(The FASEB Journal. 2006;20:1992-1999.)
© 2006 FASEB
Pivotal role of integrin 
5ß1 in hypotonic stress-induced responses of human endothelium
Masakazu Hirakawa,
Masahiro Oike1,
Michi Watanabe,
Yuji Karashima and
Yushi Ito
Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
1Correspondence: Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. E-mail: moike{at}pharmaco.med.kyushu-u.ac.jp
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ABSTRACT
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We have previously reported that both hypotonic stress (HTS) and lysophosphatidic acid (LPA) induce ATP release and a transient reorganization of actin through sequential activation of RhoA/Rho-kinase and focal adhesion kinase F-actin (FAK)/paxillin in human umbilical cord vein endothelial cells (HUVECs). LPA is known to induce the activation of RhoA via its specific receptors, but the mechanisms by which HTS initiates these intracellular signals are not known. The present study aimed to identify the molecule(s) that are unique to the sensing and/or transducing the mechanical stress. Reverse transcriptase-polymerase chain reaction revealed the expression of several integrin subunits in HUVECs. Anti-integrin 
5ß1 antibody (Ab), but not anti-integrin 2, 6, v, or ß4 antibodies, inhibited HTS-induced RhoA translocation, tyrosine phosphorylation of FAK and paxillin, ATP release, and actin reorganization. However, the LPA-induced ATP release and actin reorganization were not inhibited by any of these anti-integrin antibodies, indicating that integrin 5ß1 plays a pivotal role in the HTS-induced but not in the LPA-induced responses. It is therefore reasonable to assume that this particular subtype of integrin is involved in the initiation of the responses induced by mechanical stimuli in HUVECs.—Hirakawa, M., Oike, M., Watanabe, M., Karashima, Y., Ito, Y. Pivotal role of integrin 5ß1 in hypotonic stress-induced responses of human endothelium.
Key Words: ATP release • tyrosine kinase • RhoA • actin cytoskeleton
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INTRODUCTION
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IT IS WELL ESTABLISHED that vascular endothelial functions are controlled by mechanical stresses. We have previously demonstrated that application of hypotonic stress (HTS) to human umbilical cord vein endothelial cells (HUVECs) sequentially activates within a few minutes small GTPase family protein RhoA and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, thereby leading to ATP release and actin reorganization (1)
. Previous reports showed that tyrosine phosphorylation of FAK and RhoA/Rho kinase activation was also induced by other mechanical stresses, such as shear stress and mechanical strain (2
3
4
5)
. Furthermore, ATP release and actin reorganization are well-known mechanosensitive responses in vascular endothelium (6
,7)
. However, we have shown that lysophosphatidic acid (LPA) also induces the same intracellular signals and responses in HUVECs (1)
, suggesting that the responses evoked by HTS are not specific for mechanical stress. DNA microarray assay has also shown that the gene expression profile in HUVECs after exposure to shear stress for 24 h was similar to that after stimulation with cytokines, suggesting that most of the shear-induced changes were not specific to shear stress (8)
.
We have shown that RhoA activation is the most upstream event in the cascade initiated by HTS (1)
. Although binding of LPA to its specific receptors activates RhoA and subsequently Rho-kinase (9)
, it seems unlikely that HTS or other mechanical stresses activate LPA receptors but rather that they activate RhoA via an LPA-insensitive pathway. The family of transmembrane integrin molecules has been reported to play a role in mechanical stress-induced endothelial responses, such as cytoskeletal modulation (10)
, cell migration (11)
, endothelium-dependent vasodilatation (12)
, and activation of mitogen-activated protein kinase and FAK (13)
. Therefore, in the present study we investigated the possible involvement of integrins in the HTS-induced responses in HUVECs. For this purpose, we examined the effects of anti-integrin blocking antibodies on HTS- and LPA-induced signals and responses in HUVECs. Our results demonstrate that integrin 5ß1 is a key molecule that discriminates between HTS- and LPA-induced responses in HUVECs.
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MATERIALS AND METHODS
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Culture of HUVECs
HUVECs were purchased from Cambrex (East Rutherford, NJ). Cells were cultured in M199 medium supplemented with 15 µg/ml endothelial cell growth supplement (Sigma, St. Louis, MO), 5 U/ml heparin, and 15% FBS.
