Durham P L, Russo A F
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242, USA.
Mol Endocrinol. 1998 Jul;12(7):1002-9. doi: 10.1210/mend.12.7.0135.
We have investigated the mechanisms underlying regulation of the calcitonin gene-related peptide (CGRP) cell-specific enhancer. Recently, we reported that this enhancer is inhibited by serotonin type-1 (5-HT1) agonists, similar to currently used antimigraine drugs. We have now tested whether this repression involves a mitogen-activated protein (MAP) kinase pathway. We first demonstrate that the CGRP enhancer is strongly (10-fold) activated by a constitutively active MAP kinase kinase (MEK1), yielding reporter activities 100-fold above the enhancerless control. The involvement of a MAP kinase pathway was confirmed by down-regulation of reporter activity upon cotransfection of a dominant negative Ras. Activation of the enhancer by MEK1 was blocked in a dose-dependent manner by the 5-HT1 receptor agonist CGS 12066A (CGS). Since it is not known whether the CGRP enhancer factors are immediate targets of MAP kinases, we then used EIk-1- and c-Jun-dependent reporter genes that are directly activated by the ERK (extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinase) MAP kinases. CGS treatment repressed the activation of both of these reporters, suggesting that at least two MAP kinases are the immediate targets of CGS-mediated repression. We further demonstrate that 5-HT1 agonists inactivate ERK by dephosphorylation, even in the presence of constitutively activated MEK1. This inactivation appears to be due to a marked increase in the level of MAP kinase phosphatase-1. These results have defined a novel and general mechanism by which 5-HT1 receptor agonists can repress MAP kinase activation of target genes, such as CGRP.
我们研究了降钙素基因相关肽（CGRP）细胞特异性增强子的调控机制。最近，我们报道该增强子受到5-羟色胺1型（5-HT1）激动剂的抑制，这与目前使用的抗偏头痛药物类似。我们现在测试了这种抑制是否涉及丝裂原活化蛋白（MAP）激酶途径。我们首先证明，组成型活性MAP激酶激酶（MEK1）能强烈（10倍）激活CGRP增强子，产生的报告基因活性比无增强子对照高100倍。共转染显性负性Ras后报告基因活性下调，证实了MAP激酶途径的参与。5-HT1受体激动剂CGS 12066A（CGS）以剂量依赖性方式阻断了MEK1对增强子的激活。由于尚不清楚CGRP增强子因子是否为MAP激酶的直接靶点，我们随后使用了由细胞外信号调节激酶（ERK）和c-Jun氨基末端激酶（JNK）MAP激酶直接激活的EIk-1和c-Jun依赖性报告基因。CGS处理抑制了这两种报告基因的激活，表明至少两种MAP激酶是CGS介导的抑制的直接靶点。我们进一步证明，即使在存在组成型活性MEK1的情况下，5-HT1激动剂也能通过去磷酸化使ERK失活。这种失活似乎是由于MAP激酶磷酸酶-1水平的显著增加。这些结果确定了一种新的通用机制，通过该机制5-HT1受体激动剂可以抑制靶基因（如CGRP）的MAP激酶激活。
