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Laminar Shear Stress Up-regulates Peroxiredoxins (PRX) in Endothelial Cells

Laminar Shear Stress Up-regulates Peroxiredoxins (PRX) in Endothelial Cells THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 3, pp. 1622–1627, January 18, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Laminar Shear Stress Up-regulates Peroxiredoxins (PRX) in Endothelial Cells PRX 1 AS A MECHANOSENSITIVE ANTIOXIDANT Received for publication, September 24, 2007, and in revised form, October 23, 2007 Published, JBC Papers in Press, November 16, 2007, DOI 10.1074/jbc.M707985200 ‡ § § §¶1 ‡2 Amy L. Mowbray , Dong-Hoon Kang , Sue Goo Rhee , Sang Won Kang , and Hanjoong Jo From the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, Division of Life and Pharmaceutical Sciences and the Center for Cell Signaling and Drug Discovery Research, Department of Life Sciences, Ewha Womans University, Seoul 120-750, Korea, and Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322 Shear stress plays a significant role in endothelial cell biology esis by high levels of unidirectional laminar shear stress (LS) (1, and atherosclerosis development. Previous work by our group 2). In contrast, plaque-prone areas in curves and bifurcations of has shown that fluid flow stimulates important functional the vasculature correspond to locales exposed to low or unsta- changes in cells through protein expression regulation. Perox- ble shear stress, including oscillatory shear stress (OS) (2–4). iredoxins (PRX) are a family of antioxidant enzymes but have yet These local mechanical forces have been correlated to the to be investigated in response to shear stress. Studies have behavior of the exposed endothelium. shown that oscillatory shear stress (OS) increases reactive oxy- Endothelial cells exposed to disturbed flow experience oxi- gen species (ROS) levels in endothelial cells, whereas laminar dative stress, inflammatory molecule expression, and monocyte shear stress (LS) blocks this response. We hypothesized that recruitment as early signatures of atherosclerosis (5–9). In vitro PRX are responsible for the anti-oxidative effect of LS. To test studies have established that OS is a potent stimulator of reac- this hypothesis, bovine aortic endothelial cells (BAEC) were tive oxygen species (ROS) production in endothelial cells, and 2 2 subjected to LS (15 dyn/cm ), OS (5 dyn/cm , 1 Hz), or static quantitative measurements by our group showed a significant conditions for 24 h. Using Western blot and immunofluores- increase in both OS-dependent superoxide (O ) and hydrogen cence staining, all six isoforms of PRX were identified in BAEC. peroxide (H O ) production (9–12). We found that OS-stimu- 2 2 When compared with OS and static, exposure to chronic LS lated ROS occurs in an NADPH oxidase-dependent manner up-regulated PRX 1 levels intracellularly. LS also increased and leads to inflammatory responses (ICAM-1) (intercellular expression of PRX 5 relative to static controls, but not OS. PRX adhesion molecule 1) expression and monocyte adhesion (10, exhibited broad subcellular localization, with distribution in the 11). Conversely, LS acts to reduce ROS production and subse- cytoplasm, Golgi, mitochondria, and intermediate filaments. In quent inflammatory response (10). Nevertheless, the mecha- addition, PRX 1 knock down, using specific small interference nism by which LS restricts oxidative stress remains unclear. RNA, attenuated LS-dependent reactive oxygen species reduc- Antioxidant defense systems are critical to the protection of tion in BAEC. However, PRX 5 depletion did not. Together, cellular macromolecules. They work to maintain a reductive these results suggest that PRX 1 is a novel mechanosensitive cytosolic environment using both catalytic and non-enzymatic antioxidant, playing an important role in shear-dependent reg- processes (13). In particular, it has been hypothesized that anti- ulation of endothelial biology and atherosclerosis. oxidants are likely to regulate intracellular hydrogen peroxide in a localized manner. Production of H O occurs where 2 2 needed for intracellular signaling, while hydrogen peroxide molecules diffusing away from the site of action are destroyed Shear stress acting on the blood vessel wall plays an impor- (14). Recently, a new group of ubiquitous antioxidant proteins tant role in the development of atherosclerosis. Straight regions has been acknowledged in yeast, plant, and animal cells. The of the arterial tree are considered “protected” from atherogen- peroxiredoxins (PRX) are thiol specific-, non-selenium-con- taining enzymes that use redox-active cysteines to reduce per- oxides and eliminate ONOO . They are produced at high levels * This work was supported by National Institutes of Health Grants HL71014, 2 HL75209, and HL70531 (to H. J.) and 21C Frontier Functional Proteomics in the cell, having been reported to comprise 0.1–1% of soluble Project (FPR05C2-510) from the Korean Ministry of Science and Technol- protein in mammalian cells (15). Based on conserved cysteine ogy (to D. H. K. and S. W. K.) as well as an American Heart Association pre- residues, six isoforms of peroxiredoxins (PRX 1–6) have been doctoral fellowship (to A. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must there- identified in mammalian systems, and a variety of investiga- fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec- tions have described their functional roles in vascular remodel- tion 1734 solely to indicate this fact. 1 ing, cancer, and pulmonary and neurodegenerative diseases To whom correspondence may be addressed: Dept. of Life Sciences, 11-1 Daehyun-dong, Seodaemoon-gu, Seoul 120-750, Korea. Tel.: 82-2-3277- 3352; Fax: 82-2-3277-3760; E-mail: [email protected]. 2 3 To whom correspondence may be addressed: Wallace H. Coulter Dept. of The abbreviations used are: LS, laminar shear stress; OS, oscillatory shear Biomedical Engineering at Georgia Tech and Emory University, 2005 WMB, stress; ROS, reactive oxygen species; PRX, peroxiredoxin; BAEC, bovine aor- Atlanta, GA 30322. Tel.: 404-712-9654; Fax: 404 –727-3330; E-mail: tic endothelial cell; ST, static; PBS, phosphate-buffered saline; siRNA, small [email protected]. interference RNA. 1622 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 3 •JANUARY 18, 2008 This is an Open Access article under the CC BY license. Peroxiredoxins as Mechanosensitive Antioxidants (16–19). However, no study has yet explored the role of shear Immunocytochemistry—Following shear stress exposure or stress on PRX regulation in endothelial cells. static culture, BAEC in 100-mm tissue culture dishes were In this study, we tested the hypothesis that shear stress alters washed three times with phosphate-buffered saline. Cells were PRX function by regulating protein expression and localization, fixed with 2% paraformaldehyde and permeabilized in 0.2% Tri- which in turn affect the redox status of endothelial cells. Our ton X-100. Primary antibody in 3% bovine serum albumin was studies show that all six forms of PRX are abundantly expressed applied overnight at 4 °C, followed by incubation with second- in bovine aortic endothelial cells (BAEC) and that atheropro- ary antibody conjugated to Alexa Fluor 488 or 568 (Molecular tective LS increased intracellular PRX 1 levels compared with Probes) for1hat room temperature. Nuclei were labeled with atherogenic OS. In addition, PRX 1 knockdown experiments Hoechst stain in 3% bovine serum albumin for 15 min at room implicated PRX 1, but not PRX 5, as an important regulator of temperature. To identify mitochondria, cells