RNA preparation and reverse transcriptase-polymerase chain reaction analysis of integrin mRNA expression
Total RNA was prepared from HUVECs and converted into first-strand cDNA by using a commercial kit (RNAqueousTM-4PCR kit, Ambion, Austin, TX and Life Technologies). The following primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA expression of integrin subunits: 5'-TTGACCTATCCACTGCCACA-3' (forward) and 5'-GTCAGAACACACACCCGTTG-3' (reverse) for 2; 5'-AGAGAGACCCTGCAAGACCA-3' (forward) and 5'-CTCAAATGGGCCTTAGGACA-3' (reverse) for 3; 5'-AAAATGGATGGCCTTCTGTG-3' (forward) and 5'-TCTGCTGGACACCTGTATGC-3' (reverse) for 4; 5'-CTACAATGATGTGGCCATCG-3' (forward) and 5'-GGATATCCATTGCCATCCAG-3' (reverse) for 5; 5'-TGTGAGCTCGG-AAATCCTTT-3' (forward) and 5'-CAACTCCCGAGACCGATAAA-3' (reverse) for 6; 5'-GGGTTGTGGAGTTGCTCAGT-3' (forward) and 5'-AATGCCCCAGGTGACATTAG-3' (reverse) for v; 5'-GGCCTTGCATTACTGCTGAT-3' (forward) and 5'-CAGTGTTGTGGGATTTGCAC-3' (reverse) for ß1; 5'-TGGTCCTGCTCTCAGTGATG-3' (forward) and 5'-AGCATCTGCTGCTTCCACTT-3' (reverse) for ß3; 5'-TGGAAGTACTGTGCCTGCTG-3' (forward) and 5'-TGCATGTTGTTGGTGACCTT-3' (reverse) for ß4; and 5'-CTGGAACAA-CGGTGGAGATT-3' (forward) and 5'-CCCAATCTTCAGACCCTCAC-3' (reverse) for ß5. Expression of GAPDH mRNA was examined as an internal control with the primers 5'-GGGTCATCATCTCTGCACCT-3' (forward) and 5'-ATCCACAGTCTTCTGGGTGG-3' (reverse).
Immunofluorescence staining of integrin 5ß1
Cellular localization of integrin 5ß1 was examined by immunofluorescence staining with a monoclonal anti-human integrin 5ß1 antibody (Ab; clone JBS5, Chemicon International, Temecula, CA). Cells were washed twice with PBS and incubated with the anti-integrin 5ß1 Ab (10 µg/ml) for 15 min. The Ab bound to the cell surface integrin 5ß1 was then visualized with FITC-conjugated anti-mouse immunoglobulin (Ig) Ab (Sigma). Fluorescence images were captured with a digital microscope camera (VB6000, Keyence, Osaka, Japan) connected to a fluorescence microscope (Eclipse E600, Nikon, Tokyo, Japan).
Block of integrins with monoclonal anti-integrin antibodies
Monoclonal integrin antibodies were used for blocking integrins on cell membrane surface. HUVECs were incubated either with monoclonal anti-human integrin 2 (clone A2-IIE10, 10 µg/ml, Upstate Biotechnology, Lake Placid, NY), 5ß1 (5 µg/ml), 6 (clone NKI-GoH3, 10 µg/ml, Chemicon International), v (clone AV1, 10 µg/ml, Chemicon International), or ß4 (clone ASC-3, 10 µg/ml, Chemicon International) antibodies at 37°C for 30 (5ß1) or 60 (2, 6, v, and ß4) min. HTS or LPA was then applied to the cells, and assays were performed.
Luciferin-luciferase bioluminescence assay
The extracellular ATP concentration ([ATP]o) was measured by using luciferin-luciferase bioluminescence. Cells were seeded on 96-well plates and cultured for 2 days before the assay. Each well contained before the experiment on average 5000 cells. After careful removal of the culture medium, we added 50 µl of isotonic or hypotonic Krebs solution or LPA-containing Krebs solution containing 10 mg/ml luciferase-luciferin (Wako, CO, Osaka, Japan) to each well and counted the emitted photons during 10 min by a luminescence detection system (Argus-50/2D luminometer, Hamamatsu Photonics, Hamamatsu, Japan). Standard curves for converting photon counts into [ATP]o were obtained with the same solutions to avoid possible artifacts of ionic composition and drugs on the luciferin bioluminescence.
