Effects of Src on HNE-induced COX-2 expression in YPEN cells.

<p>(A) YPEN-1 cells were stimulated with 5 μM HNE for the indicated times (0–12 h). COX-2 protein expressions in cell lysates were analyzed by immunoblotting. (B) After pretreatment with dasatinib (300 nM) for 30 min, cells were stimulated with 5 μM 4-HNE for 9 h. (C) YPEN-1 cells were transfected with Src siRNA for 48 h and then treated with 5 μM HNE for 9 h. COX-2 protein expression levels were determined by immunoblotting. CON, control; NC, negative control; si, Src siRNA-treated cells; HNE, 4-hydroxy-2-nonenal; COX-2, cyclooxygenase-2.</p

Simulation and conformational modeling of Src modified by 4-HNE.

<p>(A) Conformational modeling of Src modified by 4-HNE at Cys248 in its SH2 domain. (B) Docking simulation for the interaction between Src and 4-HNE using AutoDock 4.2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129244#pone.0129244.ref042" target="_blank">42</a>]. The SH2 domain in the crystal structure of human active c-Src constitutes a 4-HNE binding pocket. The binding energy of the 4-HNE/Src interaction was -4.13 kcal/mol. (C) hydrogen bonding between Src and 4-HNE as determined using the Ligand Scout program based on docking simulation results. It was predicted that certain residues of Src, notably Cys248, were mainly responsible for hydrogen bonding between 4-HNE and Src.</p

Src Tyrosine Kinase Activation by 4-Hydroxynonenal Upregulates p38, ERK/AP-1 Signaling and COX-2 Expression in YPEN-1 Cells

<div><p>4-Hydroxynonenal (4-HNE), a major end product of lipid peroxidation, is highly reactive and involved in various cellular processes, such as inflammatory signaling. However, to date, the mechanistic roles of 4-HNE in inflammatory signaling related to protein tyrosine kinases have not been elucidated. In the present study, we investigated the interaction between 4-HNE and Src (a non-receptor tyrosine kinase) for its involvement in the molecular modulation of the inflammatory signaling pathway utilizing the YPEN-1 cell system. Immunoprecipitation experiments showed that 4-HNE phosphorylates (activates) Src at Tyr416 via adduct formation. In addition, LC-MS/MS and a docking simulation model revealed an addiction site at the Cys248 residue of Src, resulting in the stimulation of downstream p38, ERK/AP-1 and cyclooxygenase-2 (COX-2) signaling in YPEN-1 cells. The role of 4-HNE-activated Src in downstream inflammatory signaling was further investigated using dasatinib (a Src inhibitor) and by siRNA knockdown of Src. p38 and ERK were directly regulated by Src, as revealed by immunoblotting of the phosphorylated forms of mitogen-activated protein kinases (MAPKs), which are key elements in the signaling transduction pathway initiated by Src. The study also shows that Src modulates the HNE-enhanced activation of AP-1 and the expression of COX-2 (a target gene of AP-1). Together, the results of this study show that 4-HNE stimulates Src tyrosine kinase in activation of the inflammation process.</p></div

Identification of the site of Src modification by 4-HNE using LC-MS/MS.

<p>Sequence coverage: 70%; protein score: 28842; ion search cut off: 10. Src (10μg) was exposed to 4-HNE at a molar ratio of 1:1, digested with trypsin, and analyzed by LC MS/MS as described in Methods. Among ten ion search cut off, 4-HNE was detected in 236–260 range of amino acid. The peptide sequence containing 4-HNE adduct at amino acids, His236, Cys241, and Cys248 is denoted in bold font.</p><p>^a Observed values are those actually measured by LC MS/MS.</p><p>^b Mr(expt) are the experimental values of charge states.</p><p>^c Mr(calc) are relative molecular mass values calculated from the matched peptide sequence.</p><p>^d MASCOT scores are MS/MS ion score based on calculated probabilities.</p><p>Identification of the site of Src modification by 4-HNE using LC-MS/MS.</p

Mediation of Src in 4-HNE-induced enhancement of AP-1 activity in YPEN-1 cells

<p>(A) YPEN-1 cells were transfected with Src siRNA for 48 h, treated with HNE (10 μM), and immunoblotted for c-Jun and phosphor-c-Jun. (B) The nuclear translocation of c-Jun was visualized by immunocytochemical staining. Nuclei were stained with Hoechst dye (blue) and alexa-488-conjugated (green) goat anti-rabbit IgG to detect c-Jun antibody. Merged images are shown on the right. (D) AP-1 activities were measured by luciferase reporter assay in cells transfected with AP-1 luciferase reporter constructs. The Src inhibitor dasatinib (200 nM) was pretreated for 30 min before 4-HNE stimulation. Bars represent means±SEs (n = 4).*p<0.05 <i>vs</i>. control, #p<0.05 <i>vs</i>. vehicle. AP-1, activator protein-1; CON, control; HNE, 4-hydroxy-2-nonenal; NC, negative control; si, Src siRNA treated; TFIIB, transcription factor II B.</p

Phosphorylation and activation of Src by direct adduction with 4-HNE.

