Activation of proinflammatory signaling by 4-hydroxynonenal-Src adducts in aged kidneys

In our previous study, reactive 4-hydroxy-2-nonenal (4-HNE) was shown to activate Src (a non-receptor tyrosine kinase) by forming an adduct on binding with a specific residue of Src, leading to the activation of proinflammatory signaling pathways in cultured cells. However, to date, the deleterious roles of 4-HNE in inflammatory signaling activation in kidneys during aging have not been explored. The purpose of the present study was to document the mechanisms by which 4-HNE induces inflammation in the kidney during aging. Initial experiments revealed that activated nuclear factor-κB (NF-κB) expression was caused by 4-HNE activation, which suppressed transcriptional activity in the aged kidney. Treatment of human umbilical vein endothelial cells with 4-HNE revealed that Src caused senescence via NF-κB activation. Furthermore, our immunohistochemistry data showed that 4-HNE-adducted Src significantly increased in aged kidney tissues. The data showed age-related upregulation of downstream signaling molecules such as mitogen activated protein kinases (MAPKs), activator protein-1 (AP-1), NF-κB, and COX-2 in a cell culture cell system. Taken together, the results of this study show that the formation of adducts between 4-HNE and Src activates inflammatory signaling pathways in the aged kidney, contributing to age-related nephropathy.


INtrODUctION
The molecular inflammation hypothesis provides a molecular link between oxidative stress-induced chronic inflammation and many age-related pathologic processes involving increased lipid peroxidation during aging.
HNE is a byproduct of n-6 fatty acid peroxidation [1,5]; it has been identified as a potent cytotoxic agent and can accumulate to levels of up to 10 µM-5 mM both in vivo and in vitro [2,6]. 4-HNE generated by oxidative stress is thought to lead to apoptosis [4,7], pulmonary edema [8], and cardiovascular disorders such as atherosclerosis [9].
Although there have been several studies showing that 4-HNE is increased in aged tissue or plasma, a study on the specific relationship between 4-HNE and aging has not yet been made reported. Among several hypotheses of aging, the oxidative stress hypothesis currently offers the best mechanistic description of the aging process and of age-related chronic disease processe [10]. Recent research reports provide evidence that oxidative processes

Research Paper: Gerotarget
Oncotarget 50865 www.impactjournals.com/oncotarget are a major factor in the activation of redox-sensitive inflammatory processes, and that they act as a bridge between the normal aging process and age-related chronic diseases [11,12].
Inflammation represents the main driving force in the progression of a large majority of human chronic diseases, as well as aging. The data available strongly suggest that 4-HNE is a key molecule in inflammationrelated cell signaling, suggesting the involvement of 4-HNE in human pathologies. Specifically, several studies have shown that 4-HNE-induced inflammation is involved in COX-2 expression. [13].
A previous study revealed that the balance of protein tyrosine kinase and protein tyrosine phosphatase is important for regulating inflammatory processes and aging [13]. Furthermore, it was found that 4-HNE-activated Src (a non-receptor tyrosine kinase), among various protein tyrosine kinases and downstream signaling, directly formed adducts with a specific residue of Src, suggesting the importance of Src in inflammatory signaling pathways [14,15].
On the basis of these findings, the present study examined changes in 4-HNE and Src as well as downstream signaling proteins related to aging in vivo. As the aging model, rats at 6 and 24 months of age were used and kidneys were selected as the experimental tissue, because kidneys are among the tissues most activated by aging. The data obtained from the present study provide further molecular insights into the roles of 4-HNE and Src in age-related inflammation, leading to an accelerated aging process.

Increase in 4-HNE-adducted proteins in aged kidneys
To confirm whether 4-HNE increases in aged kidneys, 4-HNE-adducted proteins were quantified indirectly by western blotting and immunohistochemistry using 4-HNE antibody. 4-HNE was highly adducted to proteins around 60 kD in old kidneys, and densitometric analysis showed a 2-fold increase in the levels of 4-HNE-adducted proteins ( Figure 1A). Furthermore, immunohistochemistry showed that 4-HNE was strongly stained in old kidney sections ( Figure 1B). These results demonstrate that 4-HNE-adducted proteins, especially 60 kD proteins, increase in aged kidneys.

