FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1985773558> ?p ?o ?g. }

• W1985773558 endingPage "40306" @default.
• W1985773558 startingPage "40301" @default.
• W1985773558 abstract "Mesangial cells (MC) grown on extracellular matrix protein-coated plates and exposed to cyclic strain/relaxation proliferate and produce extracellular matrix protein, providing an in vitro model of signaling in stretched MC. Intracellular transduction of mechanical strain involves mitogen-activated protein kinases, and we have shown that p42/44 mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) is activated by cyclic strain in MC. In vivo studies show that increased production of nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing glomerular capillary pressure, and we have observed that NO attenuates stretch-induced ERK activity in MC via generation of cyclic guanosine monophosphate (cGMP). Accordingly, we sought to determine whether NO affects strain-induced ERK activity after strain and how this is mediated. Strain-induced ERK activity was dependent on time and magnitude of stretch and was maximal after 10 min at −27 kilopascals. Actin cytoskeleton disruption with cytochalasin D abrogated this. The non-metabolizable cGMP analogue 8-bromo cyclic GMP (8-Br-cGMP) dose-dependently attenuated strain-induced ERK activity. Cytoskeletal stabilization with jasplakinolide prevented this inhibitory effect of 8-Br-cGMP. Cyclic strain increased nuclear translocation of phospho-ERK by immunofluorescent microscopy, again attenuated by 8-Br-cGMP. Jasplakinolide prevented the inhibitory effect of 8-Br-cGMP on activated ERK nuclear translocation after strain. Strain increased ERK-dependent AP-1 nuclear protein binding, which was attenuated by cytochalasin D and 8-Br-cGMP. These data indicate that cGMP can inhibit cyclic strain-induced ERK activity, nuclear translocation, and AP-1 nuclear protein binding. Cytoskeletal disruption leads to the same effect, whereas cytoskeleton stabilization reverses the effect of 8-Br-cGMP. Thus, NO inhibits strain-induced ERK activity by cytoskeletal destabilization. Mesangial cells (MC) grown on extracellular matrix protein-coated plates and exposed to cyclic strain/relaxation proliferate and produce extracellular matrix protein, providing an in vitro model of signaling in stretched MC. Intracellular transduction of mechanical strain involves mitogen-activated protein kinases, and we have shown that p42/44 mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) is activated by cyclic strain in MC. In vivo studies show that increased production of nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing glomerular capillary pressure, and we have observed that NO attenuates stretch-induced ERK activity in MC via generation of cyclic guanosine monophosphate (cGMP). Accordingly, we sought to determine whether NO affects strain-induced ERK activity after strain and how this is mediated. Strain-induced ERK activity was dependent on time and magnitude of stretch and was maximal after 10 min at −27 kilopascals. Actin cytoskeleton disruption with cytochalasin D abrogated this. The non-metabolizable cGMP analogue 8-bromo cyclic GMP (8-Br-cGMP) dose-dependently attenuated strain-induced ERK activity. Cytoskeletal stabilization with jasplakinolide prevented this inhibitory effect of 8-Br-cGMP. Cyclic strain increased nuclear translocation of phospho-ERK by immunofluorescent microscopy, again attenuated by 8-Br-cGMP. Jasplakinolide prevented the inhibitory effect of 8-Br-cGMP on activated ERK nuclear translocation after strain. Strain increased ERK-dependent AP-1 nuclear protein binding, which was attenuated by cytochalasin D and 8-Br-cGMP. These data indicate that cGMP can inhibit cyclic strain-induced ERK activity, nuclear translocation, and AP-1 nuclear protein binding. Cytoskeletal disruption leads to the same effect, whereas cytoskeleton stabilization reverses the effect of 8-Br-cGMP. Thus, NO inhibits strain-induced ERK activity by cytoskeletal destabilization. mesangial cell(s) mitogen-activated protein kinase extracellular signal-regulated kinase pascal(s) 8-bromo cyclic guanosine monophosphate phosphate-buffered saline Tris-buffered saline S-nitroso-N-acetylpenicillamine Glomerular mesangial cells (MC)1 are positioned as architectural supports for capillary loops and are therefore exposed to pulsatile stretch/relaxation (1Akai Y. Homma T. Burns K.D. Yasuda T. Badr K.F. Harris R.C Am. J. Physiol. 