protein tyrosine phosphatase balance in the regulation of NF-κB signaling in the inflammatory process and aging

Free Radical Biology & Medicine 47 (2009) 983–991

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Significance of protein tyrosine kinase/protein tyrosine phosphatase balance in the regulation of NF-κB signaling in the inflammatory process and aging Kyung Jin Jung a,1, Eun Kyeong Lee a,1, Byung Pal Yu b,c, Hae Young Chung a,b,⁎ a b c

Department of Pharmacy, College of Pharmacy and Molecular Inflammation Research Center for Aging Intervention, Pusan National University, Busan 609-735, Korea Longevity Life Science and Technology Institutes, Pusan National University, Busan 609-735, Korea Department of Physiology, The University of Texas Health Science Center, San Antonio, TX 78229, USA

a r t i c l e

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Article history: Received 17 April 2009 Revised 4 June 2009 Accepted 2 July 2009 Available online 22 July 2009 Keywords: PTK/PTP imbalance NF-κB Molecular inflammation Aging Reactive species Free radicals

a b s t r a c t Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) are well recognized for the essential roles they play in signal transduction by maintaining proper balance under redox status. One of the major transcription factors known to be involved in aging is the redox-sensitive proinflammatory NF-κB, which could be modulated by the activities of PTKs and PTPs. This study delved into a molecular inquiry of the PTK/PTP balance, which is affected by oxidative stress-induced redox changes during aging. To obtain the underlying molecular clues, we assessed the PTK/PTP status in the aged rat kidney utilizing aging-retarding calorie restriction and inflammation-triggering LPS paradigms. The results suggest that reactive species increase PTK activation, which is counterbalanced by decreased PTP, leading to a shift in the PTK/PTP balance. This shift in the balance was confirmed by free radical-generating AAPH injected into mouse or HEK293T cells, which led to NF-κB activation. To strengthen the findings, we perturbed the PTK/PTP balance using a PTP inhibitor, Na3VO4, which caused NF-κB activation through phosphorylated NIK/IKK and MAPKs. Thus, our data suggest, for the first time, that the delicate balance between PTK and PTP is disturbed during aging and inflammation, both of which lead to NF-κB activation via NIK/IKK and MAPKs. © 2009 Elsevier Inc. All rights reserved.

Cellular signaling involving kinases generally begins with tyrosine kinase/phosphatase located in proximity to the plasma membrane and then travels to serine/threonine kinase/phosphatase located close to the nuclei. It was recently recognized that protein phosphorylation and dephosphorylation play key roles in the regulation of all cellular functions [1,2]. For instance, within higher eukaryotic organisms, protein tyrosine phosphorylation is essential for cell proliferation, differentiation, migration, and transformation. The importance of a balance between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) was recognized recently because the proper maintenance of balance is needed for the regulation of cellular homeostasis [3,4]. On the other hand, defective or inappropriate shifts due to changes in either PTKs or PTPs would lead to aberrant tyrosine phosphorylation that contributes to the pathogenesis of many diseases, including cancers and diabetes [5,6].

Abbreviations: AAPH, 2,2-azobis-(2-amidinopropane) dihydrochloride; AL, ad libitum; CR, calorie restriction; DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; ERK, extracellular signal-regulated kinase; FDP, 3,6-fluorescein diphosphate; HEK293T, human embryonic kidney 293T; IKK, IκB kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; Na3VO4, sodium orthovanadate; NF-κB, nuclear factor-κB; NIK, NF-κB-inducing kinase; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; RS, reactive species; RTK, receptor tyrosine kinase. ⁎ Corresponding author. Fax: +82 51 518 2821. E-mail address: [email protected] (H.Y. Chung). 1 These authors contributed equally to this work. 0891-5849/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2009.07.009

