Calcineurin-NFAT signaling, together with GABP and peroxisome PGC-1α, drives utrophin gene expression at the neuromuscular junction
Calcineurin-NFAT signaling, together with GABP and peroxisome PGC-1α, drives utrophin gene expression at the neuromuscular junction
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DOI：
10.1152/ajpcell.00196.2005
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发表时间：
2005-10-01
影响因子：
5.5
通讯作者：
Jasmin, BJ
中科院分区：
文献类型：
--
作者：
Angus, LM;Chakkalakal, JV;Jasmin, BJ
我们研究了钙调神经磷酸酶-NFAT（活化T细胞的核因子）信号传导是否在特异性指导肌纤维突触区室中utrophin的表达中起作用。免疫荧光实验揭示了神经钙调磷酸酶-NFAT信号级联的成分在神经肌肉接头的突触后膜结构域内的积累。使用肌纤维的突触与突触外区域的RT-PCR分析通过显示突触隔室内钙调神经磷酸酶转录物的积累证实了这些发现。我们还研究了钙调神经磷酸酶对utrophin基因表达的影响。药理学抑制钙调神经磷酸酶在小鼠与环孢素A或FK 506导致在utrophin A在突触部位的表达显着减少，而钙调神经磷酸酶的组成性激活有相反的效果。先前确定的NFAT结合位点的utrophin A启动子区域的突变，随后在小鼠肌肉中的直接基因转移研究，导致在lacZ报告基因构建体的突触表达的抑制。用培养的肌源性细胞进行的转染试验表明，钙调神经磷酸酶与GA结合蛋白（GABP）起相加作用，以反式激活utrophin A基因表达。由于GABP和钙调神经磷酸酶介导的途径都是由过氧化物酶体增殖物激活受体-γ辅激活因子-1 α（PGC-1 α）靶向的，我们研究了这种辅激活因子是否有助于utrophin基因表达。体外和体内转染实验表明，单独的PGC-1 α诱导utrophin A启动子的转录。有趣的是，这种诱导在很大程度上通过PGC-1 α与GABP的共表达而增强。总之，这些研究表明utrophin的突触表达也由钙调神经磷酸酶-NFAT信号传导驱动，并与涉及GABP和PGC-1 α的信号传导事件一起发生。
We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1 alpha alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1 alpha with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1 alpha.