Mechanotransduction by integrin is essential for IL-6 secretion from endothelial cells in response to uniaxial continuous stretch

Akitoshi Sasamoto,1,2 Masato Nagino,1 Satoshi Kobayashi,1 Keiji Naruse,2,3 Yuji Nimura,1 and Masahiro Sokabe2,3,4

1Division of Surgical Oncology, Department of Surgery, and 2Department of Physiology, Nagoya University Graduate School of Medicine; and 3Cell Mechanosensing Project, International Cooperative Research Project, Japan Science and Technology Agency, Nagoya; and 4Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan

Submitted 8 July 2004 ; accepted in final form 17 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported that uniaxial continuous stretch in human umbilical vein endothelial cells (HUVECs) induced interleukin-6 (IL-6) secretion via I{kappa}B kinase (IKK)/nuclear factor-{kappa}B (NF-{kappa}B) activation. The aim of the present study was to clarify the upstream signaling mechanism responsible for this phenomenon. Stretch-induced IKK activation and IL-6 secretion were inhibited by application of {alpha}5{beta}1 integrin-inhibitory peptide (GRGDNP), phosphatidylinositol 3-kinase inhibitor (LY-294002), phospholipase C-{gamma} inhibitor (U-73122), or protein kinase C inhibitor (H7). Although depletion of intra- or extracellular Ca2+ pool using thapsigargin (TG) or EGTA, respectively, showed little effect, a TG-EGTA mixture significantly inhibited stretch-induced IKK activation and IL-6 secretion. An increase in the intracellular Ca2+ concentration ([Ca2+]i) upon continuous stretch was observed even in the presence of TG, EGTA, or GRGDNP, but not in a solution containing the TG-EGTA mixture, indicating that both integrin activation and [Ca2+]i rise are crucial factors for stretch-induced IKK activation and after IL-6 secretion in HUVECs. Furthermore, while PKC activity was inhibited by the TG-EGTA mixture, GRGDNP, LY-294002, or U-73122, PLC-{gamma} activity was retarded by GRGDNP or LY-294002. These results indicate that continuous stretch-induced IL-6 secretion in HUVECs depends on outside-in signaling via integrins followed by a PI3-K-PLC-{gamma}-PKC-IKK-NF-{kappa}B signaling cascade. Another crucial factor, [Ca2+]i increase, may at least be required to activate PKC needed for NF-{kappa}B activation.

nuclear factor-{kappa}B; phosphatidylinositol 3-kinase; phospholipase C-{gamma}; protein kinase C; intracellular Ca2+ concentration


VASCULAR ENDOTHELIAL CELLS (ECs) that line the vascular system are subjected to three major hemodynamic forces, shear stress, transmural pressure, and mechanical stretch. Major hemodynamic forces loading ECs are shear stress by blood flow and circumferential (uniaxial) cyclic tension by vessel expansion. On the other hand, hemodynamic changes associated with clinical morbidity often cause an abrupt and sustained dilatation of vessels, leading to uniaxial continuous mechanical stretch on ECs (44). Blood congestion due to acute heart failure or to acute obstruction of vessels produces a rapid and sustained stretch load on ECs followed by secretions of a variety of chemical mediators, which in turn modify the morbid conditions (12, 26, 29). We previously reported that an ~150% uniaxial sustained stretch in sinusoidal ECs after portal vein embolization might be a trigger for liver regeneration via interleukin-6 (IL-6) secretion (21). Furthermore, we demonstrated that IL-6 secretion from ECs in response to sustained stretch is mediated by a sequential activation of I{kappa}B kinase (IKK) and nuclear factor (NF)-{kappa}B (22). However, the signal transduction pathway leading to IKK activation after sustained stretch in ECs remained to be elucidated.

Experimentally applied continuous stretch to ECs consists of a single immediate transient load followed by a sustained tonic load. We previously showed that the sustained tonic component of uniaxial continuous stretch is more important than the single immediate transient component in the stretch-induced IL-6 secretion from ECs (22). Therefore, mechanotransduction mechanisms activated by tonic force during sustained stretch seem be crucial to understanding the signaling mechanism causing IL-6 secretion. Integrins, which comprise a major family of transmembrane receptors that mediate cell adhesion to extracellular matrices (ECMs), are one of the potential candidate molecules to transduce tonic force into intracellular biochemical signals (4, 23, 25). Bhullar et al. (4) reported that the integrin-mediated signaling pathway regulates NF-{kappa}B through the activation of IKK in bovine aortic endothelial cells in response to shear stress, although its detailed signaling events have not been clarified yet.

Several studies of mechanical force-evoked integrin signaling have indicated that integrin activates NF-{kappa}B in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner (9, 37). PI3-kinase is a lipid kinase that phosphorylates phosphoinositides at the 3'-position of the inositol ring. The principal products of PI3-kinase, PI(3,4)P2 and PI(3,4,5)P3, act as second messengers that activate the serine/threonine kinase Akt/PKB (15). Alternatively, the binding of the produced PI(3,4,5)P3 to the src homology domain 2 (SH2) or to the pleckstrin homology domain (PH) of phospholipase C-{gamma} (PLC-{gamma}) leads to the activation of PLC-{gamma} (2, 11, 35). PLC-{gamma} plays an important role in the regulation of various intracellular signaling mechanisms. Activated PLC-{gamma} hydrolyzes PI(4,5)P2 to inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which contribute to an increase in intracellular Ca2+ concentration ([Ca2+]i) and activation of protein kinase C (PKC), respectively (16, 33). PKC is involved in stretch-induced reactive oxygen species (ROS) production, such as H2O2, lipid peroxides, and O2 (5, 7, 14, 27), followed by activation of NF-{kappa}B (13, 49). We also previously reported that an antioxidant inhibits the IL-6 production by suppressing NF-{kappa}B (22).

