exhibited positive staining for vimentin (indicatingorigin of mesenchyma), but negative for von Willebrand’sfactor (indicating little contamination of vascular endothe-lial cells) and α-smooth muscle actin (indicating little con-tamination of smooth muscle cells). Subconfluent cells weremade quiescent in serum-free DMEM for 24 h. The firstpassage cells were used in the present study.

1.2 [3H]-thymidine incorporation

Adult rat CFs were seeded on 96-well plates at a density of5×103 cells/well for 24 h in 10% FBS/DMEM, then starvedand exposed to various concentrations of AVP or/and aninhibitor of the upstream kinase of extracellular signal-regulated kinase 1/2 (ERK1/2), PD98059, for 24 h. DNAsynthesis was assessed by the addition of 1 μCi/mL of[3H]-thymidine (Atomic Energy Institute, Chinese Acad-emy of Science) 6 h prior to the end of the treatment period.Cells were then washed twice with cold PBS (4 ºC), andincubated with 4 ºC 10% trichloroacetic acid (TCA) for 30min. The precipitates were rinsed twice with 95% ethanoland solubilized in 0.5 mol/L NaOH. The radioactivity inaliquots was measured in a liquid scintillation spectrometer(Beckman DU640, USA).

1.3 Cell cycle analysis

2×105 CFs were plated in 100 mL cell culture flasks and incu-bated in DMEM containing 10% FBS for 18 h. Afterstarvation, cells were treated with DMEM containing 2.5%FBS in the presence or absence of AVP or/and PD98059for 24 h. Cells were detached with 0.25% trypsin and fixedwith 70% ethanol. Cell cycles were analyzed with flowcytometry after rinsing with PBS.

1.4 ERK1/2 activity assay

ERK1/2 activity was measured by using myelin basic

1 MATERIALS AND METHODS

1.1 Culture and characterization of adult rat CFsAll the experiments were carried out under the regulationsof the National Institutes of Health Guidelines on the Useof Laboratory Animals. CFs from adult Sprague-Dawley(SD) rats (200-250 g, male, from the Experimental AnimalCenter of the Fourth Military Medical University) werecultured in Dulbecco’s modified Eagle’s medium (DMEM;

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用。我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。本研究旨在进一步观察AVP是否对成年大鼠CFs增殖具有促

HE Yan-Ping et al: AVP Stimulates Proliferation of Adult Rat CFs via PKC-ERK1/2 Pathway335

protein (MBP) as a substrate, as described previously[13]with modification. After AVP treatment for intervals of 2,5, 10, 30, 60 and 120 min, CFs were washed twice withice-cold PBS and lysed with 0.5 mL lysis buffer, whichcontained 2 mmol/L EDTA, 20 mmol/L HEPES (pH 7.2),5 mmol/L EGTA, 20 mmol/L β-glycerophosphate, 20 mmol/LNaF, 50 mmol/L NaCl, 0.2 mmol/L Na3VO4, 0.01% TritonX-100, and 0.5 mmol/L fresh phenylmethylsulfonyl fluoride.Cell lysates were prepared by freezing in liquid nitrogen,thawing on ice, scraping, and sonicating (5 s). After cen-trifugation for 10 min (10 000 g, 4 ºC), 5 μL supernatantof the extract was added to 30 μL of reaction buffer con-taining 20 mmol/L HEPES (pH 7.2), 10 mmol/L MgCl2, 2mmol/L MnCl2, 0.5 mmol/L Na3VO4, 2 mmol/Ldithiothreitol, 2 mmol/L TTTAAPIASGATGAAAAIHA(cAMP-dependent protein kinase inhibitor peptide, Sigma,USA), 7.4×104 Bq γ-32P ATP and 25 μg MBP. After incu-bation at 30 ºC for 15 min, the reaction was stopped byadding 10 μL of stopping solution containing 0.6% HCl, 1mmol ATP, and 1% bovine serum albumin. 20 μL of thereaction mixture was spotted on 1.5 cm × 1.5 cm squaresof P81 phosphocellulose filter paper, washed three timeswith deionized water containing 1% orthophosphoric acid,and dried. Radioactivity was then measured by scintillationcounting. ERK1/2 activity was expressed as fold increasecompared to the control.

