Tao Lin, Zhu Yue
Department of Orthopaedics, First Hospital, China Medical UniversityShenyang 110001, Liaoing, China.
Am J Transl Res. 2018 Jan 15;10(1):86-100. eCollection 2018.
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.
多项研究表明褪黑素与特发性脊柱侧凸之间存在关联，包括我们之前的研究工作，该研究表明褪黑素可抑制成骨细胞增殖；然而，其机制仍不清楚。在此，我们利用MTT法表明褪黑素以浓度和时间依赖性方式显著降低成骨细胞增殖。通过结合实时PCR、MTT法、免疫荧光和荧光素酶测定等技术，我们证实褪黑素诱导的磷酸化环磷酸腺苷反应元件结合蛋白（CREB）变化以褪黑素受体依赖性方式降低转录活性。令人惊讶的是，用丝裂原活化蛋白激酶/细胞外信号调节激酶激酶（MEK）抑制剂PD98059处理成骨细胞会上调CREB上游的其他级联反应。接下来，我们用蛋白激酶A（PKA）和Src抑制剂处理细胞，观察到褪黑素还可激活PKA和Src途径。为了检查Src是否在cAMP-PKA途径的上游，我们在有和没有Src抑制剂（PP2）的情况下测量了对褪黑素反应的cAMP水平，发现PP2没有额外影响。因此，参与CREB磷酸化的转录依赖性机制以及褪黑素通过与PKA不同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）的表达和成骨细胞增殖减少。这些结果表明CREB和PCNA是褪黑素信号的下游靶点，并且通过PKA和Src途径调节的CREB下调有助于褪黑素诱导的成骨细胞增殖抑制。