A number of studies have shown that Ca2+ is essential for production of ROS (1, 3, 34). Elevation of [Ca2+]i is responsible for the activation of ROS-generating enzymes and the formation of free radicals. Generally, there are two major mechanisms for mechanically induced intracellular Ca2+ mobilization: 1) Ca2+ influx through Ca2+-permeable, stretch-activated (SA) channels and 2) Ca2+ release from intracellular Ca2+ stores (38, 51). Previously, we reported that SA channels play a crucial role in the mechanotransduction to cyclic stretch (1, 6, 18, 50). Cyclic stretch-induced Ca2+ influx through SA channels mediates O2 production (1) and NF-{kappa}B activation (18). On the other hand, Pahl et al. (34) showed that both release of Ca2+ from endoplasmic reticulum (ER) and subsequent generation of ROS are required for ER overload-mediated NF-{kappa}B activation. However, it is still unclear which is more important, Ca2+ influx from extracellular pools or Ca2+ release from intracellular stores, in generating ROS that leads to NF-{kappa}B activation during uniaxial continuous stretch.

In the present study, we have investigated the upstream signaling mechanism of continuous stretch-induced IKK/NF-{kappa}B activation that leads to IL-6 secretion from ECs, with special attention to the PI3-kinase-PLC-{gamma}-PKC pathway regulated by integrins. In addition, we examined the importance of [Ca2+]i levels modulated by continuous stretch in activating this signal cascade.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and chemicals. Gadolinium (III) chloride hexahydrate was purchased from Aldrich Chemical (Milwaukee, WI). Upon arrival, it was dissolved in distilled water at 1 M and stored at –80°C. Because Gd3+ is unstable, the concentrated Gd3+ was first diluted at 10 mM in distilled water and then diluted at the desired concentration in standard external solution (SES; in mM: 140 NaCl, 5 KCl, 2 CaCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.40) just before use. Human plasma fibronectin was purified according to the method of Regnault et al. (36). PI3-kinase inhibitor (LY-294002) was purchased from Cell Signaling Technology (Beverly, MA). PLC inhibitor (U-73122) and the peptide Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP) were acquired from Biomol International (Plymouth Meeting, PA). PKC inhibitor (H7), PKA inhibitor (H89), thapsigargin (TG), phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], and phosphatidylethanolamine (PE) were obtained from Sigma (St. Louis, MO). {gamma}-[32P]ATP (3,000 Ci/mmol) and [3H]PI(4,5)P2 (5.45 Ci/mmol) were purchased from Amersham Biosciences (Arlington Heights, IL). Mouse anti-focal adhesion kinase (FAK) monoclonal antibody, mouse anti-paxillin monoclonal antibody, and mouse anti-phosphotyrosine monoclonal antibody (clone PY20) were purchased from Transduction Laboratories (Lexington, KY). Glutathione-S-transferase (GST)-I{kappa}B-{alpha}(1–54) was a kind gift from Dr. Makoto Nakanishi (Department of Biochemistry, Nagoya City University Medical School, Nagoya, Japan). Other chemicals used were of special grade.

Cell culture. Primary culture of human umbilical vein endothelial cells (HUVECs) was prepared from a human umbilical cord vein according to the method described by Shirinsky et al. (42). In brief, human umbilical cords were aseptically removed from the placenta just after birth. The veins were washed with phosphate-buffered saline (PBS; in mM: 137 NaCl, 8.10 Na2HPO4, 2.68 KCl, and 1.47 KH2PO4, pH 7.40), followed by treatment with 0.2% trypsin for 10 min. The perfusate was centrifuged at 200 g for 10 min. The resulting cells were washed with PBS again and then plated in 25-cm2 flasks and maintained in Humedia-EG2 medium (Kurabo, Osaka, Japan) supplemented with 10% fetal calf serum (FCS). HUVECs were incubated in a tissue culture incubator at 37°C in a humidified atmosphere of 5% CO2-95% air. HUVECs used in this study were within two passages.