The regulation of tyrosine phosphorylation is mediated by the agonistic or antagonistic activities of PTKs and PTPs, respectively [7]. Although substantial work has been directed to elucidate the regulation of PTKs, at present little is known about the underlying mechanism. PTPs are also known to exert both positive and negative effects on signaling pathways and play crucial physiological roles in a variety of mammalian tissues and cells [8]. It is also known that dysregulation of PTP activity contributes to the pathogenesis of many human diseases [9,10]. PTPs are a family of enzymes whose structural diversity and complexity rival those of PTKs. Unlike PTKs, which share sequence homology with protein serine/threonine kinases, PTPs show no sequence similarity with the serine/threonine phosphatases [11]. However, a recent estimation from the nearly completed human genome sequence suggested that humans have 112 PTPs, which include both the tyrosine-specific and the dual-specific phosphatases [12]. One well-known way to modulate PTK and PTP activity is through change in the redox status; PTKs are activated by oxidation, whereas PTPs are suppressed by oxidation. The activation and inactivation of kinases and phosphatases are due to cysteine oxidation, which then leads to incremental kinase activity or decremented phosphatase activity [4]. Oxidation-dependent activation of PTKs may engage with membrane-bound kinases, such as with receptor tyrosine kinases (RTKs); with intracellular kinases, such as with Src tyrosine kinases or FAK; or jointly with serine/threonine or with dual-specificity kinases, such as extracellular signal-regulated kinase (ERK), Act, or Ask [13].

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Oxidation of RTKs has been reported for insulin receptor kinase, epidermal growth factor receptor, platelet-derived growth factor receptor, and Ret kinase. In contrast to PTKs, but in agreement with its opposing role in the modulation of protein tyrosine phosphorylation, PTPs are inactivated by oxidation. Oxidation of the PTP active-site cysteine to a cysteine sulfenic derivative by various oxidative agents, including H2O2, leads to the PTP's enzymatic inactivation, which can be reversed by thiol compounds [14–16]. These observations strongly indicate that the oxidation of catalytic cysteine of PTPs may occur in vivo in response to reactive species (RS) or to an increase in redox potential [17]. Oxidative stress has been studied as a fundamental force influencing the global aging phenomenon [18–20]. Cellular oxidative stressinduced redox imbalance that modulates PTK and PTP activities is considered a major cause of cellular aging by altering homeostasis. Recently, oxidative stress has been implicated in the aging process and many age-related diseases through the activation of nuclear factor-κB (NF-κB), which plays a central role in the inflammatory process [21–23]. Also, signaling cascades that activate NF-κB such as NF-κBinducing kinase (NIK)/IκB kinase (IKK) and mitogen-activated protein kinases (MAPKs) are closely related to the oxidative stress-induced aging process [24]. To highlight the importance of and to delineate the underlying molecular events of the chronic inflammatory process in aging, a molecular inflammation hypothesis was recently proposed from this laboratory [24,25]. Our current study further expanded on the assessments of NF-κB activation and closely related NF-κB signals by utilizing both oxidative stress and sodium orthovanadate (Na3VO4), which have been extensively used as experimental probes in the manipulation of PTPs because most tyrosine phosphorylation/dephosphorylation-dependent signal transduction pathways can be modulated by the intracellular redox status [26–28]. In this study, the extent of the PTK/PTP imbalance was investigated based on molecular changes due to altered redox status and the upregulation of age-related inflammatory signaling.

the kidneys were obtained after 5 h. The tissue was immediately immersed in liquid nitrogen and stored at −80°C. The kidney tissue was selected for the study based on its sensitivity to redox change, high metabolic activity, inflammatory responsiveness, and acceptance as an aging model.

Materials and methods

Methods

Animals

Kidney tissue preparations Whole kidney tissue was homogenized with 5 volumes of homogenate buffer [100 mM Tris, 1 mM EDTA, 0.5 mM PMSF, 1 μM pepstatin, 80 mg/L trypsin inhibitor, pH 7.4] with or without phosphatase inhibitors (such as 20 mM β-glycerophosphate, 20 mM NaF, and 2 mM Na3VO4) for Western blot or enzymatic assay, respectively. The homogenates were centrifuged at 900g at 4°C for 15 min. The supernatants were recentrifuged at 12,000g at 4°C for 15 min to yield a sedimented mitochondrial fraction and a postmitochondrial supernatant fraction. The supernatants were used as the cytosol fraction.

