CREB 依赖性 LPA 诱导的信号转导在小气道基底细胞和成纤维细胞之间引发了一个促纤维化的反馈回路。
CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts.
机构信息
Department of Genetic Medicine, Weill Cornell Medical College, 1300 York Avenue, Box 164, New York, NY, 10065, USA.
出版信息
Respir Res. 2021 Apr 1;22(1):97. doi: 10.1186/s12931-021-01677-0.
BACKGROUND
Lysophosphatidic acid (LPA), generated extracellularly by the action of autotaxin and phospholipase A2, functions through LPA receptors (LPARs) or sphingosine-1-phosphate receptors (S1PRs) to induce pro-fibrotic signaling in the lower respiratory tract of patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that LPA induces changes in small airway epithelial (SAE) basal cells (BC) that create cross-talk between the BC and normal human lung fibroblasts (NHLF), enhancing myofibroblast formation.
METHODS
To assess LPA-induced signaling, BC were treated with LPA for 2.5 min and cell lysates were analyzed by phosphokinase array and Western blot. To assess transcriptional changes, BC were treated with LPA for 3 h and harvested for collection and analysis of RNA by quantitative polymerase chain reaction (qPCR). To assess signaling protein production and function, BC were washed thoroughly after LPA treatment and incubated for 24 h before collection for protein analysis by ELISA or functional analysis by transfer of conditioned medium to NHLF cultures. Transcription, protein production, and proliferation of NHLF were assessed.
RESULTS
LPA treatment induced signaling by cAMP response element-binding protein (CREB), extracellular signal-related kinases 1 and 2 (Erk1/2), and epithelial growth factor receptor (EGFR) resulting in elevated expression of connective tissue growth factor (CTGF), endothelin-1 (EDN1/ET-1 protein), and platelet derived growth factor B (PDGFB) at the mRNA and protein levels. The conditioned medium from LPA-treated BC induced NHLF proliferation and increased NHLF expression of collagen I (COL1A1), smooth muscle actin (ACTA2), and autotaxin (ENPP2) at the mRNA and protein levels. Increased autotaxin secretion from NHLF correlated with increased LPA in the NHLF culture medium. Inhibition of CREB signaling blocked LPA-induced changes in BC transcription and translation as well as the pro-fibrotic effects of the conditioned medium on NHLF.
CONCLUSION
Inhibition of CREB signaling may represent a novel target for alleviating the LPA-induced pro-fibrotic feedback loop between SAE BC and NHLF.
背景
溶血磷脂酸（LPA）由自分泌酶和磷脂酶 A2 的作用在细胞外产生，通过 LPA 受体（LPAR）或鞘氨醇-1-磷酸受体（S1PR）发挥作用，在特发性肺纤维化（IPF）患者的下呼吸道诱导促纤维化信号。我们假设 LPA 诱导小气道上皮（SAE）基底细胞（BC）发生变化，从而在 BC 和正常人肺成纤维细胞（NHLF）之间产生细胞间通讯，增强肌成纤维细胞的形成。
方法
为了评估 LPA 诱导的信号，用 LPA 处理 BC 2.5 分钟，通过磷酸激酶阵列和 Western blot 分析细胞裂解物。为了评估转录变化，用 LPA 处理 BC 3 小时，收获细胞用于收集和通过定量聚合酶链反应（qPCR）分析 RNA。为了评估信号蛋白的产生和功能，在用 LPA 处理后彻底洗涤 BC，并在收集用于通过 ELISA 分析蛋白质或通过将条件培养基转移到 NHLF 培养物中来进行功能分析之前孵育 24 小时。评估 NHLF 的转录、蛋白产生和增殖。
结果
LPA 处理诱导 cAMP 反应元件结合蛋白（CREB）、细胞外信号相关激酶 1 和 2（Erk1/2）和表皮生长因子受体（EGFR）的信号，导致结缔组织生长因子（CTGF）、内皮素-1（EDN1/ET-1 蛋白）和血小板衍生生长因子 B（PDGFB）的 mRNA 和蛋白水平升高。来自 LPA 处理的 BC 的条件培养基诱导 NHLF 增殖，并增加 NHLF 的胶原 I（COL1A1）、平滑肌肌动蛋白（ACTA2）和自分泌酶（ENPP2）的 mRNA 和蛋白表达。NHLF 中自分泌酶的分泌增加与 NHLF 培养基中 LPA 的增加相关。抑制 CREB 信号阻断了 LPA 诱导的 BC 转录和翻译变化以及条件培养基对 NHLF 的促纤维化作用。
结论
抑制 CREB 信号可能代表缓解 SAE BC 和 NHLF 之间 LPA 诱导的促纤维化反馈环的新靶点。
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