Stretch apparatus and cell treatments. The cells were stretched as previously described (21). Briefly, cells were removed from the flask with 0.01% EDTA-0.02% trypsin and transferred onto an elastic silicone (polydimethylsiloxane elastomer) chamber precoated with 50 µg/ml fibronectin (FN) for 12 h. After 24 h of incubation, HUVECs were found to be confluent. The medium was replaced with starvation medium [Dulbecco's modified Eagle's medium (DMEM) containing 1% FCS, pH 7.4] 6 h before the cells were subjected to stretch. Both ends of the chamber were firmly attached to fixed metallic frames to produce a uniaxial continuous stretch by 150% of their original length. The stretched chamber with the frames was placed in an incubator. The silicone membrane was kept uniformly stretched over the whole membrane area during the incubation. To examine the intracellular signal transduction pathway leading from the continuous mechanical stimulus, we switched starvation medium to DMEM containing 20 µM Gd3+, a potent blocker for Ca2+-permeable SA channels, or nominally Ca2+-free medium (CFM): DMEM containing 5 mM ethylene glycol-bis({beta}-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) just before the stretch. Inhibitors of several signaling pathways were preincubated in the following manner. Fifty millimolar LY-294002 (PI3-kinase inhibitor) was added to the serum-starved medium 1 h before the stretch. Five micromolar TG (ER-resident Ca2+-ATPase inhibitor), 5 µM U-73122 (PLC-{gamma} inhibitor), 50 µM H-7 (PKC inhibitor), and 300 nM H89 [protein kinase A (PKA) inhibitor] were added 30 min before the stretch. Fifty micromolar GRGDNP, which competitively inhibits fibronectin binding, was added 3 h before the stretch application.

Determination of IKK activity. HUVECs were subjected to 150% continuous stretch for the indicated times after preincubation in starvation medium (DMEM with 1.0% FCS) for 6 h. For immunoprecipitation (IP), the cells were washed with ice-cold PBS and lysed in immunoprecipitated kinase buffer (50 mM HEPES, pH 8.0; 150 mM NaCl, 25 mM EGTA, 1 mM EDTA, 0.1% Tween 20, and 10% glycerol) containing a cocktail of protease inhibitors [20 µg/ml soybean trypsin inhibitor, 2 µg/ml aprotinin, 5 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride (PMSF)] and phosphatase inhibitors (50 mM NaF and 0.1 mM Na3VO4). The cell lysates were centrifuged at 20,600 g for 10 min. Polyclonal anti-IKK-{alpha} antibody (2 µl; Santa Cruz, CA) were added, and samples were incubated at 4°C for 1 h with gentle agitation. Next, 20 µl of protein G-Sepharose were added, and samples were incubated at 4°C for 1 h with gentle agitation. The resulting immunoprecipitates were washed three times with immunoprecipitated kinase buffer, then once with kinase assay buffer (50 mM HEPES, pH 8.0, 10 mM MgCl2, 2.5 mM EGTA, 1 mM DTT, 10 µM {beta}-glycerophosphate, 1 mM NaF, 0.1 mM PMSF, and 0.1 mM Na3VO4). The immunocomplex was then reacted in 20 µl of the kinase assay buffer containing 10 µM ATP, 3 µCi of {gamma}-[32P]ATP and 2 µl of GST-I{kappa}B-{alpha}(1–54) at 30°C for 30 min. The reaction was terminated by adding 3x sodium dodecyl sulfate (SDS) sample buffer (62 mM Tris·HCl, pH 6.8, 2% SDS, 1% 2-mercaptoethanol, 0.003% bromophenol blue, and 5% glycerol) to the sample and boiling for 10 min. The samples were separated on a 12.5% SDS-PAGE gel, and the autoradiogram was analyzed using the BAS 2000 radioactivity imaging system (Fuji Photo Film; Fuji, Tokyo, Japan).

Measurement of IL-6 mRNA expression. HUVECs were subjected to 150% continuous stretch for the indicated times after preincubation in starvation medium for 6 h. Total RNA was extracted from stretched cells using the RNeasy Mini kit (Qiagen, Cologne, Germany). The samples were subjected to first-strand synthesis using oligo(dT) primer and reverse transcriptase (Superscript II; Invitrogen, Carlsbad, CA). Real-time polymerase chain reaction (PCR) was performed using TaqMan Assays-on-Demand gene expression products and the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) according to the manufacturer's suggested protocol. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for each sample.

Western blot analysis. HUVECs were subjected to 150% continuous stretch for the indicated times after preincubation in starvation medium for 6 h. For immunoprecipitation, the cells were washed with ice-cold PBS and lysed using buffer A (10 mM Tris·HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 1% SDS, pH 7.4). Cell lysates were centrifuged twice at 20,600 g for 20 min at 4°C. Two microliters of 125-kDa monoclonal anti-FAK (40) or 68-kDa anti-paxillin antibody (47) were added, and the samples were incubated at 4°C for 1 h. Next, 20 µl of protein G-Sepharose were added, and the samples were incubated at 4°C for 1 h with gentle agitation. The resulting immunoprecipitates were washed five times with buffer A, and 30 µl of SDS sample buffer were added and boiled for 5 min. Proteins were then separated by SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). The membranes were blocked with 5% BSA in PBS-Tween-20 (PBS containing 0.5% Tween 20) and subsequently probed with monoclonal antibodies in blocking buffer for 1 h at room temperature. The antibody-antigen complexes were detected using horseradish peroxidase-conjugated goat anti-mouse IgG (1:1,000 dilution). Immunoreactivity was determined using the ECL Plus enhanced chemiluminescence reaction system (Amersham, Little Chalfont, UK).