Rat maintenance procedures for specific-pathogen-free (SPF) status and dietary composition of chow have been previously reported [29]. Briefly, male SPF Fischer 344 rats were fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15% α-methionine, 0.2% choline chloride, 5% salt mix, 2% vitamin mix, and 3% Solka-Floc. The ad libitum (AL)-fed group had free access to both food and water. The animals designated as calorie restriction (CR) were fed 60% of the food intake of their AL-fed littermates, beginning at 6 weeks of age. Rats at 12 and 24 months of age (n = 5) were sacrificed by decapitation and the kidneys were quickly removed and rinsed in iced-cold buffer [100 mM Tris, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM pepstatin, 2 μM leupeptin, 80 mg/L trypsin inhibitor, 20 mM β-glycerophosphate, 20 mM NaF, 2 mM Na3VO4 (pH 7.4)]. Then, the tissue was immediately frozen in liquid nitrogen and stored at −80°C. Histopathological examination revealed no evidence of nephritic lesions detected in these soy-protein-fed Fischer rats even at the advanced age of 24 months [30]. The animal protocols complied with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. In addition, SPF male Fischer 344 rats were separated into young (13 months) and old (31 months) groups. To investigate the effects of inflammation on the aging process, two additional groups were used for the injection of bacterial lipopolysaccharide (LPS). LPS was injected intraperitoneally (ip) in 5 mg/kg body wt doses into young and old rats. After 5 h, the rats (n = 5) were sacrificed by decapitation and the kidneys quickly removed. In addition, SPF C57BL/6 male mice (n = 5) were also injected (ip) with 50 mg/kg 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH) to test the effects of RS, and

Cell culture system HEK293T cells (human embryonic kidney epithelial cell line) were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were grown in Dulbecco's modified Eagle medium (Nissui, Tokyo, Japan) containing 2 mM L-glutamine, 100 mg/ml streptomycin, 2.5 mg/L amphotericin B, and 10% heat-inactivated fetal bovine serum. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2/95% air. Cells were discarded after 3 months, at which time new cells were obtained from frozen stock. Cells at exponential phase were used for all experiments. Reagents All chemical reagents were obtained from Sigma (St. Louis, MO, USA), except where noted. PP2, also called AG 1879, was purchased from Calbiochem (San Diego, CA, USA). The Antibody Beacon tyrosine kinase assay kit and 3,6-fluorescein diphosphate (FDP) were obtained from Molecular Probes (Eugene, OR, USA). Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Cell Signaling (New England BioLabs, Hertfordshire, UK). Horseradish peroxidaseconjugated anti-rabbit IgG antibody, anti-mouse IgG–horseradish peroxidase antibody, and enhanced chemiluminescence (ECL) reagents were obtained from Amersham (Buckinghamshire, UK). Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore (Bedford, MA, USA). All other materials were obtained in the highest available grade.

RS measurement RS generation was measured by utilizing a fluorescent probe, 2′,7′dichlorodihydrofluorescein diacetate (DCFDA). For tissue homogenates, 25 μM DCFDA was added to the homogenates in 50 mM phosphate buffer. For cellular RS, cells were seeded, incorporated with 2.5 μM DCFDA for 1 h, and treated with chemicals. Changes in fluorescence intensity were measured every 5 min for 30 min on a microplate reader (GENios; Tecan Instruments, Salzburg, Austria) with excitation and emission wavelengths at 485 and 530 nm, respectively. Gel shift assay (NF-κB DNA-binding activity) For tissue homogenates, the NF-κB activity was determined by gel shift assay [31]. Nuclear extracts were prepared, mixed with radiolabeled NF-κB consensus oligonucleotide (5′-GAGAGGCAAGGGGATTCCCTTAGTTAGGA-3′), and run through a 5% native polyacrylamide gel. After the sample's mobility shifted, the gel was dried and exposed to X-ray film at −80°C for 12 h. Data are represented with the NF-κB activity, which was quantified with spot density by using a densitometer (Fluorochem; Applied Biosystems, Foster City, CA, USA).

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Cell lysis Cells were washed with PBS, and then 1 ml of ice-cold PBS was added. Pellets were harvested at 785g at 4°C for 5 min. The pellets were suspended in 10 mM Tris (pH 8.0) with 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1% Nonidet-P40, and protease inhibitors, and incubated on ice for 15 min. Nuclei were separated from the cytosol by centrifugation at 12,000g at 4°C for 15 min. The supernatants were used as the cytosolic fraction and the pellets were resuspended in 10 mM Tris (pH 8.0) with 50 mM KCl, 100 mM NaCl, and protease inhibitors; incubated on ice for 30 min; and then were centrifuged at 12,557g at 4°C for 30 min. The resultant supernatants were used as the nuclear fraction. PTK activity PTK activity in the tissue homogenates and cell lysates was assayed with the Antibody Beacon tyrosine kinase assay kit (Molecular Probes). Briefly, to detect the tyrosine kinase activity, samples were prepared in 1× kinase buffer (100 mM Tris–HCl, 20 mM MgCl2, 2 mM EGTA, 2 mM DTT, 0.02% Brij 35, pH 7.5) and mixed with the Antibody Beacon detection complex plus substrate in 96-well microplates. ATP reagent was then added to the plate and continuously incubated at the reaction temperature. Fluorescence was measured at multiple time points on the GENios (Tecan Instruments) with excitation and emission wavelengths set at 485 and 535 nm, respectively.