Induction of cellular senescence by 4-HNE
To elucidate whether 4-HNE influences aging, 4-HNE-induced cellular senescence was investigated indirectly. Cellular senescence is a state of irreversible growth arrest. Senescent cells stop dividing but remain viable. A senescent cell phenotype includes apoptosis resistance, growth arrest, and altered gene expression, which is related to the inhibition of cell cycle proteins, such as p53, p21, pRB, and p16. SA-β-gal staining is the primary marker of cellular senescence: senescent cells are dyed blue. Figure 2A shows that cells were dyed blue when treated with 4-HNE. p21 is a cyclin-dependent kinase (CDK) inhibitor that inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes, and thus functions as a regulator of cell cycle progression at the G1 and S phases. The expression of p21 is increased by p53. When cells were treated with 4-HNE, p21 and p53 increased and p21 was upregulated, even after a night ( Figure 2B). Cells in the G1 phase were increased about 6 percent in the flow cytometric analysis, which meant that G1 arrest had partially occurred ( Figure 2C). These findings suggest that 4-HNE, which increases during aging, can also inversely promote the aging process.

Upregulation of gene expression and increased activation of src in aged kidneys
According to previous studies, [15] Src is believed to play an important role in 4-HNE-induced inflammatory signaling as well as aging. Furthermore, because changes in Src during aging have not yet been identified, the level of Src in aged kidneys was examined. The selected binding proteins of 4-HNE in aged kidneys, total Src and active Src, which is phosphorylated on Tyr 418, were detected by western blotting analysis in young and old kidneys ( Figure 3). The level of total Src was highly increased in old kidneys. However, by densitometric analysis, there was no significant difference in the ratios of p-Src and Src. These results confirm that both the activity and amount of Src, as well as 4-HNE, are increased in old kidneys.

binding of 4-HNE with src in aged kidneys
To confirm the interaction between Src and 4-HNE, both of which are increased during aging, an immunoprecipitation assay was performed in vivo. Albumin was selected as a positive control because albumin is known to form adducts with 4-HNE [16]. 4-HNE was more clearly detected in precipitates of Src than of albumin ( Figure 4A). Because the level of adducted 4-HNE was increased relative to that of Src, the difference of 4-HNE/Src between young and old tissues was not seen in the densitometry analysis. Double immunofluorescence staining allowed visualization of the location and amount of 4-HNE and Src ( Figure 4B). The merged picture showed that Src and 4-HNE were mostly co-localized in kidney tissue. Compared to young kidneys, the green fluorescence of 4-HNE staining was stronger in old kidneys. These findings suggest that 4-HNE and Src Oncotarget 50866 www.impactjournals.com/oncotarget exist in a bound form in old kidney tissue.

Increase in downstream signaling of src in aged kidneys
Downstream of activated Src, there are several pathways that are related to cell motility, cell proliferation, and inflammation. Among these, MAPK signaling pathways, which are related to Src-and 4-HNE-induced inflammation, were confirmed. Contrary to the results for IKKβ, another binding molecule of Src, MAPK signaling molecules such as MEK1/2, MEK3/6, p38, and ERK were in a state of activation (phosphorylation) in old kidneys; the one exception was JNK ( Figure 5A). Furthermore, COX-2, which is involved in 4-HNEinduced inflammation, was significantly increased in aged kidneys. AP-1 (c-Jun) and NF-κB (p65), the upstream transcription factors of COX-2, were also elevated in old kidneys ( Figure 5B). These findings suggest that increased inflammatory signaling molecules in aged kidneys may be regulated by Src and 4-HNE.

Effect of src inhibition on cell senescence by 4-HNE in HUVEc cells
To further illustrate the importance of Src in cell senescence, we used the siRNA-mediated gene-silencing approach to Src knockdown in HUVEC cells, which was performed by treating the cells with Src-siRNA. Levels of p53 and p21 as cell senescence markers increased in 4-HNE treated groups, but were reduced by treating the cells with Src-siRNA ( Figure 6A). Furthermore, we examined the SA-β-gal staining which was the primary marker of cellular senescence in Src knockdown condition. As a result, treatment with 4-HNE only showed higher dyed blue color, compared with 4-HNE treated Src-siRNA group ( Figure 6B). This data indicate 4-HNE induces cellular senescence through Src signaling.

