Control of Cell Cycle Progression by Fibronectin Matrix Architecture

Developmental patterning and differentiation, maintenance of parenchymal cell function, and the size, shape, and invasiveness of tumors are all orchestrated by cell interactions with the extracellular matrix. Here we show that the fibrillar structure of fibronectin (FN) matrix encodes essential regulatory cues and controls cell proliferation and signaling through changes in matrix architecture. A matrix assembled from native FN stimulated cell growth. In contrast, a mutant FN (FNΔIII1–7) that contains all known cell binding motifs but forms a structurally distinct matrix inhibited progression from G0/G1 into S phase. Furthermore, FNΔIII1–7 suppressed the stimulatory capacity of native FN and induced different levels of tyrosine phosphorylation of pp125FAK. The differential effects on cell growth were ablated by blocking formation of matrix fibrils. Thus, modification of matrix architecture provides a novel approach to control cell proliferation. Developmental patterning and differentiation, maintenance of parenchymal cell function, and the size, shape, and invasiveness of tumors are all orchestrated by cell interactions with the extracellular matrix. Here we show that the fibrillar structure of fibronectin (FN) matrix encodes essential regulatory cues and controls cell proliferation and signaling through changes in matrix architecture. A matrix assembled from native FN stimulated cell growth. In contrast, a mutant FN (FNΔIII1–7) that contains all known cell binding motifs but forms a structurally distinct matrix inhibited progression from G0/G1 into S phase. Furthermore, FNΔIII1–7 suppressed the stimulatory capacity of native FN and induced different levels of tyrosine phosphorylation of pp125FAK. The differential effects on cell growth were ablated by blocking formation of matrix fibrils. Thus, modification of matrix architecture provides a novel approach to control cell proliferation. fibronectin plasma FN recombinant FN lacking repeats III1–7 SV40-transformed 3T3 cells fluorescence-activated cell sorting bromodeoxyuridine enzyme-linked immunosorbent assay polyacrylamide gel electrophoresis. In tissues and tumors, cells live within a multidimensional fibrillar extracellular matrix. The surrounding matrix fibrils thus are uniquely poised to supply environmental signals to cells, suggesting that the structural organization of the matrix itself contributes to the control of cell behavior. Extracellular matrix regulation of cell adhesion, migration, and gene expression occurs through binding to integrins and other cell surface receptors (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 2Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar, 3Boudreau N. Myers C. Bissell M.J. Trends Cell Biol. 1995; 5: 1-4Abstract Full Text PDF PubMed Scopus (147) Google Scholar). As an integral component of extracellular matrices, FN1 matrix fibrils interact with integrin receptors through well characterized binding sites (4Hynes R.O. Fibronectins. Springer-Verlag, New York1990Crossref Google Scholar,5Mosher D.F. Curr. Opin. Struct. Biol. 1993; 3: 214-222Crossref Scopus (84) Google Scholar). Multiple FN domains are required to maintain the integrity of the matrix and deletion or mutation of specific sites in FN can affect matrix structure and assembly (6McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar, 7McDonald J.A. Quade B.J. Broekelman T.J. LaChance R. Forsman K. Hasegawa E. Akiyama S. J. Biol. Chem. 1987; 262: 2957-2967Abstract Full Text PDF PubMed Google Scholar, 8Chernousov M.A. Fogerty F.J. Koteliansky V.E. Mosher D.F. J. Biol. Chem. 1991; 266: 10851-10858Abstract Full Text PDF PubMed Google Scholar, 9Schwarzbauer J.E. J. Cell Biol. 1991; 113: 1463-1473Crossref PubMed Scopus (177) Google Scholar, 10Morla A. Ruoslahti E. J. Cell Biol. 1992; 118: 421-429Crossref PubMed Scopus (143) Google Scholar, 11Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar). In particular, a recombinant FN, FNΔIII1–7, that contains all known cell binding sites but lacks the first seven type III repeats (Fig. 1 A) exhibits an altered rate of matrix assembly with unique intermediates as compared with native FN (11Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar). FNΔIII1–7 and FN matrices also differ in their capacities to re-organize the actin cytoskeleton (12Sechler J.L. Schwarzbauer J.E. Cell Adhes. Commun. 