were incubated shear-dependent cellular redox state. with 100 nM Mitotracker Red CMXRos (Molecular Probes) in growth medium for 15 min at 37 °C prior to fixation. All cells EXPERIMENTAL PROCEDURES were mounted using the Prolong Antifade kit (Molecular Endothelial Cell Culture—Bovine aortic endothelial cells Probes), and fluorescence images were collected via confocal were obtained from Cell Applications Inc. Cells were main- microscope (Zeiss LSM 510). Primary antibodies specific for tained in a standard humidified incubator (37 °C, 5% CO )in PRX 1, 2, 3, 4, 5, and 6 (Lab Frontier), the Golgi marker GM 130 Dulbecco’s minimum Eagle’s medium (Invitrogen) supple- (Transduction Laboratories), and the intermediate filament mented with 10% fetal bovine serum (Atlanta Biologicals), hep- marker vimentin (Sigma) were used. arin sodium (American Pharmaceutical Partners), endothelial Small Interfering RNA (siRNA)—Annealed siRNA duplexes cell growth supplement (isolated by us), and minimum non- and Oligofectamine (Invitrogen) transfection agent were essential amino acids (Invitrogen). BAEC from passage 8–15 applied to BAEC for 48–72 h according to the manufacturer’s were used in the following experimental protocols. recommendation. Control non-silencing siRNA (sense, Shear Stress Studies—BAEC were grown to confluent mono- 5-UUCUCCGAACGUGUCACGUtt; antisense, 5-ACG- layers in 100-mm tissue culture dishes (Falcon) and were sub- UGACACGUUCGGAGAAtt) (Qiagen), Alexa Fluor 546-la- sequently exposed to static (ST) culture conditions or arterial beled control non-silencing siRNA (Qiagen), bovine PRX 1 levels of shear stress via cone-and-plate shear apparatus. siRNA (sense, 5-GUGCUUCUGUGGAUUCUCAtt; anti- Atheroprotective LS at 15 dynes/cm was simulated by rotating sense: 5-PhoUGAGAAUCCACAGAAGCACtt) (MWG), and a Teflon cone (0.5 cone angle) unidirectionally in medium as bovine PRX 5 siRNA (sense, 5-GUGGCAUGUCUGACCGU- previously described by us (20). To mimic unstable atherogenic UAtt; antisense, 5-PhoUAACGGUCAGACAUGCCACtt) OS, the cone was rotated bidirectionally in medium using a (MWG) were used. stepping motor (Servo Motor) and computer program (DC Hydrogen Peroxide Detection—Using a horseradish peroxi- Motor Company). Endothelial cells were exposed to OS at 5 dase-linked Amplex Red fluorescence assay, intracellular dynes/cm with directional changes of flow at 1 Hz frequency hydrogen peroxide production was measured via extracellular (7). All shear stress studies were performed in low serum (0.5% leakage of H O from conditioned BAEC as previously 2 2 fetal bovine serum) growth medium for 24 h. described (12). Briefly, cells were exposed to either static cul- Preparation of Whole Cell Lysate—Following experimental ture conditions or shear stress in low serum culture medium. treatment, cells were washed three times with ice-cold phos- After 24 h, cells were washed twice with Krebs-Ringer phos- phate-buffered saline and lysed with 600 l of lysis buffer (50 phate buffer and incubated with 5 M Amplex UltraRed mM Tris-HCl, pH 7.4, at 4 °C, 1% Nonidet P40, 0.25% sodium (Molecular Probes) and 0.1 unit/ml horseradish peroxidase deoxycholate, 150 mM NaCl, 1 mM EDTA, 30 mM NaF, 40 mM type II (Sigma-Aldrich) in Krebs-Ringer phosphate for 40 min. -glycerophosphate, 10 mM Na P O ,2mM Na VO ,1mM To identify the hydrogen peroxide-specific signal, control sam- 4 2 7 3 4 phenylmethylsulfonyl fluoride, 0.1% SDS). The lysate was fur- ples were coincubated with 500 units/ml catalase. Triplicate ther homogenized by sonication. The protein content of each readings were taken in a 96-well plate using 100-l samples of sample was determined by Bio-Rad DC assay. medium, and fluorescence was detected via plate reader at exci- Immunoblotting—Aliquots of cell lysate (20–40 g of pro- tation and emission of 530 and 580 nm, respectively. Hydrogen tein each) were resolved by size on 12.5 or 15% SDS-polyacryl- peroxide levels were calculated in terms of catalase-inhibitable amide gels and subsequently transferred to a polyvinylidene signal and were normalized to cellular protein as measured by difluoride membrane (Millipore). The membrane was incu- the Bio-Rad DC assay. H O concentrations were estimated 2 2 bated with primary antibody overnight at 4 °C, followed by using a standard curve. incubation with an alkaline phosphatase-conjugated secondary Statistical Analysis—For all quantitative data collected, sta- antibody for 1 h at room temperature. Protein expression was tistical analysis was assessed by Student’s t test using the Micro- detected by a chemiluminescence method, and the intensities cal Origin statistical package. A significant difference between of immunoreactive bands were determined via densitometry control and treatment groups was defined as p  0.05 for three using the NIH Image program (21). Primary antibodies specific or more independent experiments. for PRX 1, 2, 3, 4, 5, and 6 (Lab Frontier), phospho-endothelial RESULTS nitric-oxide synthase (Ser1177) (Cell Signaling Technology), total endothelial nitric-oxide synthase (BD Biosciences), and LS Up-regulates PRX 1 Expression in Cell Lysates—Our pre- -actin (Santa Cruz Biotechnology) were used. vious work established that fluid shear stress critically affects JANUARY 18, 2008• VOLUME 283 • NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1623 Peroxiredoxins as Mechanosensitive Antioxidants endothelial cell function by regulating protein expression pat- metric analysis indicated the level of PRX 1 was significantly terns. The PRX family is undeniably significant to cellular phys- increased by LS compared with OS and static controls. In addi- iology and pathology but remains understudied in the fluid flow tion, PRX 5 expression was significantly increased by LS with field. This led us to investigate whether shear stress regulates respect to static conditions but was not statistically different the intracellular expression of mammalian PRX in BAEC. To from OS (Fig. 1). Endothelial cell alignment and phosphoryla- this end, total BAEC lysates were collected after 24 h of static ted endothelial nitric-oxide synthase and total endothelial 2 2 culture, LS (15dynes/cm ), or OS (5dynes/cm ) and analyzed nitric-oxide synthase were included as internal controls. These by Western blot using PRX-specific antibodies. In these studies, findings suggest that PRX 1 is mechanosensitive and likely to ST, cells cultured under no shear stress, were used as a control play an important role in shear-dependent cell biology. for the shear system. As previously described, physiologically PRX Exist in Various Subcellular Locations throughout BAEC— “normal” arterial endothelial cells are not exposed to chronic Via Western blots, we have thus far found that shear stress static conditions but, rather, experience continuous fluid flow. regulates PRX 1 and 5 expression in BAEC. Based on the large Therefore, LS is a more relevant control, representing a healthy number of PRX family members, we hypothesized that individ- state, which we will compare with OS, the disease state. With ual PRX likely play important roles in specific subcellular this in mind, all six PRX were detected in BAEC, and densito- compartments. To investigate the intracellular distribution of PRX, confocal immunofluorescence staining studies were performed. This study revealed data consistent with West- ern blot analysis of shear-induced protein expression in BAEC. Image analysis of staining intensities indicated that PRX 1 increased after 24 h of LS compared with OS and static conditions (Fig. 2A). In addition to the shear dependence, Fig. 2 also shows that PRX members