FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1978163952> ?p ?o ?g. }

• W1978163952 endingPage "20617" @default.
• W1978163952 startingPage "20612" @default.
• W1978163952 abstract "Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. Studies on cells in three-dimensional collagen matrices have shown that fibroblasts switch between proliferative and quiescence phenotypes, depending upon whether matrices are attached or floating during matrix remodeling. Previous work showed that cell signaling through the ERK pathway was decreased in fibroblasts in floating matrices. In the current research, we extend the previous findings to show that serum stimulation of fibroblasts in floating matrices does not result in ERK translocation to the nucleus. In addition, there was decreased serum activation of upstream members of the ERK signaling pathway, MEK and Raf, even though Ras became GTP loaded. The findings suggest that quiescence of fibroblasts in floating collagen matrices may result from a defect in Ras coupling to its downstream effectors. Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. Studies on cells in three-dimensional collagen matrices have shown that fibroblasts switch between proliferative and quiescence phenotypes, depending upon whether matrices are attached or floating during matrix remodeling. Previous work showed that cell signaling through the ERK pathway was decreased in fibroblasts in floating matrices. In the current research, we extend the previous findings to show that serum stimulation of fibroblasts in floating matrices does not result in ERK translocation to the nucleus. In addition, there was decreased serum activation of upstream members of the ERK signaling pathway, MEK and Raf, even though Ras became GTP loaded. The findings suggest that quiescence of fibroblasts in floating collagen matrices may result from a defect in Ras coupling to its downstream effectors. Form and function of multicellular organisms depends on cell proliferation, migration, and differentiation. Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. Studies on cells in three-dimensional matrices suggest that reciprocal and adaptive mechanical interactions play a role in the regulation of morphogenesis. That is, fibroblasts cultured in collagen matrices switch between a proliferative, activated phenotype on one hand and a quiescent, resting phenotype on the other, depending upon whether the matrices are attached or floating during matrix remodeling (1Grinnell F. J. Cell Biol. 1994; 124: 401-404Crossref PubMed Scopus (977) Google Scholar, 2Kessler D. Dethlefsen S. Haase I. Plomann M. Hirche F. Krieg T. Eckes B. J. Biol. Chem. 2001; 276: 36575-36585Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). These differences in phenotype have been attributed to changes in mechanical interactions. As a consequence of remodeling, fibroblasts develop isometric tension in attached matrices but remain mechanically unloaded in floating matrices (3Grinnell F. Trends Cell Biol. 2000; 10: 362-365Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 4Tomasek J.J. Gabbiani G. Hinz B. Chaponnier C. Brown R.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3159) Google Scholar). In addition to becoming quiescent, fibroblasts in floating matrices also begin to enter apoptosis (5Grinnell F. Zhu M. Carlson M.A. Abrams J.M. Exp. Cell Res. 1999; 248: 608-619Crossref PubMed Scopus (239) Google Scholar, 6Zhu Y.K. Umino T. Liu X.D. Wang H.J. Romberger D.J. Spurzem J.R. Rennard S.I. In Vitro Cell. Dev. Biol. Anim. 2001; 37: 10-16Crossref PubMed Scopus (94) Google Scholar, 7Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 8Graf R. Freyberg M. Kaiser D. Friedl P. Apoptosis. 2002; 7: 493-498Crossref PubMed Scopus (47) Google Scholar), and apoptosis has been implicated in the disappearance of wound fibroblasts at the end of repair (9Lorena D. Uchio K. Costa A.M. Desmouliere A. Wound Repair Regen. 2002; 10: 86-92Crossref PubMed Scopus (51) Google Scholar). The proliferation of normal (untransformed) cells typically requires a combination of signals generated by growth factor stimulation and cell adhesion. One target for these signals is the canonical ERK 1The abbreviations used are: ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; siRNA, small interfering RNA. signaling pathway, Ras-Raf-MEK-ERK, which has been suggested in many cells to participate in a wide range of cell functions from proliferation to differentiation to senescence (10Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1218) Google Scholar, 11Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3548) Google Scholar). Cell adhesion, shape, and cytoskeletal organization influence the strength and duration of signals through the MAP kinase pathway, whose sustained activation is required for cell cycle progression (12Huang S. Ingber D.E. Nat. Cell Biol. 1999; 1: E131-E138Crossref PubMed Scopus (650) Google Scholar, 13Schwartz M.A. Ginsberg M.H. Nat. Cell Biol. 2002; 4: E65-E68Crossref PubMed Scopus (681) Google Scholar, 14Howe A.K. Aplin A.E. Juliano R.L. Curr. Opin. Genet. Dev. 2002; 12: 30-35Crossref PubMed Scopus (249) Google Scholar, 15Roovers K. Assoian R.K. Bioessays. 