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(1% nonfat milk) in 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20. The blot was allowed to block at room temperature for 1 h. The membrane was incubated with specific primary antibody at 25°C for 2 h, followed by a horseradish peroxidase-conjugated anti-mouse antibody (1:5000) or anti-rabbit antibody (1:5000) at 25°C for 2 h. Antibody labeling was detected using ECL per the manufacturer's instructions. Prestained protein markers were used for molecular weight determinations. The density of each band was detected with FluorChem (Alpha Innotech, San Leandro, CA, USA). Statistical analysis For Western blotting, one representative blot was shown from triplicate independent experiments. For the other assays, the results are

PTP activity FDP is a very sensitive fluorogenic substrate for assaying PTP activity. To detect the tyrosine phosphatase activity, samples were prepared in PTP assay buffer (50 mM Tris–HCl, 2 mM EGTA, 5 mM DTT, 100 μM CaCl2, pH 6.3) and mixed with 100 μM FDP in 96-well microplates. The plates were incubated at the reaction temperature for 5 min and then fluorescence was measured at multiple time points on the GENios (Tecan Instruments) with excitation and emission wavelengths set at 485 and 535 nm, respectively. Transient transfection and luciferase assay For luciferase assay, the pNF-κB-Luc vector was purchased from Clontech (Palo Alto, CA, USA). Expression plasmids pCMV-FLAG containing NIK, IKK, or p65 (transactivation subunit of NF-κB) were kindly provided by Dr. Bharat B. Aggarwal (The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA). The effects of chemicals on NIK-, IKK-, and p65-induced NF-κBdependent reporter gene transcription were measured as previously described [32,33]. Briefly, the proper combination with 0.1 μg plasmids and 0.05 μg pCMV expression vector was prepared for 2 × 104 HEK293T or YPEN-1 cells per 48-well dish using the FuGENE 6 Reagent (Roche, Indianapolis, IN, USA). Cells were transiently transfected using the FuGENE 6 reagent containing plasmids for NIK, IKK, or p65 along with NF-κB promoter DNA linked to the luciferase reporter gene. After transfection (36–48 h), the conditioned medium was removed, and chemicals were applied after the plate was changed with serum-free medium. After additional incubation for 8 h, the cells were washed with PBS and the Steady-Glo luciferase assay system (Promega, Madison, WI, USA) was added to the plate. Luciferase activity was measured by a luminometer (GENios; Tecan Instruments). Raw luciferase activities were normalized to protein concentration per well. Western blotting Western blotting was carried out as described previously [34]. Homogenized samples were boiled for 5 min with a gel-loading buffer (0.125 M Tris–HCl, 4% SDS, 10% 2-mercaptoethanol, pH 6.8, 0.2% bromophenol blue) at a ratio of 1:1. Total protein equivalents for each sample were separated by SDS–PAGE using 10% acrylamide gels and transferred to PVDF membrane at 15 V for 1 h in a semidry transfer system. The membrane was immediately placed into blocking buffer

Fig. 1. Effects of RS on NF-κB activity during aging. To measure RS level, kidney homogenates were analyzed by the DCFDA method. To quantify NF-κB binding activity, gel shift assay was performed and band density was analyzed with a densitometer. Data show that the RS levels (lines charted by right y axis) reflect the DNA-binding activity of NF-κB (bars charted by left y axis). (A) Effects of aging and CR on RS and NF-κB activity. 12- and 24-month-old rats from the AL and CR groups were used (n = 5). (B) Effects of inflammatory factor LPS on RS and NF-κB activity during aging. 13- and 31-month-old rats were injected with saline or LPS (5 mg/kg) dissolved in saline (n = 5). (C) Effects of RS producer AAPH on NF-κB activity. 6-month-old mice were injected with saline or AAPH (50 mg/kg) dissolved in saline (n = 5). AL, ad libitum group; CR, calorie restriction group; CON, untreated control group; LPS, lipopolysaccharide; AAPH, 2,2azobis-(2-amidinopropane) dihydrochloride; Mo, month; RS, reactive species.

