Original article

M. BAR-SHAI, E. HASNIS, Z. WIENER-MEGNAZI, A.Z. REZNICK


THE ROLE OF REACTIVE NITROGEN SPECIES AND CIGARETTE SMOKE
IN ACTIVATION OF TRANSCRIPTION FACTOR NF-B AND IMPLICATION
TO INFLAMMATORY PROCESSES


Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion-Israel Institution of Technology, Haifa, Israel


  Using the electromobility shift assay (EMSA) in the rat myoblast system, the activation of transcription factor NF-B by reactive nitrogen species was evaluated. Two distinct patterns of activation were demonstrated. Whereas NO donor, SNAP, activated NF-B in the classical pathway, which led to a transient response, NF-B activation by peroxynitrite donor, SIN-1, was mediated by an alternative pathway, which has been demonstrated in previous works to involve tyrosine nitration of the NF-B inhibitory protein I-Balpha. This led to a constitutive non-transient activation of NF-B and a prolonged inflammatory reaction. Lymphocytes exposed to mild intensity of cigarette smoke for 8 h, which activated NF-B, exhibited a decrease in the fraction of apoptotic cells from 27% to 19% compared with lymphocytes exposed to atmospheric air, using the FACS Annexin V assay. This also has been shown in previous works to be mediated by peroxynitrite. Thus, mild exposure to cigarette smoke induces NF-B activation, which can attenuate apoptosis in human lymphocytes and lead to prolonged inflammatory response. A possible proposed mechanism for induction of chronic inflammatory response may involve peroxynitrite-induced activation of NF-B.

Key words: apoptosis, cigarette smoke, lymphocytes, myoblast, NF-B, nitric oxide, peroxynitrite, reactive nitrogen species (RNS), reactive oxygen species (ROS), SNAP, SIN-1



INTRODUCTION

NF-B is a cytoplasmic transcription factor, which is activated by a variety of inducers including hormones, growth factors, viruses, chemicals, reactive oxygen species (ROS), reactive nitrogen species (RNS), and cytokines. NF-B triggers genes associated with processes such as inflammation, inhibition of apoptosis, growth and differentiation. In the unstimulated state, NF-B is bound in the cytosol to its inhibitory protein, I-Balpha. The classical pathway of NF-B activation involves phosphorylation of I-Balpha by IK kinase-1, release of I-aBa from NF-B subunits P65 and P50, which undergo nuclear translocation and modulation of gene expression. Concomitantly, NF-B activates the I-Balpha gene, resulting in synthesis and nuclear translocation of unphosphorylated I-Balpha that binds to NF-B and translocates it back to the cytoplasm.

Cigarette smoke (CS) has been associated with a variety of human pathologies including cardiovascular diseases and atherosclerosis, lung diseases, cancer, and infertility (1). In the pulmonary system, CS is implicated in the development and exacerbation of chronic bronchitis, asthma, and lung cancer (2). One of the proposed mechanisms by which CS exerts its effect on the airways and lungs is chronic activation of inflammatory processes by CS toxins (3, 4). Even mild environmental exposure to CS ("passive smokers") causes pulmonary disease to develop due to activation of the inflammatory system (5). This activation involves continuous recruitment of neutrophils and macrophages (5). Superoxide and NO present in CS react to form peroxynitrite anion (ONOO-), a RNS involved in protein nitration and nitrosylation, which has been associated with a variety of pathological conditions (6). Our previous work (unpublished) has shown that human lymphocytes exposed to mild, but not high, intensity CS exhibited activation of NF-B, which is mediated by formation of peroxynitrite through a reaction between smoke derived NO and endogenously produced superoxide. In the current study our aim was to examine the activation of NF-B by SNAP, S-nitroso-N-acetylpenicilamine (NO donor) and SIN-1, 3-morpholine-sydnoimine (peroxynitrite donor) and evaluate the effect of CS-induced NF-B activation on lymphocyte apoptosis, as an indicator of chronic inflammation.


MATERIAL AND METHODS

The study was approved by an institutional Ethics Committee. A L8 rat myoblast cell culture system was used for the evaluation of NF-B activation by SNAP and SIN-1, as previously described (7, 8). Human lymphocytes were used for the evaluation of cigarette smoke-induced NF-B activation and were cultured and grown, as previously described (9, 10).

Electromobility shift assay (EMSA).