1994; 267: C482-C490Crossref PubMed Google Scholar). Whereas little resident glomerular cell proliferation or sclerosis is demonstrable in normal animals (2Harris R.C. Haralson M.A. Badr K.F. Lab. Invest. 1992; 66: 548-554PubMed Google Scholar), MC proliferation and matrix production, eventually resulting in sclerosis, can be induced by maneuvers that increase intraglomerular pressure by 10 mmHg (3Brenner B.M. Kidney Int. 1983; 23: 647-655Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 4Dworkin L.D. Feiner H.D. J. Clin. Invest. 1986; 77: 797-809Crossref PubMed Scopus (199) Google Scholar, 5Dworkin L.D. Hostetter T.H. Rennke H.G. Brenner B.M. J. Clin. Invest. 1984; 73: 1448-1461Crossref PubMed Google Scholar). Moreover, in these models, preventing the intraglomerular pressure rise attenuates sclerosis (5Dworkin L.D. Hostetter T.H. Rennke H.G. Brenner B.M. J. Clin. Invest. 1984; 73: 1448-1461Crossref PubMed Google Scholar, 6Meyer T.W. Anderson S. Rennke H.G. Brenner B.M. Am. J. Med. 1985; 79: 31-36Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 7Anderson S. Meyer T.W. Rennke H.G. Brenner B.M. J. Clin. Invest. 1985; 76: 612-619Crossref PubMed Scopus (842) Google Scholar). We and others have shown reduction of sclerosis and MC proliferation in remnant glomeruli by oral l-arginine supplementation to increase NO production (8Ingram A. Parbtani A. Thai K. Ly H. Shankland S.J. Morrissey G. Scholey J.W. Kidney Int. 1995; 48: 1857-1865Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 9Reyes A.A. Purkerson M.L. Karl I. Klahr S. Am. J. Kidney Dis. 1992; 20: 168-176Abstract Full Text PDF PubMed Scopus (128) Google Scholar). l-Arginine increases nitric oxide production by the remnant kidney and reduces glomerular endothelin-1 expression but does not lower glomerular capillary pressure (8Ingram A. Parbtani A. Thai K. Ly H. Shankland S.J. Morrissey G. Scholey J.W. Kidney Int. 1995; 48: 1857-1865Abstract Full Text PDF PubMed Scopus (34) Google Scholar).The effects of mechanical forces on MC in vitro can be modelled by culturing cells in wells with deformable bottoms and then applying a vacuum to the wells to generate alternating cycles of strain and relaxation. Initial experiments using this methodology showed induction of mRNA for the proto-oncogene and AP-1 transcription factor component c-fos at 30 min (1Akai Y. Homma T. Burns K.D. Yasuda T. Badr K.F. Harris R.C Am. J. Physiol. 1994; 267: C482-C490Crossref PubMed Google Scholar). Subsequently, increases in both MC proliferation (2Harris R.C. Haralson M.A. Badr K.F. Lab. Invest. 1992; 66: 548-554PubMed Google Scholar) and collagenous and non-collagenous extracellular matrix protein synthesis were observed by 48 h, the sine qua non of sclerotic injury (10Yasuda T. Akai Y. Kondo S. Becker B.N. Homma T. Owada S. Ishida M. Harris R.C. Contrib. Nephrol. 1996; 118: 222-228Crossref PubMed Google Scholar, 11Riser B.L. Cortes P. Zhao X. Bernstein J. Dumler F. Narins R.G. J. Clin. Invest. 1992; 90: 1932-1943Crossref PubMed Scopus (247) Google Scholar).We and others have studied the link between mechanical stress and c-fos induction in stressed MC (12Hirakata M. Kaname S. Chung U.G. Joki N. Hori Y. Noda M. Takuwa Y. Okazaki T. Fujita T. Katoh T. Kurokawa K. Takuwa Y.X.O.T. Kidney Int. 1997; 51: 1028-1036Abstract Full Text PDF PubMed Scopus (95) Google Scholar, 13Kawata Y. Mizukami Y. Fujii Z. Sakamura T. Yoshida K. Matsuzaki M. J. Biol. Chem. 1998; 273: 16905-16912Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 14Ingram A. Thai K. Ly H. Kang M. Scholey J.W. Kidney Int. 1999; 55: 476-485Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). We demonstrated increases in all three canonical mitogen-activated protein kinase (MAPK) pathways in response to strain (14Ingram A. Thai K. Ly H. Kang M. Scholey J.W. Kidney Int. 1999; 55: 476-485Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The most well described mammalian MAPK cascade, p42/44 or ERK, is well recognized to lie upstream of AP-1 (15Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 16Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). ERK-dependent AP-1 induction has been demonstrated in endothelial cells in response to shear (17Jalali S. Li Y.S. Sotoudeh M. Yuan S. Li S. Chien S. Shyy J.Y. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 227-234Crossref PubMed Scopus (217) Google Scholar).In vivo, cyclic strain in the aortic wall activates ERK and AP-1 (18Xu Q. Liu Y. Gorospe M. Udelsman R. Holbrook N.J. J. Clin. Invest. 1996; 97: 508-514Crossref PubMed Scopus (184) Google Scholar), and glomerular ERK activation and AP-1 nuclear protein binding have been shown in response to angiotensin II infusion (19Hamaguchi A. Kim S. Yano M. Yamanaka S. Iwao H. J. Am. Soc. Nephrol. 1998; 9: 372-380PubMed Google Scholar). AP-1 activation may be important in the pathogenesis of glomerular sclerosis, because AP-1 activation has been shown to mediate transforming growth factor beta-1 induction (20Kim S.J. Angel P. Lafyatis R. Hattori K. Kim K.Y. Sporn M.B. Karin M. Roberts A.B. Mol. Cell. Biol. 1990; 10: 1492-1497Crossref PubMed Google Scholar, 21Mulder K.M. Cytokine Growth Factor Rev. 2000; 11: 23-35Crossref PubMed Scopus (383) Google Scholar).Recent data indicates that the actin cytoskeleton is important for ERK signaling. Cytochalasin-D prevented strain-induced ERK activity in vascular smooth muscle cells (22Numaguchi K. Eguchi S. Yamakawa T. Motley E.D. Inagami T. Circ. Res. 1999; 85: 5-11Crossref PubMed Scopus (183) Google Scholar). Interestingly, LIM kinase-1 is intimately involved in induction of serum response factor by serum, suggesting a central role for the actin cytoskeleton in intracellular signaling (23Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). ERK does interact with the actin cytoskeleton (24Leinweber B.D. Leavis P.C. Grabarek Z. Wang C.L. Morgan K.G. Biochem. J. 1999; 344: 117-123Crossref PubMed Scopus (124) Google Scholar), and ERK signaling in response to lysophosphatidic acid (25Della Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) or epidermal growth factor (26Arnautov A.M. Nikol'skii N.N. Tsitologiya. 1998; 40: 639-647PubMed Google Scholar) is dependent on the presence of an intact actin cytoskeleton.Recent studies indicate a role for NO in inhibition of cytoskeletal organization. Several NO donors and a constitutively active form of cyclic guanosine monophosphate (cGMP) inhibited MC adhesion to extracellular matrix protein via inhibition of focal adhesion kinase phosphorylation and actin cytoskeletal disruption (27Yao J. Schoecklmann H.O. Prols F. Gauer S. Sterzel R.B. Kidney Int. 1998; 53: 598-608Abstract Full Text PDF PubMed Scopus (44) Google Scholar). In vascular smooth muscle cells, cGMP-dependent kinase phosphorylated and inactivated RhoA, thereby disrupting the actin cytoskeleton (28Sauzeau V. Le Jeune H. Cario-Toumaniantz C. Smolenski A. Lohmann S.M. Bertoglio J. Chardin P. Pacaud P. Loirand G. J. Biol. Chem. 2000; 275: 21722-21729Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Consequently, we hypothesized that nitric oxide, via cyclic GMP, would limit MC ERK signaling in response to mechanical strain through cytoskeletal disruption.EXPERIMENTAL PROCEDURESCell CultureHarlan Sprague-Dawley rat mesangial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum (Life Technologies, Inc.), streptomycin (100 μg/ml), penicillin (100 units/ml), and 2 mm glutamine at 37 °C in 95% air, 5% CO2. Experiments were carried out in cells between passage 15–20.Application of Strain/RelaxationMesangial cells (5 × 104/well) were plated on to 6-well plates with flexible bottoms coated with bovine type I collagen (Flexcell International Corp., McKeesport, PA). Cells were grown to confluence for 72 h and then rendered quiescent by incubation for 24 h in Dulbecco's modified Eagle's medium with 0.5% fetal calf serum. To characterize the time of maximum response, cells were initially exposed to cycles of strain/relaxation for periods of 2, 5, 10, 30, and 60 min generated by a cyclic vacuum produced by a computer-driven system (Flexercell Strain Unit 2000, Flexcell International Corp.). Plates were exposed to continuous cycles of strain/relaxation, with each cycle being 0.5 s of strain and 0.5 s of