2000; 22: 818-826Crossref PubMed Scopus (424) Google Scholar, 16Danen E.H. Yamada K.M. J. Cell. Physiol. 2001; 189: 1-13Crossref PubMed Scopus (395) Google Scholar, 17Murphy L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 4: 556-564Crossref PubMed Scopus (761) Google Scholar, 18Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3821) Google Scholar). In previous studies, we compared ERK activation in fibroblasts in attached and floating collagen matrices. We found that, in floating matrices, there was decreased signaling through the ERK pathway (19Rosenfeldt H. Grinnell F. J. Biol. Chem. 2000; 275: 3088-3092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) along with down-regulation of the cell cycle regulatory protein cyclin D1 and an increase in the cyclin-dependent kinase inhibitor p27Kip1 (20Fringer J. Grinnell F. J. Biol. Chem. 2001; 276: 31047-31052Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In the current research, we have extended the previous findings to show that serum stimulation of fibroblasts in floating matrices does not cause ERK translocation to the nucleus. Moreover, when upstream members of the ERK signaling were analyzed, we observed that fibroblasts in floating matrices showed robust activation of Ras (GTP loading) but markedly reduced phosphorylation of Raf and MEK. These findings suggest that the quiescence of fibroblasts in floating collagen matrices may result from a defect in Ras coupling to its downstream effectors. Materials—Dulbecco's modified Eagle's medium (DMEM) was purchased from Invitrogen. Fetal bovine serum (FBS) was purchased from Intergen Co. (Purchase, NY). Type I collagen (Vitrogen) was purchased from Cohesion Corp. (Palo Alto, CA). Platelet-derived growth factor (PDGF) (BB isotype) was obtained from Upstate Biotechnology. Antibodies were obtained as follows: phospho-MEK1/2, MEK 1/2, and phospho-p44/42 MAP kinase from Cell Signaling Inc; Cdk4, ERK2, and Raf-1 (monoclonal) from Santa Cruz Biotechnology; Ras from Upstate Biotechnology; and phospho-Raf-1 (Y340,Y341) from Calbiochem. Horseradish peroxidase-goat anti-rabbit and anti-mouse were purchased from ICN Pharmaceuticals. Fluorescein isothiocyanate (FITC) goat anti-rabbit was purchased from Molecular Probes. Agarose-conjugated Raf-1 RBD was purchased from Upstate Biotechnology. Oligo-fectAMINE and Opti-MEM1, were purchased from Invitrogen. Collagen Matrix and Monolayer Culture—Fibroblasts from human foreskin specimens (<10 passages) were maintained in Falcon 75-cm2 tissue culture flasks in DMEM supplemented with 10% FBS (DMEM/10% FBS). Collagen matrix cultures were prepared using Vitrogen 100 collagen as described previously (19Rosenfeldt H. Grinnell F. J. Biol. Chem. 2000; 275: 3088-3092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 20Fringer J. Grinnell F. J. Biol. Chem. 2001; 276: 31047-31052Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The cell/collagen mixture containing 106 fibroblasts/ml and 1.5 mg/ml collagen in DMEM without serum was prewarmed to 37 °C for 3–4 min, after which aliquots (0.2 ml) were polymerized in Corning 24-well culture plates for 60 min at 37 °C in a humidified incubator with 5% CO2. After polymerization, 1.0 ml of DMEM containing 10% fetal bovine serum and 50 μg/ml ascorbic acid was added to each well. Floating matrices were gently released from the underlying culture dish with a spatula immediately after polymerization. Unless indicated otherwise, matrices were incubated for 8 h in DMEM/10% FBS followed by incubation overnight in low serum medium (DMEM/0.5% FBS) and then stimulated with DMEM containing serum or growth factors as indicated. SDS-PAGE and Immunoblotting—SDS-PAGE and immunoblotting were performed as described previously (19Rosenfeldt H. Grinnell F. J. Biol. Chem. 2000; 275: 3088-3092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 20Fringer J. Grinnell F. J. Biol. Chem. 2001; 276: 31047-31052Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Cells were extracted in Nonidet P-40 extraction buffer (0.5% Nonidet P-40, 150 mm NaCl, 3 mm KCl, 6 mm Na2HPO4, 1 mm KH2PO4, 0.5 mm MgCl2, 1 mm CaCl2, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mm 