<p>(A) YPEN-1 cells were incubated in serum free medium with 10 μM 4-HNE for 15–60 min. Src phosphorylation was quantified densitometrically. Results are representative of at least three separate experiments. Statistical significance: *p < 0.05 <i>vs</i>. CON. (B) 4-HNE and Src were immunoblotted after immunoprecipitating Src in cells treated or not treated with 4-HNE 10 μM for 30 min. CON, control; HNE, 4-hydroxy-2-nonenal.</p

The regulation of NF-kB by heptadecane via NIK/IKK and MAPKs induced by oxidative stress.

<p>YPEN-1 cells were grown until 80% confluent in 100 mm dishes in DMEM medium. Cells were pre-treated (1 hr) with heptadecane (1, 10, or 20 µM) or NAC (2 mM), then stimulated with 10 µM t-BHP. (A) After stimulation with t-BHP (1 hr for NF-kB) in the absence (-) or presence (+) of heptadecane (1, 10, or 20 µM), cells lysed and total nuclear and cytosolic proteins were extracted. Western blot was performed for p50, p65 and IκB. (B) After stimulation with t-BHP (10 min for phosphorylated NIK and 20 min for phosphorylated IKK) in the absence (-) or presence (+) of heptadecane (1, 10 or 20 µM), cells were lysed and p-NIK and p-IKKα/β levels were determined. (C) After stimulation with t-BHP (10 min for phosphorylated MEK1/2 and 20 min for phosphorylated MAPKs) in the absence (-) or presence (+) of heptadecane (1, 10, or 20 µM), cells were lysed and p-MEK1/2, p-ERK1/2, p-p38, and p-JNK levels were determined. One representative experimental blot of each protein is shown from three experiments that yielded similar results.</p

Heptadecane inhibited oxidative stress-induced NF-kB-dependent reporter gene (luciferase) expression.

<p>YPEN-1 cells were transiently transfected with a NF-kB-containing plasmid linked to the luciferase gene, then pre-incubation with heptadecane (1, 5, 10 µM and 20 µM) for 1 hr and co-treated with t-BHP for 5 hr. Results are presented in relative luminescence units (RLU). One-way analysis of variance (ANOVA) was used to determine the significances of NF-kB-dependent luciferase activity differences between untreated controls and treated groups: ^##p<0.01 vs. vector control, *p<0.05 vs. treated 0.2 µM t-BHP group, respectively.</p

Heptadecane suppressed age-related increases in NF-kB activity.

<p>(A) RS generation in aged rat was determined by observing the effects of age and heptadecane using the DCFHDA method and kidney homogenates. The values shown are means ± SEs for 5 rats. Young = 9-month-old baicalin untreated rats; Aged = 20-month-old, heptadecane untreated rats. Values are means ± SEs for 5 rats. ^###p<0.001 vs. Young rats; **p<0.01 vs. age-matched rats; ***p<0.001 vs. age-matched rats by one factor ANOVA. Western blot was performed to detect (B) nuclear p50 and p65 protein levels in nuclear extracts (30 µg protein) and age-related IkBα, IkBβ, and IkBα phosphorylation degradations in cytoplasmic extracts (40 µg protein) in young, aged, and aged rats fed heptadecane. (C) EMSA method was used to compare the nuclear NF-kB binding activities of aged rat fed heptadecane and aged counterparts. One representative result of three experiments that yielded similar results is shown. Young rats (9 months of age) and aged (20 months of age) were utilized. Heptadecane was fed to the aged rat at 2 mg or 4 mg/Kg per day for 10 days. Statistical significance: results of one-factor ANOVA: ***p<0.001 vs. young rat; ^##p<0.01, ^###p<0.001 vs. old non-heptadecane-fed rats, respectively. (D) Immunoreactivity was determined by NF-kB (p65) in renal tissue of age and heptadecane (Immunohistochemistry, ×100).</p

Possible mechanism of the effect of aging and heptadecane on the NIK/MAPKs/NF-kB pathway.

<p>RS, reactive species; NF-kB, nuclear factor kappa B; NIK, NF-kB-inducing kinase; IKK, IkB kinase; COX-2, cyclooxygenase-2.R.</p