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Role of paxillin in the early phase of orientation of the vascular endothelial cells exposed to cyclic stretching
Biochemical and Biophysical Research Communications 418 (2012) 708–713
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Role of paxillin in the early phase of orientation of the vascular endothelial cells exposed to cyclic stretching Wenjing Huang a,⇑, Naoya Sakamoto b, Ryotaro Miyazawa c, Masaaki Sato a a
Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Sendai, Japan c Department of Mechanical and Aerospace Engineering, School of Engineering, Tohoku University, Sendai, Japan b
a r t i c l e
i n f o
Article history: Received 13 January 2012 Available online 24 January 2012 Keywords: Paxillin Endothelial cells Orientation Cyclic stretching
a b s t r a c t Paxillin, a structural and signaling scaffold molecule in focal adhesions (FAs), is considered to be important in intracellular signaling transduction and the cell shape changes in response to cyclic stretching. However, the detailed role of paxillin in stretch-induced morphological changes of endothelial cells (ECs) has not fully determined until date. In this study, in order to understand the role of paxillin in the orientation of ECs exposed to cyclic stretching, we examined the time course of changes in the shape and distribution of FA proteins of paxillin knockdown ECs. Non-treated ECs subjected to 20% cyclic stretching at 0.5 Hz oriented perpendicularly to the direction of stretching after 10 min of exposure. On the other hand, the orientation of paxillin knockdown ECs was abolished at 10 min, but it was observed after 60 min of cyclic stretching exposure. Immunofluorescent microscopy revealed that accumulation and redistribution of FA proteins, including focal adhesion kinase (FAK) and integrin b1, were observed at 10 min of exposure to cyclic stretching in non-treated ECs. The accumulation of FAK and integrin b1 was not prominent in paxillin knockdown ECs under static conditions and after 10 min of exposure to cyclic stretching. However, we found that accumulation of FA proteins in paxillin knockdown ECs at 30 and 60 min was similar to that in non-transfected ECs. Because paxillin is an adaptor protein offering binding sites for FAK and integrin b1, which are critical molecules for the early signaling events of focal adhesion formation in ECs, these results suggest that paxillin is required for the early phase of EC orientation in response to cyclic stretching by scaffolding for accumulation of FA proteins. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Endothelial cells (ECs) are located on the luminal surface of blood vessel walls and are exposed to hemodynamic forces including shear stresses by blood flow and cyclic stretching due to vessel expansion. ECs adapt to such mechanical environments by altering their morphology. For instance, after exposure to cyclic stretching, ECs orient nearly perpendicular to the direction of stretching [1–3]. In addition, because cyclic stretching induces redistribution of focal adhesions (FAs), which connect the cells to the extracellular matrix and consist of integrins and many FA-associated proteins such as paxillin, focal adhesion kinase (FAK), and vinculin [4], the redistribution of FAs has been implicated in the morphological changes of ECs [5–7]. However, the detailed mechanism of FA redistribution and its role in the orientation of ECs exposed to cyclic stretching is still unclear. ⇑ Corresponding author. Address: Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6-01, Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan. Fax: +81 22 795 6945. E-mail address: [email protected] (W. Huang). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.083
Paxillin is an adaptor protein [8] that acts as a scaffold for other FA proteins, particularly for FAK and vinculin [9]. Schaller et al. reported that paxillin associates directly with the integrin b1 [10], which is necessary for the morphological responses of ECs exposed to cyclic stretching [6,11]. FAK has emerged as a key signaling component at FAs [12], and it is required for the morphological responses of ECs exposed to cyclic stretching [1]. Vinculin is regarded as a protein involved in the linkage of integrin adhesion molecules to the actin cytoskeleton through paxillin, which plays a central role in force transduction and mechanical stabilization of FAs [13]. Paxillin is the first component to appear visibly organized in protrusive regions of the cell [14], and cyclic stretching led to increased incorporation of FA proteins such as vinculin and paxillin in the cell membrane from the soluble cytoplasmic pool after only 1 min of stretching [7]. Moreover, our group has found that paxillin in ECs increased in the regions perpendicular to the direction of stretching after 2 min of exposure to cyclic stretching [15]. These findings suggest that paxillin contributes to the early phase of cell orientation in response to cyclic stretching. However, the detailed role of paxillin in the coordination of FAs and orientation of ECs exposed to cyclic stretching is still unclear.
