大鼠甲状旁腺素对体外培养心室肌细胞的促肥大作用

目的 观察不同浓度大鼠甲状旁腺素1-34（rat parathyroid hormone 1-34,rPTH1-34）对体外培养心室肌细胞的致肥大作用,观察细胞外信号调节激酶1/2（extracellular signal regulated kinases 1/2,ERK1/2）的变化。方法 体外培养新生大鼠心室肌细胞,以1×10-8～1×10-6 mol/L rPTH1-34分别孵育24 h,测定细胞直径、3H-亮氨酸(3H-Leu)掺入率、心房钠尿肽（atrial natriuretic peptide,ANP）mRNA、ERK1/2、磷酸化ERK1/2(p-ERK1/2)蛋白的表达。结果 与正常组相比,1×10-8 mol/L rPTH 1-34可使体外培养的心肌细胞直径、细胞蛋白合成速率轻度增加,ANP mRNA表达轻度升高,但无统计学意义;1×10-7,1×10-6 mol/L rPTH1-34 均可显著增加心肌细胞直径（P<0.01）,细胞蛋白合成速率（P<0.01）和ANP mRNA表达（P<0.01）。与正常组比较,PTH呈浓度依赖性地增加了ERK1/2和p-ERK1/2的表达（P<0.01）。结论 1×10-7,1×10-6 mol/L rPTH1-34能在体外诱导新生大鼠心室肌细胞发生肥大反应,且呈一定的浓度依赖性;PTH能呈浓度依赖性地刺激心肌ERK1/2过度表达。     

ERK1/2蛋白在17β-雌二醇抑制睾酮诱导的心肌细胞肥大反应中的作用

目的 观察17β-雌二醇对睾酮诱导的心肌肥大反应的影响,探讨细胞外信号调节激酶(ERK1/2蛋白)在性激素对心肌肥大影响中的作用.方法 采用差速贴壁法分离、纯化培养新生SD大鼠心肌细胞,以Bradford法测定心肌细胞蛋白质含量,同位素法分析3H-亮氨酸(3H-Leu)掺入,以免疫印迹法检测心肌细胞ERK1/2蛋白表达水平的变化.结果 10-10 ～ 10-6 mol/L 17β-雌二醇可明显对抗睾酮诱导的心肌细胞蛋白质含量和3H-Leu掺入的增加,其中以10-8 mol/L 17β-雌二醇作用最为明显；预先给予10-6 mol/L雌激素受体拮抗剂他莫昔芬作用2h,可部分取消17β-雌二醇对睾酮诱导的心肌细胞蛋白质含量增加的抑制效应.用50μmol/L ERK1/2上游激酶MEK1/2的特异性抑制剂PD98059预处理2h不能增强17β-雌二醇对睾酮诱导的心肌细胞3H-Leu掺入的抑制作用；10-8 mol/L 17β-雌二醇可以对抗睾酮诱导的心肌细胞ERK1/2蛋白表达增加；10-6mol/L他莫昔芬预处理2 h可部分取消17β-雌二醇对睾酮诱导的心肌细胞ERK1/2蛋白表达增加的抑制作用.结论 17β-雌二醇经雌激素受体介导下调睾酮诱导的心肌细胞ERK1/2蛋白表达,从而抑制由睾酮诱导的心肌细胞肥大反应.     

