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

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

Heptadecane attenuated NF-kB-responsive COX-2 and iNOS via NIK/IKK and MAPKs.

<p>YPEN-1 cells were grown to 80% confluent in 100 mm dishes in DMEM. Cells were pre-treated (1 hr) with heptadecane (20 µM) and inhibitors and then stimulated with 10 µM t-BHP. After stimulation with t-BHP in the absence (−) or presence (+) of heptadecane and each kinase inhibitor, COX-2 and iNOS gene expressions were determined in cell extracts.</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

Heptadecane suppressed expressions of NF-kB-dependent genes in aged rats.

<p>Western blot was performed to detect renal COX-2 and iNOS levels in cytoplasmic extracts (40 µg protein) from young, aged, and aged rats fed heptadecane. One representative blot of each protein is shown from three experiments that yielded similar results, respectively. Young rats (9 months of age) and aged (20 months of age) were utilized. Heptadecane was fed to aged rats 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<0.05, ^##p<0.01 vs. old non-heptadecane-fed rats, respectively.</p

Involvement of Src in the 4-HNE-induced phosphorylation of p38, ERK and JNK.

<p>(A) YPEN-1 cells were pre-incubated with 300 nM of dasatinib (a Src inhibitor) for 30 min. After refreshing medium, 10 μM 4-HNE was added for 1 h. phospho-Src (Tyr416), phospho-ERK (Tyr204), phospho-p38 (Tyr182), phosphor-JNK (Thr183/Tyr185), the total form of each MAPK, and c-Src levels were analyzed by Western blotting. (B) YPEN-1 cells were transfected with Src siRNA(Si) or negative control siRNA(NC) for 48 h, and then exposed to 10 uM 4-HNE for 1 h. Phosphorylated MAPK and Src levels were evaluated by immunoblotting. AP-1, activator protein-1; COX-2, cyclooxygenase-2; ERK, extracellular signal-regulated kinase; HNE, 4-hydroxy-2-nonenal; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NC, negative control; si, Src siRNA treated.</p

Heptadecane suppressed the age-related activations of NIK/IKK and MAPKs.

<p>Western blot on renal cytoplasmic extracts (40 µg protein) from young, aged, and aged rats fed heptadecane. (A) Phosphorylations of P38 and JNK are designated p-p38 and p-JNK1/2. (B) Phosphorylations of NIK and IKK are designated p-NIK and p-IKKα/β. (C) Phosphorylations of MEK1/2 and ERK1/2 were detected using antibodies of p-MEK1/2 and p-ERK1/2. One representative blot of each protein from 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 aged rats at 2 mg or 4 mg/Kg per day for 10 days.</p

Heptadecane protected YPEN-1 cells from t-BHP-induced oxidative stress and cytotoxicity.

<p>(A) Cells were treated with or without/heptadecane at concentrations of 1, 5, 10, and 20 µM for 1 hr and then with vehicle or 10 µM t-BHP. (B) Cells were treated with or without heptadecane at concentrations of 1, 5, 10 and 20 µM for 1 hr and then with t-BHP (10 µM) for 6 hr. Viable cell numbers were determined using an MTT assay. ^##p<0.01 vs. vehicle treated control group; **p<0.01, ***p<0.001 vs. the 10 µM t-BHP group by one-factor ANOVA.</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