1.5 Western blot analysis

CFs were treated with AVP or/and PD98059 for 5 min (fortotal- and phospho-ERK1/2) or 24 h (for p27Kip1, cyclinsD1, A and E) and lysed with the same lysis buffer as men-tioned above. Protein content was measured by Bradfordassay with Bio-Photometer (BioPhotometer 6131 GB/HK,Eppendorf) at 595 nm. Equal amount of proteins in eachsample were separated by SDS-PAGE gel electrophoresis,transferred onto a nitrocellulose membrane, and incubatedin 5% defatted milk dissolved in TBS (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.05% Tween-20). Pri-mary antibodies against p27Kip1, cyclins D1, A, E, total-and phospho-ERK1/2 (Sigma, USA) were diluted in TBSusing the appropriate dilutions. The membranes wereprobed with the indicated primary antibodyes and followedby the proper secondary horseradish peroxidase (HRP)-conjugated antibodies (Santa Cruz; diluted from 1:3 000 to1:10 000 in TBS). The membranes were then washed4 times with TBS at room temperature. To re-probe an-other primary antibody, membranes were incubated in strip-ping buffer (62.5 mmol/L Tris, pH 6.7, 100 mmol/L of 2-mercaptoethanol, and 2% SDS) for 30 min, washed, and

then used for further study. The reactive proteins werevisualized using enhanced chemiluminescence (ECL).1.6 Statistical analysis

Data were presented as mean±SEM. Comparisons wereconducted using one-way analysis of variance (ANOVA)followed by Bonferroni’s post hoc test. P<0.05 was con-sidered statistically significant .

2 RESULTS

2.1 AVP stimulates DNA synthesis in adult rat CFsvia V1 receptor

Consistent with previous work in our laboratory in neona-tal rat CFs[12], AVP (0.001-1 μmol/L) increased DNA syn-thesis as measured by [3H]-thymidine incorporation in adultrat CFs in a dose-dependent manner (Fig.1A). And 0.1μmol/L was chosen as AVP working concentration for sub-sequent analysis. Further investigation revealed that theeffect of AVP on DNA synthesis was abolished by incubat-ing adult rat CFs with a V1 receptor antagonist, d(CH2)5[Tyr2(Me), Arg8]-vasopressin (0.1 μmol/L), but not by aV2 receptor antagonist, desglycinamide-[d(CH2)5, D-Ile2,Ile4, Arg8]-vasopressin (0.1 μmol/L) (Fig.1B), indicatingthat the growth-stimulating effect of AVP on adult rat CFsis mediated via the V1 receptor.

2.2 PKC-ERK1/2 pathway is involved in AVP-induced DNA synthesis in adult rat CFs

To explore the potential involvement of ERK1/2 activation,which is known to mediate the mitogenic response inducedby growth-stimulating factors in many cell types, we firstmeasured ERK1/2 activity in adult rat CFs exposed to AVP.ERK1/2 activity assay showed that cellular ERK1/2 activa-tion could readily be induced by AVP (0.1 μmol/L). Theactivation was detectable at 2 min (1.5-fold induction),peaked at 5 min (2.7-fold induction) and sustained through1 h, with subsequent decline nearly to baseline level at 2 hafter the initiation of stimulation (Fig.2A).

Since the PKC-ERK1/2 pathway has been implicated inthe mitogenic effect of AVP on rat intestinal epithelial cells[10],we further tested the possible involvement of PKC-ERK1/2pathway in AVP-induced adult rat CF proliferation. Incu-bating CFs with an inhibitor of the upstream kinase of ERK1/2,PD98059 (30 μmol/L), abolished the mitogenic effect ofAVP, while the basal cell DNA uptake was not signifi-cantly altered (Fig.2B). Exposing cells to the pharmaco-logical PKC activator, phorbol 12-myristate 13-acetate(PMA, 30 nmol/L), as well as AVP for 5 min activatedERK1/2 phosphorylation as measured by Western blot.

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用。我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。本研究旨在进一步观察AVP是否对成年大鼠CFs增殖具有促

336Fig. 1. AVP stimulates adult DNA synthesis in rat CFs via V1 receptor.A: DNA synthesis measured by [3H]-thymidine incorporation inadult rat CFs treated with the indicated concentrations of AVP for 24h. B: [3H]-thymidine incorporation into cells treated with AVP (0.1μmol/L) in the presence or absence of a V1 receptor antagonist(V1RA), d(CH2)5[Tyr2(Me), Arg8]-vasopressin, or a V2 receptorantagonist (V2RA), desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (both at 0.1 μmol/L), for 24 h. mean±SEM, n=6. *P<0.05 vs control, #P<0.05 vs AVP.

Depleting PKC by chronic PMA incubation (2.5 μmol/L,24 h)[14] abolished the stimulating effect of AVP on ERK1/2phosphorylation (Fig.2C), suggesting that the observedERK1/2 activation induced by AVP in adult rat CFs is PKC-dependent.