PKC kinase activity assay. HUVECs were subjected to 150% continuous stretch for the indicated times after preincubation in starvation medium for 6 h. A nonradioactive PKC assay was performed using a commercially available MESACUP kit (MBL, Nagoya, Japan). PKC present in samples catalyzed phosphorylation of phosphatidylserine (PS) peptide coated on microwells. The biotinylated monoclonal antibody 2B9 was bound to phospho-PS peptide and was subsequently detected with streptavidin conjugated to peroxidase. Peroxidase substrate was then added to the microwell, and the fluorescence intensity was measured photometrically at 492 nm.

Measurement of PIP2-specific PLC activity. HUVECs were subjected to a 150% continuous stretch for the indicated times after preincubation in starvation medium for 6 h. The cells were washed with ice-cold PBS twice and lysed using buffer B (in mM: 50 Tris·HCl, pH 7.2, 1 NaHCO3, 1 NaHSO3, 1 benzamidine, and 0.1 PMSF). The assay mixture for PIP2 hydrolysis (50 µl) contained an enzyme source (4 µM protein concentration measured using DC Protein Assay, Bio-Rad, Richmond, CA), 35 mM HEPES (pH 7.2), 0.04% octylgricemide, 0.4 mM DTT, 110 mM KCl, 3.2 mM EGTA, and 8.3 mM CaCl2 to obtain a 100 nM free Ca2+ concentration and sonicated micelles of 3 nM PI(4,5)P2, 30 nM PE, and 3 nM [3H]PI(4,5)P2. An assay was performed at 30°C for 60 min and stopped by adding 0.25 ml of CHCl3/CH3OH/HCl (100:100:0.6 volume ratio) and 0.1 ml of 1 N HCl containing 5 mM EGTA. The mixture was centrifuged at 100 g for 5 min, and the radioactivity of the hydrophilic products and [3H]IP3 in the aqueous phase were measured using a liquid scintillation counter. Enzymatic activity was expressed as nanomolar PIP2 hydrolysis per milligram of protein per minute.

Measurement of [Ca2+]i. [Ca2+]i was measured as follows: HUVECs on silicone membranes were incubated for 30 min in EGM-UV2 medium containing the Ca2+ fluorescence indicator 10 µM fura-2 (Molecular Probes, Eugene, OR) and for another 10 min in a solution containing SES (in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.40) as described previously (32). [Ca2+]i was measured using the fura-2 method with a fluorescence microscope system (ARUGAS/HiSCA; Hamamatsu Photonic KK, Hamamatsu, Japan) with a x20 lens objective (Fluor 20; Zeiss) as described previously (32). Fluorescence ratio (R) was calculated using the following equation: R = (F340 B340)/(F380B380), where F340 and F380 are the emission intensities at 510 nm excited at 340 and 380 nm, respectively, and B340 and B380 are the corresponding autofluorescence values. All experiments were performed at room temperature (22 ± 3°C).

Measurement of IL-6 concentrations. After preincubation with various inhibitors, the cells were subjected to 150% continuous stretch for 6 h and then the supernatants were collected and frozen. IL-6 concentration was measured using a two-step sandwich enzyme immunoassay (R & D Systems, Minneapolis, MN).

Statistical analysis. Results are expressed as means (SD). Statistical analysis was performed with paired and unpaired Student's t-tests where appropriate. When analyzing two of three groups, P values were calculated according to Scheffé's method for multiple comparisons. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Morphological changes in HUVECs. HUVECs cultured on an elastic silicone membrane coated with FN showed a cobblestone appearance in the nonstretched condition. When the membrane was stretched 150%, the cells on the membrane were stretched to the same extent (Fig. 1A). To estimate the magnitude of cell elongation, a length of the long axis of the cells was measured at various time points. The length was increased to 150% immediately after chamber stretch and maintained for 4 h after stretch onset (Fig. 1B). HUVECs preincubated with 50 µM GRGDNP, an {alpha}5{beta}1 integrin-inhibitory peptide, for 3 h before stretch were also maintained at their stretched length for 4 h without significant cell detachment (data not shown).



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Fig. 1. Changes in cell appearance of human umbilical vein endothelial cells (HUVECs) subjected to uniaxial continuous stretch. A: phase-contrast photomicrographs of HUVECs cultured on an elastic silicone membrane in the nonstretched state (N-ST) and at 30 min, 2 h, and 4 h after 150% uniaxial continuous stretch. Bar, 30 µm; double-headed arrow, stretch axis. B: changes in the length of long axes of the cells before and during uniaxial continuous stretch. Each point represents mean (SD) of five determinations. *P < 0.05 vs. nonstretched control.

 
IKK phosphorylation and IL-6 mRNA expression in response to continuous stretch. Analysis of the promoter region of the IL-6 gene revealed the presence of a binding site for NF-{kappa}B (24, 53). NF-{kappa}B is sequestered in a latent form in the cytoplasm by the interaction with the inhibitory I{kappa}B proteins. In response to proper signals, I{kappa}B is phosphorylated by activated IKK and degraded. This leads to the release of active NF-{kappa}B that eventually is translocated in the nucleus and binds to DNA (19, 41). We have previously reported that IL-6 secretion in response to continuous stretch is dependent on IL-6 gene transcription via sequential activation of IKK and NF-{kappa}B (22). The time course of IKK activation using an in vitro kinase assay demonstrated that IKK phosphorylation started at 5 min after continuous stretch and peaked at 15 min, followed by a gradual decrease (Fig. 2A). Furthermore, we examined the time course of IL-6 mRNA expression using quantitative real-time PCR. The result indicated that the transcription of the IL-6 gene peaked at 2 h after stretching and gradually attenuated (Fig. 2B).