DIscUssION
To our knowledge, this study is the first to show that 4-HNE forms adducts with Src, thereby promoting proinflammatory NF-κB, especially in rat kidneys. In summary, we show that age-related, 4-HNE-induced Src activation causes NF-κB activation leading to senescence during the inflammatory process in aging.
In a previous study of 4-HNE and its age-related changes in serum of young and old rats, the levels of 4-HNE-modified proteins were found to increase with age [21]. Many studies have demonstrated that 4-HNE or 4-HNE-modified proteins are accumulated in aged tissues. Our group reported 4-HNE-induced apoptosis in endothelial cells and aged kidneys [4]. However, whether 4-HNE directly induces aging has not been studied because of experimental limitations. Thus, cellular senescence was applied as a model. Although still debated, cellular senescence recapitulates aspects of organism aging and mimics in vivo aging phenotypes. The results from this study showed 4-HNE-induced cellular senescence and verified that 4-HNE can indirectly accelerate the aging process ( Figure 2).
Oxidized LDL and its related lipid peroxidation products, including 4-HNE, have been reported to interact with EGF and PDGF receptors and to activate downstream signaling pathways [22,23]. Moreover, we previously found that 4-HNE binds to Src, an NRTK, and were involved with various pathways in endothelial cells [15]. This previous work supports our findings that the stimulation of senescence in HUVEC with 4-HNE increased Src activity, and immunoprecipitation verified the direct binding between 4-HNE and Src within cells. In addition, LC-MS/MS analysis identified the specific 4-HNE binding site as Cys248 in the SH2 domain of Src. The Src knockdown experiments and dasatinib (a Src inhibitor) studies showed that reductions in Src activity inhibited p38, ERK, and AP-1 activation, as well as COX-2 expression, suggesting that Src mediates 4-HNE-induced inflammatory signaling.
Because 4-HNE is known to activate MAPK, that is, ERK, JNK, and p38, we sought to determine whether Src activation stimulates these pro-inflammatory transcriptional factors. Therefore, in the present study, it was a meaningful finding that Src increased and existed in a 4-HNE-bound form, and downstream signaling of Src, indicated by MEKs and MAPKs (ERK, p38, and JNK), that led to NF-B activation or was increased in aged kidneys shed light on the potential roles of Src in 4-HNE-induced inflammation and the aging process Oncotarget 50869 www.impactjournals.com/oncotarget ( Figure 5). Thus, Src could be a novel therapeutic target molecule to inhibit chronic inflammatory states, including cardiovascular disease and cancer, as well as aging.
Oncotarget 50871 www.impactjournals.com/oncotarget Src actually plays a fundamental role in 4-HNE-induced cellular senescence and age-related disease in vivo. It is necessary to identify the response to 4-HNE using Srcknockout mouse.
The present study demonstrated that 4-HNE-Src adducts activate downstream inflammatory signaling pathways in aged kidney tissue. Thus, the results revealed a plausible mechanism for chronic inflammation and aging in the kidney due to the direct binding between 4-HNE and Src, as schematically depicted in Figure 6C.

Animals
Young (6-month-old) and old (24-month-old) specific pathogen-free male Sprague-Dawley (SD) rats were obtained from Samtako (Osan, Korea) and housed in a controlled room (23 o C ± 1 o C, 55% ± 5% relative Figure 6: Inhibition of 4-HNE-induced cell senescence by src-knockdown. A. Western blot analysis was used to assess p53 and p21 protein levels in 4-HNE treatment group 48 hr after pre-treated with or without Src-siRNA cells. Samples loaded on gels were probed with β-actin and TFIIB. b. HUVECs (3 × 10 4 cells) were treated with 4-HNE (5 μM) for 4 hr, 24 hr after pre-treated with Src-siRNA, and medium was changed daily. After 4 days, SA-β-gal staining was detected under electron microscope. The arrows indicate representative cells that are stained blue. c. Possible mechanism underlying the effect of NF-κB on 4-HNE-stimulated aging. NF-κB, nuclear transcription factor κB; COX-2, cyclooxygenase-2.
Oncotarget 50872 www.impactjournals.com/oncotarget humidity, 12 h light/dark cycle) with free access to water and an ad libitum standard laboratory diet. After an acclimation period (1 week), rats at 6 (n = 6) and 24 (n= 6) months of age were sacrificed by decapitation and the kidneys were quickly removed and rinsed in ice-cold buffer [100 mM Tris, 1 mM EDTA, 0.2 mM phenylmethylsulfonylfluoride (PMSF), 1 μM pepstatin, 2 μM leupeptin, 80 mg/L trypsin inhibitor, 20 mM glycerophosphate, 20 mM sodium fluoride, and 2 mM sodium orthovanadate (pH 7.4)]. The tissue was immediately frozen in liquid nitrogen and stored at −80°C. cell culture conditions HUVEC (human umbilical vein endothelial cells) were obtained from Lonza (Basel, Switzerland). The cells were grown in EGM™-2 (endothelial cell growth medium-2, Lonza) containing hydrocortisone, GA-1000 (gentamicin, amphotericin B), 10 ml fetal bovine serum (FBS), VEGF, hFGF-B, R3-IGF-1, ascorbic acid, and heparin. Cells were maintained at 37 o C in a humidified 5% CO2 chamber with a 95% air atmosphere. Medium was replaced daily to remove non-adherent cells and cell debris. Cells were discarded after 3 months and new cells were obtained from frozen stock.