W. Huang et al. / Biochemical and Biophysical Research Communications 418 (2012) 708–713
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Control Non-targeting Knockdown Paxillin (68 kD)
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(B) Fig. 1. HUVEC transfection with siRNA targeting paxillin. (A) Representative result of western blotting analysis of paxillin expression in HUVECs. (B) Confocal fluorescent images of HUVECs immunostained for paxillin. Green, paxillin; Blue, nucleus. Bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
In the present study, we examined the time course of changes in the orientation of paxillin knockdown ECs. In addition, we investigated the changes in accumulation and redistribution of FA proteins in knockdown ECs to reveal the detailed role of paxillin in the orientation of ECs exposed to cyclic stretching. 2. Materials and methods 2.1. Cell culture ECs were isolated from human umbilical veins with trypsin treatment, as described in a previous study [16]; they were grown in medium 199 (Invitrogen, USA) containing 20% heat-inactivated fetal bovine serum (JRH Biosciences, USA), 10 ng/ml basic fibroblast growth factor (Austral Biologicals, USA), and 1% penicillin–streptomycin (Invitrogen). Study protocols were officially approved by the Ethics Committee for Clinical Research with Human Subjects of Tohoku University. ECs were maintained at 37 °C in a humidified atmosphere and grown to confluence. Cells at passages 4–7 were used in experiments. 2.2. siRNA-based knockdown of paxillin in ECs ECs were transfected with gene-specific siRNA duplexes (as previously described) to deplete the paxillin protein [17]. The sequences of pre-designed paxillin-specific siRNA used were as follows: 50 -CCCUGACGAAAGAGAAGCCUAUU-30 and 50 -UAGGCUUCUCUUUCGUCAGGGUU-30 . Non-specific, non-targeting siRNA from Ambion (USA) was used to ensure that the decrease in paxillin expression levels was related to a specific RNAi event. Cells not treated with siRNA were used as the control. siRNA transfection (final concentration, 50 nM) was performed using the invitrogen transfection reagent (Invitrogen) according to the manufacturer’s
protocol. After 48 h, the cells were harvested and used for experiments. The reduction level of the targeting protein paxillin was assessed by western blotting and immunofluorescence analysis. 2.3. Western blotting Cells were washed with cold PBS three times. Whole cell lysates were prepared in 50 mM Tris–HCl, pH 7.3, 150 mM NaCl, 1% Triton X-100, 1% NP-40, 6.5 IU/ll aprotinin, 1 mM EDTA, 100 lM phenylmethylsulfonyl fluoride, 5 lg/ml leupeptin, and 1 lg/ml pepstatin. Next, 10 lg of proteins from each cell lysate was subjected to SDS– polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride membranes (GE Healthcare, UK), and probed with a primary antibody against paxillin (BD Transduction, USA). An alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, USA) was detected by an Amplified Alkaline Phosphatase Immun-Blot kit (Bio-Rad, USA). Equal loading was assessed by protein staining with a GAPDH antibody (Ambion). 2.4. Cyclic stretching ECs were seeded at a concentration of 20 104 cells/ml as confluent cells on fibronectin-coated membranes in a stretching chamber (Strex, Japan); they were maintained at 37 °C in a humidified atmosphere for 24 h. ECs were then subjected to 20% stretching at 0.5 Hz using a stretching apparatus (Strex) [1,15]. During all experiments, the stretching apparatus was maintained at 37 °C in an incubator. 2.5. Immunofluorescence staining and image analysis For immunofluorescent staining of integrin b1 and vinculin, ECs grown on fibronectin-coated membrane were fixed after treatment
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(B) Fig. 2. (A) Typical fluorescent images of actin filaments and nuclei in non-transfected ECs and knockdown ECs under static conditions and 10, 30, and 60 min after exposure to 20% cyclic stretching at 0.5 Hz. Red, actin filament; Blue, nucleus. Bar = 50 lm. (B) Histograms of the orientation angle of non-transfected and paxillin knockdown HUVECs under static conditions and exposed to 20% cyclic stretching at 0.5 Hz for 10, 30, and 60 min. Numbers in parentheses indicate the number of cells studied. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
in 4% paraformaldehyde solution in PBS for 10 min at room temperature (RT), washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and blocked with Block Ace (Snow Brand Milk Products, Japan) for 60 min. Incubation with an antibody of integrin b1 or vinculin (Sigma–Aldrich, USA) was performed in the blocking solution for 60 min at RT, followed by staining with an Alexa 488-conjugated secondary antibody (Invitrogen). Actin filaments were stained with Alexa fluor 546-conjugated phalloidin (Invitrogen) for 20 min; the nuclei were then stained with 40 ,6diamidino-2-phenylindole (Invitrogen) for 2 min at RT. For the immunostaining of FAK, ECs were fixed with methanol (Wako Pure Chemical Industries, Japan) for 10 min at 20 °C and blocked with
Block Ace (Snow Brand Milk Products) for 60 min. After staining with an FAK antibody (BD Transduction, USA) and the Alexa 488conjugated secondary antibody (Invitrogen), the nuclei of ECs were stained. After immunostaining, stained cells were observed under a confocal laser scanning microscope (Olympus, Japan). From the actin staining images, the orientation angle of cells was measured using the ImageJ software (NIH, USA). We determined the orientation angle of ECs, which is defined as the angle between the direction of the long axis of an ellipse equivalent to cell shape and the stretching direction. ECs aligned at angles between 60° and 90° were defined as oriented cells.