4-(2-aminoethyl)-benzene-sulfonyl fluoride, 50 mm NaF, 1 mm Na3VO4,and1mm (NH4)2MoO4,pH 7.4) (50 μl/matrix and 1 ml/60-mm tissue culture dish) by homogenization (50 strokes) using a Dounce homogenizer (pestle B; Wheaton Scientific, Millville, NJ). Samples used for the Ras-GTP pull-down assay were extracted in Mg2+ lysis buffer purchased from Upstate Biotechnology. Samples were clarified by centrifugation for 10 min at 16,000 × g (Beckman Microfuge), and the supernatants were either incubated with reagents for the Ras-GTP pull-down assay or else dissolved in 1× reducing sample buffer (250 mm Tris, 2% SDS, 40% glycerol, 20% mercaptoethanol, 0.04% bromphenol blue) and boiled for 5 min. Equal amounts of protein (equalized by the lactate dehydrogenase assay) were subjected to SDS-PAGE electrophoresis using 10% acrylamide minislab gels. After transfer to polyvinylidene difluoride membranes, Western blotting was performed according to the primary antibody manufacturers' specifications. Immunofluorescence Microscopy—Collagen matrix samples were fixed for 10 min at 22 °C with 3% paraformaldehyde in DPBS (150 mm NaCl, 3 mm KCl, 6 mm Na2HPO4, 1 mm KH2PO4, 1 mm CaCl2, and 0.5 mm MgCl2, pH 7.2), washed for 2× 10 min with DPBS, blocked for 30 min with DPBS containing 1% bovine serum albumin and 1% glycine, and then permeabilized for 10 min with –20 °C methanol. Subsequently, the samples were washed 2× 10 min with DPBS and then blocked with 1.5% goat serum/DPBS. Primary antibodies against ERK2 or Raf-1 were diluted in 1.5% goat serum/DPBS (1:100 and 1:25, respectively) and added to cells for 2 h at 37 °C. The samples were then washed and stored overnight in DPBS at 4 °C. Subsequently, samples were washed 6× 10 min with DPBS, then the secondary antibody in 1.5% goat serum/DPBS was added for 1 h at 37 °C. Final washing consisted of 6× 20 min with DPBS. Monolayer samples were treated similar to above except permeabilization was carried out with –20 °C methanol for 5 min, and incubation with primary and secondary antibodies was for 30 min at 22 °C. Samples were mounted on glass slides with Fluoromount G, and observations and images were made using a Leica TCS-SP confocal laser-scanning inverted microscope and TCSNT workstation. DNA Synthesis—DNA synthesis was determine as previously (19Rosenfeldt H. Grinnell F. J. Biol. Chem. 2000; 275: 3088-3092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 20Fringer J. Grinnell F. J. Biol. Chem. 2001; 276: 31047-31052Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Cells in collagen matrices and monolayer culture were incubated in DMEM/10% FBS containing 5 μCi/ml [3H]thymidine (specific activity, 5 Ci/mmol) for 1 h. Subsequently, cells were harvested from matrices and monolayer culture and treated with 10% trichloroacetic acid in phosphate saline containing 125 μg/ml bovine serum albumin for 20 min at 4 °C. Precipitates were collected on Whatman 2.5-cm glass microfiber filters, washed, and transferred to scintillation vials containing 10 ml of Budget Solve. Radioactivity was counted using a Beckman scintillation counter (LS 6000 SC). Radioactive counts were adjusted for equal cell numbers by measuring the lactate dehydrogenase activity of an aliquot of the harvested cells with the lactate dehydrogenase diagnostic kit (Sigma). Raf-1 Gene Silencing—Raf-1 gene silencing was accomplished using siRNA (21Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8162) Google Scholar). Oligonucleotide sequences used were 5′-GACGUUCCUGAAGCUUGCCTT-3′ and 5′-GGCAAGCUUCAGGAACGUCTT-3′ (prepared by the University of Texas Southwestern siRNA core facility). To accomplish high efficiency transfection, fibroblast cultures (50–60% confluent) were first rinsed with antibiotic-free DMEM and then treated with trypsin-EDTA for 1 min to elicit cell rounding but not detachment. Subsequently, antibiotic-free 10% FBS/DMEM was added 4:1 to quench the trypsin. After cells were rinsed with antibiotic-free DMEM, they were incubated with AB solution (1:1 ratio; A = 1 μm siRNA annealed oligonucleotides in Opti-MEM1; B = 7% Oligo-fectAMINE, 93% opti-MEM1) diluted 1 to 5 with antibiotic-free DMEM. After 48 h, transfection medium was removed and replaced with 10% FBS/DMEM (plus antibiotics) for an additional 24 h. Phosphorylation of ERK and MEK—Fibroblasts incubated overnight in attached or floating collagen matrices were stimulated with medium containing 10% FBS. Fig. 1 shows that, before serum stimulation, levels of phospho-ERK and phospho-MEK detected by immunoblotting with specific antibodies were very low. Within 10 min following stimulation, MEK and ERK became phosphorylated in fibroblasts in attached collagen matrices, and phosphorylation persisted for at least 60 min. On the other hand, there was markedly less stimulation of ERK and MEK phosphorylation in fibroblasts in floating collagen matrices. ERK phosphorylation is required for nuclear translocation (22Lenormand P. Sardet C. Pages G. L'Allemain G. Brunet A. Pouyssegur J. J. Cell Biol. 1993; 122: 1079-1088Crossref PubMed Scopus (585) Google Scholar, 23Seth A. Gonzalez F.A. Gupta S. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 24796-24804Abstract Full Text PDF PubMed Google Scholar). To learn whether the extent of ERK phosphorylation observed in Fig. 1 was sufficient to cause ERK translocation to the nucleus, immunofluorescence-staining experiments were carried out. Fig. 2, A and B show monolayer controls in which it can be observed that translocation of endogenous ERK2 from the cytoplasm to the nucleus results in a loss of the “empty” appearance of the nucleus. Translocation of ERK also occurred in fibroblasts in attached collagen matrices (Fig. 2, C and D) but not in cells in floating matrices (Fig. 2, E and F). Counts made on cells in six microscope fields selected at random showed that nuclear translocation of ERK occurred in 29 of 30 cells in attached matrices, but only 2 of 30 cells in floating matrices. GTP-Loading of Ras—The findings above demonstrated that, in floating matrices, the ERK signaling pathway of fibroblasts was disrupted somewhere upstream of MEK. Consequently, subsequent studies focused on Ras and Raf. Pulldown assays to detect GTP-loaded Ras were carried out using the Ras binding domain of Raf. Fig. 3A shows a representative blot, and Fig. 3B shows data from three experiments combined and quantified. Serum stimulation caused transient Ras activation, which amounted to about a 3-fold increase compared with unstimulated cells in attached collagen matrices and a ∼7-fold increase compared with unstimulated cells in floating matrices. Higher stimulation of RasGTP in floating matrices compared with attached matrices was not unique to serum. For instance, Fig. 3C shows that RasGTP stimulation by 50 ng/ml PDGF had a similar pattern. These results demonstrated that cells in floating matrices could respond to serum-stimulation by robust activation of Ras. Higher levels of Ras-GTP loading in floating matrices compared with attached matrices may have occurred because of the absence of feedback down-regulation of Ras-GTP by activated ERK (24Haugh J.M. Huang A.C. Wiley H.S. Wells A. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 34350-34360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 25Foschi M. Chari S. Dunn M.J. Sorokin A. EMBO J. 1997; 16: 6439-6451Crossref PubMed Scopus (142) Google Scholar, 26Fucini R.V. Okada S. Pessin J.E. J. Biol. Chem. 1999; 274: 18651-18658Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) Studies also were carried out to compare Ras activation in fibroblasts in attached collagen matrices compared with a monolayer culture. Fig. 4 shows that the Ras-GTP loading response of cells in attached matrices to serum stimulation was less than that for fibroblasts in monolayer cultures even if the culture dishes were coated with collagen. Similarly, when we tested the DNA synthetic response 24 h after serum stimulation (Fig. 4, bottom row), fibroblasts in attached collagen matrices showed less of in increase in DNA synthesis than cells in monolayer cultures, consistent with the lower level of Ras activation. Raf Phosphorylation—Having demonstrated that Ras activation occurred in both attached and floating matrices, we then analyzed Raf-1 activation. Ideally, Raf-1 activity should have been measured directly by in vitro kinase assay. Although the assay worked for cells in monolayer culture, we were unable to adapt the method to the fibroblast collagen matrix culture model. Therefore, other methods were employed. Activation of Raf-1 involves phosphorylation on several residues (10Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1218) Google Scholar, 27Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar), including Tyr-341 (28Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell. Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (304) Google Scholar, 29Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (527) Google Scholar, 30Tilbrook P.A. Colley S.M. McCarthy D.J. Marais R. Klinken S.P. Arch. Biochem. Biophys. 