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
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ABSTRACT |
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nuclear factor-B; phosphatidylinositol 3-kinase; phospholipase C-
; protein kinase C; intracellular Ca2+ concentration
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-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-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-
(PLC-
) leads to the activation of PLC-
(2, 11, 35). PLC-
plays an important role in the regulation of various intracellular signaling mechanisms. Activated PLC-
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-
B (13, 49). We also previously reported that an antioxidant inhibits the IL-6 production by suppressing NF-
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-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-
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-
B activation during uniaxial continuous stretch.
In the present study, we have investigated the upstream signaling mechanism of continuous stretch-induced IKK/NF-B activation that leads to IL-6 secretion from ECs, with special attention to the PI3-kinase-PLC-
-PKC pathway regulated by integrins. In addition, we examined the importance of [Ca2+]i levels modulated by continuous stretch in activating this signal cascade.
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EXPERIMENTAL PROCEDURES |
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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(-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-
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- 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
-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
-[32P]ATP and 2 µl of GST-I
B-
(154) 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)/(F380 B380), 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.
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RESULTS |
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DISCUSSION |
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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-, 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-
, 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-
activities to confirm our hypothetical signaling pathway. Continuous stretch-induced PKC and PLC-
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-
-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-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-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--[Ca2+]i increase-PKC-IKK-NF-
B signaling pathway, leading to IL-6 secretion.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bae YS, Cantley LG, Chen CS, Kim SR, Kwon KS, and Rhee SG. Activation of phospholipase C- by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 273: 44654469, 1998.
3. Balasubramanyam M, Koteswari AA, Kumar RS, Monickaraj SF, Maheswari JU, and Mohan V. Curcumin-induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications. J Biosci 28: 715721, 2003.[ISI][Medline]
4. Bhullar IS, Li YS, Miao H, Zandi E, Kim M, Shyy JY, and Chien S. Fluid shear stress activation of IB kinase is integrin-dependent. J Biol Chem 273: 3054430549, 1998.
5. Cheng JJ, Chao YJ, and Wang DL. Cyclic strain activates redox-sensitive proline-rich tyrosine kinase 2 (PYK2) in endothelial cells. J Biol Chem 277: 4815248157, 2002.
6. Danciu TE, Adam RM, Naruse K, Freeman MR, and Hauschka PV. Calcium regulates the PI3K-Akt pathway in stretched osteoblasts. FEBS Lett 536: 193197, 2003.[CrossRef][ISI][Medline]
7. Dang PMC, Fontayne A, Hakim J, El Benna J, and Périanin A. Protein kinase C phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst. J Immunol 166: 12061213, 2001.
8. Davies PF, Robotewskyj A, and Griem ML. Quantitative studies of endothelial cell adhesion: directional remodeling of focal adhesion sites in response to flow forces. J Clin Invest 93: 20312038, 1994.[ISI][Medline]
9. Dimmeler S, Assmus B, Hermann C, Haendeler J, and Zeiher AM. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res 83: 334341, 1998.
10. D'Souza SE, Ginsberg MH, and Plow EF. Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem Sci 16: 246250, 1991.[CrossRef][ISI][Medline]
11. Falasca M, Logan SK, Lehto VP, Baccante G, Lemmon MA, and Schlessinger J. Activation of phospholipase C by PI 3-kinase-induced PH domain-mediated membrane targeting. EMBO J 17: 414422, 1998.
12. Fee D, Grzybicki D, Dobbs M, Ihyer S, Clotfelter J, Macvilay S, Hart MN, Sandor M, and Fabry Z. Interleukin 6 promotes vasculogenesis of murine brain microvessel endothelial cells. Cytokine 12: 655665, 2000.[CrossRef][ISI][Medline]
13. Flohé L, Brigelius-Flohé R, Saliou C, Traber MG, and Packer L. Redox regulation of NF-B activation. Free Radic Biol Med 22: 11151126, 1997.[CrossRef][ISI][Medline]
14. Fontayne A, Dang PMC, Gougerot-Pocidalo MA, and El Benna J. Phosphorylation of p47phox sites by PKC,
II,
, and
: effect on binding to p22phox and on NADPH oxidase activation. Biochemistry 41: 77437750, 2002.[CrossRef][ISI][Medline]
15. Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, and Tsichlis PN. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81: 727736, 1995.[CrossRef][ISI][Medline]
16. Geiger RV, Berk BC, Alexander RW, and Nerem RM. Flow-induced calcium transients in single endothelial cells: spatial and temporal analysis. Am J Physiol Cell Physiol 262: C1411C1417, 1992.