Western blot analysis of tyrosine phosphorylation and RhoA activation
Tyrosine phosphorylation of cellular proteins and RhoA activation were assessed with chemiluminescence Western blotting using an enhanced chemiluminescence system (SuperSignal West Dura, Pierce, Rockford, IL). Cells were lysed after 1, 2, 5, or 10 min exposure to HTS. Western blot analysis for phosphotyrosine was performed by using monoclonal anti-phosphotyrosine Ab (clone PY20, Exalpha Biologicals, Watertown, MA). For the assessment of RhoA activation, the cell lysate was centrifuged for 1 h at 100,000 g, and the pellet was harvested as a membrane fraction. A constant amount of membrane fraction (50 µg protein per lane) was separated with SDS-PAGE, and RhoA was detected with monoclonal anti-RhoA Ab (Cytoskeleton, Denver, CO). The expression of ß-actin protein was assessed as an internal control, using monoclonal anti-ß-actin Ab (Sigma). The emitted chemiluminescence was detected in each experiment and analyzed with a lumino image analyzer (FAS-1000, Toyobo, Osaka, Japan).
Immunological staining of endothelial F-actin
The rearrangement of F-actin by HTS was examined by a previously reported immunological method (14)
using rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR).
Solutions and drugs
The standard extracellular solution was a modified Krebs solution (1.5 mM Ca2+ solution) containing (in mM): 132 NaCl, 5.9 KCl, 1.2 MgCl2, 1.5 CaCl2, 11.5 glucose (Glc), and 11.5 HEPES; pH adjusted to 7.3 with NaOH. Hypotonic solutions were made by adding appropriate amounts of distilled water to normal Krebs solution. We have previously confirmed that the reduction of the ionic concentrations did not influence the [Ca2+]i responses (15)
. All other drugs were purchased from Sigma.
Data analysis
Pooled data are mean ± SE, and statistical significance was determined using Student’s unpaired t test. Probabilities <5% (P<0.05) were regarded as significant.
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RESULTS
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Expression of integrin subunits in HUVECs
First, we examined which subunits of integrin and ß are expressed in the HUVECs used in the present study. RT-PCR revealed that at least mRNAs of 3, 5, 6, v, ß1, ß3, ß4, and ß5 subunits are expressed in HUVECs (Fig. 1
A). Integrin 5ß1 is one of the possible combinations of these integrin subunits that has been reported to be involved in mechanosensing in vascular endothelium (16)
. We therefore used immunofluorescence staining with an anti-integrin 5ß1 Ab to detect and localize integrin 5ß1 in HUVECs. As shown in Fig. 1B
, integrin 5ß1 did not show an apparent clustering on the cell surface but was scattered over the membrane surface.
Effects of integrin antibodies on HTS-induced RhoA activation
Next, we examined the effects of blocking antibodies against integrin 5ß1 on HTS-induced responses. We also used the blocking antibodies against integrin 6, v, and ß4 subunits, the mRNAs of which were detected in HUVECs with RT-PCR. Furthermore, to exclude possible nonspecific effects of IgG, we examined the effects of the blocking Ab against the integrin 2 subunit, which was not detected with RT-PCR. The HTS-induced membrane translocation of small G protein RhoA, a hallmark of its activation (17)
, was maximal 2 to 5 min after starting HTS in control HUVECs (Fig. 2
). The anti-integrin 5ß1 Ab inhibited the membrane translocation of RhoA induced by –30% HTS (Fig. 2)
, whereas the other anti-integrin antibodies did not affect the maximal level and the time course of the HTS-induced translocation of RhoA (Fig. 2)
.
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Figure 2. Effects of anti-integrin antibodies on HTS (–30%)-induced membrane translocation of small G protein RhoA in HUVECs. Cells were treated with anti-integrin 2 (clone A2-IIE10, 10 µg/ml, 60 min), 5ß1 (clone JBS5, 5 µg/ml, 30 min), 6 (clone NKI-GoH3, 10 µg/ml, 60 min), v (clone AV1, 10 µg/ml, 60 min), and ß4 (clone ASC-3, 10 µg/ml, 60 min) antibodies at 37°C. Cells were lysed before or after applying –30% HTS for indicated period and centrifuged as described in Materials and Methods. Band densities of RhoA and ß-actin were measured in membrane fraction, and RhoA/ß-actin values were expressed relative to that of isotonic control. Note that anti-integrin 5ß1 Ab, but not other antibodies, completely suppressed the HTS-induced change in RhoA expression in membrane fraction. Representative RhoA band images are shown in upper panel. Data in lower panel represent the densitometric analysis of bands obtained from 4 experiments. **P < 0.01 vs. control.