2001; 396: 128-132Crossref PubMed Scopus (23) Google Scholar) and Ser-338 (31King A.J. Wireman R.S. Hamilton M. Marshall M.S. FEBS Lett. 2001; 497: 6-14Crossref PubMed Scopus (35) Google Scholar). Fig. 5A shows that, in attached but not floating collagen matrices, serum stimulation resulted in a pronounced increase in Raf-1 phosphorylation at Tyr-341 as detected by immunoblotting with specific antibodies. Equal loading of the immunoblots was demonstrated by staining for Cdk4 (20Fringer J. Grinnell F. J. Biol. Chem. 2001; 276: 31047-31052Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Variability in total Raf-1 staining was unexplained but occurred with two different antibodies (Santa Cruz Biotechnology and BD Transduction Laboratories). Phosphorylation of Raf-1 also could be observed by gel shift when SDS-PAGE was carried out for a long enough time to allow Raf-1 to reach almost to the bottom of the gels. Under these conditions, as shown by Fig. 5B, the shift in Raf-1 was observed following serum stimulation of cells in attached but not floating matrices. This band shift was eliminated when samples where pretreated with λ protein phosphatase (New England Biolabs, 40 units/sample, 30 min, 37 °C) before SDS-PAGE (not shown), indicating that Raf phosphorylation was responsible for the shift. Raf-1 Localization—Taken together, the foregoing results suggested that activation of Ras but not Raf-1 occurred in fibroblasts in floating collagen matrices following serum stimulation. Besides changes in phosphorylation, Raf-1 activation also depends on changes in Raf-1 localization (10Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1218) Google Scholar, 27Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar). Fibroblasts in attached and floating collagen matrices develop markedly difference morphological features. In attached collagen matrices, cells become bipolar with prominent actin stress fibers, indicating the presence of isometric tension; in floating matrices, however, cells have rounded cell bodies with numerous fine processes and diffuse staining of the actin, indicating an absence of isometric tension (20Fringer J. Grinnell F. J. Biol. Chem. 2001; 276: 31047-31052Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Therefore, we visualized the distribution of endogenous Raf-1 in fibroblasts in attached versus floating collagen matrices before and after serum stimulation. Fig. 6A shows examples of the staining pattern of endogenous Raf-1 in fibroblasts in attached collagen matrices before and after serum stimulation. Before serum stimulation, Raf-1 distribution was punctate and in linear arrays toward the ends of the cells. After stimulation, the punctate staining pattern showed increased distribution along the cell margins, and the linear arrays were no longer evident. Fig. 6B, by contrast, shows that Raf-1 distribution in fibroblasts in floating matrices was punctate and more uniformly distributed with linear arrays absent. Moreover, no difference in the staining pattern could be detected before and after serum stimulation. There have been few studies of endogenous Raf localization. Therefore, to confirm the specificity of endogenous Raf-1 staining, cells were subjected to gene silencing using siRNA for Raf-1. Fig. 7 shows by immunoblotting that Raf-1 was markedly reduced in fibroblasts subjected to transfection with siRNA compared with mock transfected cells. Examination of these cells by immunofluorescence showed that, in the gene-silenced cells but not the mock-transfected controls, the punctate Raf-1 staining pattern was completely lost. In the current research, we confirmed our previous finding that serum stimulation causes markedly reduced ERK phosphorylation in fibroblasts in floating compared with attached collagen matrices. Moreover, we now show that serum-stimulated ERK translocation to the nucleus is inhibited in fibroblasts in floating matrices. Because nuclear translocation of ERK is required for growth factor-induced cell cycle entry (32Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google Scholar), these findings are consistent with the possibility that the quiescence of human fibroblasts in floating collagen results from decreased activity of the ERK signaling pathway (19Rosenfeldt H. Grinnell F. J. Biol. Chem. 2000; 275: 3088-3092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 20Fringer J. Grinnell F. J. Biol. Chem. 2001; 276: 31047-31052Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Additional studies were carried out to determine activation of upstream elements that have been implicated in the ERK signaling pathway, namely, Ras, Raf, and MEK. Similar to ERK, the ERK activator MEK showed decreased phosphorylation in response to serum stimulation. Therefore, attention was focused on Ras and Raf. Experiments using Ras-GTP pull-down assays demonstrated that Ras activation occurred in fibroblasts in either floating or attached matrices. Indeed, the extent of activation was higher in floating matrices. Higher activation of Ras in floating matrices was not serum specific; PDGF had a similar effect. Feedback down-regulation of Ras has been shown to depend on ERK activation (24Haugh J.M. Huang A.C. Wiley H.S. Wells A. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 34350-34360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 25Foschi M. Chari S. Dunn M.J. Sorokin A. EMBO J. 1997; 16: 6439-6451Crossref PubMed Scopus (142) Google Scholar, 26Fucini R.V. Okada S. Pessin J.E. J. Biol. Chem. 1999; 274: 18651-18658Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Consequently, higher levels of Ras-GTP loading in collagen matrices may be explained by loss of signaling to ERK in these matrices. Although serum stimulated Ras activation of fibroblasts in attached matrices, the extent of stimulation was lower than that observed for cells in a monolayer culture. Because the difference was observed with collagen-coated or uncoated culture dishes, the unique features of collagen-binding integrins probably cannot be the explanation (33Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 34Pozzi A. Wary K.K. Giancotti F.G. Gardner H.A. J. Cell Biol. 1998; 142: 587-594Crossref PubMed Scopus (251) Google Scholar). Focal adhesions of fibroblasts in attached collagen matrices typically are smaller and less numerous than those observed for cells in a monolayer culture (35Tamariz E. Grinnell F. Mol. Biol. Cell. 2002; 13: 3915-3929Crossref PubMed Scopus (204) Google Scholar, 36Vaughan M.B. Howard E.W. Tomasek J.J. Exp. Cell Res. 2000; 257: 180-189Crossref PubMed Scopus (399) Google Scholar). Because integrins and integrin-associated proteins of focal adhesions synergize with growth factor receptors and contribute to Ras signaling (37Yamada K.M. Even-Ram S. Nat. Cell Biol. 2002; 4: E75-E76Crossref PubMed Scopus (244) Google Scholar, 38Schlaepfer D.D. Hunter T. Trends Cell Biol. 1998; 8: 151-157Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 39Mettouchi A. Klein S. Guo W. Lopez-Lago M. Lemichez E. Westwick J.K. Giancotti F.G. Mol. Cell. 2001; 8: 115-127Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 40Moro L. Dolce L. Cabodi S. Bergatto E. Erba E.B. Smeriglio M. Turco E. Retta S.F. Giuffrida M.G. Venturino M. Godovac-Zimmermann J. Conti A. Schaefer E. Beguinot L. Tacchetti C. Gaggini P. Silengo L. Tarone G. Defilippi P. J. Biol. Chem. 2002; 277: 9405-9414Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), differences between focal adhesions of cells in a monolayer culture versus collagen matrices might be responsible for differences in Ras activation. In any case, lower activation of Ras in collagen matrices compared with a monolayer culture might explain the previous observation that DNA synthesis also is lower under the former conditions (41Yoshizato K. Taira T. Shioya N. Ann. Plast. Surg. 1984; 13: 9-14Crossref PubMed Scopus (48) Google Scholar, 42Nishiyama T. Tsunenaga M. Nakayama Y. Adachi E. Hayashi T. Matrix. 1989; 9: 193-199Crossref PubMed Scopus (70) Google Scholar, 43Mio T. Adachi Y. Romberger D.J. Ertl R.F. Rennard S.I. In Vitro Cell Dev. Biol. Anim. 1996; 32: 427-433Crossref PubMed Scopus (110) Google Scholar, 44Schor S.L. J. Cell Sci. 1980; 41: 159-175Crossref PubMed Google Scholar). Despite the robust serum-stimulated activation of Ras in fibroblasts in attached matrices, Raf-1 did not appear to become activated in fibroblasts in floating matrices. Ideally, Raf-1 activity should have been measured directly by in vitro kinase assay. We were unable, however, to adapt the method for monolayer cultures to the fibroblast collagen matrix model. Consequently, activation was determined indirectly. Raf-1 activation involves phosphorylation on several residues (10Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1218) Google Scholar, 27Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar) including, Tyr-341 (28Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell. Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (304) Google Scholar, 29Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (527) Google Scholar, 30Tilbrook P.A. Colley S.M. McCarthy D.J. Marais R. Klinken S.P. Arch. Biochem. Biophys. 2001; 396: 128-132Crossref PubMed Scopus (23) Google Scholar) and Ser-338 (31King A.J. Wireman R.S. Hamilton M. Marshall M.S. FEBS Lett. 2001; 497: 6-14Crossref PubMed Scopus (35) Google Scholar). For cells in floating matrices stimulated by serum, we did not observe phosphorylation at Tyr-341 (28Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell. Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (304) Google Scholar, 29Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (527) Google Scholar, 30Tilbrook P.A. Colley S.M. McCarthy D.J. Marais R. Klinken S.P. Arch. Biochem. Biophys. 2001; 396: 128-132Crossref PubMed Scopus (23) Google Scholar) or a band shift on SDS-PAGE. Moreover, there was a marked difference between Raf-1 distribution on cells in floating and attached matrices. That is, the linear punctate arrays and serum-stimulated relocalization observed in fibroblasts in attached matrices were not observed in cells in floating matrices. Specificity of staining was confirmed by Raf-1 gene silencing. The changes in Raf-1 localization in fibroblasts in attached versus floating collagen matrices may interfere with the Raf-1 translocation that normally accompanies Raf-1 activation (10Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1218) Google Scholar, 27Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar). Many studies on cell growth regulation have compared proliferating cells in a monolayer culture with quiescent cells in a suspension culture. Disruption of the ERK signaling pathway in fibroblasts in a suspension culture was reported to occur between Ras and Raf (45Lin T.H. Chen Q. Howe A. Juliano R.L. J. Biol. Chem. 1997; 272: 8849-8852Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) or Raf and MEK (46Renshaw M.W. Ren X.D. Schwartz M.A. EMBO J. 1997; 16: 5592-5599Crossref PubMed Scopus (270) Google Scholar), depending on culture conditions. In the former case, inhibition probably resulted from transient activation of protein kinase A (PKA) (47Howe A.K. Juliano R.L. Nat. Cell Biol. 2000; 2: 593-600Crossref PubMed Scopus (180) Google Scholar), which, along with Akt (protein kinase B), has been implicated in phosphorylation of Raf at Ser-259 (48Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (909) Google Scholar, 49Dhillon A.S. Pollock C. Steen H. Shaw P.E. Mischak H. Kolch W. Mol. Cell. Biol. 2002; 22: 3237-3246Crossref PubMed Scopus (190) Google Scholar). Dephosphorylation of Ser-259 is required for subsequent membrane translocation and activation (50Kubicek M. Pacher M. Abraham D. Podar K. Eulitz M. Baccarini M. J. Biol. Chem. 2002; 277: 7913-7919Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 51Dhillon A.S. Meikle S. Yazici Z. Eulitz M. Kolch W. EMBO J. 2002; 21: 64-71Crossref PubMed Scopus (229) Google Scholar). The possibility that an increase in Akt or PKA can account for our observations has yet to be studied. It should be noted, however, that others reported a decline in Akt during fibroblast culture in floating collagen matrices (7Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and, although cyclic AMP levels do increase in fibroblasts in collagen matrices switched from restrained to floating conditions (52He Y. Grinnell F. J. Cell Biol. 1994; 126: 457-464Crossref PubMed Scopus (60) Google Scholar), the increase is transient and part of the plasma membrane-wounding response (53Grinnell F. Curr. Top. Pathol. 1999; 93: 61-73Crossref PubMed Scopus (23) Google Scholar). Consequently, the mechanism that accounts for decreased Ras-Raf signaling in floating collagen matrices remains an important problem for future research. Raf is only one of several Ras effectors that have been implicated in cell proliferation; others include Ral and phosphatidylinositol 3-kinase (54Sears R.C. Nevins J.R. J. Biol. Chem. 2002; 277: 11617-11620Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 55Stacey D. Kazlauskas A. Curr. Opin. Genet. Dev. 2002; 12: 44-46Crossref PubMed Scopus (33) Google Scholar). Our studies indicating a defect in Ras-Raf coupling as well other work showing inactivation of the PI3K pathway in fibroblasts in floating collagen matrices (7Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) raise the possibility that a general loss of coupling between Ras and its downstream effectors in response to serum stimulation causes cells in these matrices to become quiescent. We are indebted to Drs. Michael White, William Snell, and David Lee and to Chin-Han Ho and HongMei Jiang for their advice and assistance." @default.
• W1978163952 created "2016-06-24" @default.
• W1978163952 creator A5043692232 @default.
• W1978163952 creator A5073869071 @default.
• W1978163952 date "2003-06-01" @default.
• W1978163952 modified "2023-09-30" @default.
• W1978163952 title "Fibroblast Quiescence in Floating Collagen Matrices" @default.
• W1978163952 cites W1487918137 @default.
• W1978163952 cites W1517998622 @default.
• W1978163952 cites W1531194664 @default.
• W1978163952 cites W1611419331 @default.
• W1978163952 cites W1655051481 @default.
• W1978163952 cites W1905619727 @default.
• W1978163952 cites W1951333968 @default.