exhibited assorted staining patterns. This result raised two interesting questions: 1) Are PRX located in specific subcellular locations and 2) does shear stress alter this subcel- lular localization? To clearly characterize the location of each PRX within specific subcellular compartments, colocalization staining was performed in static BAEC using PRX-specific anti- FIGURE 1. PRX 1 is up-regulated by LS. Confluent BAEC were exposed to LS, OS, or static conditions for 1 day, and cell lysates were obtained. A, equal bodies and organelle-specific markers for Golgi (GM 130), aliquots of protein (20 – 40 g) were analyzed by Western blot using antibod- endoplasmic reticulum (KDEL receptor, data not shown), lyso- ies specific to PRX 1– 6 phospho-endothelial nitric-oxide synthase (peNOS), some (cathepsin S, data not shown), intermediate filament total endothelial nitric-oxide synthase (teNOS), and-actin blots were used as shear stress controls and internal loading controls, respectively. B, densito- (vimentin), and mitochondria (Mitotracker). PRX 1 staining metric analysis was used to quantify the intensity of each band, and the aver- (green) overlapped with the Golgi marker staining (red), shown age values (mean S.E., n 12) are shown in bar graphs as % of static control. *, p  0.05 indicates significance compared with static control. **, p  0.05 as yellow in the merged image (Fig. 3A), suggesting that PRX 1 indicates significance compared with OS. exists in the Golgi apparatus. In addition, PRX 2, 4, 5, and 6 also appeared to be found in the Golgi (Fig. 3, B and E–G). The PRX 3 staining pattern was distinctly dif- ferent from other PRX, showing clear colocalization with the mito- chondria marker (Fig. 3D). Interest- ingly, PRX 2 staining revealed colo- calization with the intermediate filament marker (Fig. 3C). In addi- tion to these subcellular localiza- tions, PRX 1, 2, 4, 5 and 6 were also expressed in the cytosol (Fig. 3, A–C and E–G). Next, we examined whether shear stress stimulated expression of PRX members in other subcellular locations. Sub- cellular location did not appear to change in response to shear stress, although Golgi were located FIGURE 2. LS stimulated PRX 1 expression in BAEC. Confluent BAEC were exposed to LS, OS, or static condi- upstream of the direction of flow tions for 1 day as in Fig. 1. Cells were stained using antibodies specific to PRX 1– 6. Secondary antibodies after chronic LS, consistent with conjugated to Alexa Fluor 488 (green) were imaged by confocal microscopy. Nuclei were counter-stained with Hoechst dye (blue). Arrows indicate unique subcellular staining patterns of each PRX. previous reports (Fig. 2, A, B, and 1624 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 3 •JANUARY 18, 2008 Peroxiredoxins as Mechanosensitive Antioxidants Here, specific siRNAs were used to knock down either PRX 1 or PRX 5 protein levels in order to investigate PRX-dependent ROS accumula- tion. Western blots with isoform- specific PRX antibodies were used to determine the efficacy and speci- ficity of these siRNAs. Compared with non-silencing siRNA and Alexa Fluor 546-labeled non-silenc- ing siRNA (50 nM each), 48 h of treatment of BAEC with PRX 1 siRNA dramatically reduced (by 75% of non-silencing control) expression of PRX 1 at a concentra- tion as low as 10 nM (Fig. 4A). As shown in Fig. 4B,10nM PRX 5 siRNA also effectively reduced PRX 5 expression (by 90% of non-silenc- ing control). Via Western blots, the specificity of PRX 1 and 5 siRNAs was assessed by examining the expression of all other PRX family FIGURE 3. PRX are located in various subcellular organelles in BAEC. Static BAEC were co-stained for PRX members (Fig. 4, A and B). Using 1– 6 and subcellular organelles using PRX-specific antibodies (green), as in Fig. 2, and organelle-specific mark- isoform-specific PRX antibodies, we ers (red). Nuclei were counterstained with Hoechst dye (blue). Center panels: Mitochondria, Golgi, and inter- found that these siRNAs had no sig- mediate filaments are stained with Mitotracker CMXRos, GM 130 antibody, and vimentin antibody, respec- tively. GM 130 and vimentin were visualized by secondary antibodies conjugated to Alexa Fluor 568 (red). nificant effect on other PRX, indi- Merged images are shown in the right panel. Yellow staining indicates colocalization. cating that they exclusively targeted PRX 1 or 5, respectively, among all PRX family members (Fig. 4, A and B). Therefore, PRX 1 and 5 siRNAs were confidently used at 10 nM concentration in subse- quent functional studies. It has been well established that chronic exposure of endo- thelial cells to OS stimulates, while LS inhibits, ROS production (10, 11, 23, 24). Utilizing an Amplex Red assay in the presence or absence of catalase, we verified that hydrogen peroxide levels were 87% less in BAEC exposed to LS compared with those treated with OS (Fig. 5A). Using a standard hydrogen peroxide dose curve, we also found that the relative hydrogen peroxide levels following static culture, LS, and OS were consistent with expected cellular concentrations (Fig. 5B). To determine whether PRX 1 was responsible for the LS-dependent decrease in ROS levels in BAEC, we knocked down PRX 1 using PRX 1 FIGURE 4. PRX 1 and PRX 5 siRNAs specifically reduce PRX 1 and PRX 5 protein expression, respectively. BAEC were transfected with either 50 nM siRNA as indicated above (Fig. 4A). PRX 1 depletion signifi- non-silencing siRNA (NS), 50 nM Alexa Fluor 546-labeled siRNA (Alexa), or PRX cantly increased catalase-inhibitable hydrogen peroxide by 1(A)orPRX5(B) siRNAs (10, 20, 50, and 100 nM) for 48 h. Cell lysates were analyzed by Western blot with PRX-specific antibodies as indicated. -actin 2-fold above non-silencing controls in static-, LS-, and was used as an internal control. OS-treated BAEC (Fig. 5C). To investigate whether this was a global effect of mechanosensitive PRX family members, we also D–F). Taken together, these results clearly indicate that PRX knocked down PRX 5 using PRX 5-specific siRNA. However, are abundantly expressed throughout the subcellular when compared with non-silencing controls, PRX 5 depletion organelles of endothelial cells. had no significant effect on hydrogen peroxide levels in any PRX 1 Prevents Oxidative Stress in Endothelial Cells Exposed group. Taken together, these data suggest that PRX 1 is a criti- to LS—PRX 1 is a prominent antioxidant, and our data indicate cally important regulator of ROS levels in both a basal and shear- its expression is highly up-regulated by LS (22). Consequently, dependent manner. we investigated whether PRX 1 was responsible for the DISCUSSION decreased ROS levels in endothelial cells exposed to LS. The depletion of individual PRX from cellular systems provides a Through protein expression analysis and subsequent func- useful tool to study the functional role of each PRX isoform. tional studies, we have discovered PRX 1 as a mechanosensitive JANUARY 18, 2008• VOLUME 283 • NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1625 Peroxiredoxins as Mechanosensitive Antioxidants throughout the cellular milieu of BAEC, colocalizing with the cyto- plasm, Golgi apparatus, mitochon- dria, and intermediate filaments. These observations were consistent with previously reported localiza- tion studies in other cell types, but we are the first to report an apparent PRX 2 colocalization with vimentin (32, 33). This costaining of PRX 2 with vimentin suggests that it may be located in the intermediate fila- ment, but further studies will be necessary to confirm this finding. Although detection of PRX in the Golgi body likely reflects