Fibroblast Quiescence in Floating Collagen Matrices

DECREASE IN SERUM ACTIVATION OF MEK AND RAF BUT NOT RAS*

Jeanne Fringer {ddagger} and Frederick Grinnell §

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.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1, 2). 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 (3, 4). In addition to becoming quiescent, fibroblasts in floating matrices also begin to enter apoptosis (5, 6, 7, 8), and apoptosis has been implicated in the disappearance of wound fibroblasts at the end of repair (9).

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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (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 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 (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 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 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 Synthesis—DNA 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 Silencing—Raf-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 (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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.



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FIG. 1.
Phosphorylation of ERK and MEK. Fibroblasts were cultured 8 h in attached or floating collagen matrices in DMEM/10% FBS followed by incubation overnight in DMEM/0.5% FBS, after which DMEM/10% was added to the incubations. At the times indicated, cell extracts were prepared and subjected to SDS-PAGE and immunoblotted to determine levels of phospho-ERK (P-ERK), ERK, phospho-MEK (P-ERK), and MEK.

 

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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FIG. 2.
Nuclear translocation of endogenous ERK. Fibroblasts in a monolayer culture or in attached or floating collagen matches were cultured and stimulated as described in the Fig. 1 legend. After 10 min, cells were fixed and stained to determine the distribution of ERK2. Arrows point to nuclei. Bar = 32 µm for panels A and B and 20 µm for panels C–F.

 

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 (24, 25, 26)



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FIG. 3.
GTP-loading of Ras in attached and floating matrices. A, fibroblasts were cultured in attached or floating collagen matrices as described in the Fig. 1 legend. At the times indicated, cell extracts were prepared, and portions were subjected to pull-down assay analysis using the Raf RBD binding domain. Samples of the extracts and pulldown pellets were analyzed by SDS-PAGE and immunoblotting for Ras. B, results from three different experiments were quantified by measuring the band densities using NIH Image. To calculate the Ras index, the ratio of Ras-GTP to total RAS observed at time 0 in attached collagen matrices was set to 1, and all other measurements were compared with this value. C, same as described for panel A, except that the cells were stimulated with DMEM + 50 ng/ml PDGF instead of DMEM/10% FBS.

 

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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FIG. 4.
GTP loading of Ras compared with DNA synthesis. Fibroblasts were cultured in a monolayer culture or in attached collagen matches. Otherwise, details are the same as in the Fig. 3 legend. Collagen-coated culture dishes were prepared by incubation with 50 µg/ml collagen solution for 15 min and then rinsed with DPBS prior to adding the cells. The DNA synthesis index was calculated as the ratio of [3H]thymidine incorporation into stimulated and unstimulated cells measured 24 h after serum stimulation. Data shown are from duplicate determinations. Absolute values (±S.D.) for serum-stimulated cells were as follows: attached matrices, 5600 ± 500; monolayer 34500 ± 5300; collagen-coated monolayer 33500 ± 2800.

 

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 (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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FIG. 5.
Raf phosphorylation. Fibroblasts were cultured in attached or floating collagen matrices as described in Fig. 1. At the times indicated, cells extracts were prepared, and extracts were subjected to SDS-PAGE and immunoblotted to determine levels of phospho-Raf-1 (Tyr-340,Tyr-341), total Raf-1, and Cdk4 as indicated. A, SDS-PAGE was run until the dye front reached the bottom of the gel. B, SDS-PAGE was run long enough for Raf-1 to almost reach the bottom of the gel.

 

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 {lambda} 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 (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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FIG. 6.
Localization of Raf-1 in collagen culture. Fibroblasts in attached (A) or floating (B) collagen matrices were cultured and stimulated as described in the Fig. 1 legend. Samples were fixed and stained to determine distribution of anti-Raf-1 before and after stimulation as indicated. Bar = 20 µm.

 

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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FIG. 7.
Raf-1 gene silencing with siRNA. Fibroblasts were transfected for 48 in DMEM with 2 µM siRNA or without siRNA (mock transfection), after which the media was switched to DMEM/10% FBS for 24 h and then DMEM/0.5% FBS overnight. Cell cultures were used to prepared extracts that were subjected to SDS-PAGE and immunoblotted to determine levels of Raf-1 or Cdk4. Alternatively, cultures were fixed and stained to determine distribution of Raf-1. Observations and images were made with a Nikon Elipse 400 fluorescent microscope equipped with a Photometrics SenSys camera and MetaView work station. Bar = 20 µm.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (32), 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 (19, 20).

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.


    FOOTNOTES
 
* This research was supported by NIGMS, National Institutes of Health Grant GM31321. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Laboratory of Gene Regulation and Development, NICHD, National Institutes of Health, Bethesda, MD 20892. Back

§ 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. Back


    ACKNOWLEDGMENTS
 
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.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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