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W. Huang et al. / Biochemical and Biophysical Research Communications 418 (2012) 708–713
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Stretching (20%, 0.5 Hz) Fig. 3. Fluorescent images of integrin b1 in HUVECs under static conditions (0 min) and exposed to 20% cyclic stretching at 0.5 Hz for 10, 30, and 60 min. Green, Integrin b1; Blue, nucleus. Bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3. Results A typical result of western blot analysis of paxillin expression in ECs is shown in Fig. 1A. Knockdown ECs showed a significant reduction of approximately 95% in the paxillin protein level compared to non-transfected ECs and ECs transfected with non-targeting siRNA. There was no difference between non-transfected ECs and ECs transfected with non-targeting siRNA. Confocal fluorescent images of ECs immunostained for paxillin are shown in Fig. 1B. A prominent reduction in paxillin was observed in ECs transfected with paxillintargeting siRNA. This result confirmed the specificity of paxillin knockdown by siRNA transfection. Fluorescent images of actin filaments and nuclei in ECs exposed to cyclic stretching are shown in Fig. 2A. Both control and knockdown ECs showed random orientation at 0 min. ECs without paxillin siRNA treatment aligned perpendicularly to the stretching direction after stretching for 10 min and became more perpendicular to the direction of stretching at 30 and 60 min. On the other hand, orientation of ECs treated with paxillin siRNA was not observed at 10 min. After 30 min of exposure to cyclic stretching, paxillin knockdown ECs started orienting perpendicularly to the stretching direction and orientation similar to controls was observed at 60 min. After cyclic stretching, stress fibers reoriented perpendicularly to the direction of stretching from a random alignment under static conditions. In contrast, the actin filaments in knockdown ECs were significantly thinner at 0 and 10 min compared with those in controls, whereas the orientation of actin filaments was still observed after stretching for 30 and 60 min. The relative frequency of the orientation angle of ECs exposed to cyclic stretching is shown in Fig. 2B. Both control and paxillin knockdown ECs showed random orientation under static conditions. After 10 min of stretching, approximately 60% of control ECs oriented to angles ranging from 60° to 90°. However, prominent reorientation of paxillin knockdown ECs was not observed. The reorientation tendency of ECs transfected with paxillin siRNA was observed at 30 min. Approximately 70% of the knockdown
ECs showed orientation angles from 60° to 90° after 60 min of cyclic stretching exposure, similar to control cells. Immunofluorescent images for integrin b1 are shown in Fig. 3. Integrin b1 initially demonstrated a random punctuate distribution in control ECs. After 10 min of exposure to cyclic stretching, integrin b1 formed large FAs, and an increased number of FAs oriented perpendicularly to the direction of cyclic stretching were observed, similar to the alignment of actin filaments. After ECs were exposed to 30 and 60 min of stretching, integrin b1 reorganized in a linear pattern, and most FAs oriented almost perpendicularly to the direction of stretching. With paxillin knockdown treatment, we found that integrin b1 in ECs was arranged in a diffuse manner and still demonstrated limited accumulation and random orientation at 10 min in knockdown ECs. We found that after 30 and 60 min of exposure to cyclic stretching, prominent accumulation of integrin b1 was observed. Furthermore, adhesions of integrin b1 orienting perpendicularly to the direction of stretching were observed, similar to controls. Fig. 4A shows FAK staining results. FAK was located primarily in the periphery of control ECs under static conditions. After 10 min of exposure to cyclic stretching, FAK in the control cells accumulated and oriented almost perpendicularly to the direction of stretching. At 30 and 60 min, FAK became more concentrated, and FAK fibrils that aligned almost perpendicularly to the direction of stretching appeared. In contrast, only weak FAK accumulation with undefined orientation was observed in the ECs transfected with paxillin siRNA compared with control ECs under static conditions and at 10 min. However, FAK accumulation was observed at 10 min in the ECs transfected with paxillin siRNA and showed orientation perpendicular to the direction of cyclic stretching at 30 and 60 min. Fig. 4B shows vinculin staining results. After 10, 30, and 60 min of exposure to cyclic stretching, the vinculin in control ECs was observed to reorganize into a linear shape and orient perpendicularly to the direction of stretching. Unlike integrin b1 and FAK, paxillin knockdown appeared not to influence the accumulation of vinculin in ECs.