relaxation, for a total of 60 cycles per min. Initially, the vacuum pressures used were −10 to −27 kPa, inducing a 16–28% elongation in the diameter of the surface. Subsequent experiments were performed at the time and strain level of maximal response, 10 min and −27 kPa, respectively (average 28% elongation in diameter of the plates).For the experiments studying NO effects, 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP, Sigma) was added in the indicated concentrations 10 min prior to the initiation of stretch protocols. For experiments studying cytoskeletal manipulation, cytochalasin-D (Sigma) or jasplakinolide (Molecular Probes, Eugene, OR) was added prior to the initiation of stretch protocols at the indicated concentrations.p42/44 MAPK (ERK) ActivityProtein Isolation and Western BlottingInitially, the time course and concentration dependence of ERK activity in response to stretch were studied, and subsequent experiments were performed at −27 kPa at 10 min. Cultures were serum starved overnight prior to stretch protocols. After stretch protocols with or without inhibitors, medium was removed, and the cells were washed once with ice-cold PBS. Protein was isolated as described above, and 40 μg of sample was separated on a 12% SDS-polyacrylamide gel electrophoresis gel. After electroblotting to a nitrocellulose membrane, membranes were incubated for 3 h at room temperature with 25 ml of blocking buffer (1× TBS, 0.1% Tween 20 with 5% w/v nonfat dry milk) and then overnight at 4 °C with ERK1/2 MAPK (Thr202/Tyr204) polyclonal antibody (1:1000)(New England Biolabs, Beverly, MA) in 10 ml of antibody dilution buffer (1× TBS, 0.05% Tween 20 with 5% bovine serum albumin) with gentle rocking at 4 °C. Membranes were then washed three times with 1× TBS, 0.05% Tween 20 and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000) in 10 ml of blocking buffer for 45 min at room temperature. After three further washes in TBS, the membrane was incubated with LumiGlo reagent and then exposed to x-ray film.Activity Assays200 μg of total cellular protein was incubated with immobilized phospho-p44/42 MAPK (Thr202/Tyr204) monoclonal antibody (15 μl, New England Biolabs) with gentle rocking overnight at 4 °C. Lysates were microcentrifuged for 30 s at 14,000 rpm to recover the beads, and the pellet was washed twice with 0.5 ml of 1× lysis buffer. For the kinase assay, after immunoprecipitation, pellets were washed twice with 0.5 ml of kinase buffer (25 mm Tris, 5 mmβ-glycerophosphate, 2 mm dithiothreitol, 0.1 mm sodium orthovanadate, 10 mmMgCl2). ERK activity was then measured by suspending the pellet in 50 μl of 1× kinase buffer containing 200 μm ATP and 2 μg of Elk1 fusion protein as substrate. After incubation for 30 min at 30 °C, the reaction was terminated with 25 μm 3× SDS sample buffer (187.5 mmTris-HCl (pH 6.8), 6% w/v SDS, 30% glycerol, 150 mmdithiothreitol, 0.3% w/v bromphenol blue), boiled for 5 min, vortexed, and then microcentrifuged for 2 min. 20 μl of sample was then run on a 10% non-reducing SDS-polyacrylamide gel electrophoresis gel. After blotting to nitrocellulose, membranes were incubated for 3 h at room temperature with 25 ml of blocking buffer (1× TBS, 0.1% Tween 20 with 5% w/v nonfat dry milk) and then overnight at 4 °C with phospho-specific anti-Elk1 (Ser383) antibody 1:1000 in 10 ml of antibody dilution buffer. Membranes were washed three times with 1× TBS, 0.05% Tween 20 and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000) for 1 h at room temperature. After three further washes in TBS, the membrane was processed as above.Fluorescence MicroscopyERK Nuclear TranslocationAfter each strain protocol with or without inhibitors, cells were washed three times with PBS and fixed with 3.7% formaldehyde (300 μl/well) for 10 min at room temperature. Cells were washed three times with PBS, then permeabilized in 100% methanol for 5 min at −20 °C, washed again with PBS, and incubated with a 1:50 dilution of anti-phospho-ERK (Thr180/Tyr182) (New England Biolabs) in PBS for 30 min at room temperature. Cells were washed three times in PBS and incubated with a 1:50 dilution of an Alexa 488 goat anti-rabbit IgG (H + L) conjugate (Molecular Probes) in PBS for 30 min at room temperature