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Effects of integrin antibodies on HTS-induced tyrosine phosphorylation
In control HUVECs, HTS (–30%) induced tyrosine phosphorylation of 125 and 68 kDa proteins (Fig. 3
), which we have previously identified as FAK and paxillin, respectively, by using anti-focal adhesion kinase F-actin, anti-phosphorylated FAK, and anti-paxillin antibodies. In addition, we have also shown that tyrosine phosphorylation of these proteins is a downstream signal of RhoA activation in HUVECs (1)
. Because the anti-integrin 5ß1 Ab inhibited the HTS-induced RhoA translocation (Fig. 2)
, we might also expect that this Ab would inhibit the HTS-induced tyrosine phosphorylation. Indeed, the HTS-induced tyrosine phosphorylation of 125 kDa FAK (Fig. 3A
) and 68 kDa paxillin (Fig. 3B
) was significantly inhibited in HUVECs pretreated with the anti-integrin 5ß1 Ab but not by the anti-integrin 2, 6, v, and ß4 antibodies (Fig. 3)
.
Effects of integrin antibodies on HTS-induced ATP release in HUVECs
As reported previously, the sequential activation of RhoA and FAK/paxillin leads to ATP release in HUVECs (1)
. We have therefore also examined the effects of the anti-integrin antibodies on the HTS-induced ATP release, as assessed by the increase in [ATP]o. An exposure of control cells for 10 min to a –30% hypotonic solution elevated [ATP]o to 62.3 ± 1.6 nM (n=28), a value that is significantly higher than that of 18.2 ± 0.5 nM (n=29) during a 10 min exposure to isotonic solution (Fig. 4
A). None of the anti-integrin antibodies affected the values of [ATP]o in isotonic solution (P>0.05 vs. untreated control; Fig. 4A
). In contrast, the anti-integrin 5ß1 Ab significantly inhibited the HTS-induced increase in [ATP]o (28.9±1.1 nM, n=7, P<0.01 vs. untreated control), but the anti-integrin 2, 6, v, or ß4 antibodies did not affect the HTS-induced ATP release (P>0.05 vs. untreated control; Fig. 4A
).
Effects of integrin antibodies on HTS-induced actin reorganization
We have also demonstrated that HTS induces a transient reorganization of the actin cytoskeleton, which occurs in parallel with the ATP release (1
, 18)
. Dense actin fibers reached a maximum level 2 min after starting HTS and then converged into peripheral adhesion complexes after 10 min (Fig. 4B
). The anti-integrin 5ß1 Ab, but not the anti-integrin 2, 6, v, or ß4 antibodies, inhibited the HTS-induced actin fiber formation (Fig. 4B
). Therefore, these results suggest that integrin 5ß1 plays a central role in sensing HTS and evoking intracellular signals and responses in HUVECs
Effects of integrin antibodies on LPA-induced responses in HUVECs
As reported previously, LPA mimicked all of the HTS-induced intracellular signals and responses in HUVECs (1)
. In the present study, we first confirmed the LPA increased [ATP]o in untreated control cells (LPA, 49.5±2.4 nM, n=12; without LPA, 19.6±3.3 nM, n=12; P<0.01; Fig. 5
A). However, unlike the HTS-induced ATP release, this LPA-induced [ATP]o increase was not inhibited by the anti-integrin 5ß1 Ab (with LPA, 44.7±2.2 nM, n=4; without LPA, 26.6±5.7 nM, n=4; both P>0.05 vs. control; Fig. 5A
). Anti-integrin 2, 6, v, and ß4 antibodies also did not affect the LPA-induced increase in [ATP]o (Fig. 5A
).
LPA also induced a transient reorganization of actin filaments in control cells (Fig. 5Ba
) and its time course was similar to the HTS-induced one, i.e., actin fibers appeared after 2 min and converged into peripheral local adhesion complexes in 10 min. The anti-integrin 5ß1 Ab did not interfere with the LPA-induced actin reorganization (Fig. 5Bb
). Furthermore, anti-integrin 2, 6, v, and ß4 antibodies also did not affect LPA-induced actin reorganization (not shown). Therefore, these results suggest that integrins do not play any role in the LPA-induced responses in HUVECs.