α-烯醇化酶在内皮素-1诱导的肥大心肌细胞中的表达和调控通路

目的 观察内皮素-1(ET-1)诱导的心肌细胞肥大模型中细胞外信号调节激酶(ERK1/2)、磷酸化细胞外信号调节激酶(p-ERK1/2)、低氧诱导因子(HIF-1α),α-烯醇化酶(α-enolase)蛋白表达情况,探讨肥大心肌细胞α-enolase高表达的调控机制. 方法 建立ET-1诱导的心肌细胞肥大模型,从细胞表面积、细胞蛋白合成速率和肌原纤维的重排3方面进行验证;将原代培养心肌细胞随机分为4组:(1)对照组;(2)PD98059干预组;(3)ET-1刺激组;(4)PD98059+ET-1刺激组.免疫印迹方法 检测ERK1/2、p-ERK1/2、HIF-1α、α-enolase的蛋白表达. 结果 ET-1刺激后心肌细胞表面积为(1350.7±107.5)μm2,较对照组(896.1±70.2)μm2增加(P<0.05);ET-1刺激后心肌细胞[3H]亮氨酸掺入量较对照组增加[分别为(1387.9±14.8)dpm和(787.7±10.2)dpm,P<0.013;肌原纤维染色可见ET-1刺激屙心肌细胞肌原纤维排列较对照组紧密、染色浓,表明ET-1能诱导心肌细胞肥大.ERK1/2抑制剂PD98059预处理的心肌细胞在ET-1刺激后细胞表面积和细胞的[3H]亮氨酸掺入量均较ET-1刺激组减少[细胞表面积分别为(907.0±92.5)μm2和(1350.7±107.5)μm2,P<0.05;[3H]亮氨酸掺入量:(841.5±10.5)dpm和(1387.9±14.8)dpm,P<0.05].ET-1刺激后心肌细胞有p-ERK1/2表达,其抑制剂PD98059在抑制ERK1/2活化的同时也部分抑制了HIF-1α、α-enolase蛋白的表达. 结论 ERK1/2的活化与ET-1诱导的细胞肥大关系密切,在ET-1促心肌细胞肥大过程中,从MAPK/ERK1/2至HlF-1α及α-enolase这条信号通路町能参与α-enolase高表达的调控.     

结缔组织生长因子诱导大鼠心肌细胞肥大的作用及其与细胞外信号调节激酶1/2的关系

目的 观察结缔组织生长因子(CTGF)诱导大鼠心肌细胞肥大的作用,探讨其作用与细胞外信号调节激酶1/2(ERK1/2)的关系.方法 以培养的新生Sprague-Dawley(SD)大鼠心肌细胞为实验模型,用图象分析法测定心肌细胞表面积,用[3H]-亮氨酸掺入法测定心肌细胞蛋白合成速率,用考马斯亮兰法测定心肌细胞蛋白含量,用蛋白免疫印迹法(Western blot)测定心肌细胞总ERK1/2(t-ERK1/2)与磷酸化ERK1/2(p-ERK1/2)的蛋白表达水平.结果 (1)随着CTGF浓度的增加,心肌细胞表面积呈剂量依赖性增加,其中10、25、50、100 μg/L的CTGF组心肌细胞表面积分别为(929.9±132.2)、(1411.3±129.2)、(1732.0±153.0)、(2040.6±205.4)μm2,均明显高于对照组心肌细胞表面积[(606.3±72.7)μm2,P均＜0.01];100 μmol/L的PD98059明显减少CTGF诱导的心肌肥大(P＜0.01).(2)随着CTGF浓度的增加,心肌细胞蛋白合成速率与蛋白含量呈剂量依赖性增加,CTGF组心肌细胞[3H]-亮氨酸掺入率明显高于对照组(P＜0.01);ERK1/2抑制剂PD98059明显减少CTGF诱导的心肌细胞[3H]-亮氨酸掺入率及蛋白含量(P＜0.01).(3)随着CTGF浓度的增加,心肌细胞p-ERK1/2表达呈剂量依赖性增高,5、10、25、50、100 μg/L的CTGF组的心肌细胞p-ERK1/2表达明显高于对照组,而t-ERK1/2在各组表达差异不明显.结论 CTGF可诱导心肌细胞肥大,该作用可能是通过ERK1/2的磷酸化来实现的.     

