Norford D, Koo J S, Gray T, Alder K, Nettesheim P
Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Exp Lung Res. 1998 May-Jun;24(3):355-66. doi: 10.3109/01902149809041540.
The retinoic acid (RA) and differentiation dependence of constitutive expression of the nitric oxide synthase (NOS) isoforms, iNOS, eNOS, and bNOS, was examined by reverse transcriptase polymerase chain recitation (RT-PCR) in cultured, normal, human, tracheobronchial epithelial (NHTBE) cells. In the presence of RA (RA+), early passage NHTBE cells grown in air-liquid interface (ALI) cultures undergo mucous differentiation; in the absence of RA (RA-), they undergo metaplastic squamous differentiation. Under both conditions the respective differentiated phenotype develops around day 10 of culture. We found that iNOS mRNA levels were much higher in RA+ cultures, expressing the mucous phenotype, than in RA- cultures, expressing the metaplastic squamous phenotype. In contrast, eNOS mRNA levels were much higher in RA- cultures than in RA+ cultures. Expression of bNOS was not significantly affected by the RA status. The pattern of expression of NOS isoforms was then studied during the course of development of the two cellular phenotypes. During the early stages of differentiation, expression of iNOS (RA+) and eNOS (RA-) was very low, indicating that the expression of these two isoforms was not only dependent on the presence or absence of RA, but also on the degree of differentiation. The differentiation dependence of bNOS mRNA was less obvious. Four days of RA treatment of RA- cultures, which reverses the squamous phenotype and restores mucous differentiation, induced iNOS expression in a concentration-dependent manner. eNOS expression was depressed by 10(-8) M RA, while bNOS mRNA levels were slightly reduced by 10(-6) M RA. No NOS proteins were detected in unstimulated RA+ and RA- cultures. iNOS protein was induced by cytokine treatment in RA+ cultures, in contrast to eNOS and bNOS protein levels, which were unaffected. Our studies show that constitutive expression of the NOS isoforms is differentially regulated and that iNOS and eNOS mRNA levels are dependent on the stage of mucous and squamous differentiation, respectively. bNOS expression was only marginally affected by the RA or differentiation status.
通过逆转录聚合酶链反应（RT-PCR），在培养的正常人气管支气管上皮（NHTBE）细胞中检测了视黄酸（RA）以及一氧化氮合酶（NOS）亚型（诱导型NOS、内皮型NOS和脑型NOS）组成型表达对分化的依赖性。在存在RA（RA+）的情况下，在气液界面（ALI）培养中生长的早期传代NHTBE细胞会发生黏液分化；在不存在RA（RA-）的情况下，它们会发生化生鳞状分化。在这两种条件下，各自的分化表型在培养第10天左右形成。我们发现，表达黏液表型的RA+培养物中的诱导型NOS mRNA水平远高于表达化生鳞状表型的RA-培养物。相反，内皮型NOS mRNA水平在RA-培养物中比在RA+培养物中高得多。脑型NOS的表达不受RA状态的显著影响。然后在两种细胞表型的发育过程中研究了NOS亚型的表达模式。在分化早期，诱导型NOS（RA+）和内皮型NOS（RA-）的表达非常低，这表明这两种亚型的表达不仅取决于RA的存在与否，还取决于分化程度。脑型NOS mRNA的分化依赖性不太明显。对RA-培养物进行4天的RA处理可逆转鳞状表型并恢复黏液分化，以浓度依赖的方式诱导诱导型NOS表达。10^(-8) M的RA可抑制内皮型NOS表达，而10^(-6) M的RA可使脑型NOS mRNA水平略有降低。在未刺激的RA+和RA-培养物中未检测到NOS蛋白。与内皮型NOS和脑型NOS蛋白水平未受影响相反，细胞因子处理可在RA+培养物中诱导诱导型NOS蛋白。我们的研究表明，NOS亚型的组成型表达受到不同的调节，诱导型NOS和内皮型NOS mRNA水平分别取决于黏液和鳞状分化阶段。脑型NOS的表达仅受到RA或分化状态的轻微影响。
