Ji R R, Rupp F
Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Neurosci. 1997 Mar 1;17(5):1776-85. doi: 10.1523/JNEUROSCI.17-05-01776.1997.
The involvement of cAMP-responsive element-binding protein (CREB) signaling in tissue injury-induced inflammation and hyperalgesia has been characterized by measuring phosphorylation of CREB at serine-133 (CREB Ser133) using a specific antibody. In the unstimulated state, unphosphorylated CREB was observed in most nuclei of spinal neurons except for motor neurons, where only a small portion of neurons were stained. A few dorsal root ganglion (DRG) neurons were also CREB-positive. After a unilateral injection of formalin into the hindpaw, a strong and bilateral phosphorylation of CREB Ser133 was induced, as assessed by both immunohistochemistry and Western blot. PhosphoCREB (pCREB)-positive neurons were found in laminae I, II, V, and X of spinal cord on both sides. CREB phosphorylation was very rapid and reached peak levels within 10 min of formalin treatment, whereas few pCREB-positive neurons were seen in unstimulated spinal cord. The induction of pCREB was predominantly postsynaptic, because only 5% of DRG neurons were labeled after inflammation. In contrast to CREB phosphorylation, the induction of c-Fos expression reached peak levels 2 hr after formalin treatment and c-Fos induction was mainly ipsilateral. Both formalin-evoked CREB phosphorylation and c-Fos expression in the spinal cord were suppressed by pretreatment with the NMDA receptor antagonist MK-801 (3.5 mg/kg, i.p.) or halothane anesthesia. These results suggest that CREB signaling may play a role in the long-term facilitation of spinal cord neurons after hyperalgesia. Furthermore, our results indicate that CREB phosphorylation may be necessary but not sufficient for c-fos induction.
通过使用特异性抗体检测丝氨酸133位点的环磷腺苷反应元件结合蛋白（CREB）磷酸化，已对CREB信号通路在组织损伤诱导的炎症和痛觉过敏中的作用进行了表征。在未受刺激状态下，除运动神经元外，在脊髓神经元的大多数细胞核中观察到未磷酸化的CREB，在运动神经元中只有一小部分神经元被染色。少数背根神经节（DRG）神经元也呈CREB阳性。在后爪单侧注射福尔马林后，通过免疫组织化学和蛋白质印迹法评估，诱导了CREB丝氨酸133的强烈双侧磷酸化。在脊髓两侧的I、II、V和X层中发现了磷酸化CREB（pCREB）阳性神经元。CREB磷酸化非常迅速，在福尔马林处理后10分钟内达到峰值水平，而在未受刺激的脊髓中很少见到pCREB阳性神经元。pCREB的诱导主要是突触后性的，因为炎症后只有5%的DRG神经元被标记。与CREB磷酸化相反，c-Fos表达的诱导在福尔马林处理后2小时达到峰值水平，且c-Fos诱导主要是同侧性的。脊髓中福尔马林诱发的CREB磷酸化和c-Fos表达均被NMDA受体拮抗剂MK-801（3.5mg/kg，腹腔注射）预处理或氟烷麻醉所抑制。这些结果表明，CREB信号通路可能在痛觉过敏后脊髓神经元的长期易化中发挥作用。此外，我们的结果表明，CREB磷酸化可能是c-fos诱导所必需的，但并不充分。
