Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131, Mainz, Germany.
Department of Molecular Embryology, Institute for Anatomy and Cell Biology, Freiburg, Germany.
Cell Commun Signal. 2022 Apr 7;20(1):47. doi: 10.1186/s12964-022-00855-x.
NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA isoform is detected. However, the expression of NOS2 in differentiating human pluripotent stems (hPSCs) has not been analyzed yet.
Public available RNA-seq databases were screened for data of hPSCs during differentiation to different target cells. An isoform specific algorithm was used to analyze NOS2 mRNA isoform expression. In addition, we differentiated four different human iPSC cell lines toward cortical neurons and analyzed NOS2 mRNA expression by qRT-PCR and 5'-RACE. The functionality of the NOS2-2 protein was analyzed by transient transfection of expression clones in human DLD1 cells and nitrate measurement in the supernatant of these cells.
In RNA-seq databases we detected a transient expression of the NOS2 mRNA during the differentiation of hPSCs to cardiomyocytes, chondrocytes, mesenchymal stromal cells, neurons, syncytiotrophoblast cells, and trophoblasts. NOS2 mRNA isoform specific analyses showed, that the transiently expressed NOS2 mRNA in differentiating hPSC (NOS2-2; "diff-iNOS") differ remarkably from the already described NOS2 transcript found in colon or induced islets (NOS2-1; "immuno-iNOS"). Also, analysis of the NOS2 mRNA- and protein expression during the differentiation of four different hiPSC lines towards cortical neurons showed a transient expression of the NOS2 mRNA and NOS2 protein on day 18 of the differentiation course. 5'-RACE experiments and isoform specific qRT-PCR analyses revealed that only the NOS2-2 mRNA isoform was expressed in these experiments. To analyze the functionality of the NOS2-2 protein, we transfected human DLD-1 cells with tetracycline inducible expression clones encoding the NOS2-1- or -2 coding sequence. After induction of the NOS2-1 or -2 mRNA expression by tetracycline a similar nitrate production was measured proofing the functionality of the NOS2-2 protein isoform.
Our data show that a differentiation specific NOS2 isoform (NOS2-2) is transiently expressed during differentiation of hPSC. Video Abstract.
NOS2 表达主要存在于暴露于细菌或细胞因子处理的组织中，主要与先天免疫反应有关。NOS2 有三种同工型（NOS2-1 至 -3）。在 RNA-seq 数据集中，分析炎症基因表达时，仅检测到 NOS2-1 mRNA 同工型的表达。然而，尚未分析分化中的人类多能干细胞（hPSC）中 NOS2 的表达。
筛选公共可用的 RNA-seq 数据库，以获取 hPSC 在向不同靶细胞分化过程中的数据。使用同工型特异性算法分析 NOS2 mRNA 同工型表达。此外，我们将四种不同的人 iPSC 细胞系分化为皮质神经元，并通过 qRT-PCR 和 5'-RACE 分析 NOS2 mRNA 表达。通过在人 DLD1 细胞中转染表达克隆并测量这些细胞上清液中的硝酸盐来分析 NOS2-2 蛋白的功能。
在 RNA-seq 数据库中，我们在 hPSC 向心肌细胞、软骨细胞、间充质基质细胞、神经元、合胞滋养层细胞和滋养层细胞分化过程中检测到 NOS2 mRNA 的瞬时表达。NOS2 mRNA 同工型特异性分析表明，分化中的 hPSC 中瞬时表达的 NOS2 mRNA（NOS2-2；“分化-iNOS”）与已在结肠或诱导胰岛中发现的描述性 NOS2 转录本明显不同（NOS2-1；“免疫-iNOS”）。此外，在将四种不同的 hiPSC 系分化为皮质神经元的过程中分析 NOS2 mRNA 和蛋白表达表明，在分化过程的第 18 天，NOS2 mRNA 和 NOS2 蛋白出现瞬时表达。5'-RACE 实验和同工型特异性 qRT-PCR 分析表明，在这些实验中仅表达 NOS2-2 mRNA 同工型。为了分析 NOS2-2 蛋白的功能，我们用 tetracycline 诱导表达克隆转染人 DLD-1 细胞，该克隆编码 NOS2-1 或 -2 编码序列。在用 tetracycline 诱导 NOS2-1 或 -2 mRNA 表达后，测量类似的硝酸盐生成量，证明 NOS2-2 蛋白同工型的功能。
我们的数据表明，在 hPSC 分化过程中，NOS2 的一种分化特异性同工型（NOS2-2）是瞬时表达的。
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