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expressed as means ± SE (n = 5). The statistical significance of the difference between the groups (age and dietary supplemented) was determined by one-factor ANOVA followed by the Fischer's protected LSD post hoc test. Values of p b 0.05 were considered statistically significant.

Results Effects of RS on NF-κB activity during aging To characterize a possible role for PTK and PTP in age-related molecular inflammation by RS, we first analyzed the molecular inflammatory NF-κB activity with age-related RS. The NF-κB activity was significantly increased in aged AL rats but not in the CR rats, experiencing the antioxidative and anti-inflammatory actions of calorie restriction, which was exactly the same trends seen in RS levels during aging with AL and CR (Fig. 1A). To further examine specific correlations between NF-κB activity and RS-related molecular inflammation during aging, we used LPS (5 mg/kg)-injected aged rats as a model for induced molecular inflammation and AAPH (50 mg/ kg)-injected mice as a model for induced genuine RS levels. Results showed that both LPS and AAPH increased NF-κB activity and RS levels (Figs. 1B and C) and similar trends were observed in age-related NF-κB activity and RS levels. From the results, there was a positive correlation

Fig. 2. Effects of age and CR on PTK activity. (A) PTK activity was measured in kidney homogenates from AL and CR rats, ages 12 and 24 months (n = 5), and verified using the PTK inhibitor PP2. PP2 was applied at 20 μM to kidney homogenates and then incubated for 10 min. The activity was measured with the Antibody Beacon tyrosine kinase assay kit as described in the manufacturer's manual. (B) PTP activity was measured using a fluorogenic substrate in kidney homogenates from AL and CR rats, ages 12 and 24 months (n = 5) and verified using the PTP inhibitor Na3VO4 (20 μM). (C) PTK/PTP ratio was measured in kidney homogenates from ready-measured activity of PTK and PTP in AL and CR rats, ages 12 and 24 months. The ratio balance point is set to the value of 1.0 at 12 months of age. Each value is the mean ± SE of five rats in each group. Results of onefactor ANOVA: ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 vs 12-month-old AL control; #p b 0.05, ## p b 0.01, ###p b 0.001 vs age-matched AL rats; §p b 0.05, §§p b 0.01, §§§p b 0.001 vs untreated group with same condition. AL, ad libitum group; CR, calorie restriction group; PP2, specific inhibitor of tyrosine kinase; Mo, month.

Fig. 3. Effects of age and LPS on PTK/PTP balance. The effects of proinflammatory LPS on PTK and PTP activities in aged rats were evaluated. Each activity, (A) PTK and (B) PTP, was applied to measure (C) the PTK/PTP ratio from kidney homogenates in control and LPS-treated rats (5 mg/kg), ages 13 and 31 months (n = 5). Each value is the mean ± SE of five rats in each group. The ratio balance point is set to the value of 1.0 at 13 months of age. Results of one-factor ANOVA: ⁎p b 0.05 vs 13-month-old untreated control; ## p b 0.01, ###p b 0.001 vs age-matched untreated rats.

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between NF-κB activity and RS levels during age-related inflammatory processes. Changes in activities of PTK and PTP during aging To assess overall age-related changes in PTK activity, first, total PTK was measured with an assay kit for kidney homogenates. The results showed an increase in PTK activity with age, especially in 24month-old rats, which showed a significant increase in fluorescence intensity compared with 12-month-old-rats in the AL group. However, CR rats showed consistently reduced PTK activity compared with age-matched AL counterparts. Measurement of PTK enzymatic assay was verified by using a specific inhibitor of PTK, PP2 (Fig. 2A). To explore age-related changes in PTP activity, total PTP was measured with the fluorogenic substrate FDP in kidney homogenates. The result show a decrease in PTP activity with age, whereas CR rats showed no decrease in PTP activity. Measurements of the PTP enzymatic assay were verified using a specific inhibitor of PTP, Na3VO4 (Fig. 2B). From these results, the PTK/PTP ratio was obtained as depicted in Fig. 2C. Data in the figure show an increase in the PTK/PTP ratio with age for the AL groups, suggesting a shift in the PTK/PTP balance during aging, whereas CR rats showed little change in the ratio for the aged group. Effects of age, LPS, and AAPH treatment on PTK/PTP balance To substantiate the influence of age-related inflammation on the PTK/PTP balance, the activity of PTK and PTP was quantified in kidney homogenates. As shown in Fig. 3A, the age-related increase in PTK

Fig. 4. Effects of AAPH on PTK/PTP balance. The effects of RS on PTK and PTP activities were examined using the RS inducer AAPH. Each activity, (A) PTK and (B) PTP, was applied to measure (C) the PTK/PTP ratio from kidney homogenates in AAPH-treated mice (50 mg/kg, males, 6 months of age). The ratio balance point is set to the value of 1.0 for the control group. Each value is the mean ± SE (n = 5). Results of one-factor ANOVA: ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vs untreated control. CON, untreated control group; AAPH, 2,2-azobis-(2-amidinopropane) dihydrochloride.