in the dark. Cells were washed and then mounted by removing the flexible base from each well and placing it directly on a glass slide using one drop of anti-fade mount medium (Slow Fade, Molecular Probes). A drop of mount medium was then placed on top of the cells and covered with a glass slip. Slides were stored at 4 °C in the dark until confocal laser scanning microscopy was performed using a Bio-Rad MRC-600 confocal microscope (Bio-Rad) within 10 days.F-actin StainingMC were fixed with formaldehyde exactly as described above and then permeabilized by dipping in acetone for 5 min at −20 °C. Subsequently, Texas Red phalloidin solution (Molecular Probes) was applied for 20 min at room temperature. Cells were then washed, mounted, and analyzed exactly as described above.Nuclear Protein Binding to AP-1 Consensus SequencesAfter each strain protocol, MC were washed in cold PBS, and nuclear extracts were prepared by lysis in hypotonic buffer (20 mm Hepes, pH 7.9, 1 mm EDTA, 1 mmEGTA, 20 mm NaF, 1 mmNa3VO4, 1 mmNa4P2O7, 1 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 0.6% Nonidet P-40), homogenized, and sedimented at 16,000 ×g for 20 min at 4 °C. Pelleted nuclei were resuspended in hypotonic buffer containing 0.42 m NaCl2, 20% glycerol and rotated for 30 min at 4 °C. After centrifugation for 20 min at 16,000 × g, the supernatant containing nuclear proteins was collected, and the protein concentration was measured with the Bio-Rad assay kit.Radiolabeled AP-1 consensus oligonucleotides were prepared by incubating 2 μl of consensus oligonucleotide (1.75 pmol/μl, Promega), 1 μl of T4 polynucleotide kinase 10× buffer, 1 μl of [32P]ATP (3,000 Ci/ml) (Amersham Pharmacia Biotech), and 5 μl of nuclease-free water for 10 min at 37 °C. The reaction was stopped by adding 1 μl of 0.5 m EDTA. Unlabeled [32P]ATP was removed from the oligonucleotide mixture with Chroma-Spin STE-10 columns (CLONTECH).Nuclear proteins (3 μg) were incubated with 2 μg of poly(dI-dC)·poly(dI·dC) (Amersham Pharmacia Biotech) in binding buffer (20 mm HEPES pH 7.9, 1.8 mmMgCl2, 2 mm dithiothreitol, 0.5 mm EDTA, 0.5 mg/ml bovine serum albumin) for 30 min at room temperature and then reacted with radiolabeled consensus oligonucleotides at room temperature for 20 min (50,000–100,000 cpm). Reaction mixtures were electrophoresed in a 6% polyacrylamide gel and autoradiographed. Competition experiments were performed with 100× excess unlabeled AP-1 consensus oligonucleotides.DISCUSSIONIn the best-characterized animal model of chronic renal failure, the subtotally nephrectomized rat, increased glomerular capillary pressure (as little as 20%) triggers MC responses that ultimately result in glomerulosclerosis (10Yasuda T. Akai Y. Kondo S. Becker B.N. Homma T. Owada S. Ishida M. Harris R.C. Contrib. Nephrol. 1996; 118: 222-228Crossref PubMed Google Scholar, 11Riser B.L. Cortes P. Zhao X. Bernstein J. Dumler F. Narins R.G. J. Clin. Invest. 1992; 90: 1932-1943Crossref PubMed Scopus (247) Google Scholar, 31Yasuda T. Kondo S. Homma T. Harris R.C. J. Clin. Invest. 1996; 98: 1991-2000Crossref PubMed Scopus (144) Google Scholar). In vitro studies of the application of cyclic mechanical strain to MC have demonstrated that this stimulus results in MC proliferation (2Harris R.C. Haralson M.A. Badr K.F. Lab. Invest. 1992; 66: 548-554PubMed Google Scholar, 32Harris R.C. Akai Y. Yasuda T. Homma T. Kidney Int. Suppl. 1994; 45: S17-S21PubMed Google Scholar, 33Kawata Y. Fujii Z. Sakamura T. Kitano M. Suzuki N. Matsuzaki M. Biochim. Biophys. Acta. 1998; 1401: 195-202Crossref PubMed Scopus (23) Google Scholar) and production of collagenous protein (2Harris R.C. Haralson M.A. Badr K.F. Lab. Invest. 1992; 66: 548-554PubMed Google Scholar) and fibronectin (12Hirakata M. Kaname S. Chung U.G. Joki N. Hori Y. Noda M. Takuwa Y. Okazaki T. Fujita T. Katoh T. Kurokawa K. Takuwa Y.X.O.T. Kidney Int. 1997; 51: 1028-1036Abstract Full Text PDF PubMed Scopus (95) Google Scholar).We and others have studied how the mechanical signal is transduced in mesangial cells. The first site of transduction occurs at the cell membrane (34Ingram A.J. Scholey J.W. Curr. Opin. Nephrol. Hypertension. 