The effect of SNAP treatment on NF-B activation in L8 myoblasts was measured by the EMSA assay, as previously described (9). L8 cells were treated with 10 mM SNAP for 10, 20, 30, 60, and 120 min and with 20 mM SNAP for 10, 20, and 30 min. Equal amounts of nuclear extracts were added to each lane. Nuclear extracts were analyzed by EMSA using 32P-labeled kB consensus oligonucleotides sequence of NF-B promoter. The effect of SIN-1 treatment on NF-B activation was also evaluated by EMSA in the identical conditions as those described for SNAP.

FACS-Annexin V assay

NF-B is activated by cigarette smoke and is also known to inhibit apoptosis in various cell lines, including human lymphocytes. Thus, we evaluated the effect of smoke-induced activation of NF-B on apoptosis of human lymphocytes after exposure of lymphocytes to low intensity cigarette smoke for 8 h. Control lymphocytes were exposed to atmospheric air for the same period. Then, cells were collected, stained for Annexin V and proprium iodide, and then underwent counting and analysis by FACS assay.


RESULTS

NF-B was activated by SNAP in a time and concentration-dependent manner. Using both 10 mM and 20 mM SNAP concentrations, NF-B activation and nuclear translocation was identified already after 10 min, followed by a gradual decrease (Fig. 1). Using SIN-1, NF-B activation and nuclear translocation was identified after 10 min, but no decrease was noted through 120 min (Fig. 2). In the FACS-Annexin V assay, lymphocytes exposed to mild intensity of cigarette smoke for 8 h, which activated NF-B, exhibited a decrease in the fraction of apoptotic cells from 22% to 13% compared with lymphocytes exposed to atmospheric air (Fig. 3A). The mean decrease in apoptotic cells in 3 identical experiments was from 27% to 19% (Fig. 3B).

Fig. 1. Time-dependent activation of NF-B by two concentrations of SNAP using EMSA. SNAP concentrations in µM.

Fig. 2. Time-dependent activation of NF-B by two concentrations of SIN-1. SIN-1 concentrations in µM.

Fig. 3. A - Staining for Annexin V and propidium iodide as an index for lymphocyte apoptosis following exposure to low intensity cigarette smoke. In each square, cells in the lower right quadrant (Annexin V positive, Propidium Iodide negative) represent apoptotic cells. Left square - cells immediately after collection, right square - following 8 h of exposure to mild intensity cigarette smoke, middle square - following 8 h of exposure to atmospheric air. MECS - Mild Exposed Cigarette Smoke. B - Mean ±SD of apoptotic cells from three different experiments.


DISCUSSION

SNAP activates NF-B in the classical pathway in the rat myoblasts system, which may lead to transient inflammatory response. As shown in Fig 1, upon SNAP treatment, the NO produced activates IKK, which phosphorylates I-Balpha, leading to its ubiquitination and subsequent degradation. The released activated NF-B translocates to the nucleus and activates a variety of genes, including the gene for I-Balpha itself, leading to its de novo synthesis, which causes feedback inhibition and deactivation of NF-B (Fig 4).

Fig. 4. Proposed mechanism for the two distinct patterns of NF-B activation by SNAP and SIN-1.

On the other hand, NF-B activation by SIN-1 is mediated by an alternative pathway, leading to a prolonged inflammatory reaction. In previous works it was shown to involve I-Balpha nitration rather than phosphorylation. Upon activation of NF-B by SIN-1, the prolonged exposure of ONOO- causes nitration of I-Balpha, rather than phosphorylation. This prevents its ubiquitination and further degradation, and ultimately leads to non-transient, prolonged activation of NF-B (8) (Fig. 4). Mild exposure to cigarette smoke induces NF-B activation, which can attenuate apoptosis in human lymphocytes and leads to increased inflammatory response.

In summary, chronic inflammatory response evoked by peroxynitrite-induced activation of NF-B may lead to tissue-specific deleterious biological consequences. In human lymphocytes, this may result in interference with apoptosis regulation and increased risk for chronic inflammation and malignant diseases. In rat myocytes, it may lead to increased protein breakdown and muscle atrophy. Therefore, we assume that peroxynitrite may be one of key mediators in physiological and pathological inflammatory processes.

Acknowledgments: This work was supported by the Krol foundation of Lakewood, NJ, and a grant from the Vice President of Research, Technion, Israel.


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Author’s address: A.Z. Reznick, Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion-Israel Institution of Technology, Haifa, 31096 Israel;
e-mail: reznick@tx.technion.ac.il