2.3 p27Kip1 and cyclins D1, A and E are the down-stream targets of ERK1/2 in mediating AVP-inducedDNA synthesis in adult rat CFs

Next, we explored the downstream effectors of ERK1/2in mediating AVP-induced DNA synthesis in adult rat CFs.Western blot analysis revealed that the protein expressionof p27Kip1, which negatively regulates mammalian cell cycleprogression, was markedly attenuated upon AVP treatment,accompanied by increased expressions of cyclins D1, Aand E. Consistent with the changes observed in DNA

synthesis, inhibiting ERK1/2 activation by PD98059 (30

Acta Physiologica Sinica, June 25, 2008, 60 (3): 333-340

Fig. 2. PKC-ERK1/2 pathway is involved in AVP-induced DNAsynthesis in adult rat CFs. A: CFs were incubated with AVP (0.1μmol/L) for the indicated time. The activity of ERK1/2 was mea-sured by myelin basic protein (MBP) phosphotransferase activity.

*

P<0.05 vs the baseline level. B: [3H]-thymidine incorporation into

cells treated with AVP (0.1 μmol/L) alone for 24 h or in the presenceof PD98059 (PD, 30 μmol/L) for 24 h before and during AVPstimulation. #P<0.05 vs AVP. C: Western blot analysis of phospho-rylated and total ERK1/2 protein expressions in cells exposed toPMA (2.5 μmol/L) for 24 h and stimulated with AVP (0.1 μmol/L)for 5 min, or to acute stimulation by PMA (30 nmol/L) or AVP (0.1μmol/L) for 5 min. Upper panel showed a representative immunoblot,and lower panel provided the pooled relative values of densitometricscanning. Immunoblotting of β-actin confirmed equal loading.Mean±SEM, n=6. *P<0.05 vs control, #P<0.05 vs AVP.

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用。我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。本研究旨在进一步观察AVP是否对成年大鼠CFs增殖具有促

HE Yan-Ping et al: AVP Stimulates Proliferation of Adult Rat CFs via PKC-ERK1/2 Pathway337

μmol/L) abolished the effect of AVP on protein expres-sions of p27Kip1 as well as cyclins D1, E and A (Fig.3).2.4 AVP induces cell cycle progression in adult ratCFs and ERK1/2 is involved

To gain a more direct insight into whether AVP induces cellcycle progression in adult rat CFs, we employed flowcytometry to analyze cell cycle distribution. Cell growthwas synchronized by serum starvation. As shown in Table1, AVP induced cell cycle progression from G0/G1 into Sphase, accompanied by increased proliferation index (PI).The effects of AVP on G0/G1-S phase transition and PI wereinhibited by PD98059 (30 μmol/L), which alone was insuf-ficient to induce any detectable change in cell cycle distri-bution and PI value. These results indicate that ERK1/2 is

Fig. 3. p27Kip1 and cyclins D1, A and E are the downstream effectors of ERK1/2 in mediating AVP-induced DNA synthesis in adult rat CFs.Cells were incubated with AVP (0.1 μmol/L) alone for 24 h or in the presence of PD98059 (30 μmol/L) 24 h before and during AVP stimulation.The protein expressions of p27Kip1 and cyclins D1, A and E were measured by Western blot analysis. A: Representative immunoblots of theindicated proteins. B and C: Relative values of densitometric scanning. Immunoblotting of β-actin confirmed equal loading. Mean±SEM, n=6.

*

P<0.05 vs control, #P<0.05 vs A

VP.

Table 1. Cell cycle distribution in adult rat CFs incubated with AVP or/and PD98059

Group

Cell cycle distribution (%) G0/G1

Control0.1 μmol/L AVP

0.1 μmol/L AVP + 30 μmol/L PD9805930 μmol/L PD98059

86.75±1.2770.50±1.3584.07±1.3787.05±1.25

S 8.72±1.0718.53±1.08*10.40±1.20# 8.57±0.82

G2/M 4.53±0.8310.97±1.16 5.53±1.42 4.38±1.38

PI (%)13.25±1.2729.50±1.35*15.93±1.37#12.95±1.25

mean±SEM, n=6. Proliferation index (PI) = S+G2/M. *P<0.05 vs control, #P<0.05 vs AVP.

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用。我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。本研究旨在进一步观察AVP是否对成年大鼠CFs增殖具有促

338indispensable for cell cycle progression through G1 phaseand the subsequent cellular proliferation.

3 DISCUSSION

The chronic elevation of AVP levels in patients with hyper-tension or heart failure ultimately leads to detrimentalmyocardial effects. For example, the survival and ventricularenlargement (SAVE) trial has demonstrated that high AVPlevels are associated with poor long-term prognosis[15], whichmight indicate its role not only in the pathophysiology of theheart failure but also in disease progression. Thus AVP hasemerged as an important candidate for therapeutic inter-vention in the treatment of cardiac hypertrophy.