1997; 4: 413-424Crossref PubMed Scopus (32) Google Scholar), suggesting that the structurally distinct architecture of native and altered FN matrices may have significantly different intracellular consequences. Matrix engagement of specific receptors represents an important control point for determining cell shape, cytoskeletal geometry, and the organization of intracellular components (13Folkman J. Moscona A. Nature. 1978; 273: 345-349Crossref PubMed Scopus (1939) Google Scholar, 14Chen C.S. Mrksich M. Huang S. Whitesides G.M. Ingber D.E. Science. 1997; 276: 1425-1428Crossref PubMed Scopus (4114) Google Scholar) as well as initiating the signaling events that lead to cell cycle progression (15Guadagno T.M. Ohtsubo M. Roberts J.M. Assoian R.K. Science. 1993; 262: 1572-1575Crossref PubMed Scopus (365) Google Scholar, 16Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (353) Google Scholar, 17Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (403) Google Scholar). We now show that the architecture of the FN matrix can also regulate this process. FN matrices with distinct morphologies had opposite effects on cell growth by specifically altering the rate of G0/G1 to S phase progression. These effects required FN fibril formation demonstrating that the structure of the matrix plays an active role in modulating cell signaling. SVT2 (SV40-transformed 3T3) cells were grown in Dulbecco's modified Eagle's medium plus 10% calf serum. Growth conditions for CHOα5 cells and expression and purification of pFN and recombinant FNs were as described previously (11Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar). FNΔIII1–2 is a baculovirus-expressed full-length recombinant FN lacking the first two type III repeats. SVT2 and CHOα5 cells were seeded at a density of 2 × 105 cells in either four-well (Nunc) plastic chamber slides (for SVT2 cells) or 24-well dishes with glass coverslips (CHOα5), allowed to attach and spread for 16–24 h, then incubated in serum-free medium for 16–22 h to obtain a population of cells in G0. As is the case for most transformed cell lines, serum starvation was not sufficient to totally synchronize SVT2 or CHOα5 cells but did enrich a G0 population as determined by propidium iodide staining and FACS analysis. For synchronization at G1/S, CHOα5 and SVT2 cells were seeded in 24-well dishes at a concentration of 2.5 × 105cells/well. After a 16-h incubation in complete medium, SVT2 cells were cultured with 0.5 mm hydroxyurea for 14 h and CHOα5 cells in 1 mm hydroxyurea for 18 h to synchronize cells at late G1/S phase. Cells were released from hydroxyurea, washed, and refed with complete medium containing FN-depleted serum and 50 μg/ml pFN or FNΔIII1–7. Cells were stained with propidium iodide (Cycle Test Plus DNA Reagent Kit, Becton Dickinson), and 3 × 104 cells were analyzed by FACS 10 h after release from hydroxyurea to monitor progression through S phase and G2/M. A set of nonsynchronized cells and cells after hydroxyurea incubation were also stained and analyzed by FACS using Cell Quest software (Becton Dickinson). After synchronization, cells were transferred into complete medium containing FN-depleted serum plus 50 μg/ml pFN, 50 μg/ml FNΔIII1–7, or other recombinant FNs or without added FN. For the mixture of FNΔIII1–7 with pFN, 50 μg/ml of each protein were added. For 70-kDa inhibition of fibril formation, 50 μg/ml pFN or FNΔIII1–7 was mixed with 250 μg/ml 70-kDa fragment. Cells were allowed to assemble FN matrix for the indicated time periods. For immobilized proteins, 96-well microtiter plates were coated overnight at 4 °C with 10 μg/ml pFN or FNΔIII1–7 for BrdUrd incorporation or 5, 10, and 15 μg/ml for adhesion. Serum-starved CHOα5 cells were trypsinized and plated onto coated wells at a concentration of 2 × 104 cells/well and then cultured in medium containing FN-depleted serum for a 10–20 h time course. BrdUrd was added to a final concentration of 10 μm and incubated for 30 min. BrdUrd-positive cells were detected with an anti-BrdUrd antibody followed by an alkaline phosphatase-conjugated secondary antibody and developed with nitro blue tetrazolium and X-phosphate substrate solution (Boehringer Mannheim). The numbers