protein processing or packaging, localiza- tion within other organelles indi- cates that PRX may act both globally and in a site-specific manner to reg- ulate ROS in endothelial cells. In the endothelium, ROS, such as O and H O arise from several sources,, 2 2, including NADPH oxidase, xan- FIGURE 5. PRX 1 knock down increases H O production in BAEC, whereas PRX 5 knock down does not. 2 2 thine oxidase, mitochondrial oxi- Catalase-inhibitable hydrogen peroxide levels were assessed via Amplex Red assay, and average values (mean dase, cytochrome P450, and uncou- S.E., n  6–12) are shown in bar graphs as % of non-silencing (NS) siRNA-treated static controls. A, confluent BAEC pled nitric-oxide synthase (34, 35). were exposed to ST, LS, or OS for 24 h prior to assay. B, a hydrogen peroxide dose curve was used to estimate relative hydrogen peroxide concentrations in cells conditioned with shear stress. C, BAEC were transfected with either At relatively low concentrations, non-silencing or PRX 1 siRNA (10 nM) for 48 h and then exposed to ST, LS, or OS for 24 h prior to assay. D, BAEC were ROS play critical roles in redox sig- transfected with either non-silencing or PRX 5 siRNA (10 nM) for 48 h and then exposed to ST, LS, or OS for 24 h prior to assay. *, p 0.05 designates significance between indicated groups. naling and normal cell function. However, higher concentrations of antioxidant. Data to support this concept include: 1) PRX 1 is ROS induce oxidative damage of DNA, proteins, carbohy- up-regulated intracellularly by chronic LS compared with OS, drates, and lipids (36–38). This damage has been shown to 2) PRX exhibit broad staining patterns in BAEC and localize in critically affect cellular function and apoptosis when it occurs in important cellular structures, 3) ROS production is signifi- mitochondria, lysosomes, and nuclei (39–41). In addition, cantly reduced in cells exposed to chronic LS, and 4) this effect cytosolic proteins modified by ROS have been shown to affect can be attenuated by PRX 1 depletion, but not PRX 5 depletion. local cell signaling and, collectively, the redox status of the cell Through this work, we reveal for the first time that PRX are (11, 42, 43). Ubiquitous distribution of PRX in BAEC may regulated by shear stress in endothelial cells. Previous studies reflect diverse sources of ROS throughout the cells and provide have shown that other antioxidants are also controlled by shear protection for important macromolecules and structures stress. LS has been shown to up-regulate antioxidant genes, against local ROS production. In addition, widespread alloca- including endothelial nitric-oxide synthase, CuZn superoxide tion of PRX may be important for comprehensive management dismutase, manganese superoxide dismutase, glutathione per- of the overall oxidative state of cells. oxidase, glutathione, and thioredoxin (25–29). In addition, Several studies have shown that oxidative stress is regulated Chen et al. (30) observed that many genes protective against by shear stress in endothelial cells (10–12, 24, 26). We have oxidative stress are induced by exposure to prolonged LS. They previously published that both LS and OS stimulate ROS pro- have also noted that such genes are regulated through a con- duction acutely but the ROS transiently elevated by LS returns served, shear-sensitive antioxidant response element. In sup- to basal levels within a few hours (10). However, unlike LS, OS port of our finding that PRX 1 expression is LS-dependent, continues to increase ROS production, maintaining elevated recent work has shown that PRX 1 is a target gene of nuclear levels as long as cells are exposed to OS (10, 24, 26). The mech- factor (erythroid-derived 2)-related factor 2 (Nrf2), a key tran- anism by which ROS levels are lowered in cells exposed to scription factor that binds to antioxidant response element chronic LS is undefined. This study demonstrates that endothe- (31). Collectively, these studies indicate that cells possess an lial cells exposed to chronic LS express much more PRX 1 com- elaborate system of shear-responsive antioxidants and that pared with OS and static conditions. These findings suggest each may play an independent role to mediate oxidative stress that PRX 1 is up-regulated by LS and that this may be respon- and modulate redox-sensitive signaling pathways. sible for LS-mediated decrease in ROS levels. The ubiquitous nature of the PRX family itself exemplifies As previously determined by electron spin resonance spec- this concept. Immunofluorescence microscopy revealed PRX trometry and dichlorofluorescein-diacetate methods, endothe- 1626 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 3 •JANUARY 18, 2008 Peroxiredoxins as Mechanosensitive Antioxidants (2003) J. Biol. Chem. 278, 47291–47298 lial cells exposed to OS produce significantly more superoxide 11. Sorescu, G. P., Song, H., Tressel, S. L., Hwang, J., Dikalov, S., Smith, D. A., and hydrogen peroxide than those in static culture (10–12). In Boyd, N. L., Platt, M. O., Lassegue, B., Griendling, K. K., and Jo, H. (2004) contrast, endothelial cells treated with chronic, unidirectional Circ. Res. 95, 773–779 high shear generate considerably less O . Here, we used 12. McNally, J. S., Davis, M. E., Giddens, D. P., Saha, A., Hwang, J., Dikalov, S., Amplex Red assay as an independent method to measure ROS Jo, H., and Harrison, D. G. (2003) Am. J. Physiol. 285, H2290–H2297 levels in BAEC. Consistent with our previous reports, OS 13. Mates, J. M., Perez-Gomez, C., and De Castro, I. N. (1999) Clin. Biochem. increased and LS decreased ROS production (Fig. 5A). The ROS 32, 595–603 14. Rhee, S. G., Chae, H. Z., and Kim, K. (2005) Free Radic. Biol. Med. 38, measured by this assay was inhibitable by catalase, indicating that 1543–1552 H O is the primary ROS component. 2 2 15. Wood, Z. A., Schroder, E., Robin Harris, J., and Poole, L. B. (2003) Trends PRX 1 is the most abundant and ubiquitously distributed Biochem. Sci. 28, 32–40 member of mammalian PRX (22). Our current study demon- 16. Kinnula, V. L., Lehtonen, S., Sormunen, R., Kaarteenaho-Wiik, R., Kang, strated that PRX 1 is dramatically up-regulated by chronic LS S. W., Rhee, S. G., and Soini, Y. (2002) J. Pathol. 196, 316–323 compared with OS and is located in the cytoplasm and Golgi. 17. Kinnula, V. L., Lehtonen, S., Kaarteenaho-Wiik, R., Lakari, E., Paakko, P., Kang, S. W., Rhee, S. G., and Soini, Y. (2002) Thorax 57, 157–164 Knock down of PRX 1 using siRNA resulted in significantly 18. Krapfenbauer, K., Engidawork, E., Cairns, N., Fountoulakis, M., and Lu- higher ROS levels in BAEC exposed to LS, OS, and static con- bec, G. (2003) Brain Res. 967, 152–160 ditions, whereas PRX 5 depletion did not. Although PRX 1 19. Choi, M. H., Lee, I. K., Kim, G. W., Kim, B. U., Han, Y. H., Yu, D. Y., Park, knock down did not fully abolish the antioxidative outcome of H. S., Kim, K. Y., Lee, J. S., Choi, C., Bae, Y. S., Lee, B. I., Rhee, S. G., and LS, its significant effect was somewhat surprising considering Kang, S. W. (2005) Nature 435, 347–353 the presence of other PRX family members and additional 20. Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. 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Traub, O., and Berk, B. C. (1998) Arterioscler. Thromb. Vasc. Biol. 18, Leopold, J. A. (2004) J. Biol. Chem. 279, 4425–4432 677–685 9. Harrison, D., Griendling, K. K., Landmesser, U., Hornig, B., and Drexler, 41. Martinet, W., Knaapen, M. W., De Meyer, G. R., Herman, A. G., and H. (2003) Am. J. Cardiol. 91, 3A, 7A–11A Kockx, M. M. (2002) Circulation 106, 927–932 10. Hwang, J., Saha, A., Boo, Y. C., Sorescu, G. P., McNally, J. S., Holland, S. M., 42. Finkel, T. (1999) J. Leukocyte Biol. 65, 337–340 Dikalov, S., Giddens, D. P., Griendling, K. K., Harrison, D. G., and Jo, H. 43. Sykes, M. C., Mowbray, A. L., and Jo, H. (2007) Circ. Res., 100, 152–154 JANUARY 18, 2008• VOLUME 283 • NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1627 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Laminar Shear Stress Up-regulates Peroxiredoxins (PRX) in Endothelial Cells

