Kluge Milena, Namkoong Eun, Khakipoor Shokoufeh, Park Kyungpyo, Roussa Eleni
Department of Molecular Embryology, Faculty of Medicine, Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg, Germany.
Department of Physiology, School of Dentistry, Seoul National University, Seoul, South Korea.
J Cell Physiol. 2019 Sep;234(9):15061-15079. doi: 10.1002/jcp.28147. Epub 2019 Jan 15.
Bicarbonate concentration in saliva is controlled by the action of acid-base transporters in salivary duct cells. We show for the first time expression of ATP6V1B1 in submandibular gland and introduce transforming growth factor-beta (TGF-β) as a novel regulator of V-ATPase subunits. Using QRT-PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, chromatin immunoprecipitation, and intracellular H( ) recording with H( )-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein we show that in the human submandibular gland (HSG) cell line, activation of TGF-β signaling upregulates ATP6V1E1 and ATP6V1B2, downregulates ATP6V1B1, and has no effect on ATP6V1A. TGF-β1 effects on ATP6V1B1 are mediated through the canonical, the soluble adenylate cyclase, and ERK signaling. A CREB binding sequence was identified in the ATP6V1B1 promoter and CREB binding decreased after TGF-β1 treatment. Following acidosis, a bafilomycin-sensitive and Na -independent cell pH recovery was observed in HSG cells, an effect that was not influenced after disruption of acidic lysosomes. Moreover, neutralization of TGF-βs, inhibition of TGF-β receptor, or inhibition of the canonical pathway decreased membrane expression of ATP6V1A and prevented the acidosis-induced increased V-ATPase activity. The results suggest multiple modes of action of TGF-β1 on V-ATPase subunits in HSG cells: TGF-β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context-dependent manner. Moreover, surface V-ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.
唾液中的碳酸氢盐浓度受唾液腺导管细胞中酸碱转运体的作用控制。我们首次展示了ATP6V1B1在下颌下腺中的表达，并引入转化生长因子-β（TGF-β）作为V-ATP酶亚基的新型调节因子。使用定量逆转录聚合酶链反应（QRT-PCR）、免疫印迹、表面蛋白生物素化、免疫荧光、染色质免疫沉淀以及用氢离子敏感染料2',7'-双（羧乙基）-5-（和-6）-羧基荧光素进行细胞内氢离子记录，我们发现，在人下颌下腺（HSG）细胞系中，TGF-β信号激活会上调ATP6V1E1和ATP6V1B2，下调ATP6V1B1，并且对ATP6V1A没有影响。TGF-β1对ATP6V1B1的作用是通过经典途径、可溶性腺苷酸环化酶和细胞外信号调节激酶（ERK）信号介导的。在ATP6V1B1启动子中鉴定出一个环磷腺苷效应元件结合蛋白（CREB）结合序列，TGF-β1处理后CREB结合减少。酸中毒后，在HSG细胞中观察到一种对巴弗洛霉素敏感且不依赖钠离子的细胞pH恢复，酸性溶酶体破坏后该效应不受影响。此外，中和TGF-β、抑制TGF-β受体或抑制经典途径会降低ATP6V1A的膜表达，并阻止酸中毒诱导的V-ATP酶活性增加。结果表明TGF-β1对HSG细胞中V-ATP酶亚基有多种作用模式：TGF-β1可能以上下文依赖的方式调节某些亚基的转录或蛋白质合成以及其他亚基的运输。此外，表面V-ATP酶在唾液腺导管细胞中具有活性，并参与酸中毒后的细胞内pH调节。