Fig. 5. Effects of RS on PTK/PTP balance and NF-κB activation in HEK293T cells. Either LPS (1 mg/ml) or AAPH (200 μM) was used as a proinflammatory RS generator. (A) PTK and (B) PTP activities are shown as graphs. (C) PTK/PTP balance was assessed in RS-treated HEK293T cells. Each activity, PTK and PTP, was applied to measure the PTK/PTP ratio in cell lysates. Each value is the mean ± SE from triplicate independent experiments. Results (A, B, and C) of one-factor ANOVA: ⁎⁎⁎p b 0.001 for PTK, #p b 0.05 ###p b 0.001 for PTP vs untreated control. (D) HEK293T cells were transiently transfected with a plasmid containing the NF-κB binding site linked to the luciferase gene and then treated with the indicated concentrations. Results of one-factor ANOVA: ⁎⁎p b 0.01 vs the vector-onlytransfected group; #p b 0.05 vs NF-κB reporter-transfected group without treatment; $ p b 0.05 vs NF-κB reporter-transfected group with LPS treatment. CON, untreated control group; VT, vector-only-transfected group; LPS, lipopolysaccharide; AAPH, 2,2azobis-(2-amidinopropane) dihydrochloride; NAC, N-acetylcysteine.

activity in the LPS (5 mg/kg)-injected group was more pronounced in the 31-month-old group, compared to the 13-month-old group. On the other hand, for the LPS-injected groups, the age-related decrease

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group, whereas PTP activity was decreased in the AAPH-treated group, compared to the untreated control group. The PTK/PTP activities are depicted as a ratio and the results show the imbalanced state of PTK/ PTP (Fig. 4C). Effects of LPS and AAPH on PTK/PTP balance and NF-κB activity in HEK293T cells

Fig. 6. Comparison of RS-generating capacity with Na3VO4 and AAPH. HEK293T cells were incorporated with 2.5 μM DCFDA and treated with AAPH (50 μM) and Na3VO4 (50 μM) in the presence or absence of a potent antioxidant, NAC (100 μM). Fluorescence intensity was measured every 5 min for 30 min. Data show the fold increase in RS generation, compared to the untreated control group. Each value is the mean ± SE. Results of onefactor ANOVA: ⁎⁎p b 0.01 vs untreated control, #p b 0.05 vs AAPH-treated group. CON, untreated control group; AAPH, 2,2-azobis-(2-amidinopropane) dihydrochloride.

in PTP activity was more significant in the old group (Fig. 3B). These data clearly show that the LPS-induced inflammation perturbed the PTK/PTP balance, resulting in an imbalanced state (Fig. 3C). To obtain in vivo evidence of the effect of increased oxidative stress on the PTK/PTP status, the RS generator AAPH was injected into mice (50 mg/kg) to mimic the situation of aging and inflammatory conditions with PTK/PTP imbalance. As shown in Figs. 4A and B, PTK activity in kidney homogenates was increased in the AAPH-treated

The effects of proinflammatory RS on the cellular PTK/PTP balance in HEK293T cells were assessed by treatment with LPS (1 μg/ml) or AAPH (200 μM). As shown in Fig. 5A, PTK activity was increased in the LPS- and AAPH-treated groups, whereas cotreatment with the antioxidant N-acetylcysteine (NAC) resulted in inhibited PTK activity. In contrast, PTP activity was decreased in the LPS- and AAPH-treated groups compared to untreated control, and NAC treatment restored the decrease in PTP activity (Fig. 5B). Accordingly, the results in Fig. 5C show that RS induced a PTK/PTP imbalance in the in vitro cell system. To further elucidate whether these changes lead to NF-κB activation, the NF-κB-dependent reporter gene (luciferase) expression was examined. As shown in Fig. 5D, NF-κB binding activity was significantly increased by treatment with LPS and AAPH, but was blocked by treatment with the antioxidant NAC. Effects of PTK/PTP imbalance on NF-κB activation by PTP inhibitor Na3VO4 in HEK293T cells Before examining the effects of Na3VO4 on NF-κB activity, we characterized the RS-generating capacity of Na3VO4 compared to AAPH before further experiments (Fig. 6). In HEK293T cells, during