结缔组织生长因子诱导大鼠心肌细胞肥大的作用及其与细胞外信号调节激酶1/2的关系

目的观察结缔组织生长因子(CTGF)诱导大鼠心肌细胞肥大的作用,探讨其作用与细胞外信号调节激酶1/2(ERK1/2)的关系。方法以培养的新生Sprague-Dawley(SD)大鼠心肌细胞为实验模型,用图象分析法测定心肌细胞表面积,用[3H]亮氨酸掺入法测定心肌细胞蛋白合成速率,用考马斯亮兰法测定心肌细胞蛋白含量,用蛋白免疫印迹法(Westernblot)测定心肌细胞总ERK1/2(t-ERK1/2)与磷酸化ERK1/2(p-ERK1/2)的蛋白表达水平。结果(1)随着CTGF浓度的增加,心肌细胞表面积呈剂量依赖性增加,其中10、25、50、100μg/L的CTGF组心肌细胞表面积分别为(929·9±132·2)、(1411·3±129·2)、(1732·0±153·0)、(2040·6±205·4)μm2,均明显高于对照组心肌细胞表面积[(606·3±72·7)μm2,P均<0·01];100μmol/L的PD98059明显减少CTGF诱导的心肌肥大(P<0·01)。(2)随着CTGF浓度的增加,心肌细胞蛋白合成速率与蛋白含量呈剂量依赖性增加,CTGF组心肌细胞[3H]亮氨酸掺入率明显高于对照组(P<0·01);ERK1/2抑制剂PD98059明显减少CTGF诱导的心肌细胞[3H]亮氨酸掺入率及蛋白含量(P<0·01)。(3)随着CTGF浓度的增加,心肌细胞p-ERK1/2表达呈剂量依赖性增高,5、10、25、50、100μg/L的CTGF组的心肌细胞p-ERK1/2表达明显高于对照组,而t-ERK1/2在各组表达差异不明显。结论CTGF可诱导心肌细胞肥大,该作用可能是通过ERK1/2的磷酸化来实现的。     

MEK信号通路参与PTEN对心肌细胞肥大的负性调控

目的探讨第10号染色体上同源丢失的磷酸酶张力蛋白（PTEN）基因的过度表达对血管紧张素Ⅱ（AngⅡ）诱导的心肌肥大的负性调控机制。方法通过携带野生型PTEN基因的腺病毒（Ad-PTEN）感染,构建过度表达PTEN的原代培养的心肌细胞模型。以AngⅡ为促心肌肥大的刺激剂,采用RT-PCR检测受感染细胞内心房钠尿肽（ANP）、β肌球蛋白重链（β-MHC）mRNA的表达,用Western blot检测细胞外信号调节激酶1/2（ERK1/2）和磷酸化ERK1/2（pERK1/2）蛋白的表达。结果Ad-PTEN感染后,心肌细胞内过度表达的PTEN mRNA和其蛋白,可明显抑制AngⅡ刺激所致心肌细胞肥大标志基因的表达。AngⅡ刺激可使ERK1/2与pERK1/2蛋白的表达明显增高;但PTEN的过度表达能明显抑制AngⅡ引起的ERK1/2与pERK1/2蛋白的表达。结论PTEN能够负性调控AngⅡ刺激引起的心肌细胞肥大,MEK1/ERK1/2信号通路可能参与了PTEN的负性调控过程。     

血管紧张素-(1-7)在拮抗血管紧张素Ⅱ诱导心肌细胞Cx43间隙连接上调中的作用

目的探讨血管紧张素-(1-7)[Ang-(1-7)]在血管紧张素Ⅱ(AngⅡ)诱导心肌细胞Cx43间隙连接中作用。方法AngⅡ处理培养心肌细胞24h。PD98059和Ang-(1-7)在AngⅡ刺激细胞前1h加到培养基中,对照组加等体积药物溶剂DMSO。用Western blot分析和电镜观察心肌细胞Cx43表达和间隙连接。结果Western blot分析显示用10-9~10-6mol/L AngⅡ刺激细胞24h,Cx43的表达与对照组相比呈浓度依赖性增加;用AngⅡ0.1μmol/L刺激心肌细胞24h,与对照组相比Cx43表达上调、磷酸化ERK1/2活性增加(P<0.01),ERK1/2激酶特异性抑制剂1μmol/LPD98059和0.1μmol/L Ang-(1-7)能阻断AngⅡ上调Cx43表达和磷酸化ERK1/2活性增加。电镜观察证明用AngⅡ0.1μmol/L刺激心肌细胞24h,AngⅡ处理组细胞间隙连接数目和大小较对照组增加(P<0.05),0.1μmol/L Ang-(1-7)能阻断AngⅡ增加心肌细胞间隙连接数目和大小。结论Ang-(1-7)通过抑制磷酸化ERK1/2活性增加,从而拮抗AngⅡ上调培养新生鼠心肌细胞Cx43间隙连接。     

