钙离子/钙调素依赖蛋白激酶激酶β在脂联素促进下颌下腺分泌中的作用及机制研究

My previous study has shown that adiponectin functions as a promoter of salivary secretion in rat submandibular glands via activation of AMPK and paracellular permeability. Ca2+ plays an important role in gland secretion, and calmodulin-dependent protein kinase kinase β (CaMKKβ) is an important upstream molecular and an AMPK activator. However, whether Ca2+/CaMKKβ are participated in secretion induced by adiponectin is unknown. In this study, we will use physiological and pharmacological methods to clarify that adiponectin could increase intracellular Ca2+ ([Ca2+]i), and adiponectin-induced secretion might be dependent on the increased [Ca2+]i, to reveal that CaMKKβ regulated by increased [Ca2+]i could activate AMPK and participate in adiponectin-induced secretion, and to propose that AMPK, activated by Ca2+/CaMKKβ signalling, could ubiquitin degradationCa2+-released-activated Ca2+ channel modulator 1 (Orai1), inhibit the Ca2+-released-activated Ca2+ channel, and reduce [Ca2+]i. This study will provide better understanding in the molecular mechanism of adiponectin-induced secretion in rat submandibular glands, and provide a potential therapeutic strategy to modulate salivary gland dysfunction.

申请者前期研究发现脂联素可激活腺苷酸活化蛋白激酶（AMPK），增加旁细胞途径通透性，进而促进大鼠下颌下腺分泌。Ca2+在唾液腺分泌中发挥重要作用，且钙调素依赖蛋白激酶激酶β（CaMKKβ）作为Ca2+的重要下游分子，也可激活AMPK。然而，Ca2+/CaMKKβ是否参与脂联素激活AMPK的促分泌作用，尚不清楚。本课题拟从器官水平，组织水平和细胞水平，明确脂联素可以升高细胞内Ca2+（[Ca2+]i）；揭示脂联素的促分泌作用依赖于[Ca2+]i的增加；明确Ca2+通过CaMKKβ激活AMPK，参与脂联素的促分泌作用；揭示AMPK被Ca2+/CaMKKβ途径激活后，通过泛素化降解Ca2+释放激活Ca2+通道蛋白1，抑制Ca2+释放激活Ca2+通道开放，反馈降低[Ca2+]i。该研究将有助于全面认识脂联素和Ca2+在唾液腺分泌中的作用，为临床使用脂联素作为调节唾液腺分泌相关疾病的靶点提供依据。

申请者前期研究发现脂联素可激活腺苷酸活化蛋白激酶（AMPK），增加旁细胞途径通透性，进而促进大鼠下颌下腺分泌。Ca2+在唾液腺分泌中发挥重要作用，且钙调素依赖蛋白激酶激酶β（CaMKKβ）作为Ca2+的重要下游分子，也可激活AMPK。然而，Ca2+/CaMKKβ是否参与脂联素激活AMPK的促分泌作用，尚不清楚。本项目按照原定计划内容和进度安排顺利进行，在项目执行期间，我们从器官水平，组织水平和细胞水平，明确了脂联素可以升高细胞内Ca2+（[Ca2+]i）；揭示了脂联素促进大鼠下颌下腺分泌的作用依赖于[Ca2+]i的增加；明确了Ca2+可通过其下游信号分子CaMKKβ激活AMPK，参与脂联素的促分泌作用；进一步在体外水平证实了AMPK被Ca2+/CaMKKβ途径激活后，通过泛素化降解Ca2+释放激活Ca2+通道蛋白1（Orai1），抑制Ca2+释放激活Ca2+通道的开放，负反馈降低[Ca2+]i。该研究将有助于全面认识脂联素和Ca2+在唾液腺分泌中的作用，为临床使用脂联素作为调节唾液腺分泌相关疾病的靶点提供依据。