Fig. 7. Effects of Na3VO4 on NF-κB in HEK293T cells. (A) To detect the NF-κB-dependent reporter gene (luciferase) expression, HEK293T cells were transiently transfected with an NFκB-containing plasmid linked to the luciferase gene and then incubated for 40 h, before treatment with the Na3VO4. Then, the cells were incubated with the indicated concentrations of Na3VO4 for 8 h. Each value is the mean ± SE. Results of one-factor ANOVA: ⁎⁎⁎p b 0.001 vs the vector-only-transfected group; ###p b 0.001 vs the NF-κB reporter-transfected group without treatment. UT, untransfected group; VT, vector-only-transfected group; Reporter, plasmid with NF-κB-dependent luciferase. (B) Cells were incubated with the indicated concentrations of Na3VO4 for 30 min. Western blotting was performed to detect nuclear p-p65, p65, and p50 levels, and one representative blot is shown from three experiments that yielded similar results. Histone H1 was used as loading control for nuclear fractionated samples. Blot density was detected with FluorChem and standardized to % of untreated group. Results of one-factor ANOVA: ##p b 0.01, ###p b 0.001 vs untreated control group.

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30 min, Na3VO4 did not significantly change the cellular RS levels, whereas AAPH increased the RS levels. This supports the notion that any result from the following experiments with Na3VO4 is not caused directly by RS generation. To induce a PTK/PTP imbalance, the PTP inhibitor Na3VO4 was used. Because PTPs are antagonistic regulators of PTKs, the use of a PTP inhibitor can be effective in perturbing the balance of PTK/PTP. To investigate whether NF-κB is activated by the PTK/PTP imbalance, NFκB and NF-κB-dependent signaling were investigated in HEK293T cells in the absence or presence of Na3VO4. First, Na3VO4-induced NF-κB-dependent reporter gene expression was examined after transient transfection with an NF-κB-regulated luciferase reporter construct. As shown in Fig. 7A, luciferase activity increased with Na3VO4 in a dose-dependent manner at 12, 25, and 50 μM. Also, in the nuclear fraction of the HEK293T cells, an increase in phosphorylated p65 and nuclear translocation of both p65 and p50 were observed, as Fig. 7B shows. Second, NF-κB signaling was examined because NF-κB activation is mediated through the sequential kinases of NIK/IKK and MAPKs. To delineate the action of Na3VO4 in the NF-κB signaling pathway and on NF-κB activation, cells were transfected with NIK-, IKK-, and p65-expressing plasmids, and then NF-κB-dependent luciferase expression was monitored. As shown in Fig. 8A, all of the plasmidtransfected cells induced NF-κB–luciferase gene expression, especially those with Na3VO4, which stimulated NIK-, IKK-, and p65induced NF-κB reporter gene expression. To examine the active status of NIK/IKK, phosphorylated NIK and IKK were observed. The levels of phosphorylated NIK and IKK were high increment by

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treatment with Na3VO4 (Fig. 8B). In addition, to determine the effects of Na3VO4 on MAPKs linked to NF-κB activation, Na3VO4induced phosphorylation of ERK, p38, and JNK in a dose-dependent manner was detected (Fig. 8C). These results contribute to the understanding of how phosphorylated p65 is increased by treatment with Na3VO4, which means a broad impact on the activation of NF-κB and NF-κB signaling, NIK/IKK, and MAPKs due to PTK/PTP imbalance. Discussion The molecular mechanisms of signal transduction have been the focus of intense research during the past decade. Much of this work, however, has explored PTK-mediated signaling pathways, whereas the study of PTPs has lagged behind [9]. Nevertheless, many PTPs are now recognized to play equally important roles in transduction activity in which no kinase-regulated system would work without the counterbalancing participation of phosphatase. In this study, we attempted to show the importance of the proper balance between PTK and PTP in the modulation of NF-κB in inflammation and the aging process. We found evidence that a PTK/ PTP imbalance due to activated PTK and inactivated PTP occurred from increased oxidative stress during aging. The results further showed that a PTK/PTP imbalance triggered the phosphorylation of NF-κB signaling, including NIK/IKK and MAPKs (ERK, p38, and JNK), that leads to eventual NF-κB activation. These findings contrasted with those of CR animals with reduced oxidative stress, suppressed NF-κB signaling, and NF-κB inactivation.