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Fig. 4. (A) Fluorescent images of FAK in HUVECs under static conditions and exposed to 20% cyclic stretching at 0.5 Hz for 10, 30, and 60 min. Green, FAK; Blue, nucleus. Bar = 50 lm. (B) Fluorescent images of vinculin with nucleus in HUVECs under static conditions and exposed to 20% cyclic stretching at 0.5 Hz for 10, 30, and 60 min. Green: vinculin, Blue: nucleus. Bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion ECs exposed to cyclic stretching exhibit rapid changes in the orientation of actin filaments and orient perpendicular to the direction of stretching after 10 min of exposure [15,18]. In addition, FA reorganization is known to be crucial for the morphological responses of ECs exposed to cyclic stretching [6,19–21]. Our group has previously shown that after 2 min of exposure to cyclic stretching, paxillin levels in ECs were significantly higher in the regions perpendicular to the direction of stretching [15]. In the present study, we investigated the roles of paxillin in the orientation changes of ECs exposed to cyclic stretching for 10, 30, and
60 min; the orientation of ECs transfected with paxillin siRNA was not observed at 10 min, but the orientation was observed after 60 min of exposure to cyclic stretching. These results showed for the first time that paxillin is required for the early phase of orientation of ECs exposed to cyclic stretching. Yano et al. [6] reported that cyclic stretching-induced FAK phosphorylation occurred concomitant with the redistribution of integrin b1. Because integrin b1 itself cannot be phosphorylated, recruitment of tyrosine kinases such as FAK was thought to initiate biochemical signaling. Moreover, Naruse et al. reported that cyclic stretching induced phosphorylation and redistribution of paxillin [1]. As a scaffold protein, paxillin has binding sites for integrin b1 and FAK [8].
W. Huang et al. / Biochemical and Biophysical Research Communications 418 (2012) 708–713
In addition, paxillin can target to FAs without interactions with FAK [9] and is regarded as a switch to regulate the assembly and form of cell-matrix adhesions [22]. In this study, we found that knockdown of paxillin resulted in the weak accumulation of integrin b1 and FAK in ECs after 10 min of exposure to cyclic stretching. These results suggest that paxillin is a key protein for the signaling pathway between integrin b1 and FAK in ECs in the early phase of cell orientation. On the other hand, we found that knockdown of paxillin did not change the accumulation and redistribution of vinculin. Talin, another FA protein, is first recruited to the membrane; vinculin is then recruited to adhesion plaques by binding to talin [23]. In addition, vinculin is not associated with FAK [24] and integrin b1 [10]. Thus, an alternative possibility for the localization of vinculin to focal adhesions is that it is recruited by binding to talin. In this study, we found that the ECs transfected with paxillin siRNA oriented perpendicularly to the direction of stretching after 60 min of exposure, while the orientation of ECs was abolished at 10 min. Accumulation of FA proteins, integrin b1, and FAK similar to that of control cells was also observed after 30 and 60 min of exposure to cyclic stretching even in paxillin knockdown ECs. These results suggest that there are compensatory mechanisms by which the accumulation and redistribution of FA proteins occurs. Furthermore, the recruitment of FA proteins is a considerably complex process that may be regulated by FA proteins other than paxillin. For example, Src tyrosine kinase is an important molecule for stretching-induced orientation response in cells [25] and leads to the phosphorylation of p130Cas, another scaffold protein in FAs [12]. The p130Cas phosphorylation level was low in unstretched cells, and stretching, which can lead to the extension of p130Cas, increases its tyrosine phosphorylation [26]. p130Cas can bind to several different proteins such as FAK [27], and p130Cas phosphorylation is crucial for its association with other signaling molecules [28]. Therefore, we suppose that such stretching-dependent signaling is involved in the accumulation of FAs and cell orientation after 30 min of exposure to cyclic stretching, but the detailed mechanisms should be investigated in further studies. In conclusion, in the present study, we investigated the time course of EC orientation when subjected to cyclic stretching. Control ECs oriented after 10 min of exposure to cyclic stretching. In contrast, the orientation of ECs transfected with paxillin siRNA was abolished at 10 min, but the orientation of transfected ECs was observed after 60 min of exposure to cyclic stretching. Furthermore, we found that the knockdown of paxillin led to delayed accumulation of integrin b1 and FAK, distribution of which is crucial for the morphological changes of ECs exposed to cyclic stretching. These findings suggest that paxillin is involved in the early phase of EC orientation by playing a key role in the redistribution of integrin b1 and FAK. Acknowledgments The authors thank Dr. Makoto Takahashi and Dr. Ikuo Takahashi for providing human umbilical cords; in addition, the authors thank Ms. Eri Inoue and Mr. Shinya Chubachi for kind help in some experiments. This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) in Japan (No. 20001007). References [1] K. Naruse, T. Yamada, X.R. Sai, M. Hamaguchi, M. Sokabe, Pp125FAK is required for stretch dependent morphological response of endothelial cells, Oncogene 17 (1998) 455–463. [2] Y. Katanosaka, J.H. Bao, T. Komatsu, T. Suemori, A. Yamada, S. Mohri, K. Naruse, Analysis of cyclic-stretching responses using cell-adhesion-patterned cells, J. Biotechnol. 133 (2008) 82–89.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Role of paxillin in the early phase of orientation of the vascular endothelial cells exposed to cyclic stretching Wenjing Huang a,⇑, Naoya Sakamoto b, Ryotaro Miyazawa c, Masaaki Sato a a
Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Sendai, Japan c Department of Mechanical and Aerospace Engineering, School of Engineering, Tohoku University, Sendai, Japan b
a r t i c l e
i n f o
Article history: Received 13 January 2012 Available online 24 January 2012 Keywords: Paxillin Endothelial cells Orientation Cyclic stretching
a b s t r a c t Paxillin, a structural and signaling scaffold molecule in focal adhesions (FAs), is considered to be important in intracellular signaling transduction and the cell shape changes in response to cyclic stretching. However, the detailed role of paxillin in stretch-induced morphological changes of endothelial cells (ECs) has not fully determined until date. In this study, in order to understand the role of paxillin in the orientation of ECs exposed to cyclic stretching, we examined the time course of changes in the shape and distribution of FA proteins of paxillin knockdown ECs. Non-treated ECs subjected to 20% cyclic stretching at 0.5 Hz oriented perpendicularly to the direction of stretching after 10 min of exposure. On the other hand, the orientation of paxillin knockdown ECs was abolished at 10 min, but it was observed after 60 min of cyclic stretching exposure. Immunofluorescent microscopy revealed that accumulation and redistribution of FA proteins, including focal adhesion kinase (FAK) and integrin b1, were observed at 10 min of exposure to cyclic stretching in non-treated ECs. The accumulation of FAK and integrin b1 was not prominent in paxillin knockdown ECs under static conditions and after 10 min of exposure to cyclic stretching. However, we found that accumulation of FA proteins in paxillin knockdown ECs at 30 and 60 min was similar to that in non-transfected ECs. Because paxillin is an adaptor protein offering binding sites for FAK and integrin b1, which are critical molecules for the early signaling events of focal adhesion formation in ECs, these results suggest that paxillin is required for the early phase of EC orientation in response to cyclic stretching by scaffolding for accumulation of FA proteins. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Endothelial cells (ECs) are located on the luminal surface of blood vessel walls and are exposed to hemodynamic forces including shear stresses by blood flow and cyclic stretching due to vessel expansion. ECs adapt to such mechanical environments by altering their morphology. For instance, after exposure to cyclic stretching, ECs orient nearly perpendicular to the direction of stretching [1–3]. In addition, because cyclic stretching induces redistribution of focal adhesions (FAs), which connect the cells to the extracellular matrix and consist of integrins and many FA-associated proteins such as paxillin, focal adhesion kinase (FAK), and vinculin [4], the redistribution of FAs has been implicated in the morphological changes of ECs [5–7]. However, the detailed mechanism of FA redistribution and its role in the orientation of ECs exposed to cyclic stretching is still unclear. ⇑ Corresponding author. Address: Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6-01, Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan. Fax: +81 22 795 6945. E-mail address: [email protected] (W. Huang). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.083
Paxillin is an adaptor protein [8] that acts as a scaffold for other FA proteins, particularly for FAK and vinculin [9]. Schaller et al. reported that paxillin associates directly with the integrin b1 [10], which is necessary for the morphological responses of ECs exposed to cyclic stretching [6,11]. FAK has emerged as a key signaling component at FAs [12], and it is required for the morphological responses of ECs exposed to cyclic stretching [1]. Vinculin is regarded as a protein involved in the linkage of integrin adhesion molecules to the actin cytoskeleton through paxillin, which plays a central role in force transduction and mechanical stabilization of FAs [13]. Paxillin is the first component to appear visibly organized in protrusive regions of the cell [14], and cyclic stretching led to increased incorporation of FA proteins such as vinculin and paxillin in the cell membrane from the soluble cytoplasmic pool after only 1 min of stretching [7]. Moreover, our group has found that paxillin in ECs increased in the regions perpendicular to the direction of stretching after 2 min of exposure to cyclic stretching [15]. These findings suggest that paxillin contributes to the early phase of cell orientation in response to cyclic stretching. However, the detailed role of paxillin in the coordination of FAs and orientation of ECs exposed to cyclic stretching is still unclear.