丹参酮ⅡA磺酸钠对血管紧张素Ⅱ诱导的大鼠心肌细胞外信号调节激酶1/2的作用

目的研究丹参酮ⅡA磺酸钠(STS)对血管紧张素Ⅱ(AngⅡ)诱导的心肌肥大反应中细胞外信号调节激酶1/2(ERK1/2)是否有抑制作用。方法培养新生大鼠心肌细胞,考马斯亮蓝法测定心肌细胞蛋白含量、[3H]-亮氨酸掺入法测定蛋白合成速率作为心肌肥大指标;用免疫荧光标记法和Western-blot测定磷酸化ERK1/2蛋白(p-ERK1/2)表达。结果1)AngⅡ(1μmol/L)处理24h,心肌细胞[3H]-亮氨酸掺入率、蛋白含量明显增加,STS能明显抑制AngⅡ介导心肌细胞[3H]-亮氨酸掺入率、蛋白含量的增加;2)AngⅡ刺激心肌细胞可见胞核内出现磷酸化ERK1/2荧光染色,丹参酮ⅡA可阻断AngⅡ引起的ERK1/2活化、入核过程。3)用AngⅡ(1μmol/L)处理心肌细胞5min,磷酸化ERK1/2蛋白(p-ERK1/2)表达即开始增加,10min左右时最明显。4)STS剂量依赖性抑制AngⅡ诱导的心肌细胞磷酸化ERK1/2蛋白表达。结论STS可以抑制AngⅡ诱导的心肌肥厚,其机制与抑制磷酸化ERK1/2表达有关。     

丹参酮ⅡA磺酸钠对血管紧张素Ⅱ诱导的大鼠心肌细胞外信号调节激酶1/2的作用

目的 研究丹参酮ⅡA磺酸钠(STS)对血管紧张素Ⅱ(Ang Ⅱ)诱导的心肌肥大反应中细胞外信号调节激酶1/2(ERK1/2)是否有抑制作用.方法 培养新生大鼠心肌细胞,考马斯亮蓝法测定心肌细胞蛋白含量、[3H]-亮氨酸掺人法测定蛋白合成速率作为心肌肥大指标;用免疫荧光标记法和Western-blot测定磷酸化ERK1/2蛋白(p-ERK1/2)表达.结果 1)Ang Ⅱ(1 μmol/L)处理24 h,心肌细胞[3H]-亮氨酸掺入率、蛋白含量明显增加,STS能明显抑制Ang Ⅱ介导心肌细胞[3H]-亮氨酸掺人率、蛋白含量的增加;2)Ang Ⅱ刺激心肌细胞可见胞核内出现磷酸化ERK1/2荧光染色,丹参酮ⅡA可阻断AngⅡ引起的ERK1/2活化、入核过程.3)用Ang Ⅱ(1 μmol/L)处理心肌细胞5 min,磷酸化ERK1/2蛋白(p-ERK1/2)表达即开始增加,10 min左右时最明显.4)STS剂量依赖性抑制Ang Ⅱ诱导的心肌细胞磷酸化ERK1/2蛋白表达.结论 STS可以抑制Ang Ⅱ诱导的心肌肥厚,其机制与抑制磷酸化ERK1/2表达有关.     

阿托伐他汀通过ERK1/2通路抑制内皮微粒诱导的人脐静脉内皮细胞ICAM-1的表达

目的探讨阿托伐他汀对内皮细胞微粒(EMPs)诱导人脐静脉内皮细胞(HUVECs)细胞间黏附分子-1(ICAM-1)表达的影响及其与ERKl/2信号通路的关系。方法将HUVECs分为不同浓度EMPs作用组与阿托伐他汀干预组。应用Western印迹检测磷酸化ERK1/2和ICAM-1蛋白的表达,实时荧光定量PCR(qRT-PCR)检测ICAM-1 mRNA的表达。结果 EMPs可诱导HUVECs ICAM-1 mRNA和蛋白及磷酸化ERK1/2蛋白表达增加,且具有浓度和时间依赖关系(均P<0.01);阿托伐他汀及ERK1/2特异性抑制剂PD98059显著抑制EMPs诱导的HUVECs ICAM-1 mRNA和蛋白及磷酸化ERK1/2蛋白的表达(均P<0.01)。结论阿托伐他汀通过ERK1/2信号通路抑制EMPs诱导HUVECs ICAM-1表达。     

Angiotensin II stimulates DNA and protein synthesis in vascular smooth muscle cells from human arteries: role of extracellular signal-regulated kinases.