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DISCUSSION
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The present study shows that the HTS-induced membrane translocation of RhoA (Fig. 2)
, tyrosine phosphorylation of FAK and paxillin (Fig. 3)
, ATP release (Fig. 4A
), and actin reorganization (Fig. 4B
) in HUVECs are suppressed by the anti-integrin 5ß1 Ab. These effects were not due to nonspecific effects of the treatment with Ab, since higher concentrations of the blocking antibodies against integrin 2, 6, v, and ß4 subunits did not affect any of these HTS-induced intracellular signals and responses. We have previously reported that HTS induces a sequential activation of RhoA/Rho-kinase and FAK/paxillin and that the activation of this cascade leads to ATP release and actin reorganization (1)
. The present results, therefore, indicate that the activation of integrin 5ß1 is located upstream of these HTS-induced sequential responses. In addition to their involvement in cell attachment and migration, integrins have been supposed to play a role in endothelial mechanotransduction (16)
. Urbich et al. (11)
reported that integrin 5ß1 mediates the shear stress-induced cell migration and the activation of MAPK, Akt, and FAK in HUVECs. In contrast, Tzima et al. (10)
reported that the shear stress-induced activation of integrin vß3 in bovine aortic endothelial cells leads to a transient suppression of Rho activity and disruption of actin filaments. Other groups also reported the involvement of integrin ß1 subunit (19
, 20)
and vß3 (19)
in shear stress-mediated responses in endothelium. Integrin 5ß1 and vß3 share the same extracellular matrix ligand, fibronectin, and it was reported that the interaction between these integrins and fibronectin was required for the shear stress-induced association of integrin with the adapter protein Shc in HUVECs (21)
. Therefore, both 5ß1 and vß3 integrins may play a role in endothelial mechanotransduction. However, as described in the present study, the clone AV1 of the anti-integrin v Ab, which was developed using human integrin vß3 as an immunogen and was shown to successfully block integrin vß3-mediated cell adhesion (22)
, did not affect any of the HTS-induced responses in HUVECs. Our conclusion is therefore that integrin 5ß1 plays a pivotal role in the HTS-induced responses in HUVECs.
We have shown in a previous study that LPA mimicked the HTS-induced responses (1)
. It is well established that LPA binds to its specific receptors and activates RhoA via LPA1 and LPA2 receptors (23)
, but evidence for the involvement of integrins in the activation of LPA receptors has not been presented so far. However, since none of the anti-integrin antibodies examined in this study suppressed the LPA-induced ATP release (Fig. 5A
) and actin reorganization (Fig. 5B
), it is unlikely that they are involved in the activation of LPA receptors. Various candidate mechanosensing molecules, such as heparan sulfate proteoglycan (24)
, or molecular complexes, including PECAM-1, cadherin, and VEGFR2, have been proposed for endothelium (25)
. If they are also involved in HTS-induced responses, these sensor molecules may activate integrin 5ß1 and intracellular signals originating from RhoA activation. Therefore, the present results indicate that LPA and HTS induce identical intracellular signals and that only a few initial mechanosensing molecules, including integrin 5ß1 and LPA receptor(s), are actually stimulus-specific (Fig. 6
).
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Figure 6. Schematic diagram of role of integrin 5ß1 in endothelial mechanotransduction. See text for detailed explanation.
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ATP release has been considered as one of the central mechanosensitive responses in endothelium (15)
and also in other cell types (26
27
28)
. ATP release is not only evoked by hypotonic cell swelling (15)
but also by shear stress (29)
and membrane stretch (27)
. The present study revealed for the first time that integrin 5ß1 is involved in HTS-induced ATP release. Although we did not examine the possible role of integrin 5ß1 in the ATP release induced by other mechanical stresses, its involvement might be likely since this particular subtype of integrin has been reported to mediate shear stress-induced responses in vascular endothelium (10
, 11
, 16
, 21
, 30)
. ATP release is also induced by nonmechanical stimuli, such as agonist stimulation (31)
, third generation ß blockers (32)
, lipopolysaccharide (LPS; 33
), and LPA (1)
in vascular endothelium, but until now it has not been clear whether these responses are mediated by the same pathways as the mechanosensitive ATP release. The results shown in this study and in our previous report (1)
may resolve this issue: ATP release is one of the common endothelial responses evoked by RhoA-mediated intracellular signals, and any stimulus that evokes these signals would also induce ATP release. In conclusion, HTS-induced but not LPA-induced responses are mediated by integrin 5ß1, and therefore integrin 5ß1 plays a pivotal role in the initiation of HTS-induced responses in HUVECs.
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ACKNOWLEDGMENTS
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We thank Dr. Guy Droogmans for comments and help with the manuscript. This study was carried out as a part of "Ground Research Announcement for the Space Utilization" promoted by Japan Aerospace Exploration Agency and Japan Space Forum. This study was also supported in part by a grant-in-aid from the Japan Society for the Promotion of Science.
Received for publication January 10, 2006.
Accepted for publication May 25, 2006.
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