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(B) Fig. 1. HUVEC transfection with siRNA targeting paxillin. (A) Representative result of western blotting analysis of paxillin expression in HUVECs. (B) Confocal fluorescent images of HUVECs immunostained for paxillin. Green, paxillin; Blue, nucleus. Bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
In the present study, we examined the time course of changes in the orientation of paxillin knockdown ECs. In addition, we investigated the changes in accumulation and redistribution of FA proteins in knockdown ECs to reveal the detailed role of paxillin in the orientation of ECs exposed to cyclic stretching. 2. Materials and methods 2.1. Cell culture ECs were isolated from human umbilical veins with trypsin treatment, as described in a previous study [16]; they were grown in medium 199 (Invitrogen, USA) containing 20% heat-inactivated fetal bovine serum (JRH Biosciences, USA), 10 ng/ml basic fibroblast growth factor (Austral Biologicals, USA), and 1% penicillin–streptomycin (Invitrogen). Study protocols were officially approved by the Ethics Committee for Clinical Research with Human Subjects of Tohoku University. ECs were maintained at 37 °C in a humidified atmosphere and grown to confluence. Cells at passages 4–7 were used in experiments. 2.2. siRNA-based knockdown of paxillin in ECs ECs were transfected with gene-specific siRNA duplexes (as previously described) to deplete the paxillin protein [17]. The sequences of pre-designed paxillin-specific siRNA used were as follows: 50 -CCCUGACGAAAGAGAAGCCUAUU-30 and 50 -UAGGCUUCUCUUUCGUCAGGGUU-30 . Non-specific, non-targeting siRNA from Ambion (USA) was used to ensure that the decrease in paxillin expression levels was related to a specific RNAi event. Cells not treated with siRNA were used as the control. siRNA transfection (final concentration, 50 nM) was performed using the invitrogen transfection reagent (Invitrogen) according to the manufacturer’s
protocol. After 48 h, the cells were harvested and used for experiments. The reduction level of the targeting protein paxillin was assessed by western blotting and immunofluorescence analysis. 2.3. Western blotting Cells were washed with cold PBS three times. Whole cell lysates were prepared in 50 mM Tris–HCl, pH 7.3, 150 mM NaCl, 1% Triton X-100, 1% NP-40, 6.5 IU/ll aprotinin, 1 mM EDTA, 100 lM phenylmethylsulfonyl fluoride, 5 lg/ml leupeptin, and 1 lg/ml pepstatin. Next, 10 lg of proteins from each cell lysate was subjected to SDS– polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride membranes (GE Healthcare, UK), and probed with a primary antibody against paxillin (BD Transduction, USA). An alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, USA) was detected by an Amplified Alkaline Phosphatase Immun-Blot kit (Bio-Rad, USA). Equal loading was assessed by protein staining with a GAPDH antibody (Ambion). 2.4. Cyclic stretching ECs were seeded at a concentration of 20 104 cells/ml as confluent cells on fibronectin-coated membranes in a stretching chamber (Strex, Japan); they were maintained at 37 °C in a humidified atmosphere for 24 h. ECs were then subjected to 20% stretching at 0.5 Hz using a stretching apparatus (Strex) [1,15]. During all experiments, the stretching apparatus was maintained at 37 °C in an incubator. 2.5. Immunofluorescence staining and image analysis For immunofluorescent staining of integrin b1 and vinculin, ECs grown on fibronectin-coated membrane were fixed after treatment
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(B) Fig. 2. (A) Typical fluorescent images of actin filaments and nuclei in non-transfected ECs and knockdown ECs under static conditions and 10, 30, and 60 min after exposure to 20% cyclic stretching at 0.5 Hz. Red, actin filament; Blue, nucleus. Bar = 50 lm. (B) Histograms of the orientation angle of non-transfected and paxillin knockdown HUVECs under static conditions and exposed to 20% cyclic stretching at 0.5 Hz for 10, 30, and 60 min. Numbers in parentheses indicate the number of cells studied. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
in 4% paraformaldehyde solution in PBS for 10 min at room temperature (RT), washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and blocked with Block Ace (Snow Brand Milk Products, Japan) for 60 min. Incubation with an antibody of integrin b1 or vinculin (Sigma–Aldrich, USA) was performed in the blocking solution for 60 min at RT, followed by staining with an Alexa 488-conjugated secondary antibody (Invitrogen). Actin filaments were stained with Alexa fluor 546-conjugated phalloidin (Invitrogen) for 20 min; the nuclei were then stained with 40 ,6diamidino-2-phenylindole (Invitrogen) for 2 min at RT. For the immunostaining of FAK, ECs were fixed with methanol (Wako Pure Chemical Industries, Japan) for 10 min at 20 °C and blocked with
Block Ace (Snow Brand Milk Products) for 60 min. After staining with an FAK antibody (BD Transduction, USA) and the Alexa 488conjugated secondary antibody (Invitrogen), the nuclei of ECs were stained. After immunostaining, stained cells were observed under a confocal laser scanning microscope (Olympus, Japan). From the actin staining images, the orientation angle of cells was measured using the ImageJ software (NIH, USA). We determined the orientation angle of ECs, which is defined as the angle between the direction of the long axis of an ellipse equivalent to cell shape and the stretching direction. ECs aligned at angles between 60° and 90° were defined as oriented cells.