OBJECTIVE: This study investigates the growth effects and associated signaling pathways of angiotensin II (Ang II) in human vascular smooth muscle cells. METHODS: Cultured vascular smooth muscle cells derived from resistance arteries (< 300 microm diameter) from subcutaneous gluteal biopsies of healthy subjects (n = 6) and human aortic vascular smooth muscle cells were used. Cells were studied between passages 3 and 6. Both 3H-thymidine and 3H-leucine incorporation were measured as indices of vascular smooth muscle cell hyperplasia (DNA synthesis) and cell hypertrophy (protein synthesis), respectively. Growth effects of Ang II (10(-12) - 10(-6) mol/l), in the absence and presence of 10(-5) mol/l losartan (AT1 antagonist) and PD123319 (AT2 antagonist), were determined. Ang II-induced effects were compared to those of endothelin-1. To determine whether extracellular signal-regulated kinase (ERK)-dependent pathways play a role in Ang II-mediated growth, cells were pretreated with the selective ERK kinase (MEK) inhibitor, PD98059 (10(-5) mol/l). ERK activation was determined by Western blot in the absence and presence of PD98059. RESULTS: Ang II dose-dependently increased 3H-thymidine incorporation in cells from aorta (Emax = 276 +/- 10.4% of control) and resistance arteries (Emax = 284 +/- 5.1% of control). Ang II also stimulated 3H-leucine incorporation in cells from aorta (Emax = 162 +/- 11.6 of control) and resistance arteries (Emax 175 +/- 10% of control). Unlike Ang II, endothelin-1 failed to significantly alter cellular growth, except at high concentrations (> 10(-7) mol/l), where it had a weak stimulatory effect Losartan, but not PD123319, blocked Ang II-stimulated growth responses. Ang II significantly increased phosphorylation of ERK-1 and ERK-2, with maximum responses obtained at 5 min. PD98059 inhibited Ang II-stimulated ERK activity and abrogated agonist-induced DNA and protein synthesis. Losartan, but not PD123319 inhibited Ang II-induced phosphorylation of ERK-1 and ERK-2. CONCLUSIONS: Ang II stimulates both hyperplasia and hypertrophy in vascular smooth muscle cells from human arteries. These growth effects are mediated via Ang II receptors of the AT1 subtype that are linked to ERK-dependent signaling pathways.     

MEK1/2-ERK1/2 mediates alpha1-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes

We examined the relative roles of the mitogen-activated protein kinases (MAPK) in mediating the alpha1-adrenergic receptor (alpha1-AR) stimulated hypertrophic phenotype in adult rat ventricular myocytes (ARVM). Norepinephrine (NE; 1 microM) in the presence of the beta -AR antagonist propranolol (Pro; 2 microM) caused activation of Ras (>six-fold), MAPK/ERK kinase 1 and 2 (MEK1/2, >10-fold) and extracellular signal-regulated kinases 1 and 2 (ERK1/2, approximately 30-fold) within 5 min, as determined by kinase activity assays and Western blots using phospho-specific antibodies. Conversely, p38 and c-Jun amino-terminal kinases (JNK) were not activated by NE/Pro. Activated MEK1/2 signals remained detectable at 2 h, and activated ERK1/2 remained detectable at 48 h. The alpha1-AR selective inhibitor prazosin (100 nM) completely inhibited the NE/Pro-stimulated activation of Ras, MEK1/2 and ERK1/2. The MEK inhibitor PD98059 caused a concentration-dependent inhibition of NE/Pro-stimulated protein synthesis (as assessed by [3H]leucine incorporation and cellular protein accumulation) and ERK1/2 activation, with approximately 50% inhibition at a concentration between 10 and 50 microM, which is consistent with the known IC50 values of PD98059 for MEK1 (4 microM) and MEK2 (50 microM). Thus, these data show that alpha1-AR stimulated hypertrophy in ARVM is dependent on the MEK1/2-ERK1/2 signaling pathway.     