Mowbray, Amy L.; Kang, Dong-Hoon; Rhee, Sue Goo; Kang, Sang Won; Jo, Hanjoong
Journal of Biological Chemistry – Jan 1, 2008
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 3, pp. 1622–1627, January 18, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Laminar Shear Stress Up-regulates Peroxiredoxins (PRX) in Endothelial Cells PRX 1 AS A MECHANOSENSITIVE ANTIOXIDANT Received for publication, September 24, 2007, and in revised form, October 23, 2007 Published, JBC Papers in Press, November 16, 2007, DOI 10.1074/jbc.M707985200 ‡ § § §¶1 ‡2 Amy L. Mowbray , Dong-Hoon Kang , Sue Goo Rhee , Sang Won Kang , and Hanjoong Jo From the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, Division of Life and Pharmaceutical Sciences and the Center for Cell Signaling and Drug Discovery Research, Department of Life Sciences, Ewha Womans University, Seoul 120-750, Korea, and Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322 Shear stress plays a significant role in endothelial cell biology esis by high levels of unidirectional laminar shear stress (LS) (1, and atherosclerosis development. Previous work by our group 2). In contrast, plaque-prone areas in curves and bifurcations of has shown that fluid flow stimulates important functional the vasculature correspond to locales exposed to low or unsta- changes in cells through protein expression regulation. Perox- ble shear stress, including oscillatory shear stress (OS) (2–4). iredoxins (PRX) are a family of antioxidant enzymes but have yet These local mechanical forces have been correlated to the to be investigated in response to shear stress. Studies have behavior of the exposed endothelium. shown that oscillatory shear stress (OS) increases reactive oxy- Endothelial cells exposed to disturbed flow experience oxi- gen species (ROS) levels in endothelial cells, whereas laminar dative stress, inflammatory molecule expression, and monocyte shear stress (LS) blocks this response. We hypothesized that recruitment as early signatures of atherosclerosis (5–9). In vitro PRX are responsible for the anti-oxidative effect of LS. To test studies have established that OS is a potent stimulator of reac- this hypothesis, bovine aortic endothelial cells (BAEC) were tive oxygen species (ROS) production in endothelial cells, and 2 2 subjected to LS (15 dyn/cm ), OS (5 dyn/cm , 1 Hz), or static quantitative measurements by our group showed a significant conditions for 24 h. Using Western blot and immunofluores- increase in both OS-dependent superoxide (O ) and hydrogen cence staining, all six isoforms of PRX were identified in BAEC. peroxide (H O ) production (9–12). We found that OS-stimu- 2 2 When compared with OS and static, exposure to chronic LS lated ROS occurs in an NADPH oxidase-dependent manner up-regulated PRX 1 levels intracellularly. LS also increased and leads to inflammatory responses (ICAM-1) (intercellular expression of PRX 5 relative to static controls, but not OS. PRX adhesion molecule 1) expression and monocyte adhesion (10, exhibited broad subcellular localization, with distribution in the 11). Conversely, LS acts to reduce ROS production and subse- cytoplasm, Golgi, mitochondria, and intermediate filaments. In quent inflammatory response (10). Nevertheless, the mecha- addition, PRX 1 knock down, using specific small interference nism by which LS restricts oxidative stress remains unclear. RNA, attenuated LS-dependent reactive oxygen species reduc- Antioxidant defense systems are critical to the protection of tion in BAEC. However, PRX 5 depletion did not. Together, cellular macromolecules. They work to maintain a reductive these results suggest that PRX 1 is a novel mechanosensitive cytosolic environment using both catalytic and non-enzymatic antioxidant, playing an important role in shear-dependent reg- processes (13). In particular, it has been hypothesized that anti- ulation of endothelial biology and atherosclerosis. oxidants are likely to regulate intracellular hydrogen peroxide in a localized manner. Production of H O occurs where 2 2 needed for intracellular signaling, while hydrogen peroxide molecules diffusing away from the site of action are destroyed Shear stress acting on the blood vessel wall plays an impor- (14). Recently, a new group of ubiquitous antioxidant proteins tant role in the development of atherosclerosis. Straight regions has been acknowledged in yeast, plant, and animal cells. The of the arterial tree are considered “protected” from atherogen- peroxiredoxins (PRX) are thiol specific-, non-selenium-con- taining enzymes that use redox-active cysteines to reduce per- oxides and eliminate ONOO . They are produced at high levels * This work was supported by National Institutes of Health Grants HL71014, 2 HL75209, and HL70531 (to H. J.) and 21C Frontier Functional Proteomics in the cell, having been reported to comprise 0.1–1% of soluble Project (FPR05C2-510) from the Korean Ministry of Science and Technol- protein in mammalian cells (15). Based on conserved cysteine ogy (to D. H. K. and S. W. K.) as well as an American Heart Association pre- residues, six isoforms of peroxiredoxins (PRX 1–6) have been doctoral fellowship (to A. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must there- identified in mammalian systems, and a variety of investiga- fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec- tions have described their functional roles in vascular remodel- tion 1734 solely to indicate this fact. 1 ing, cancer, and pulmonary and neurodegenerative diseases To whom correspondence may be addressed: Dept. of Life Sciences, 11-1 Daehyun-dong, Seodaemoon-gu, Seoul 120-750, Korea. Tel.: 82-2-3277- 3352; Fax: 82-2-3277-3760; E-mail: [email protected]. 2 3 To whom correspondence may be addressed: Wallace H. Coulter Dept. of The abbreviations used are: LS, laminar shear stress; OS, oscillatory shear Biomedical Engineering at Georgia Tech and Emory University, 2005 WMB, stress; ROS, reactive oxygen species; PRX, peroxiredoxin; BAEC, bovine aor- Atlanta, GA 30322. Tel.: 404-712-9654; Fax: 404 –727-3330; E-mail: tic endothelial cell; ST, static; PBS, phosphate-buffered saline; siRNA, small [email protected]. interference RNA. 1622 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 3 •JANUARY 18, 2008 This is an Open Access article under the CC BY license. Peroxiredoxins as Mechanosensitive Antioxidants (16–19). However, no study has yet explored the role of shear Immunocytochemistry—Following shear stress exposure or stress on PRX regulation in endothelial cells. static culture, BAEC in 100-mm tissue culture dishes were In this study, we tested the hypothesis that shear stress alters washed three times with phosphate-buffered saline. Cells were PRX function by regulating protein expression and localization, fixed with 2% paraformaldehyde and permeabilized in 0.2% Tri- which in turn affect the redox status of endothelial cells. Our ton X-100. Primary antibody in 3% bovine serum albumin was studies show that all six forms of PRX are abundantly expressed applied overnight at 4 °C, followed by incubation with second- in bovine aortic endothelial cells (BAEC) and that atheropro- ary