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Fig. 2. Activation of I{kappa}B kinase (IKK) phosphorylation and interleukin-6 (IL-6) mRNA expression in response to 150% uniaxial continuous stretch. A: autoradiogram of in vitro kinase assay of IKK-{alpha} using glutathione-S-transferase (GST)-I{kappa}B-{alpha}(1–54) and {gamma}-[32P]ATP as substrates at various time points. Bottom: activity of IKK relative to the nonstretched control. Each point represents the mean (SD) of four experiments. *P < 0.05 vs. nonstretched control. B: changes in the amount of IL-6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA during continuous stretch were analyzed using quantitative real-time PCR. Relative expression levels of IL-6 mRNA normalized to GAPDH mRNA expression are shown. Each point represents the mean (SD) of four experiments. *P < 0.05 vs. nonstretched control.

 
Changes in [Ca2+]i. To examine whether uniaxial continuous stretch results in an increase in [Ca2+]i in HUVECs, the cells cultured on an elastic silicone membrane were stretched by 150% continuously, and changes in [Ca2+]i were measured. As shown in Fig. 3A, in the presence of 2 mM extracellular Ca2+ (SES solution), onset of stretch elicited a transient increase in [Ca2+]i that slowly declined to the initial [Ca2+]i level at ~3 min. To determine whether the increase in [Ca2+]i observed in response to sustained stretch originated from extracellular Ca2+, we measured [Ca2+]i in cells stretched in SES containing 5 mM EGTA (CFM) or 20 µM Gd3+. A tiny rise in [Ca2+]i that might be released from intracellular Ca2+ stores was observed when extracellular Ca2+ was depleted by EGTA (Fig. 3B) and when Ca2+ influx was blocked by Gd3+ (data not shown). To investigate a possible contribution of intracellular Ca2+ releases, intracellular Ca2+ stores were depleted by pretreating the cells with 5 µM TG, an ER Ca2+ pump inhibitor, for 30 min before the stretch. This treatment showed little effect on the stretch induced Ca2+ transient (Fig. 3C), however, suggesting that the stretch-induced Ca2+ transient was caused mainly by Ca2+ influx through SA channels. When extracellular Ca2+ was removed by EGTA after depletion of the intracellular Ca2+ stores by TG, the [Ca2+]i rise was almost completely inhibited (Fig. 3D).



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Fig. 3. Changes in continuous-stretch induced increases in intracellular Ca2+ concentration ([Ca2+]i) under various pharmacological conditions. Confluent HUVECs in an elastic silicone chambers were subjected to 150% continuous stretch, and the change in [Ca2+]i level was measured as a ratio of 340- to 380-nm values (see EXPERIMENTAL PROCEDURES) in the presence of 2 mM extracellular Ca2+ (A), 5 mM EGTA (B), 5 µM thapsigargin (TG) (C), and a mixture of 5 µM TG and 5 mM EGTA (D). Each trace is representative of at least four independent repeatable experiments. Bottom traces indicate time course of stretch stimulus.

 
Effects of [Ca2+]i on continuous stretch-induced IKK activation and IL-6 mRNA expression. To investigate whether the stretch-induced change in [Ca2+]i plays an important role for the activation of IKK and expression of IL-6 mRNA, we examined the effects of SA channel blocker, extracellular Ca2+ depletion, intracellular Ca2+ depletion, and extra- and intracellular Ca2+ depletion. Under these conditions, continuous stretch-induced IKK activation at 15 min, at which point IKK was maximally activated, was inhibited only when 5 µM TG and 5 mM EGTA were coapplied (Fig. 4A). Similar to the results of IKK activation, IL-6 mRNA expression at 2 h, a peak time point of IL-6 mRNA expression, was inhibited only by the coapplication of TG and EGTA (Fig. 4B).



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Fig. 4. Requirement of [Ca2+]i increases in the continuous stretch-induced IKK phosphorylation and IL-6 mRNA expression. A: autoradiogram of in vitro kinase assay of IKK-{alpha} using GST-I{kappa}B-{alpha}(1–54) and {gamma}-[32P]ATP as substrates 15 min after stretch onset in the presence of 20 µM Gd3+, 5 mM EGTA, 5 µM TG, or a mixture of 5 µM TG and 5 mM EGTA. Bottom: kinase activity of IKK relative to the nonstretched control. Bars, means (SD) of four experiments. *P < 0.05 vs. 150% stretched in the absence of inhibitors. B: IL-6 and GAPDH mRNA levels 2 h after stretch onset under the same conditions as above analyzed using quantitative real-time PCR. Relative expression levels of IL-6 mRNA are shown. Bars, means (SD) of four experiments. *P < 0.05 vs. 150% stretched in absence of inhibitors. ST, stretch.