of total and BrdUrd-positive cells were counted for several fields (750–1000 total cells) and the percentage of positive cells calculated. Alternatively, cells were labeled with 10 μmBrdUrd and processed for ELISA using BrdU Detection Kit III and ABTS substrate (Boehringer Mannheim). Plates were read on a microtiter plate reader at 405 nm with a reference wavelength of 490 nm. For adhesion, serum-starved CHOα5 cells were trypsinized and plated at a concentration of 2 × 105cells onto a 48-well dish coated with either 10 μg/ml pFN or 10 μg/ml FNΔIII1–7. Cells were lysed (50 mmTris-HCl, pH 7.5, 250 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 50 mm sodium fluoride, and 0.1 mm sodium orthovanadate) 0.5, 1, 3, 6, and 9 h after plating. For matrix assembly, CHOα5 cells were seeded, serum-starved, and incubated with FNs. Cells were lysed as described 3, 9, 16 and 24 h after release from serum-free medium. The concentration of total protein in each lysate was determined by BCA assay (Pierce). 30 μg/lane of total protein was separated by 6% SDS-PAGE, transferred to nitrocellulose, blocked overnight in 5% bovine serum albumin, Buffer A, and probed with anti-phosphotyrosine antibody PT66 (Sigma) at a concentration of 1:3000. For adhesion, blots were developed by ECL (Pierce) and exposed to x-ray film. For matrix assembly, blots were incubated with 125I-protein A then analyzed and quantitated using a Molecular Dynamics PhosphorImager as described (11Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar). The level of phosphorylation after serum starvation (time 0) was set to 1.0, and all other values are expressed relative to that value. p130 was detected by immunoprecipitation of 150 μg of total cell lysates with 20 μg of polyclonal anti-p130 antibody (Santa Cruz). Immunoprecipitated proteins were separated by 6% SDS-PAGE and the level of phosphorylation detected with anti-phosphotyrosine antibody PT66 and developed by ECL. Native FN and FNΔIII1–7 matrices have markedly different effects on cell proliferation as shown in Fig. 1 B. CHOα5 cells, which fail to synthesize a FN matrix (11Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar), were serum-starved to obtain an enriched population of quiescent cells in G0. Cells were then released from serum-free conditions and incubated with medium containing native pFN, baculovirus-expressed mutant FNΔIII1–7, or no exogenous FN. During the incubation, CHOα5 cells bind and assemble exogenous FN into a fibrillar matrix at the cell surface (11Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar). Labeling of newly synthesized DNA by incorporation of BrdUrd was then used to monitor cell cycle progression. Compared with cells without FN matrix, pFN stimulated and FNΔIII1–7 inhibited entry into S phase (Fig. 1 B). Twice as many cells with a native FN matrix were BrdUrd-positive 16 h after release from serum-free conditions as compared with cells with FNΔIII1–7 matrix (Fig. 1 C). With native FN matrix, cells entered S phase at least 8 h earlier than cells with FNΔIII1–7 matrix. Cells with no FN matrix progressed into S phase at a rate intermediate to native FN and FNΔIII1–7. BrdUrd incorporation by cells with a full-length recombinant FN matrix was the same as cells with pFN, indicating that differences in growth were not due to the source of the baculovirus-expressed recombinant protein. Not only does FNΔIII1–7 matrix inhibit cell growth, it also suppresses the stimulatory effects of native FN. A matrix composed of equal proportions of pFN and FNΔIII1–7 reduced BrdUrd incorporation by 30% relative to cells in the presence of native FN matrix (Fig. 1 D). This result shows that the FNΔIII1–7 matrix has a dominant-negative effect on growth stimulation by native FN. FNΔIII1–7 matrix also slowed the proliferation of SVT2 cells that assemble a matrix using endogenously produced FN (Fig. 2 A). Incubation of cells with exogenous pFN or FNΔIII1–7 results in co-assembly with endogenous SVT2 FN as well as a significant increase in the overall level of matrix-associated FN (9Schwarzbauer J.E. J. Cell Biol. 1991; 113: 1463-1473Crossref PubMed Scopus (177) Google Scholar). The G0 to S phase interval of SVT2 cells with FNΔIII1–7-containing matrix was 4 h longer than that of cells assembling a pFN matrix, suggesting a dominant