antibody conjugated to Alexa Fluor 488 or 568 (Molecular tective LS increased intracellular PRX 1 levels compared with Probes) for1hat room temperature. Nuclei were labeled with atherogenic OS. In addition, PRX 1 knockdown experiments Hoechst stain in 3% bovine serum albumin for 15 min at room implicated PRX 1, but not PRX 5, as an important regulator of temperature. To identify mitochondria, cells were incubated shear-dependent cellular redox state. with 100 nM Mitotracker Red CMXRos (Molecular Probes) in growth medium for 15 min at 37 °C prior to fixation. All cells EXPERIMENTAL PROCEDURES were mounted using the Prolong Antifade kit (Molecular Endothelial Cell Culture—Bovine aortic endothelial cells Probes), and fluorescence images were collected via confocal were obtained from Cell Applications Inc. Cells were main- microscope (Zeiss LSM 510). Primary antibodies specific for tained in a standard humidified incubator (37 °C, 5% CO )in PRX 1, 2, 3, 4, 5, and 6 (Lab Frontier), the Golgi marker GM 130 Dulbecco’s minimum Eagle’s medium (Invitrogen) supple- (Transduction Laboratories), and the intermediate filament mented with 10% fetal bovine serum (Atlanta Biologicals), hep- marker vimentin (Sigma) were used. arin sodium (American Pharmaceutical Partners), endothelial Small Interfering RNA (siRNA)—Annealed siRNA duplexes cell growth supplement (isolated by us), and minimum non- and Oligofectamine (Invitrogen) transfection agent were essential amino acids (Invitrogen). BAEC from passage 8–15 applied to BAEC for 48–72 h according to the manufacturer’s were used in the following experimental protocols. recommendation. Control non-silencing siRNA (sense, Shear Stress Studies—BAEC were grown to confluent mono- 5-UUCUCCGAACGUGUCACGUtt; antisense, 5-ACG- layers in 100-mm tissue culture dishes (Falcon) and were sub- UGACACGUUCGGAGAAtt) (Qiagen), Alexa Fluor 546-la- sequently exposed to static (ST) culture conditions or arterial beled control non-silencing siRNA (Qiagen), bovine PRX 1 levels of shear stress via cone-and-plate shear apparatus. siRNA (sense, 5-GUGCUUCUGUGGAUUCUCAtt; anti- Atheroprotective LS at 15 dynes/cm was simulated by rotating sense: 5-PhoUGAGAAUCCACAGAAGCACtt) (MWG), and a Teflon cone (0.5 cone angle) unidirectionally in medium as bovine PRX 5 siRNA (sense, 5-GUGGCAUGUCUGACCGU- previously described by us (20). To mimic unstable atherogenic UAtt; antisense, 5-PhoUAACGGUCAGACAUGCCACtt) OS, the cone was rotated bidirectionally in medium using a (MWG) were used. stepping motor (Servo Motor) and computer program (DC Hydrogen Peroxide Detection—Using a horseradish peroxi- Motor Company). Endothelial cells were exposed to OS at 5 dase-linked Amplex Red fluorescence assay, intracellular dynes/cm with directional changes of flow at 1 Hz frequency hydrogen peroxide production was measured via extracellular (7). All shear stress studies were performed in low serum (0.5% leakage of H O from conditioned BAEC as previously 2 2 fetal bovine serum) growth medium for 24 h. described (12). Briefly, cells were exposed to either static cul- Preparation of Whole Cell Lysate—Following experimental ture conditions or shear stress in low serum culture medium. treatment, cells were washed three times with ice-cold phos- After 24 h, cells were washed twice with Krebs-Ringer phos- phate-buffered saline and lysed with 600 l of lysis buffer (50 phate buffer and incubated with 5 M Amplex UltraRed mM Tris-HCl, pH 7.4, at 4 °C, 1% Nonidet P40, 0.25% sodium (Molecular Probes) and 0.1 unit/ml horseradish peroxidase deoxycholate, 150 mM NaCl, 1 mM EDTA, 30 mM NaF, 40 mM type II (Sigma-Aldrich) in Krebs-Ringer phosphate for 40 min. -glycerophosphate, 10 mM Na P O ,2mM Na VO ,1mM To identify the hydrogen peroxide-specific signal, control sam- 4 2 7 3 4 phenylmethylsulfonyl fluoride, 0.1% SDS). The lysate was fur- ples were coincubated with 500 units/ml catalase. Triplicate ther homogenized by sonication. The protein content of each readings were taken in a 96-well plate using 100-l samples of sample was determined by Bio-Rad DC assay. medium, and fluorescence was detected via plate reader at exci- Immunoblotting—Aliquots of cell lysate (20–40 g of pro- tation and emission of 530 and 580 nm, respectively. Hydrogen tein each) were resolved by size on 12.5 or 15% SDS-polyacryl- peroxide levels were calculated in terms of catalase-inhibitable amide gels and subsequently transferred to a polyvinylidene signal and were normalized to cellular protein as measured by difluoride membrane (Millipore). The membrane was incu- the Bio-Rad DC assay. H O concentrations were estimated 2 2 bated with primary antibody overnight at 4 °C, followed by using a standard curve. incubation with an alkaline phosphatase-conjugated secondary Statistical Analysis—For all quantitative data collected, sta- antibody for 1 h at room temperature. Protein expression was tistical analysis was assessed by Student’s t test using the Micro- detected by a chemiluminescence method, and the intensities cal Origin statistical package. A significant difference between of immunoreactive bands were determined via densitometry control and treatment groups was defined as p  0.05 for three using the NIH Image program (21). Primary antibodies specific or more independent experiments. for PRX 1, 2, 3, 4, 5, and 6 (Lab Frontier), phospho-endothelial RESULTS nitric-oxide synthase (Ser1177) (Cell Signaling Technology), total endothelial nitric-oxide synthase (BD Biosciences), and LS Up-regulates PRX 1 Expression in Cell Lysates—Our pre- -actin (Santa Cruz Biotechnology) were used. vious work established that fluid shear stress critically affects JANUARY 18, 2008• VOLUME 283 • NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1623 Peroxiredoxins as Mechanosensitive Antioxidants endothelial cell function by regulating protein expression pat- metric analysis indicated the level of PRX 1 was significantly terns. The PRX family is undeniably significant to cellular phys- increased by LS compared with OS and static controls. In addi- iology and pathology but remains understudied in the fluid flow tion, PRX 5 expression was significantly increased by LS with field. This led us to investigate whether shear stress regulates respect to static conditions but was not statistically different the intracellular expression of mammalian PRX in BAEC. To from OS (Fig. 1). Endothelial cell alignment and phosphoryla- this end, total BAEC lysates were collected after 24 h of static ted endothelial nitric-oxide synthase and total endothelial 2 2 culture, LS (15dynes/cm ), or OS (5dynes/cm ) and analyzed nitric-oxide synthase were included as internal controls. These by Western blot using PRX-specific antibodies. In these studies, findings suggest that PRX 1 is mechanosensitive and likely to ST, cells cultured under no shear stress, were used as a control play an important role in shear-dependent cell biology. for the shear system. As previously described, physiologically PRX Exist in Various Subcellular Locations throughout BAEC— “normal” arterial endothelial cells are not exposed to chronic Via Western blots, we have thus far found that shear stress static conditions but, rather, experience continuous fluid flow. regulates PRX 1 and 5 expression in BAEC. Based on the large Therefore, LS is a more relevant control, representing a healthy number of PRX family members, we hypothesized that individ- state, which we will compare with OS, the disease state. With ual PRX likely play important roles in specific subcellular this in mind, all six PRX were detected in BAEC, and densito- compartments. To investigate the intracellular