2000; 9: 49-55Crossref PubMed Scopus (15) Google Scholar). Initial studies of mechanical strain in MC noted increased proliferation in concert with induction of expression of the proto-oncogene and AP-1 component c-fos (1Akai Y. Homma T. Burns K.D. Yasuda T. Badr K.F. Harris R.C Am. J. Physiol. 1994; 267: C482-C490Crossref PubMed Google Scholar). Both down-regulation of protein kinase C (32Harris R.C. Akai Y. Yasuda T. Homma T. Kidney Int. Suppl. 1994; 45: S17-S21PubMed Google Scholar) and calcium chelation (32Harris R.C. Akai Y. Yasuda T. Homma T. Kidney Int. Suppl. 1994; 45: S17-S21PubMed Google Scholar) were shown to attenuate c-fos expression induced by strain. Studies of the proliferative effects of mechanical strain showed matrix dependence. Cells adherent to fibronectin showed the greatest strain-induced proliferative response, and this was inhibited by coincubation with RGD peptides (35Wilson E. Sudhir K. Ives H.E. J. Clin. Invest. 1995; 96: 2364-2372Crossref PubMed Scopus (268) Google Scholar). Integrin-focal adhesion complex interactions have been studied, and tyrosine phosphorylation of the focal adhesion-associated kinase pp125FAK was seen in stretched MC (36Hamasaki K. Mimura T. Furuya H. Morino N. Yamazaki T. Komuro I. Yazaki Y. Nojima Y. Komuro I.X.Y.Y. Biochem. Biophys. Res. Commun. 1995; 212: 544-549Crossref PubMed Scopus (41) Google Scholar). Integrin binding to extracellular matrix protein leads to clustering and the formation of a signaling complex termed a focal adhesion that associates with actin filaments, leading to their reorganization into filamentous stress fibers (37Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3784) Google Scholar). Stretched MC elongate and align in the direction of stress, and actin filaments coalesce and orient themselves along this long axis (2Harris R.C. Haralson M.A. Badr K.F. Lab. Invest. 1992; 66: 548-554PubMed Google Scholar). Various tyrosine kinases associate with focal adhesions after their formation, in particular focal adhesion kinase and the Src family kinases (38Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar). In mesangial cells, cyclic strain-induced increases of vascular permeability factor mRNA are Src-dependent (39Gruden G. Thomas S. Burt D. Lane S. Chusney G. Sacks S. Viberti G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12112-12116Crossref PubMed Scopus (112) Google Scholar), providing evidence that the assembly of the focal adhesion complex and its association with kinases are important in the transduction of mechanical signals in MC. Further downstream, Src and focal adhesion kinase activate Ras via the Shc-Grb2-Sos complex (40Schlaepfer D.D. Hunter T. J. Biol. Chem. 1997; 272: 13189-13195Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Ras is activated in response to cyclic strain in vascular smooth muscle cells (41Komuro I. Kudo S. Yamazaki T. Zou Y. Shiojima I. Yazaki Y. FASEB J. 1996; 10: 631-636Crossref PubMed Scopus (206) Google Scholar, 42Li C. Hu Y. Mayr M. Xu Q. J. Biol. Chem. 1999; 274: 25273-25280Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar).Signaling of mechanical stimuli to the cell nucleus after membrane events involves the ubiquitous MAPK cascades. ERK interacts with the actin cytoskeleton (24Leinweber B.D. Leavis P.C. Grabarek Z. Wang C.L. Morgan K.G. Biochem. J. 1999; 344: 117-123Crossref PubMed Scopus (124) Google Scholar), which is indispensible for signaling in response to lysophosphatidic acid (25Della Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) or epidermal growth factor (26Arnautov A.M. Nikol'skii N.N. Tsitologiya. 1998; 40: 639-647PubMed Google Scholar). Each of the MAPK cascades consists of three protein kinases acting sequentially, a mitogen-activated protein kinase kinase activator (MKK), a mitogen-activated protein kinase activator (MEK), and a mitogen-activated protein kinase (15Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). We have shown activation of all three cascades in MC in response to cyclic strain (14Ingram A. Thai K. Ly H. Kang M. Scholey J.W. Kidney Int. 1999; 55: 476-485Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). More recently, we have demonstrated inhibition of strain-induced ERK and stress-activated protein kinase/c-Jun NH2-terminal kinase by NO donors (43Ingram A.J. James L. Ly H. Thai K. Cai L. Scholey J.W. Kidney Int. 2000; 58: 1067-1077Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In the nucleus, both ERK and stress-activated protein kinase/c-Jun NH2-terminal kinase may induce AP-1 (15Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 16Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). As noted, cyclic strain in the aortic wall activates ERK and AP-1 (18Xu Q. Liu Y. Gorospe M. Udelsman R. Holbrook N.J. J. Clin. Invest. 