17. Gloe T, Sohn HY, Meininger GA, and Pohl U. Shear stress-induced release of basic fibroblast growth factor from endothelial cells is mediated by matrix interaction via integrin v
3. J Biol Chem 277: 2345323458, 2002.
18. Inoh H, Ishiguro N, Sawazaki S, Amma H, Miyazu M, Iwata H, Sokabe M, and Naruse K. Uni-axial cyclic stretch induces the activation of transcription factor nuclear factor B in human fibroblast cells. FASEB J 16: 405407, 2002.
19. Karin M. The beginning of the end: IB kinase (IKK) and NF-
B activation. J Biol Chem 274: 2733927342, 1999.
20. Kato T, Ishiguro N, Iwata H, Kojima T, Ito T, and Naruse K. Up-regulation of COX2 expression by uni-axial cyclic stretch in human lung fibroblast cells. Biochem Biophys Res Commun 244: 615619, 1998.[CrossRef][ISI][Medline]
21. Kawai M, Naruse K, Komatsu S, Kobayashi S, Nagino M, Nimura Y, and Sokabe M. Mechanical stress-dependent secretion of interleukin 6 by endothelial cells after portal vein embolization: clinical and experimental studies. J Hepatol 37: 240246, 2002.[CrossRef][ISI][Medline]
22. Kobayashi S, Nagino M, Komatsu S, Naruse K, Nimura Y, Nakanishi M, and Sokabe M. Stretch-induced IL-6 secretion from endothelial cells requires NF-B activation. Biochem Biophys Res Commun 308: 306312, 2003.[CrossRef][ISI][Medline]
23. Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T, Chien S, and Shyy JY. Fluid shear stress activation of focal adhesion kinase: linking to mitogen-activated protein kinases. J Biol Chem 272: 3045530462, 1997.
24. Libermann TA and Baltimore D. Activation of interleukin-6 gene expression through the NF-B transcription factor. Mol Cell Biol 10: 23272334, 1990.[ISI][Medline]
25. Liu Y, Chen BP, Lu M, Zhu Y, Stemerman MB, Chien S, and Shyy JY. Shear stress activation of SREBP1 in endothelial cells is mediated by integrins. Arterioscler Thromb Vasc Biol 22: 7681, 2002.
26. Maruo N, Morita I, Shirao M, and Murota S. IL-6 increases endothelial permeability in vitro. Endocrinology 131: 710714, 1992.[Abstract]
27. Matsushita H, Lee K, and Tsao PS. Cyclic strain induces reactive oxygen species production via an endothelial NAD(P)H oxidase. J Cell Biochem 81, Suppl 36: 99106, 2001.
28. Muller JM, Chilian WM, and Davis MJ. Integrin signaling transduces shear stress-dependent vasodilation of coronary arterioles. Circ Res 80: 320326, 1997.
29. Nakamura T, Suzuki S, Ushiyama C, Shimada N, and Koide H. Effect of phosphodiesterase III inhibitor on plasma concentrations of endothelin-1 and tumour necrosis factor in patients with acute heart failure. Acta Cardiol 57: 1921, 2002.[ISI][Medline]
30. Naruse K, Sai X, Yokoyama N, and Sokabe M. Uniaxial cyclic stretch induces c-src activation and translocation in human endothelial cells via SA channel activation. FEBS Lett 441: 111115, 1998.[CrossRef][ISI][Medline]
31. Naruse K, Yamada T, Sai XR, Hamaguchi M, and Sokabe M. Pp125FAK is required for stretch dependent morphological response of endothelial cells. Oncogene 17: 455463, 1998.[CrossRef][ISI][Medline]
32. Naruse K, Yamada T, and Sokabe M. Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch. Am J Physiol Heart Circ Physiol 274: H1532H1538, 1998.