distribution of PRX, confocal immunofluorescence staining studies were performed. This study revealed data consistent with West- ern blot analysis of shear-induced protein expression in BAEC. Image analysis of staining intensities indicated that PRX 1 increased after 24 h of LS compared with OS and static conditions (Fig. 2A). In addition to the shear dependence, Fig. 2 also shows that PRX members exhibited assorted staining patterns. This result raised two interesting questions: 1) Are PRX located in specific subcellular locations and 2) does shear stress alter this subcel- lular localization? To clearly characterize the location of each PRX within specific subcellular compartments, colocalization staining was performed in static BAEC using PRX-specific anti- FIGURE 1. PRX 1 is up-regulated by LS. Confluent BAEC were exposed to LS, OS, or static conditions for 1 day, and cell lysates were obtained. A, equal bodies and organelle-specific markers for Golgi (GM 130), aliquots of protein (20 – 40 g) were analyzed by Western blot using antibod- endoplasmic reticulum (KDEL receptor, data not shown), lyso- ies specific to PRX 1– 6 phospho-endothelial nitric-oxide synthase (peNOS), some (cathepsin S, data not shown), intermediate filament total endothelial nitric-oxide synthase (teNOS), and-actin blots were used as shear stress controls and internal loading controls, respectively. B, densito- (vimentin), and mitochondria (Mitotracker). PRX 1 staining metric analysis was used to quantify the intensity of each band, and the aver- (green) overlapped with the Golgi marker staining (red), shown age values (mean S.E., n 12) are shown in bar graphs as % of static control. *, p  0.05 indicates significance compared with static control. **, p  0.05 as yellow in the merged image (Fig. 3A), suggesting that PRX 1 indicates significance compared with OS. exists in the Golgi apparatus. In addition, PRX 2, 4, 5, and 6 also appeared to be found in the Golgi (Fig. 3, B and E–G). The PRX 3 staining pattern was distinctly dif- ferent from other PRX, showing clear colocalization with the mito- chondria marker (Fig. 3D). Interest- ingly, PRX 2 staining revealed colo- calization with the intermediate filament marker (Fig. 3C). In addi- tion to these subcellular localiza- tions, PRX 1, 2, 4, 5 and 6 were also expressed in the cytosol (Fig. 3, A–C and E–G). Next, we examined whether shear stress stimulated expression of PRX members in other subcellular locations. Sub- cellular location did not appear to change in response to shear stress, although Golgi were located FIGURE 2. LS stimulated PRX 1 expression in BAEC. Confluent BAEC were exposed to LS, OS, or static condi- upstream of the direction of flow tions for 1 day as in Fig. 1. Cells were stained using antibodies specific to PRX 1– 6. Secondary antibodies after chronic LS, consistent with conjugated to Alexa Fluor 488 (green) were imaged by confocal microscopy. Nuclei were counter-stained with Hoechst dye (blue). Arrows indicate unique subcellular staining patterns of each PRX. previous reports (Fig. 2, A, B, and 1624 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 3 •JANUARY 18, 2008 Peroxiredoxins as Mechanosensitive Antioxidants Here, specific siRNAs were used to knock down either PRX 1 or PRX 5 protein levels in order to investigate PRX-dependent ROS accumula- tion. Western blots with isoform- specific PRX antibodies were used to determine the efficacy and speci- ficity of these siRNAs. Compared with non-silencing siRNA and Alexa Fluor 546-labeled non-silenc- ing siRNA (50 nM each), 48 h of treatment of BAEC with PRX 1 siRNA dramatically reduced (by 75% of non-silencing control) expression of PRX 1 at a concentra- tion as low as 10 nM (Fig. 4A). As shown in Fig. 4B,10nM PRX 5 siRNA also effectively reduced PRX 5 expression (by 90% of non-silenc- ing control). Via Western blots, the specificity of PRX 1 and 5 siRNAs was assessed by examining the expression of all other PRX family FIGURE 3. PRX are located in various subcellular organelles in BAEC. Static BAEC were co-stained for PRX members (Fig. 4, A and B). Using 1– 6 and subcellular organelles using PRX-specific antibodies (green), as in Fig. 2, and organelle-specific mark- isoform-specific PRX antibodies, we ers (red). Nuclei were counterstained with Hoechst dye (blue). Center panels: Mitochondria, Golgi, and inter- found that these siRNAs had no sig- mediate filaments are stained with Mitotracker CMXRos, GM 130 antibody, and vimentin antibody, respec- tively. GM 130 and vimentin were visualized by secondary antibodies conjugated to Alexa Fluor 568 (red). nificant effect on other PRX, indi- Merged images are shown in the right panel. Yellow staining indicates colocalization. cating that they exclusively targeted PRX 1 or 5, respectively, among all PRX family members (Fig. 4, A and B). Therefore, PRX 1 and 5 siRNAs were confidently used at 10 nM concentration in subse- quent functional studies. It has been well established that chronic exposure of endo- thelial cells to OS stimulates, while LS inhibits, ROS production (10, 11, 23, 24). Utilizing an Amplex Red assay in the presence or absence of catalase, we verified that hydrogen peroxide levels were 87% less in BAEC exposed to LS compared with those treated with OS (Fig. 5A). Using a standard hydrogen peroxide dose curve, we also found that the relative hydrogen peroxide levels following static culture, LS, and OS were consistent with expected cellular concentrations (Fig. 5B). To determine whether PRX 1 was responsible for the LS-dependent decrease in ROS levels in BAEC, we knocked down PRX 1 using PRX 1 FIGURE 4. PRX 1 and PRX 5 siRNAs specifically reduce PRX 1 and PRX 5 protein expression, respectively. BAEC were transfected with either 50 nM siRNA as indicated above (Fig. 4A). PRX 1 depletion signifi- non-silencing siRNA (NS), 50 nM Alexa Fluor 546-labeled siRNA (Alexa), or PRX cantly increased catalase-inhibitable hydrogen peroxide by 1(A)orPRX5(B) siRNAs (10, 20, 50, and 100 nM) for 48 h. Cell lysates were analyzed by Western blot with PRX-specific antibodies as indicated. -actin 2-fold above non-silencing controls in static-, LS-, and was used as an internal control. OS-treated BAEC (Fig. 5C). To investigate whether this was a global effect of mechanosensitive PRX family members, we also D–F). Taken together, these results clearly indicate that PRX knocked down PRX 5 using PRX 5-specific siRNA. However, are abundantly expressed throughout the subcellular when compared with non-silencing controls, PRX 5 depletion organelles of endothelial cells. had no significant effect on hydrogen peroxide levels in any PRX 1 Prevents Oxidative Stress in Endothelial Cells Exposed group. Taken together, these data suggest that PRX 1 is a criti- to LS—PRX 1 is a prominent antioxidant, and our data indicate cally important regulator of ROS levels in both a basal and shear- its expression is highly up-regulated by LS (22). Consequently, dependent manner. we investigated whether PRX 1 was responsible for the DISCUSSION decreased ROS levels in endothelial cells exposed to LS. The depletion of individual PRX from cellular systems provides a Through protein expression analysis and subsequent func- useful tool to study the functional role of each PRX isoform. tional studies, we have discovered PRX 1 as a mechanosensitive JANUARY 18, 2008• VOLUME 283 • NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1625 Peroxiredoxins as Mechanosensitive Antioxidants throughout the cellular milieu of BAEC, colocalizing with the cyto- plasm, Golgi apparatus, mitochon- dria, and intermediate filaments. These observations