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Stretching (20%, 0.5 Hz) Fig. 3. Fluorescent images of integrin b1 in HUVECs under static conditions (0 min) and exposed to 20% cyclic stretching at 0.5 Hz for 10, 30, and 60 min. Green, Integrin b1; Blue, nucleus. Bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3. Results A typical result of western blot analysis of paxillin expression in ECs is shown in Fig. 1A. Knockdown ECs showed a significant reduction of approximately 95% in the paxillin protein level compared to non-transfected ECs and ECs transfected with non-targeting siRNA. There was no difference between non-transfected ECs and ECs transfected with non-targeting siRNA. Confocal fluorescent images of ECs immunostained for paxillin are shown in Fig. 1B. A prominent reduction in paxillin was observed in ECs transfected with paxillintargeting siRNA. This result confirmed the specificity of paxillin knockdown by siRNA transfection. Fluorescent images of actin filaments and nuclei in ECs exposed to cyclic stretching are shown in Fig. 2A. Both control and knockdown ECs showed random orientation at 0 min. ECs without paxillin siRNA treatment aligned perpendicularly to the stretching direction after stretching for 10 min and became more perpendicular to the direction of stretching at 30 and 60 min. On the other hand, orientation of ECs treated with paxillin siRNA was not observed at 10 min. After 30 min of exposure to cyclic stretching, paxillin knockdown ECs started orienting perpendicularly to the stretching direction and orientation similar to controls was observed at 60 min. After cyclic stretching, stress fibers reoriented perpendicularly to the direction of stretching from a random alignment under static conditions. In contrast, the actin filaments in knockdown ECs were significantly thinner at 0 and 10 min compared with those in controls, whereas the orientation of actin filaments was still observed after stretching for 30 and 60 min. The relative frequency of the orientation angle of ECs exposed to cyclic stretching is shown in Fig. 2B. Both control and paxillin knockdown ECs showed random orientation under static conditions. After 10 min of stretching, approximately 60% of control ECs oriented to angles ranging from 60° to 90°. However, prominent reorientation of paxillin knockdown ECs was not observed. The reorientation tendency of ECs transfected with paxillin siRNA was observed at 30 min. Approximately 70% of the knockdown
ECs showed orientation angles from 60° to 90° after 60 min of cyclic stretching exposure, similar to control cells. Immunofluorescent images for integrin b1 are shown in Fig. 3. Integrin b1 initially demonstrated a random punctuate distribution in control ECs. After 10 min of exposure to cyclic stretching, integrin b1 formed large FAs, and an increased number of FAs oriented perpendicularly to the direction of cyclic stretching were observed, similar to the alignment of actin filaments. After ECs were exposed to 30 and 60 min of stretching, integrin b1 reorganized in a linear pattern, and most FAs oriented almost perpendicularly to the direction of stretching. With paxillin knockdown treatment, we found that integrin b1 in ECs was arranged in a diffuse manner and still demonstrated limited accumulation and random orientation at 10 min in knockdown ECs. We found that after 30 and 60 min of exposure to cyclic stretching, prominent accumulation of integrin b1 was observed. Furthermore, adhesions of integrin b1 orienting perpendicularly to the direction of stretching were observed, similar to controls. Fig. 4A shows FAK staining results. FAK was located primarily in the periphery of control ECs under static conditions. After 10 min of exposure to cyclic stretching, FAK in the control cells accumulated and oriented almost perpendicularly to the direction of stretching. At 30 and 60 min, FAK became more concentrated, and FAK fibrils that aligned almost perpendicularly to the direction of stretching appeared. In contrast, only weak FAK accumulation with undefined orientation was observed in the ECs transfected with paxillin siRNA compared with control ECs under static conditions and at 10 min. However, FAK accumulation was observed at 10 min in the ECs transfected with paxillin siRNA and showed orientation perpendicular to the direction of cyclic stretching at 30 and 60 min. Fig. 4B shows vinculin staining results. After 10, 30, and 60 min of exposure to cyclic stretching, the vinculin in control ECs was observed to reorganize into a linear shape and orient perpendicularly to the direction of stretching. Unlike integrin b1 and FAK, paxillin knockdown appeared not to influence the accumulation of vinculin in ECs.
