Fuller S J, Davies E L, Gillespie-Brown J, Sun H, Tonks N K
Cardiac Medicine, Imperial College School of Medicine at NHLI, University of London, Dovehouse Street, London SW3 6LY, UK.
Biochem J. 1997 Apr 15;323 ( Pt 2)(Pt 2):313-9. doi: 10.1042/bj3230313.
The effect of constitutive expression of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) on gene expression in response to hypertrophic agonists was examined in cultured neonatal rat ventricular myocytes. Luciferase (LUX) reporter genes linked to promoters for atrial natriuretic factor, ventricular myosin light chain 2, beta-myosin heavy chain, skeletal muscle alpha-actin (SkM alpha-actin) and serum response element-regulated c-fos (c-fos-SRE) were transfected into cardiomyocytes. Phenylephrine (PE; 10 microM), phorbol 12-myristate 13-acetate (1 microM) and endothelin 1 (10 nM) stimulated the expression of these various reporter genes by 2. 5-20-fold. MKP-1 inhibited these effects by 60-85%. In contrast, MKP-1 had no effect on the expression of a constitutively active Rous sarcoma virus-LUX reporter gene. A catalytically inactive mutant MKP-1CS (cysteine-->serine mutation) and the broad-specificity protein tyrosine phosphatase 1B (PTP-1B) had no significant effect on any reporter gene tested. MKP-1 had much less effect on the morphological features accompanying agonist-induced cardiac hypertrophy. PE (10 microM) increased myocyte area by 59% but this effect was only decreased by one-third by MKP-1 and was also partly decreased (by 25%) by expression of PTP-1B. PE also altered cell shape but this was unaffected by MKP-1. There was also no clear effect of MKP-1 on the organization of the contractile apparatus into sarcomeric structures in the presence of 10 microM PE. We conclude that the transcriptional responses accompanying cardiac myocyte hypertrophy are dependent on an MKP-1-sensitive step, presumably the activation of one or members of the MAPK family, but that cell size, shape and myofibrillar organization are much less sensitive to inhibition by MKP-1.
在培养的新生大鼠心室肌细胞中,研究了丝裂原活化蛋白激酶(MAPK)磷酸酶1(MKP-1)的组成型表达对肥厚性激动剂反应中基因表达的影响。将与心房利钠因子、心室肌球蛋白轻链2、β-肌球蛋白重链、骨骼肌α-肌动蛋白(SkMα-肌动蛋白)启动子以及血清反应元件调节的c-fos(c-fos-SRE)相连的荧光素酶(LUX)报告基因转染到心肌细胞中。苯肾上腺素(PE;10μM)、佛波酯12-肉豆蔻酸酯13-乙酸酯(1μM)和内皮素1(10 nM)使这些不同报告基因的表达增加了2.5至20倍。MKP-1抑制这些作用的60%至85%。相比之下,MKP-1对组成型活性劳氏肉瘤病毒-LUX报告基因的表达没有影响。催化失活的突变体MKP-1CS(半胱氨酸→丝氨酸突变)和广谱特异性蛋白酪氨酸磷酸酶1B(PTP-1B)对所测试的任何报告基因均无显著影响。MKP-1对伴随激动剂诱导的心脏肥大的形态学特征影响小得多。PE(10μM)使心肌细胞面积增加59%,但这种作用仅被MKP-1降低三分之一,并且也被PTP-1B的表达部分降低(25%)。PE还改变了细胞形状,但这不受MKP-1影响。在存在10μM PE的情况下,MKP-1对收缩装置组织成肌节结构也没有明显影响。我们得出结论,伴随心肌细胞肥大的转录反应依赖于MKP-1敏感步骤,推测是MAPK家族一个或多个成员的激活,但细胞大小、形状和肌原纤维组织对MKP-1抑制的敏感性要低得多。