Melatonin regulates CRE-dependent gene transcription underlying osteoblast proliferation by activating Src and PKA in parallel.
Melatonin regulates CRE-dependent gene transcription underlying osteoblast proliferation by activating Src and PKA in parallel.
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褪黑激素通过同时激活 Src 和 PKA 来调节成骨细胞增殖的 CRE 依赖性基因转录。
DOI：
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发表时间：
2018-01
影响因子：
2.2
通讯作者：
Zhu Yue
中科院分区：
文献类型：
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作者：
Tao Lin;Zhu Yue
一些研究表明褪黑激素与特发性脊柱侧凸之间存在关系，包括我们之前的研究表明褪黑激素可以抑制成骨细胞增殖；然而，其机制尚不清楚。在这里，我们利用MTT试验显示褪黑素以浓度和时间依赖的方式显著降低成骨细胞增殖。通过实时PCR、MTT测定、免疫荧光和荧光素酶测定等综合技术，我们证实褪黑激素诱导的cAMP反应元件结合蛋白（CREB）磷酸化的变化以褪黑激素受体依赖的方式降低了转录活性。令人惊讶的是，用丝裂原激活的蛋白激酶/细胞外信号调节激酶（MEK）抑制剂PD98059处理成骨细胞上调了CREB上游的其他级联反应。接下来，我们用PKA和Src抑制剂处理细胞，并观察到褪黑激素也可以激活蛋白激酶A （PKA）和Src途径。为了研究Src是否位于cAMP- pka通路的上游，我们测量了有和没有Src抑制剂（PP2）的褪黑激素对cAMP水平的反应，发现PP2没有额外的影响。因此，参与CREB磷酸化的转录依赖机制，以及褪黑激素，通过与PKA分离的平行信号通路激活Src。最后，我们用慢病毒CREB短发夹（sh） rna转染成骨细胞，发现增殖细胞核抗原（PCNA）的表达和成骨细胞的增殖都有所下降。这些结果表明，CREB和PCNA是褪黑激素信号传导的下游靶点，通过PKA和Src途径调控CREB的下调，有助于褪黑激素诱导的成骨细胞增殖抑制。
Several studies have indicated a relationship between melatonin and idiopathic scoliosis, including our previous work which demonstrated that melatonin can inhibit osteoblast proliferation; however, the mechanism remains unclear. Here, we utilized a MTT assay to show that melatonin significantly reduces osteoblast proliferation in a concentration-and time-dependent manner. Through a combination of techniques, including real-time PCR, MTT assays, immunofluorescence, and luciferase assays, we confirmed that melatonin-induced changes in phosphorylated cAMP response element-binding protein (CREB) reduced transcriptional activity in a melatonin receptor-dependent manner. Surprisingly, treatment of osteoblasts with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 up-regulated other cascades upstream of CREB. We next treated cells with PKA and Src inhibitors and observed that melatonin can also activate the protein kinase A (PKA) and Src pathways. To examine whether Src is upstream from the cAMP-PKA pathway, we measured cAMP levels in response to melatonin with and without a Src inhibitor (PP2) and found that PP2 had no additional effect. Therefore, the transcription-dependent mechanisms involved in CREB phosphorylation, along with melatonin, activated Src via a parallel signaling pathway that was separate from that of PKA. Finally, we transfected osteoblasts with lentiviral CREB short hairpin (sh) RNAs and found a decrease in the expression of proliferating cell nuclear antigen (PCNA) and osteoblast proliferation. These results suggest that CREB and PCNA are downstream targets of melatonin signaling, and that the down-regulation of CREB, which is regulated via PKA and Src pathways, contributes to the melatonin-induced inhibition of osteoblast proliferation.
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影响因子：
10.3
作者：
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