1996; 97: 508-514Crossref PubMed Scopus (184) Google Scholar), and glomerular ERK activation and AP-1 nuclear protein binding occur with angiotensin II infusion (19Hamaguchi A. Kim S. Yano M. Yamanaka S. Iwao H. J. Am. Soc. Nephrol. 1998; 9: 372-380PubMed Google Scholar), a maneuver that would be expected to increase glomerular pressure.Accordingly, we sought to determine how NO interfered with transmission of the stretch signal to ERK. Given recent data showing that 8-bromo-cGMP inhibited actin cytoskeletal organization via cyclic GMP kinase-mediated phosphorylation of RhoA (28Sauzeau V. Le Jeune H. Cario-Toumaniantz C. Smolenski A. Lohmann S.M. Bertoglio J. Chardin P. Pacaud P. Loirand G. J. Biol. Chem. 2000; 275: 21722-21729Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), we elected to study whether the effect of NO donors on stretch-induced ERK activity was through cytoskeletal disruption.We first observed that stretch led to the formation of actin stress fibers within 10 min in MC. Prevention of stretch-induced actin stress fiber formation in MC completely eliminated the usual stretch-induced ERK activity. This is consistent with a recent report in vascular smooth muscle cells using the same stretch system (22Numaguchi K. Eguchi S. Yamakawa T. Motley E.D. Inagami T. Circ. Res. 1999; 85: 5-11Crossref PubMed Scopus (183) Google Scholar). Having established cytoskeletal dependence of stretch-induced ERK activity in MC, we sought to characterize the effects of NO on the actin cytoskeleton after stretch. Phalloidin staining of F-actin revealed that either SNAP or 8-bromo-cGMP led to cytoskeletal disassembly after 10 min of incubation. This has been observed in resting aortic smooth muscle cells with 40 min of 100 μm 8-bromo-cGMP (28Sauzeau V. Le Jeune H. Cario-Toumaniantz C. Smolenski A. Lohmann S.M. Bertoglio J. Chardin P. Pacaud P. Loirand G. J. Biol. Chem. 2000; 275: 21722-21729Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Pre-incubation with 8-bromo-cGMP also inhibited stretch-induced ERK activation in MC in this study, consistent with its effects on the cytoskeleton. In accord with this, phospho-ERK induction and nuclear translocation, which was easily visualized after 10 min of stretch, was essentially prevented by pre-incubation with 8-bromo-cGMP.Whereas these data demonstrate an association between cytoskeletal disruption and prevention of strain-induced ERK activity by 8-bromo-cGMP, we sought to strengthen this link by studying cytoskeletal stabilization. A major finding of this study is that incubation with jasplakinolide in addition to 8-bromo-cGMP prior to and during stretch prevented 8-bromo-cGMP-mediated inhibition of strain-induced ERK1/2 activity. Furthermore, induction and nuclear translocation of phospho-ERK by immunofluorescent microscopy was also preserved by co-incubation with jasplakinolide. The effects of jasplakinolide on the actin cytoskeleton are complex, but jasplakinolide appears to stabilize actin stress fibers by decreasing the dissociation rate of actin subunits (29Bu" @default.
• W1985773558 created "2016-06-24" @default.
• W1985773558 creator A5014537109 @default.
• W1985773558 creator A5016117730 @default.
• W1985773558 creator A5017873319 @default.
• W1985773558 creator A5051306669 @default.
• W1985773558 creator A5079462177 @default.
• W1985773558 creator A5088373188 @default.
• W1985773558 date "2000-12-01" @default.
• W1985773558 modified "2023-09-27" @default.
• W1985773558 title "NO Inhibits Stretch-induced MAPK Activity by Cytoskeletal Disruption" @default.
• W1985773558 cites W1543126301 @default.
• W1985773558 cites W1560179018 @default.
• W1985773558 cites W1573821584 @default.
• W1985773558 cites W1576457818 @default.
• W1985773558 cites W1975755598 @default.