 
Role of integrin in continuous stretch-induced IKK activation and IL-6 mRNA expression. Integrins containing the {beta}1-subunit (e.g., {alpha}5{beta}1) may function as mechanosensors by interacting with cognate ECM proteins (e.g., fibronectin) (43). We therefore examined the involvement of integrin ({beta}1-subunit) in stretch-induced IKK activation and IL-6 mRNA expression. First, to clarify whether integrin could be activated by uniaxial continuous stretch, we examined tyrosine phosphorylation of focal adhesion proteins (FAK and paxillin) that have been used as markers for integrin activation (8). The stretch-induced increases in the tyrosine phosphorylation of FAK and paxillin were 2.1- and 2.2-fold that of baseline, respectively. The peak times of FAK and paxillin tyrosine phosphorylation were observed at ~5 min. The FAK tyrosine phosphorylation at 5 min was inhibited only by a mixture of TG and EGTA (Fig. 5A). If integrins mediate continuous stretch-induced IL-6 secretion, blocking integrins should inhibit stretch-induced IKK activation and IL-6 mRNA expression. To test this hypothesis, confluent monolayers of HUVECs were treated with the synthetic peptide GRGDNP, which competitively inhibits {alpha}5{beta}1 binding with ECM (10, 28). Preincubation of HUVECs with 50 µM GRGDNP for 3 h before stretch abolished stretch-induced IKK activation (Fig. 5B) and IL-6 mRNA expression (Fig. 5C), while the peptide showed little effect on the IKK activity and IL-6 mRNA expression in unstretched HUVECs (control). In contrast, stretch-induced [Ca2+]i transients were not affected significantly by the same treatment with GRGDNP (Fig. 5D, top). However, a tiny stretch-induced [Ca2+]i transient observed in the presence of EGTA was abolished by treatment with GRGDNP (Fig. 5D, bottom). It is suggested that stretch-induced integrin activation triggers intracellular TG-sensitive Ca2+ release that might be mediated by PLC-{gamma}-dependent IP3 production.



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Fig. 5. A: Western blot analysis using immunoprecipitation (IP) with anti-focal adhesion kinase (FAK) monoclonal antibody (top) and anti-paxillin monoclonal antibody (middle), and immunoblotting (IB) with anti-phosphotyrosine (PY20) at various time points. Western blot analysis using IP with anti-FAK monoclonal antibody at 5 min after stretch onset in the presence of 20 µM Gd3+, 5 µM TG, or a mixture of 5 µM TG and 5 mM EGTA (bottom). Each result is representative of three independent repeatable experiments. B: effects of an {alpha}5{beta}1 integrin-inhibitory peptide, GRGDNP, on IKK activity. Autoradiogram of in vitro kinase assay of IKK-{alpha} using GST-I{kappa}B-{alpha}(1–54) and {gamma}-[32P]ATP as substrates at 15 min after continuous stretch in the presence or absence of 50 µM GRGDNP. Bottom: kinase activity of IKK relative to the nonstretched control in the absence of GRGDNP. Bars, means (SD) of four experiments. *P < 0.05 vs. 150% stretched in the absence of GRGDNP. C: levels of IL-6 and GAPDH mRNA expression at 2 h after continuous stretch in the presence or absence of 50 µM GRGDNP were analyzed using quantitative real-time PCR. Expression of IL-6 mRNA relative to the nonstretched control in the absence of GRGDNP is shown. Bars, means (SD) of four experiments. *P < 0.05 vs. 150% stretched in the absence of GRGDNP. D: confluent HUVECs applied with GRGDNP for 3 h before the stretch were subjected to 150% continuous stretch, and the change in [Ca2+]i level was measured as a ratio of 340- to 380-nm values (top). Confluent HUVECs applied with GRGDNP for 3 h and with 5 mM EGTA just before the stretch were subjected to 150% continuous stretch, and the change in [Ca2+]i level was measured as a ratio of 340- to 380-nm values (bottom). Bottom: time course of stretch stimulus.

 
Involvement of PI3-kinase, PLC-{gamma}, and PKC in continuous stretch-induced IKK activation and IL-6 mRNA expression. To further characterize the signaling mechanisms responsible for continuous stretch-induced IKK activation and IL-6 mRNA expression, we examined the effects of inhibitors on the PI3-kinase-PLC-{gamma}-PKC signaling pathway that may play a major role in the endothelial response to mechanical stress. As expected, 50 µM LY-294002 (PI3-kinase inhibitor), 5 µM U-73122 (PLC-{gamma} inhibitor), or 50 µM H7 (PKC inhibitor) significantly attenuated the activation of IKK (Fig. 6A) and expression of IL-6 mRNA (Fig. 6B), while 300 nM H89 (PKA inhibitor) showed no significant effect on IKK activation and IL-6 mRNA expression in response to uniaxial continuous stretch in HUVECs (Fig. 6, A and B). These drugs showed little effect on IKK activity and IL-6 mRNA expression in unstretched HUVECs (control).