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Fig. 4. (A) Fluorescent images of FAK in HUVECs under static conditions and exposed to 20% cyclic stretching at 0.5 Hz for 10, 30, and 60 min. Green, FAK; Blue, nucleus. Bar = 50 lm. (B) Fluorescent images of vinculin with nucleus in HUVECs under static conditions and exposed to 20% cyclic stretching at 0.5 Hz for 10, 30, and 60 min. Green: vinculin, Blue: nucleus. Bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion ECs exposed to cyclic stretching exhibit rapid changes in the orientation of actin filaments and orient perpendicular to the direction of stretching after 10 min of exposure [15,18]. In addition, FA reorganization is known to be crucial for the morphological responses of ECs exposed to cyclic stretching [6,19–21]. Our group has previously shown that after 2 min of exposure to cyclic stretching, paxillin levels in ECs were significantly higher in the regions perpendicular to the direction of stretching [15]. In the present study, we investigated the roles of paxillin in the orientation changes of ECs exposed to cyclic stretching for 10, 30, and
60 min; the orientation of ECs transfected with paxillin siRNA was not observed at 10 min, but the orientation was observed after 60 min of exposure to cyclic stretching. These results showed for the first time that paxillin is required for the early phase of orientation of ECs exposed to cyclic stretching. Yano et al. [6] reported that cyclic stretching-induced FAK phosphorylation occurred concomitant with the redistribution of integrin b1. Because integrin b1 itself cannot be phosphorylated, recruitment of tyrosine kinases such as FAK was thought to initiate biochemical signaling. Moreover, Naruse et al. reported that cyclic stretching induced phosphorylation and redistribution of paxillin [1]. As a scaffold protein, paxillin has binding sites for integrin b1 and FAK [8].
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In addition, paxillin can target to FAs without interactions with FAK [9] and is regarded as a switch to regulate the assembly and form of cell-matrix adhesions [22]. In this study, we found that knockdown of paxillin resulted in the weak accumulation of integrin b1 and FAK in ECs after 10 min of exposure to cyclic stretching. These results suggest that paxillin is a key protein for the signaling pathway between integrin b1 and FAK in ECs in the early phase of cell orientation. On the other hand, we found that knockdown of paxillin did not change the accumulation and redistribution of vinculin. Talin, another FA protein, is first recruited to the membrane; vinculin is then recruited to adhesion plaques by binding to talin [23]. In addition, vinculin is not associated with FAK [24] and integrin b1 [10]. Thus, an alternative possibility for the localization of vinculin to focal adhesions is that it is recruited by binding to talin. In this study, we found that the ECs transfected with paxillin siRNA oriented perpendicularly to the direction of stretching after 60 min of exposure, while the orientation of ECs was abolished at 10 min. Accumulation of FA proteins, integrin b1, and FAK similar to that of control cells was also observed after 30 and 60 min of exposure to cyclic stretching even in paxillin knockdown ECs. These results suggest that there are compensatory mechanisms by which the accumulation and redistribution of FA proteins occurs. Furthermore, the recruitment of FA proteins is a considerably complex process that may be regulated by FA proteins other than paxillin. For example, Src tyrosine kinase is an important molecule for stretching-induced orientation response in cells [25] and leads to the phosphorylation of p130Cas, another scaffold protein in FAs [12]. The p130Cas phosphorylation level was low in unstretched cells, and stretching, which can lead to the extension of p130Cas, increases its tyrosine phosphorylation [26]. p130Cas can bind to several different proteins such as FAK [27], and p130Cas phosphorylation is crucial for its association with other signaling molecules [28]. Therefore, we suppose that such stretching-dependent signaling is involved in the accumulation of FAs and cell orientation after 30 min of exposure to cyclic stretching, but the detailed mechanisms should be investigated in further studies. In conclusion, in the present study, we investigated the time course of EC orientation when subjected to cyclic stretching. Control ECs oriented after 10 min of exposure to cyclic stretching. In contrast, the orientation of ECs transfected with paxillin siRNA was abolished at 10 min, but the orientation of transfected ECs was observed after 60 min of exposure to cyclic stretching. Furthermore, we found that the knockdown of paxillin led to delayed accumulation of integrin b1 and FAK, distribution of which is crucial for the morphological changes of ECs exposed to cyclic stretching. These findings suggest that paxillin is involved in the early phase of EC orientation by playing a key role in the redistribution of integrin b1 and FAK. Acknowledgments The authors thank Dr. Makoto Takahashi and Dr. Ikuo Takahashi for providing human umbilical cords; in addition, the authors thank Ms. Eri Inoue and Mr. Shinya Chubachi for kind help in some experiments. This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) in Japan (No. 20001007). References [1] K. Naruse, T. Yamada, X.R. Sai, M. Hamaguchi, M. Sokabe, Pp125FAK is required for stretch dependent morphological response of endothelial cells, Oncogene 17 (1998) 455–463. [2] Y. Katanosaka, J.H. Bao, T. Komatsu, T. Suemori, A. Yamada, S. Mohri, K. Naruse, Analysis of cyclic-stretching responses using cell-adhesion-patterned cells, J. Biotechnol. 133 (2008) 82–89.
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