• W1985773558 cites W1984203028 @default.
• W1985773558 cites W1994651714 @default.
• W1985773558 cites W2002367577 @default.
• W1985773558 cites W2005294216 @default.
• W1985773558 cites W2022140257 @default.
• W1985773558 cites W2023687572 @default.
• W1985773558 cites W2027318170 @default.
• W1985773558 cites W2040508577 @default.
• W1985773558 cites W2045141910 @default.
• W1985773558 cites W2048927276 @default.
• W1985773558 cites W2051292594 @default.
• W1985773558 cites W2056736540 @default.
• W1985773558 cites W2062690889 @default.
• W1985773558 cites W2063033753 @default.
• W1985773558 cites W2063928700 @default.
• W1985773558 cites W2069982781 @default.
• W1985773558 cites W2073951465 @default.
• W1985773558 cites W2087391683 @default.
• W1985773558 cites W2091031177 @default.
• W1985773558 cites W2093833100 @default.
• W1985773558 cites W2096714571 @default.
• W1985773558 cites W2101907071 @default.
• W1985773558 cites W2109436786 @default.
• W1985773558 cites W2118897660 @default.
• W1985773558 cites W2121452261 @default.
• W1985773558 cites W2125862902 @default.
• W1985773558 cites W2138852525 @default.
• W1985773558 cites W2149438199 @default.
• W1985773558 cites W2149907763 @default.
• W1985773558 cites W2150603117 @default.
• W1985773558 cites W2158439879 @default.
• W1985773558 cites W2208774147 @default.
• W1985773558 cites W2291863582 @default.
• W1985773558 cites W2325923824 @default.
• W1985773558 cites W2411195602 @default.
• W1985773558 cites W4213141530 @default.
• W1985773558 cites W4249092084 @default.
• W1985773558 doi "https://doi.org/10.1074/jbc.m007018200" @default.
• W1985773558 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10984494" @default.
• W1985773558 hasPublicationYear "2000" @default.
• W1985773558 type Work @default.
• W1985773558 sameAs 1985773558 @default.
• W1985773558 citedByCount "53" @default.
• W1985773558 countsByYear W19857735582012 @default.
• W1985773558 countsByYear W19857735582013 @default.
• W1985773558 countsByYear W19857735582014 @default.
• W1985773558 countsByYear W19857735582015 @default.
• W1985773558 countsByYear W19857735582016 @default.
• W1985773558 countsByYear W19857735582017 @default.
• W1985773558 countsByYear W19857735582020 @default.
• W1985773558 countsByYear W19857735582021 @default.
• W1985773558 countsByYear W19857735582022 @default.
• W1985773558 countsByYear W19857735582023 @default.
• W1985773558 crossrefType "journal-article" @default.
• W1985773558 hasAuthorship W1985773558A5014537109 @default.
• W1985773558 hasAuthorship W1985773558A5016117730 @default.
• W1985773558 hasAuthorship W1985773558A5017873319 @default.
• W1985773558 hasAuthorship W1985773558A5051306669 @default.
• W1985773558 hasAuthorship W1985773558A5079462177 @default.
• W1985773558 hasAuthorship W1985773558A5088373188 @default.
• W1985773558 hasBestOaLocation W19857735581 @default.
• W1985773558 hasConcept C11960822 @default.
• W1985773558 hasConcept C12554922 @default.
• W1985773558 hasConcept C142669718 @default.
• W1985773558 hasConcept C1491633281 @default.
• W1985773558 hasConcept C185592680 @default.
• W1985773558 hasConcept C55493867 @default.
• W1985773558 hasConcept C57074206 @default.
• W1985773558 hasConcept C86803240 @default.
• W1985773558 hasConcept C95444343 @default.
• W1985773558 hasConceptScore W1985773558C11960822 @default.
• W1985773558 hasConceptScore W1985773558C12554922 @default.
• W1985773558 hasConceptScore W1985773558C142669718 @default.
• W1985773558 hasConceptScore W1985773558C1491633281 @default.
• W1985773558 hasConceptScore W1985773558C185592680 @default.
• W1985773558 hasConceptScore W1985773558C55493867 @default.
• W1985773558 hasConceptScore W1985773558C57074206 @default.
• W1985773558 hasConceptScore W1985773558C86803240 @default.
• W1985773558 hasConceptScore W1985773558C95444343 @default.
• W1985773558 hasIssue "51" @default.
• W1985773558 hasLocation W19857735581 @default.
• W1985773558 hasOpenAccess W1985773558 @default.

• next