拉西地平对CTGF诱导大鼠心肌细胞肥大的影响及其与ERK1/2的关系

目的 观察拉西地平对结缔组织生长因子（CTGF）诱导大鼠心肌细胞肥大的作用,探讨其作用与细胞外信号调节激酶1/2（ERK1/2）的关系。方法 以培养的新生SD大鼠的心肌细胞为实验模型,用图象分析法、3H-亮氨酸掺入法、考马斯亮蓝染色、蛋白免疫印迹法（Western blot）等,评价拉西地平对CTGF诱导心肌细胞肥大的抑制作用及其与ERK1/2的关系。结果 ①以50 ng/L的CTGF作用24 h后,心肌细胞表面积为（1 812.52±168.73）μm2,与对照组（689.31±96.58）μm2比较显著增加（P<0.01）;5、10、25及50 μmol/L拉西地平干预组的心肌细胞表面积分别为（1 476.52±156.73）μm2、（1 120.39±149.68）μm2、（926.10±101.44）μm2及（739.81±91.55）μm2,与CTGF组比较呈浓度依赖性降低（P<0.01）。②以50 ng/L的CTGF作用24 h后,心肌细胞的3H-亮氨酸掺入率为（2 368.72±122.45）cpm/孔,与对照组（950.26±89.43）cpm/孔比较显著增加（P<0.01）。 5、10、25及50 μmol/L拉西地平干预组的心肌细胞的3H-亮氨酸掺入率,分别为（2023.12±106.15）cpm/孔、（1629.15±103.46）cpm/孔、（1302.19±98.53）cpm/孔及（1 055.72±90.96）cpm/孔,与CTGF组比较呈浓度依赖性降低（P<0.01）。③以50 ng/L的CTGF作用24 h后,心肌细胞蛋白的含量为（1.692±0.203）ng/细胞,与对照组（0.622±0.068）ng/细胞比较显著增加（P<0.01）;5、10、25及50 μmol/L拉西地平干预组的心肌细胞蛋白的含量分别为（1.269±0.167）ng/细胞、（0.923±0.119）ng/细胞、（0.766±0.085）ng/细胞及（0.682±0.063）ng/细胞,与CTGF组比较呈浓度依赖性降低（P<0.01）。④50 ng/L CTGF组的心肌细胞p-ERK1/2表达强度明显高于对照组,5 μmol/L、10 μmol/L、25 μmol/L、50 μmol/L拉西地平干预组心肌细胞p-ERK1/2的表达强度与CTGF组比较呈浓度依赖性降低。心肌细胞t-ERK1/2的表达在各组之间没有明显的差异。结论 拉西地平可抑制CTGF诱导的心肌细胞肥大,其作用机制可能与ERK1/2磷酸化有关。     

Obligate mitogen-activated protein kinase activation in parathyroid hormone stimulation of calcium transport but not calcium signaling

PTH regulates calcium homeostasis through direct actions on its cognate type I receptor in the kidney and bone. PTH inhibits phosphate transport in renal proximal (PCT) tubules and stimulates calcium absorption by distal convoluted tubules (DCT). We examined PTH activation of the mitogen-activated protein kinase (MAPK) cascade raf-MEK-ERK in PCT and DCT cells and its effects on calcium transport and signaling. In DCT cells, PTH stimulates phosphorylation of ERK2 and activation of ERK2 kinase and is blocked by the MEK inhibitor PD98059. In DCT cells, stimulation of calcium entry with ionomycin did not activate ERK2 or augment PTH-stimulated ERK2 activity, indicating that MAPK activation lies upstream of calcium entry. ERK2 activation by PTH was blocked by the protein kinase C inhibitor calphostin-C but was unaffected by the protein kinase A inhibitor Rp-cAMPs. PD98059 abolished the increase of intracellular calcium induced by PTH demonstrating that ERK2 activation is directly involved in the increase of intracellular calcium activated by PTH in the DCT. Thus, PTH- stimulated ERK2 activation is PKC dependent and calcium independent. PTH also induced ERK2 phosphorylation in PCT cells. However, this effect is not involved in the transient rise of intracellular calcium because PD98059 did not inhibit the PTH-stimulated rise of intracellular calcium but abolished ERK2 activation. In conclusion, PTH activates MAPK in both distal and proximal renal tubule cells. However, the rise of [Ca2+]i depends upon MAPK activation only in distal cells. Thus, a common PTH1R exhibits differential signaling along the nephron that contributes to the ability to regulate distinct physiological actions of PTH.     

Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy

The signal transduction mechanisms mediating hypertrophic responses in myocardial cells (MCs) remain uncertain. We investigated the role of the extracellular signal-regulated kinase (ERK) cascade in myocardial cell hypertrophy by the strategy of using the adenovirus-mediated overexpression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which is the upstream activator of ERK. We generated recombinant adenoviruses expressing constitutively active MEK1 (MEK1 EE) and dominant negative MEK1 (MEK1 DN). Overexpression of MEK1 EE in MCs activated ERK1/2 and subsequently induced atrial natriuretic peptide (ANP) mRNA expression. In addition, MEK1 EE overexpression resulted in an increase in cell size and sarcomeric reorganization. In contrast, overexpression of MEK1 DN in MCs inhibited endothelin-1 (ET-1)-, phenylephrine (PE)-, leukemia inhibitory factor (LIF)-, isoproterenol (ISP)-, and mechanical stretch-induced ERK activation and ANP mRNA expression. MEK1 DN overexpression inhibited ET-1-, PE-, LIF-, and ISP-induced increases in cell size and sarcomeric reorganization. Consistent with the observed effects on cellular morphology, overexpression of MEK1 EE resulted in an increase in amino acid incorporation, while overexpression of MEK1 DN inhibited ET-1-, PE-, LIF-, ISP-, and mechanical stretch-induced increases in amino acid incorporation. These results indicate that the ERK cascade plays an important role in the signaling pathway leading to the development of myocardial cell hypertrophy.     

Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinase-dependent pathways

Doxorubicin (Dox), an anthracyclin antineoplastic agent, causes dilated cardiomyopathy. CARP has been identified as a nuclear protein whose mRNA levels are exquisitely sensitive to Dox. In this study we investigated the molecular mechanisms underlying the repression of CARP expression by Dox in cultured neonatal rat cardiac myocytes. Dox (1 micromol/l)-mediated decrease in CARP mRNA levels was strongly correlated with BNP but not with ANP mRNA levels. Hydrogen peroxide scavenger catalase (1 mg/ml) but not hydroxyl radical scavengers dimethylthiourea (10 mmol/l) or mannitol (10 mmol/l) blunted the Dox-mediated decrease in CARP and BNP expression. Superoxide dismutase inhibitor diethyldithiocarbamic acid (10 mmol/l), which inhibits the generation of hydrogen peroxide from superoxide metabolism, attenuated the repression. PD98059 (MEK1 inhibitor, 50 micromol/l), SB203580 (p38 MAP kinase inhibitor, 10 micromol/l), calphostin C (protein kinase C (PKC) inhibitor, 1 micromol/l), non-selective protein tyrosine kinase inhibitors genistein (50 micromol/l) or herbimycin A (1 micromol/l) failed to abrogate the downregulation of CARP and BNP expression by Dox. In contrast, H7 (30 micromol/l), a potent inhibitor of serine/threonine kinase, significantly blocked Dox-mediated downregulation of CARP and BNP expression. Transient transfection of a series of 5'-deletion and site-specific mutation constructs revealed that M-CAT element located at -37 of the human CARP promoter mediates Dox-induced repression of CARP promoter activity. These results suggest that a genetic response to Dox is mediated through the generation of hydrogen peroxide, which is selectively linked to the activation of H7-sensitive serine/threonine kinase distinct from PKC and well characterized mitogen-activated protein (MAP) kinases (ERK and p38MAP kinase). Furthermore, our data implicated M-CAT element as a Dox-response element within the CARP promoter in cardiac myocytes.