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Quantitative proteomic analysis revealed N'-nitrosonornicotine-induced down-regulation of nonmuscle myosin II and reduced cell migration in cultured human skin fibroblast cells.

ABSTRACT: The association of tobacco smoke with decreased cell motility and wound healing is well documented; however, the cellular mechanisms and specific toxic tobacco constituents responsible for this effect are not well understood. Tobacco-specific N-nitrosamines (TSNAs) are among the most important classes of carcinogens found in tobacco products. The TSNA N'-nitrosonornicotine (NNN) is present at relatively high levels in tobacco and its smoke, as well as second- and third-hand smoke. To investigate the cellular pathways that are perturbed upon NNN exposure, we employed a quantitative proteomic approach, utilizing stable isotope labeling by amino acids in cell culture and mass spectrometry, to assess the NNN-induced alteration of protein expression in GM00637 human skin fibroblast cells. With this approach, we were able to quantify 2599 proteins, 191 of which displayed significantly changed expression following NNN exposure. One of the main findings from our proteomic analysis was the down-regulation of six different subunits of myosin, particularly nonmuscle myosin II heavy chain, isoforms A, B, and C. In addition, we found the altered expression of several extracellular matrix proteins and proteins involved in cellular adhesion. Together, our quantitative proteomic results suggested that NNN exposure may interfere with fibroblast motility. An in vitro scratch wound assay result supported that NNN exposure reduced the ability of dermal fibroblast to migrate into the scratched area. The results from the present study offer novel insights into the cellular mechanisms of NNN toxicity and identify NNN as a specific tobacco constituent that contributes to decreased fibroblast migration.

SUBMITTER: Prins JM

PROVIDER: S-EPMC3586308 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Quantitative proteomic analysis revealed N'-nitrosonornicotine-induced down-regulation of nonmuscle myosin II and reduced cell migration in cultured human skin fibroblast cells.

Prins John M JM Wang Yinsheng Y

Journal of proteome research 20130128 3

The association of tobacco smoke with decreased cell motility and wound healing is well documented; however, the cellular mechanisms and specific toxic tobacco constituents responsible for this effect are not well understood. Tobacco-specific N-nitrosamines (TSNAs) are among the most important classes of carcinogens found in tobacco products. The TSNA N'-nitrosonornicotine (NNN) is present at relatively high levels in tobacco and its smoke, as well as second- and third-hand smoke. To investigate ...[more]

PMID: 23305604

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Project description:BackgroundTriglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis.MethodsTo identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells.ResultsUsing SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search.ConclusionsWe performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future.

Dataset Information

Quantitative proteomic analysis revealed N'-nitrosonornicotine-induced down-regulation of nonmuscle myosin II and reduced cell migration in cultured human skin fibroblast cells.

Publications

Quantitative proteomic analysis revealed N'-nitrosonornicotine-induced down-regulation of nonmuscle myosin II and reduced cell migration in cultured human skin fibroblast cells.

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