33. Nollert MU, Eskin SG, and McIntire LV. Shear stress increases inositol trisphosphate levels in human endothelial cells. Biochem Biophys Res Commun 170: 281287, 1990.[CrossRef][ISI][Medline]
34. Pahl HL and Baeuerle PA. Activation of NF-B by ER stress requires both Ca2+ and reactive oxygen intermediates as messengers. FEBS Lett 392: 129136, 1996.[CrossRef][ISI][Medline]
35. Rameh LE, Rhee SG, Spokes K, Kazlauskas A, Cantley LC, and Cantley LG. Phosphoinositide 3-kinase regulates phospholipase C-mediated calcium signaling. J Biol Chem 273: 2375023757, 1998.
36. Regnault V, Rivat C, and Stoltz JF. Affinity purification of human plasma fibronectin on immobilized gelatin. J Chromatogr 432: 93102, 1988.[Medline]
37. Reyes-Reyes M, Mora N, Zentella A, and Rosales C. Phosphatidylinositol 3-kinase mediates integrin-dependent NF-B and MAPK activation through separate signaling pathways. J Cell Sci 114: 15791589, 2001.
38. Rosales OR, Isales CM, Barrett PQ, Brophy C, and Sumpio BE. Exposure of endothelial cells to cyclic strain induces elevations of cytosolic Ca2+ concentration through mobilization of intracellular and extracellular pools. Biochem J 326: 385392, 1997.[ISI][Medline]
39. Ruwhof C, van Wamel JT, Noordzij LA, Aydin S, Harper JC, and van der Laarse A. Mechanical stress stimulates phospholipase C activity and intracellular calcium ion levels in neonatal rat cardiomyocytes. Cell Calcium 29: 7383, 2001.[CrossRef][ISI][Medline]
40. Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, and Parsons JT. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci USA 89: 51925196, 1992.
41. Senftleben U and Karin M. The IKK/NF-B pathway. Crit Care Med 30, Suppl 1: S18S26, 2002.[CrossRef]
42. Shirinsky VP, Antonov AS, Birukov KG, Sobolevsky AV, Romanov YA, Kabaeva NV, Antonova GN, and Smirnov VN. Mechano-chemical control of human endothelium orientation and size. J Cell Biol 109: 331339, 1989.[Abstract]
43. Shyy JY and Chien S. Role of integrins in endothelial mechanosensing of shear stress. Circ Res 91: 769775, 2002.
44. Signorelli SS, Malaponte MG, Di Pino L, Costa MP, Pennisi G, and Mazzarino MC. Venous stasis causes release of interleukin 1 (IL-1
), interleukin 6 (IL-6) and tumor necrosis factor
(TNF
) by monocyte-macrophage. Clin Hemorheol Microcirc 22: 311316, 2000.[ISI][Medline]
45. Taskinen P and Ruskoaho H. Stretch-induced increase in atrial natriuretic peptide secretion is blocked by thapsigargin. Eur J Pharmacol 308: 295300, 1996.[CrossRef][ISI][Medline]
46. Tseng H, Peterson TE, and Berk BC. Fluid shear stress stimulates mitogen-activated protein kinase in endothelial cells. Circ Res 77: 869878, 1995.
47. Turner CE, Glenney JR Jr, and Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol 111: 10591068, 1990.[Abstract]
48. Walker LM, Publicover SJ, Preston MR, Said Ahmed MAA, and El Haj AJ. Calcium-channel activation and matrix protein upregulation in bone cells in response to mechanical strain. J Cell Biochem 79: 648661, 2000.[CrossRef][ISI][Medline]
49. Wang DS, Proffit D, and Tsao PS. Mechanotransduction of endothelial oxidative stress induced by cyclic strain. Endothelium 8: 283291, 2001.[Medline]
50. Wang JG, Miyazu M, Matsushita E, Sokabe M, and Naruse K. Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem Biophys Res Commun 288: 356361, 2001.[CrossRef][ISI][Medline]
51. Wirtz HR and Dobbs LG. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 250: 12661269, 1990.[ISI][Medline]
52. Yamamoto H, Teramoto H, Uetani K, Igawa K, and Shimizu E. Cyclic stretch upregulates interleukin-8 and transforming growth factor-1 production through a protein kinase C-dependent pathway in alveolar epithelial cells. Respirology 7: 103109, 2002.[CrossRef][ISI][Medline]
53. Zhang YH, Lin JX, and Vilcek J. Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a B-like sequence. Mol Cell Biol 10: 38183823, 1990.[ISI][Medline]