From the Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039
Received for publication, December 4, 2002
, and in revised form, March 27, 2003.
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ABSTRACT |
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INTRODUCTION |
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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 ERK1 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 (10, 11). 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 (12, 13, 14, 15, 16, 17, 18).
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 (19) along with down-regulation of the cell cycle regulatory protein cyclin D1 and an increase in the cyclin-dependent kinase inhibitor p27Kip1 (20). 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.
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EXPERIMENTAL PROCEDURES |
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Collagen Matrix and Monolayer CultureFibroblasts 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 (19, 20). The cell/collagen mixture containing 106 fibroblasts/ml and 1.5 mg/ml collagen in DMEM without serum was prewarmed to 37 °C for 34 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 ImmunoblottingSDS-PAGE and immunoblotting were performed as described previously (19, 20). 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 x g (Beckman Microfuge), and the supernatants were either incubated with reagents for the Ras-GTP pull-down assay or else dissolved in 1x 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 MicroscopyCollagen 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 2x 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 2x 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 6x 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 6x 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 SynthesisDNA synthesis was determine as previously (19, 20). 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 SilencingRaf-1 gene silencing was accomplished using siRNA (21). 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 (5060% 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.
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RESULTS |
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ERK phosphorylation is required for nuclear translocation (22, 23). 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.
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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 RasThe 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 (24,
25,
26)
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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.
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Raf PhosphorylationHaving 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 (10, 27), including Tyr-341 (28, 29, 30) and Ser-338 (31). 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 (20). Variability in total Raf-1 staining was unexplained but occurred with two different antibodies (Santa Cruz Biotechnology and BD Transduction Laboratories).
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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 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 LocalizationTaken 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 (10, 27). 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 (20). 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.
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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.
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DISCUSSION |
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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 (24, 25, 26). 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 (33, 34). Focal adhesions of fibroblasts in attached collagen matrices typically are smaller and less numerous than those observed for cells in a monolayer culture (35, 36). Because integrins and integrin-associated proteins of focal adhesions synergize with growth factor receptors and contribute to Ras signaling (37, 38, 39, 40), 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 (41, 42, 43, 44).
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 (10, 27) including, Tyr-341 (28, 29, 30) and Ser-338 (31). For cells in floating matrices stimulated by serum, we did not observe phosphorylation at Tyr-341 (28, 29, 30) 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 (10, 27).
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 (45) or Raf and MEK (46), depending on culture conditions. In the former case, inhibition probably resulted from transient activation of protein kinase A (PKA) (47), which, along with Akt (protein kinase B), has been implicated in phosphorylation of Raf at Ser-259 (48, 49). Dephosphorylation of Ser-259 is required for subsequent membrane translocation and activation (50, 51). 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 (7) and, although cyclic AMP levels do increase in fibroblasts in collagen matrices switched from restrained to floating conditions (52), the increase is transient and part of the plasma membrane-wounding response (53). 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 (54, 55). 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 (7) 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.
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FOOTNOTES |
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Present address: Laboratory of Gene Regulation and Development, NICHD,
National Institutes of Health, Bethesda, MD 20892.
To whom correspondence should be addressed: Dept. of Cell Biology, University
of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX
75390-9039. Tel.: 214-648-2181; Fax: 214-648-8694, E-mail:
frederick.grinnell{at}utsouthwestern.edu.
1 The 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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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