were consistent with previously reported localiza- tion studies in other cell types, but we are the first to report an apparent PRX 2 colocalization with vimentin (32, 33). This costaining of PRX 2 with vimentin suggests that it may be located in the intermediate fila- ment, but further studies will be necessary to confirm this finding. Although detection of PRX in the Golgi body likely reflects protein processing or packaging, localiza- tion within other organelles indi- cates that PRX may act both globally and in a site-specific manner to reg- ulate ROS in endothelial cells. In the endothelium, ROS, such as O and H O arise from several sources,, 2 2, including NADPH oxidase, xan- FIGURE 5. PRX 1 knock down increases H O production in BAEC, whereas PRX 5 knock down does not. 2 2 thine oxidase, mitochondrial oxi- Catalase-inhibitable hydrogen peroxide levels were assessed via Amplex Red assay, and average values (mean dase, cytochrome P450, and uncou- S.E., n  6–12) are shown in bar graphs as % of non-silencing (NS) siRNA-treated static controls. A, confluent BAEC pled nitric-oxide synthase (34, 35). were exposed to ST, LS, or OS for 24 h prior to assay. B, a hydrogen peroxide dose curve was used to estimate relative hydrogen peroxide concentrations in cells conditioned with shear stress. C, BAEC were transfected with either At relatively low concentrations, non-silencing or PRX 1 siRNA (10 nM) for 48 h and then exposed to ST, LS, or OS for 24 h prior to assay. D, BAEC were ROS play critical roles in redox sig- transfected with either non-silencing or PRX 5 siRNA (10 nM) for 48 h and then exposed to ST, LS, or OS for 24 h prior to assay. *, p 0.05 designates significance between indicated groups. naling and normal cell function. However, higher concentrations of antioxidant. Data to support this concept include: 1) PRX 1 is ROS induce oxidative damage of DNA, proteins, carbohy- up-regulated intracellularly by chronic LS compared with OS, drates, and lipids (36–38). This damage has been shown to 2) PRX exhibit broad staining patterns in BAEC and localize in critically affect cellular function and apoptosis when it occurs in important cellular structures, 3) ROS production is signifi- mitochondria, lysosomes, and nuclei (39–41). In addition, cantly reduced in cells exposed to chronic LS, and 4) this effect cytosolic proteins modified by ROS have been shown to affect can be attenuated by PRX 1 depletion, but not PRX 5 depletion. local cell signaling and, collectively, the redox status of the cell Through this work, we reveal for the first time that PRX are (11, 42, 43). Ubiquitous distribution of PRX in BAEC may regulated by shear stress in endothelial cells. Previous studies reflect diverse sources of ROS throughout the cells and provide have shown that other antioxidants are also controlled by shear protection for important macromolecules and structures stress. LS has been shown to up-regulate antioxidant genes, against local ROS production. In addition, widespread alloca- including endothelial nitric-oxide synthase, CuZn superoxide tion of PRX may be important for comprehensive management dismutase, manganese superoxide dismutase, glutathione per- of the overall oxidative state of cells. oxidase, glutathione, and thioredoxin (25–29). In addition, Several studies have shown that oxidative stress is regulated Chen et al. (30) observed that many genes protective against by shear stress in endothelial cells (10–12, 24, 26). We have oxidative stress are induced by exposure to prolonged LS. They previously published that both LS and OS stimulate ROS pro- have also noted that such genes are regulated through a con- duction acutely but the ROS transiently elevated by LS returns served, shear-sensitive antioxidant response element. In sup- to basal levels within a few hours (10). However, unlike LS, OS port of our finding that PRX 1 expression is LS-dependent, continues to increase ROS production, maintaining elevated recent work has shown that PRX 1 is a target gene of nuclear levels as long as cells are exposed to OS (10, 24, 26). The mech- factor (erythroid-derived 2)-related factor 2 (Nrf2), a key tran- anism by which ROS levels are lowered in cells exposed to scription factor that binds to antioxidant response element chronic LS is undefined. This study demonstrates that endothe- (31). Collectively, these studies indicate that cells possess an lial cells exposed to chronic LS express much more PRX 1 com- elaborate system of shear-responsive antioxidants and that pared with OS and static conditions. These findings suggest each may play an independent role to mediate oxidative stress that PRX 1 is up-regulated by LS and that this may be respon- and modulate redox-sensitive signaling pathways. sible for LS-mediated decrease in ROS levels. The ubiquitous nature of the PRX family itself exemplifies As previously determined by electron spin resonance spec- this concept. Immunofluorescence microscopy revealed PRX trometry and dichlorofluorescein-diacetate methods, endothe- 1626 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 3 •JANUARY 18, 2008 Peroxiredoxins as Mechanosensitive Antioxidants (2003) J. Biol. Chem. 278, 47291–47298 lial cells exposed to OS produce significantly more superoxide 11. Sorescu, G. P., Song, H., Tressel, S. L., Hwang, J., Dikalov, S., Smith, D. A., and hydrogen peroxide than those in static culture (10–12). In Boyd, N. L., Platt, M. O., Lassegue, B., Griendling, K. K., and Jo, H. (2004) contrast, endothelial cells treated with chronic, unidirectional Circ. Res. 95, 773–779 high shear generate considerably less O . Here, we used 12. McNally, J. S., Davis, M. E., Giddens, D. P., Saha, A., Hwang, J., Dikalov, S., Amplex Red assay as an independent method to measure ROS Jo, H., and Harrison, D. G. (2003) Am. J. Physiol. 285, H2290–H2297 levels in BAEC. Consistent with our previous reports, OS 13. Mates, J. M., Perez-Gomez, C., and De Castro, I. N. (1999) Clin. Biochem. increased and LS decreased ROS production (Fig. 5A). The ROS 32, 595–603 14. Rhee, S. G., Chae, H. Z., and Kim, K. (2005) Free Radic. Biol. Med. 38, measured by this assay was inhibitable by catalase, indicating that 1543–1552 H O is the primary ROS component. 2 2 15. Wood, Z. A., Schroder, E., Robin Harris, J., and Poole, L. B. (2003) Trends PRX 1 is the most abundant and ubiquitously distributed Biochem. Sci. 28, 32–40 member of mammalian PRX (22). Our current study demon- 16. Kinnula, V. L., Lehtonen, S., Sormunen, R., Kaarteenaho-Wiik, R., Kang, strated that PRX 1 is dramatically up-regulated by chronic LS S. W., Rhee, S. G., and Soini, Y. (2002) J. Pathol. 196, 316–323 compared with OS and is located in the cytoplasm and Golgi. 17. Kinnula, V. L., Lehtonen, S., Kaarteenaho-Wiik, R., Lakari, E., Paakko, P., Kang, S. W., Rhee, S. G., and Soini, Y. (2002) Thorax 57, 157–164 Knock down of PRX 1 using siRNA resulted in significantly 18. Krapfenbauer, K., Engidawork, E., Cairns, N., Fountoulakis, M., and Lu- higher ROS levels in BAEC exposed to LS, OS, and static con- bec, G. (2003) Brain Res. 967, 152–160 ditions, whereas PRX 5 depletion did not. Although PRX 1 19. Choi, M. H., Lee, I. K., Kim, G. W., Kim, B. U., Han, Y. H., Yu, D. Y., Park, knock down did not fully abolish the antioxidative outcome of H. S., Kim, K. Y., Lee, J. S., Choi, C., Bae, Y. S., Lee, B. I., Rhee, S. G., and LS, its significant effect was somewhat surprising considering Kang, S. W. (2005) Nature 435, 347–353 the presence of other PRX family members and additional 20. Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. 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