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Fig. 6. Effects of the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor (LY-294002), phospholipase C-{gamma} (PLC-{gamma}) inhibitor (U-73122), protein kinase C inhibitor (H7), or protein kinase A inhibitor (H89) on IKK phosphorylation and IL-6 mRNA expression. A: autoradiogram of in vitro kinase assay at 15 min after stretch onset in the presence of 50 µM LY-294002, 5 µM U-73122, 50 µM H7, or 300 nM H89. Bottom: kinase activity of IKK relative to nonstretched control. Bars, means (SD) of four experiments. *P < 0.05 vs. 150% stretched in the absence of inhibitors. B: IL-6 and GAPDH mRNA expression levels 2 h after continuous stretch in the presence of 50 µM LY-294002, 5 µM U-73122, 50 µM H7, or 300 nM H89 were analyzed using quantitative real-time PCR. Relative expression of IL-6 mRNA is shown. LY-294002 was applied at 1 h, and U-73122, H7, or H89 was applied 30 min before stretching. Bars, means (SD) of four experiments. *P < 0.05 vs. 150% stretched in the absence of inhibitors.

 
Involvement of integrin, PI3-kinase, PLC-{gamma}, and [Ca2+]i in continuous stretch-induced PKC activity. Many investigators have demonstrated that mechanical forces, including shear stress and cyclic stretch, result in PKC activation in ECs (46, 52). Our results described above strongly suggest that PKC is also activated by continuous stretch in HUVECs. We therefore performed a nonradioactive PKC assay with and without the use of several inhibitors of the integrin-dependent signaling pathway. PKC was transiently activated at 10 min after stretch onset (Fig. 7A). This activation was significantly inhibited by several drugs, including GRGDNP, LY-294002, U-73122, or a mixture of TG and EGTA (Fig. 7B).



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Fig. 7. Effects of [Ca2+]i levels, {alpha}5{beta}1 integrin-inhibitory peptide (GRGDNP), PI3-K inhibitor (LY-294002), or PLC-{gamma} inhibitor (U-73122) on protein kinase C (PKC) activity. A: time course change of PKC activity in response to continuous stretch. Bars, means (SD) of four experiments. *P < 0.05 vs. nonstretched control. B: relative kinase activity of PKC at 10 min after stretch onset in the presence of a mixture of 5 µM TG and 5 mM EGTA, 50 µM GRGDNP, 50 µM LY-294002, or 5 µM U-73122. Bars are means (SD) of four experiments. *P < 0.05 vs. 150% stretched in the absence of inhibitors.

 
Involvement of integrin and PI3-kinase in continuous stretch-induced PIP2-specific PLC activity. In cardiomyocytes, mechanical stretch leads to activation of PLC (39). Therefore, we tested the effect of continuous stretch on PLC activity with varying stretch durations ranging from 3 to 30 min. The level of [3H]IP3 products of PIP(4,5)P2 hydrolysis increased in a time-dependent manner within 5 min of stretch. The PIP2-specific PLC activity increased markedly at ~5 min, keeping almost at its level for 30 min (Fig. 8A). The PIP2-specific PLC activity at 5 min was inhibited by GRGDNP or the PI3-K inhibitor LY-294002 (Fig. 8B).



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Fig. 8. Effects of {alpha}5{beta}1 integrin-inhibitory peptide (GRGDNP) or PI3-K inhibitor (LY-294002) on PLC-{gamma} activity. A: changes in PLC-{gamma} activity under continuous stretch. Bars, means (SD) of two experiments. PIP2 hydrolysis is expressed as nmol·mg of protein–1·min–1. B: relative activity of PLC-{gamma} at 5 min after stretch onset in the presence of 50 µM GRGDNP or 50 µM LY-294002. Bars, means (SD) of four experiments. *P < 0.05 vs. 150% stretched in the absence of inhibitors.

 
Effects of various inhibitors on continuous stretch-induced IL-6 secretion. We have previously reported that IL-6 secretion in response to continuous stretch is markedly increased for the first 6 h and then gradually decreases and stabilizes for the next 12 h (22). To examine whether the various inhibitors tested above actually have effects on IL-6 production, we measured IL-6 concentration in the presence of various inhibitors using the two-step sandwich enzyme immunoassay. As shown in Table 1, IL-6 concentration in the supernatant at 6 h after the onset of continuous stretch significantly increased in the presence of Gd3+, EGTA, TG, or H89 but not in the presence of GRGDNP, LY-294002, U-73122, H7, or a coapplication of TG and EGTA, suggesting that IL-6 secretion requires an activation of integrin, PI3-K, PLC-{gamma}, or PKC and a [Ca2+]i increase. Throughout the course of the present experiments, >90% of the cells looked healthy and were adherent to the membrane even over 6 h. Moreover, treatments with these drugs did not significantly affect the basal level of IL-6 secretion, strongly indicating that pharmacological results in this study were not contaminated by nonspecific cell damage.


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Table 1. Effects of various inhibitors on continuous stretch-induced IL-6 release

 

    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we demonstrated that IL-6 secretion from HUVECs in response to continuous mechanical stretch requires outside-in signaling via integrins followed by activation of the PI3-kinase-PLC-{gamma}-PKC signaling cascade that leads to IKK/NF-{kappa}B activation.