Preparation of cytosolic and nuclear fraction from tissues
All solutions, tubes, and centrifuges were maintained at 4 o C. Kidney tissues (150 mg) were homogenized with 1 ml of homogenate buffer A [10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl 2 , 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 1 mM pepstatin, and 1 mM p-aminobenzamidine] with a tissue homogenizer for 20 s. Homogenates were kept on ice for 15 min; then, 125 μl of 10% NP40 solution was added and mixed for 15 s. The mixture was then centrifuged at 13,000 g for 2 min, and the supernatant containing the cytosolic proteins was collected. The pelleted nuclei were washed once with 400 μl of buffer A plus 25 μl of 10% NP40, centrifuged, suspended in 50 μl of buffer C [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 10% (vol/vol) glycerol], mixed for 20 min, and centrifuged at 13,000 g for 30 min. The supernatant containing the nuclear proteins was stored at −80°C.

Preparation of cytosolic and nuclear extracts of treated cells
Nuclear and cytosolic extracts were prepared as described previously [15]. Treated cells were washed and then scraped into 1.0 ml of ice-cold PBS and pelleted at 840 g for 5 min at 4 o C. Pellets were suspended in 10 mM Tris (pH 8.0) containing 1.5 mM MgCl 2 , 1 mM DTT, 0.1% NP40, and protease inhibitors, and then incubated on ice for 15 min. Nuclei were separated from cytosols by centrifugation at 13,000 g for 15 min at 4°C. Supernatants (cytosolic fractions) were removed and pellets were suspended in 10 mM Tris (pH 8.0) containing 50 mM KCl, 100 mM NaCl, and protease inhibitors, incubated on ice for 30 min, and then centrifuged at 13,000 g for 30 min at 4 o C to obtain nuclear fractions.

Western blotting
Western blotting was carried out as described previously [24]. Homogenized samples were boiled for 5 min with gel-loading buffer (125 mM Tris-Cl, 4% SDS, 10% 2-mercaptoethanol, pH 6.8, 0.2% bromophenol blue) at a ratio of 1:1. Total protein equivalents of samples were separated by SDS-PAGE using acrylamide gels as described by Laemmli [25] and then transferred to PVDF membranes at 15 V for 1 h using a semi-dry transfer system. Membranes were then immediately placed into a blocking buffer of 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween-20 containing 1% non-fat milk. Blots were blocked at room temperature for 1 h, and then membranes were incubated with the appropriate specific primary antibody at 25 o C for 1 h, followed by horseradish peroxidase-conjugated secondary antibody at 25 o C for 1 h. Antibody labeling was detected using enhanced chemiluminescence, according to the manufacturer's instructions. Molecular weights were determined using pre-stained protein markers.

Immunoprecipitation
Tissue lysates were immunoprecipitated in a buffer containing 40 mM Tris (pH 7.6), 120 mM NaCl, 5 mM EDTA, 0.1% NP40, protease inhibitors, and phosphatase inhibitors. Samples (300 μg) were precleared by incubation with a 50% slurry of protein A at 4 o C for 2 h and centrifuged at 12,000 g at 4 o C for 10 min. Pellets were