A large pool of studies have suggested that angiotensin IIand endothelin 1, two of the neurohormoral factors in-creased in patients with hypertension or congestive heartfailure apart from AVP, directly induce cardiac hypertro-phy independent of hemodynamic mechanisms. AlthoughAVP plays an important role in the development of cardiachypertrophy via increased peripheral vascular resistanceand increased cardiac afterload, the direct role of AVP incardiac hypertrophy is not firmly established. In rat models,AVP has been shown to act as a growth factor and inducecardiomyocyte hypertrophy[16-18]. In these studies, AVP wasfound to have a direct protein synthetic effect on culturedcardiomyocytes with motivation of calcium and activationof mitogen-activated protein kinase (MAPK). However,the signaling events and celluar effects of AVP with re-spect to CFs are not well defined. CF is the major cell typethat controls cardiac remodeling under physiological andpathologic conditions[19]. Unlike myocytes, CFs are highlyproliferative and sensitive to numerous hormonal factors. Com-bining the present work with our previous observation[12],we demonstrated that AVP is a potent mitogen for both neo-natal and adult rat CFs. The mitogenic effect on adult ratCFs was mediated via V1 receptors and PKC-ERK1/2pathway. Further examination revealed that ERK1/2 acti-vation decreased protein expression of cell cycle inhibitorp27Kip1 and increased expressions of cyclins D1, E and A,thus identified the cell cycle regulatory proteins linking thePKC-ERK1/2 pathway activation and the proliferation ofadult rat CFs.

The AVP receptors can be categorized into V1 and V2receptors. The binding of AVP to the V1 receptor is knownto stimulate the degradation of inositol phospholipids, toproduce the intracellular second messengers diacylglycerol,which activates PKC, and phosphatidylinositol 4,5-bisphosphate, which mobilizes Ca2+. The downstream

Acta Physiologica Sinica, June 25, 2008, 60 (3): 333-340

messengers play pivotal roles in a variety of cell type-specific responses. Although AVP has been shown to acti-vate ERK1/2 in rat cardiomyocytes[20,21], the effect of AVPon ERK1/2 activation in rat CFs has not been reported. In thepresent study, we demonstrated that AVP activates ERK1/2 inadult rat CFs and this stimulation is mediated through theV1 receptor and PKC. Interestingly, however, AVP-inducedcardiomyocyte growth can also be mediated via the PKC-ERK1/2 pathway[21], which, in line with our present work,might indicate the significance of this pathway in regulat-ing the growth of cardiac cells.

Mitogenic signaling through ERK1/2 induces cell cycleentry in many cells, including fibroblasts[22] and smoothmuscle cells[23]. ERK, a subfamily of MAPK, is a criticalregulator of cell cycle progression into S phase. Cyclin Dis a rate-limiting, step-controlling initiator of DNA replica-tion whose transcription initiation, assembly, and nucleartransport is mitogen-dependent. In the present study, wedemonstrated that ERK1/2 activation plays a pivotal role inthe induction of cyclin D1 expression upon AVP treatment.This is in agreement with previous observations showingthat the extent of cyclin D1 accumulation is correlated withthe mitogenic potential of growth factors and their abilityto induce ERK1/2 activation[24].

The protein p27Kip1 is a cyclin-dependent kinase inhibitorthat regulates cell cycle progression. It has been estab-lished that p27Kip1 is an essential component of the path-way that connects mitogenic signals to the cell cycle at therestriction point in fibroblast cell cycle progression[25]. Severalstudies have indicated that ERK1/2 activation contributesto the down-regulation of p27Kip1[26-28]. However, most ofthese observations have been focused on cancer cells. Byusing flow cytometry to analyze cell cycle distribution, itwas shown that AVP induced the G0/G1-S phase transitionin adult rat CFs, which is also a highly proliferative celltype. We also confirmed a role of endogenous ERK1/2activation in G1 phase progression via the inhibition of p27Kip1expression. Moreover, the expression of cyclin E, which isinvolved in late G1 phase progression, and cyclin A, which isrequired for the onset of DNA replication during S phase[29],was markedly induced by AVP stimulation via the media-tion of ERK1/2.

Taken together, our findings demonstrated that AVP actsas a potent mitogen for adult rat CFs and elucidated thefunctional role of PKC-ERK1/2 pathway in mediating thismitogenic effect. We also identified the essential targets ofERK1/2 and characterized their cell cycle-related effects.An understanding of the effects of AVP on CF proliferationand the underlying mechanism may provide useful insight

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用。我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。本研究旨在进一步观察AVP是否对成年大鼠CFs增殖具有促

HE Yan-Ping et al: AVP Stimulates Proliferation of Adult Rat CFs via PKC-ERK1/2 Pathway339

into the study and treatment of adverse cardiac remodeling,which is deeply implicated in the pathophysiology of hy-pertension and congestive heart failure.

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