inhibitory effect of the FNΔIII1–7matrix on cell growth. Morphologically, SVT2 cell matrices containing pFN and FNΔIII1–7 are distinct. Immunofluorescence staining of cells with exogenous pFN shows an ordered fibrillar matrix (Fig. 2 B). In contrast, FNΔIII1–7 matrix appears less uniform and is characterized by fibrils of varying length and thickness (Fig. 2, C and D). Together, these results demonstrate that co-assembly of FNΔIII1–7 with SVT2 FN has a dominant-negative effect on cell growth and, as with CHOα5 cells, this effect correlates with differences in matrix fibril organization. The effects of matrix assembly on cell growth are restricted to events controlling G0/G1 progression. CHOα5 and SVT2 cells synchronized at G1/S with hydroxyurea were released into complete medium containing pFN, FNΔIII1–7, or no FN and stained with propidium iodide to monitor DNA synthesis and cell cycle progression by FACS analysis. Neither native FN or FNΔIII1–7 matrix altered the progression of cells through S or G2/M as the percentages of cells traveling through these phases and back to G1 were identical (not shown). pFN and FNΔIII1–7 both contain all known cell binding sites and both use α5β1 integrin to initiate matrix assembly (11Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar, 18Fogerty F.J. Akiyama S.K. Yamada K.M. Mosher D.F. J. Cell Biol. 1990; 111: 699-708Crossref PubMed Scopus (187) Google Scholar, 19Wu C. Bauer J.S. Juliano R.L. McDonald J.A. J. Biol. Chem. 1993; 268: 21883-21888Abstract Full Text PDF PubMed Google Scholar), yet the two types of matrices have opposite effects on cell growth. Cell proliferation would be affected if there are different levels of integrin-mediated binding and adhesion to these two FNs (16Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (353) Google Scholar, 17Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (403) Google Scholar, 20Renshaw M.W. Ren X.D. Schwartz M.A. EMBO J. 1997; 16: 5592-5599Crossref PubMed Scopus (269) Google Scholar). To address this possibility, attachment and growth of CHOα5 cells on immobilized pFN or FNΔIII1–7protein were measured. Equal numbers of cells attached to pFN and FNΔIII1–7 substrates in 30 min (not shown). Attached cells showed identical levels of BrdUrd incorporation on the two proteins (Fig. 3 A). Therefore, cell adhesive interactions with native and mutant FNs are indistinguishable. An alternative explanation for the opposite growth responses is that the distinct architectures of the two matrices may influence cell proliferation. If fibrillar matrix structure controls the rate of growth, then inhibition of fibril formation should eliminate the differences between native FN and FNΔIII1–7 matrices. Inclusion of excess 70-kDa amino-terminal fragment of FN during matrix assembly blocks FN-FN interactions via the assembly domain (see Fig. 1 A) thus preventing fibril formation by pFN (6McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar, 7McDonald J.A. Quade B.J. Broekelman T.J. LaChance R. Forsman K. Hasegawa E. Akiyama S. J. Biol. Chem. 1987; 262: 2957-2967Abstract Full Text PDF PubMed Google Scholar) and FNΔIII1–7(11Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar). 70-kDa fragment does not interfere with FN-integrin interactions. Inhibition of fibril formation by addition of 70-kDa fragment reversed the growth stimulatory effects of native FN matrix and the inhibitory effects of FNΔIII1–7 matrix (Fig. 3 B). In both cases, a block in fibril assembly resulted in BrdUrd incorporation comparable with that of cells with no matrix. Further evidence for the role of fibrillar matrix structure in regulating cell growth was provided by experiments with two other mutant recombinant FNs, FNΔIII1–2 and FN(syn-). FNΔIII1–2 is defective in FN binding and polymerization, 2J. L. Sechler and J. E. Schwarzbauer, unpublished observations. while FN(syn-) does not bind well to α5β1 integrin (21Sechler J.L. Corbett S.A. Schwarzbauer J.E. Mol. Biol. Cell. 1997; 8: 2563-2573Crossref PubMed Scopus (125) Google Scholar). As a result of these defects, each can only form short fibrils but cannot assemble into an extensive matrix of the type shown in Fig. 2. Cells grown in the presence of either of these two mutant FNs progressed into S phase at a rate similar to cells with no FN matrix (Fig. 