Fig. 8. Effects of Na3VO4 on NF-κB signaling and NIK/IKK and MAPKs in HEK293T cells. (A) Cells were treated with Na3VO4 (25 μM) and incubated for 8 h after transient cotransfection with the NF-κB-dependent reporter gene and pCMV-NIK, IKK, or p65 for 40 h. Each value is the mean ± SE. Results of one-factor ANOVA: ⁎⁎⁎p b 0.001 vs vector-only-transfected group; ###p b 0.001 vs transfected group with NF-κB-containing plasmid only; $$$p b 0.001 vs untreated group transfected with indicated plasmids. VT, vector-only-transfected group; Reporter, plasmid with NF-κB-dependent luciferase. (B and C) Western blotting was performed for Na3VO4-induced phosphorylated (B) NIK and IKK and (C) the MAPKs phosphoERK, -p38, and -JNK. Cells were incubated with the indicated concentrations of Na3VO4 for 15 min. β-Actin was used as loading control for cytosolic fractionated samples. One representative blot is shown from three experiments that yielded similar results. Blot density was detected with FluorChem and standardized to % of untreated group. Results of onefactor ANOVA: ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 vs untreated control group.

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results from CR support the notion that the age-related alterations in NF-κB signaling toward NF-κB activation are more likely to be caused by the PTK/PTP imbalance than from an age-related redox imbalance. The importance of the current study is the finding of the modulation of NF-κB by PTK/PTP during aging, implying that the maintenance of NF-κB and its signaling through proper PTK/PTP balance could be a novel approach to anti-inflammatory intervention. Acknowledgments This work was supported by Korea Science and Engineering Foundation grants funded by the Korean government (MOST) (MRC program No. 2009-0083538 and No. R01-2007-000-20852-0). We are grateful to the Aging Tissue Bank for supplying aged tissue. We thank Dr. Bharat B. Aggarwal (The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA) for the generous gift of NIK, IKK, and p65 expression vector. References

Fig. 9. Proposed model for NF-κB signaling via PTK/PTP imbalance during aging. Hypothetical scheme from this study shows that age-related RS can increase PTK activity but decrease PTP activity, indicating a PTK/PTP imbalance. Subsequently, the PTK/PTP imbalance increases phosphorylation of NIK/IKK and MAPKs, leading to NF-κB activation and aggravating the inflammatory process during aging. The CR group was used as a positive control for the antioxidative and anti-inflammatory paradigm. PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; NIK, NF-κB-inducing kinase; IKK, IκB kinase; MAPK, mitogen-activated protein kinase containing ERK, p38, and JNK; RS, reactive species; CR, calorie restriction.

Based on this study, the PTK/PTP balance could be considered as an intrinsic proinflammatory factor linking NF-κB and RS. Recent studies on PTK/PTP balance clearly show it as a major regulator of cellular homeostasis [3,4]. Consequently, defective and/or inappropriate regulation of PTP activity could lead to aberrant tyrosine phosphorylation, which may subsequently contribute to the development of many human diseases [35]. Taking these previous reports and our present findings together, the proper maintenance of PTK/PTP balance may be an important key determinant in redox-sensitive signal transduction during the aging process. Recent reports showed that the dysregulation of protein tyrosine phosphorylation by PTKs or PTPs provokes a critical error in cellular processes relevant during diseases like cancer [36]. For an example of PTKs, the ABL tyrosine kinase in the oncogenic BCR–ABL fusion protein is constitutively active and its activity is thought to be mainly signaling over time in the progress of cancerous diseases and chronic inflammation [37,38]. In addition, dysregulation of PTPs such as PTEN, PTP1B, SHP 1/2, or Cdc25 contributes to the pathogenesis of cancer, neurodegeneration, and diabetes as well [39,40], because PTPs downstream in the signaling pathways, PI3K/Akt, MAPKs, ERKs, JNKs, and NF-κB, are all associated with the modulation of cell physiology and survival. As many PTKs and PTPs are identified as highly active targets for diseases, therapeutic PTK- or PTP-based strategies are currently been explored in the development of disease treatment. In conclusion, this study documents for the first time that an agerelated PTK/PTP imbalance led to NF-κB activation in inflammation and aging. Our data clearly indicate that this imbalance is attenuated by the antioxidative, anti-inflammatory effects of CR (Fig. 9). Thus,

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