There is increasing evidence indicating that integrins are important in mechanotransduction in ECs (43). Evidence for shear stress-induced activation of integrins has been provided by several studies in which shear-induced responses were blocked by Arg-Gly-Asp (RGD) peptide. Preincubation of HUVECs with RGD peptide abolished shear stress-induced Akt phosphorylation (9) and secretion of basic fibroblast growth factor (17). Inspired by these studies, we have investigated the contribution of integrins to continuous stretch-induced IL-6 production using RGD peptides. Our results demonstrate that the integrin-blocking RGD peptide (GRGDNP) attenuated IKK phosphorylation, IL-6 mRNA expression, and IL-6 secretion induced by continuous stretch. Although uniaxial continuous stretch caused a [Ca2+]i transient even in the presence of GRGDNP, probably via activation of SA channels, stretch-induced IL-6 production was not inhibited by the blockade of SA channels and external Ca2+ depletion. In contrast, inhibition of stretch-induced integrin activation by GRGDNP prevented IL-6 production. These results imply that the activation of integrins but not SA channels plays a pivotal role in eliciting continuous stretch-induced IL-6 secretion.

To clarify the downstream signaling molecules after integrin activation in response to continuous stretch, we investigated the involvement of PI3-kinase, PLC-{gamma}, and PKC using their inhibitors. All of the inhibitors impaired IKK activation, IL-6 mRNA expression, and IL-6 secretion in stretched HUVECs, suggesting that PI3-kinase, PLC-{gamma}, and PKC are necessary upstream effectors in IKK activation leading to IL-6 production. In addition, we examined the effects of these inhibitors on PKC and PLC-{gamma} activities to confirm our hypothetical signaling pathway. Continuous stretch-induced PKC and PLC-{gamma} activations were inhibited by an application of GRGDNP, LY-294002, or U-73122 and by GRGDNP or LY-294002, demonstrating that exposure of HUVECs to continuous strain leads to activation of the integrin-PI3-kinase-PLC-{gamma}-PKC signaling pathway.

Ca2+ often plays a critical regulatory role in signal transduction induced by mechanical stimuli. SA channels have been proposed as a potential candidate mechanotransducer because such channels have been found to induce Ca2+ influx in response to uniaxial cyclic stretch (20, 30, 31). Previously, we demonstrated that cyclic stretch-dependent Ca2+ influx is essential in several stretch-dependent signal transductions (1, 18, 50), while others have suggested that TG-sensitive intracellular Ca2+ stores serve as a mechanotransducer in stretch-induced signaling pathways (45, 48). In the present study, we have examined whether Ca2+ influx through SA channels or Ca2+ release from intracellular stores is important in continuous stretch-induced signaling that leads to IL-6 secretion. The results demonstrate that inhibition of neither Ca2+ influx nor Ca2+ release retarded the IL-6 production caused by continuous stretch. In contrast, when both intra- and extracellular Ca2+ were depleted before stretching the cells, a sustained stimulus no longer elicited a [Ca2+]i rise, PKC or IKK activation, IL-6 mRNA expression, and IL-6 secretion. One possible interpretation of these results is that a [Ca2+]i increase, regardless of its source, is necessary for stretch-induced IL-6 production. However, as shown in Fig. 5A, treatment of the cells with the combination of EGTA and TG might cause an unphysiologically low [Ca2+]i, leading to deterioration of intracellular Ca2+ homeostasis. Nevertheless, we tentatively hypothesize that a [Ca2+]i increase would be necessary for the stretch-induced IL-6 secretion because [Ca2+]i increase is generally required for ROS-generating enzymes (e.g., PKC) and the formation of free radicals (1, 3, 34). As for the mechanism of the stretch-induced [Ca2+]i increase, we prefer the idea that an integrin-dependent Ca2+ release from intracellular stores is more essential than Ca2+ influx through SA channels, because inhibition of integrin activation almost completely abolished stretch-induced IL-6 production, while blockade of Ca2+ influx had little effect on it. As mentioned in RESULTS, the inhibition of integrin activation by GRGDNP eliminated a stretch-induced [Ca2+]i transient sensitive to TG. Whereas TG blocked an integrin-dependent intracellular Ca2+ releases, IL-6 production was not inhibited by TG treatment. Under this condition, Ca2+ influx might play a critical role in ROS generation leading to NF-{kappa}B activation.

Our previous study demonstrated that upregulation of IL-6 mRNA requires >30 min of sustained stretch (22), although the activation of IKK occurred within 15 min and subsided within 30 min. This discrepancy in the time courses implies that NF-{kappa}B translocation into the nucleus and subsequent IL-6 gene transcription may involve some unknown mechanisms that remained to be resolved.

In summary, our present results suggest that continuous stretch-induced IL-6 secretion in HUVECs is dependent on both outside-in signaling via integrins, which activate the PI3-K-PLC-{gamma}-[Ca2+]i increase-PKC-IKK-NF-{kappa}B signaling pathway, leading to IL-6 secretion.


    GRANTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Grants for Scientific Research 13480216 (to M. Sokabe) and 13671297 and 15591398 (to M. Nagino), Scientific Research on Priority Areas Grant 15086270 (to M. Sokabe), Creative Scientific Research Grant 16GS0308 (to M. Sokabe) from the Ministry of Education, Culture, Sports Science and Technology, and a grant from the Japan Space Forum (to M. Sokabe).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. Makoto Nakanishi (Department of Biochemistry, Nagoya City University Medical School, Nagoya, Japan) for providing GST-I{kappa}B-{alpha}(1–54).


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Sokabe, Department of Physiology, Nagoya Univ. Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan (E-mail: msokabe{at}med.nagoya-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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