3 B). These results demonstrate that the differential effects of native FN and FNΔIII1–7 on cell growth occur only when each is interacting with cells from within a fibrillar matrix. More importantly, the opposite effects of these FNs on cell proliferation are directly attributable to the structural differences between native FN and FNΔIII1–7 matrix fibrils. Cell interactions with FN activate a number of intracellular signal transduction cascades (2Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar, 22Parsons J.T. Curr. Opin. Cell Biol. 1996; 8: 146-152Crossref PubMed Scopus (277) Google Scholar, 23Schlaepfer D.D. Hunter T. Trends Cell Biol. 1998; 8: 151-157Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). In particular, phosphorylation of focal adhesion kinase (pp125FAK) is an early biochemical response to integrin-mediated adhesion to FN substrates (24Guan J.L. Trevithick J.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (472) Google Scholar, 25Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. S. A. 1992; PubMed Scopus Google Scholar, S.K. S.K. Proc. Natl. S. A. 1992; PubMed Scopus Google Scholar). If FNΔIII1–7 matrix structure can cell cycle then intracellular signaling in response to matrix should be levels of were compared in cells assembling different fibrillar matrices. Cells assembling native FN matrix a increase in phosphorylation cells with either no FN or FNΔIII1–7 matrix (Fig. 4 A). The differential effects of these matrices were at the time point a increase in phosphorylation was all due to the addition of serum (2Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar). The levels of phosphorylation in response to native FN matrix were the time course. phosphorylation in cells with FNΔIII1–7 matrix the time point when it to within of levels with pFN (Fig. 4 A). In to the effects of fibrillar matrix assembly, cell adhesion on immobilized pFN or FNΔIII1–7protein resulted in a phosphorylation of to levels (Fig. 4 B). on both phosphorylation was with after 3 A focal adhesion p130 A. Parsons J.T. K. J. Cell 1995; PubMed Google Scholar, K. Ruoslahti E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), did not differential phosphorylation (Fig. 4 C). p130 phosphorylation was and comparable levels were with both pFN and FNΔIII1–7 during matrix assembly and cell adhesion. Therefore, cell interactions with fibrillar matrix specific responses that are not with cell binding to immobilized protein to the stimulatory effect on cell native FN matrix induced FNΔIII1–7 matrix, on the other inhibited both phosphorylation and cell cycle progression. the structure of the FN matrix can regulate signaling and Matrix architecture regulate the of cell signals by modulating the of cell binding As shown a of matrix fibrils can the of proliferation different matrix to or maintain cell growth Inhibition of cell growth by that can block FN fibril formation has also 1998; PubMed Scopus Google Scholar). unique structural on the of FN matrix fibrils from all of the cell to the organization of the actin plays a role in intracellular signaling that lead to cell cycle progression C.S. Mrksich M. Huang S. Whitesides G.M. Ingber D.E. Science. 1997; 276: 1425-1428Crossref PubMed Scopus (4114) Google Scholar, R.M. E. Assoian R.K. Mol. Biol. Cell. 1996; PubMed Scopus Google Scholar), and inhibition of to focal correlates with cell proliferation Mol. Biol. Cell. 1996; PubMed Scopus Google Scholar). of the cytoskeleton by specific in matrix structure would influence cell growth. This is by the between differences in actin and distinct native FN and FNΔIII1–7matrix (12Sechler J.L. Schwarzbauer J.E. Cell Adhes. Commun. 1997; 4: 413-424Crossref PubMed Scopus (32) Google Scholar). Extracellular matrix can the of between integrins and the cytoskeleton Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and may between matrix architecture and cell growth results also show that altering the structure of the FN matrix can be than no matrix at pFN have shown to have R. S. E. Ruoslahti E. 1996; 2: PubMed Scopus Google Scholar), and may be through effects on matrix organization. the of that can matrix architecture through dominant-negative effects be a novel for controlling cell and growth and We and for of the and Corbett and for We are to and to for their on of this