• W1978163952 cites W1964195356 @default.
• W1978163952 cites W1966260349 @default.
• W1978163952 cites W1968142651 @default.
• W1978163952 cites W1969294711 @default.
• W1978163952 cites W1971727526 @default.
• W1978163952 cites W1972811295 @default.
• W1978163952 cites W1980855212 @default.
• W1978163952 cites W1981002854 @default.
• W1978163952 cites W1987815966 @default.
• W1978163952 cites W198944900 @default.
• W1978163952 cites W1993639183 @default.
• W1978163952 cites W1997656209 @default.
• W1978163952 cites W1998777645 @default.
• W1978163952 cites W2011093418 @default.
• W1978163952 cites W2011736517 @default.
• W1978163952 cites W2014914858 @default.
• W1978163952 cites W2016193552 @default.
• W1978163952 cites W2017959339 @default.
• W1978163952 cites W2033060201 @default.
• W1978163952 cites W2036908471 @default.
• W1978163952 cites W2039983290 @default.
• W1978163952 cites W2041545682 @default.
• W1978163952 cites W2044959710 @default.
• W1978163952 cites W2046713135 @default.
• W1978163952 cites W2056764678 @default.
• W1978163952 cites W2071472168 @default.
• W1978163952 cites W2077452823 @default.
• W1978163952 cites W2078636606 @default.
• W1978163952 cites W2084980574 @default.
• W1978163952 cites W2086640219 @default.
• W1978163952 cites W2086783008 @default.
• W1978163952 cites W2090463334 @default.
• W1978163952 cites W2092067894 @default.
• W1978163952 cites W2098573045 @default.
• W1978163952 cites W2100646314 @default.
• W1978163952 cites W2109538717 @default.
• W1978163952 cites W2114646523 @default.
• W1978163952 cites W2119859567 @default.
• W1978163952 cites W2122322025 @default.
• W1978163952 cites W2133203551 @default.
• W1978163952 cites W2151170471 @default.
• W1978163952 cites W2156008029 @default.
• W1978163952 cites W2157370973 @default.
• W1978163952 cites W2162884347 @default.
• W1978163952 cites W4213141530 @default.
• W1978163952 doi "https://doi.org/10.1074/jbc.m212365200" @default.
• W1978163952 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12663662" @default.
• W1978163952 hasPublicationYear "2003" @default.
• W1978163952 type Work @default.
• W1978163952 sameAs 1978163952 @default.
• W1978163952 citedByCount "41" @default.
• W1978163952 countsByYear W19781639522012 @default.
• W1978163952 countsByYear W19781639522013 @default.
• W1978163952 countsByYear W19781639522014 @default.
• W1978163952 countsByYear W19781639522015 @default.
• W1978163952 countsByYear W19781639522019 @default.
• W1978163952 countsByYear W19781639522020 @default.
• W1978163952 crossrefType "journal-article" @default.
• W1978163952 hasAuthorship W1978163952A5043692232 @default.
• W1978163952 hasAuthorship W1978163952A5073869071 @default.
• W1978163952 hasBestOaLocation W19781639521 @default.
• W1978163952 hasConcept C185592680 @default.
• W1978163952 hasConcept C189165786 @default.
• W1978163952 hasConcept C202751555 @default.
• W1978163952 hasConcept C2780381497 @default.
• W1978163952 hasConcept C55493867 @default.
• W1978163952 hasConcept C86803240 @default.
• W1978163952 hasConcept C95444343 @default.
• W1978163952 hasConceptScore W1978163952C185592680 @default.
• W1978163952 hasConceptScore W1978163952C189165786 @default.
• W1978163952 hasConceptScore W1978163952C202751555 @default.
• W1978163952 hasConceptScore W1978163952C2780381497 @default.
• W1978163952 hasConceptScore W1978163952C55493867 @default.
• W1978163952 hasConceptScore W1978163952C86803240 @default.
• W1978163952 hasConceptScore W1978163952C95444343 @default.
• W1978163952 hasIssue "23" @default.
• W1978163952 hasLocation W19781639521 @default.
• W1978163952 hasOpenAccess W1978163952 @default.
• W1978163952 hasPrimaryLocation W19781639521 @default.
• W1978163952 hasRelatedWork W1914616719 @default.
• W1978163952 hasRelatedWork W1965221772 @default.
• W1978163952 hasRelatedWork W1993602817 @default.
• W1978163952 hasRelatedWork W2409786412 @default.
• W1978163952 hasRelatedWork W2793858243 @default.

• next