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• W2140433265 abstract "In apoptosis the tumor suppressor p53and the c-myc proto-oncogene are usually up-regulated. We show a novel alternative pathway of apoptosis in human primary cells that is mediated by transcriptionally dependent decreases in p53 and c-Myc and decreases in p21. This pathway is regulated by the alternatively spliced V region and high-affinity heparin-binding domain of fibronectin. Requirements for c-Myc, p53, and p21 signals in maintaining survival and for their decreases in inducing apoptosis were demonstrated by the ability of p53, c-Myc, and p21 ectopic expression to rescue this apoptotic phenotype, and the ability of p53-deficient and c-myc antisense conditions to trigger a faster rate of apoptosis. In apoptosis the tumor suppressor p53and the c-myc proto-oncogene are usually up-regulated. We show a novel alternative pathway of apoptosis in human primary cells that is mediated by transcriptionally dependent decreases in p53 and c-Myc and decreases in p21. This pathway is regulated by the alternatively spliced V region and high-affinity heparin-binding domain of fibronectin. Requirements for c-Myc, p53, and p21 signals in maintaining survival and for their decreases in inducing apoptosis were demonstrated by the ability of p53, c-Myc, and p21 ectopic expression to rescue this apoptotic phenotype, and the ability of p53-deficient and c-myc antisense conditions to trigger a faster rate of apoptosis. extracellular matrix fibronectin fibronectin fragments containing the alternatively spliced V region and a mutated or wild-type heparin-binding domain, respectively glutaraldehyde-3-phosphate dehydrogenase chloramphenicol acetyltransferase Rous sarcoma virus Multiple signaling pathways of apoptosis are triggered by various external insults to or stimuli on the cell. In the case of the extracellular matrix (ECM),1it appears that depriving cells of anchorage and/or appropriate survival signals by disrupting the signals mediated by ECM-integrin interactions induces a pathway of apoptosis for which some signaling components have been identified (1Ilic D. Almeida E.A.C. Schlaepfer D.D. Dazin P. Aizawa S. Damsky C.H. J. Cell Biol. 1998; 143: 547-560Crossref PubMed Scopus (437) Google Scholar, 2Kapila Y.L. Niu J. Johnson P.W. J. Biol. Chem. 1997; 272: 18932-18938Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). This pathway seems to be initiated with the loss of phosphorylation of the integrin-associated signaling molecule focal adhesion kinase (pp125FAK) (1Ilic D. Almeida E.A.C. Schlaepfer D.D. Dazin P. Aizawa S. Damsky C.H. J. Cell Biol. 1998; 143: 547-560Crossref PubMed Scopus (437) Google Scholar, 2Kapila Y.L. Niu J. Johnson P.W. J. Biol. Chem. 1997; 272: 18932-18938Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 3Hungerford J.E. Compton M.T. Matter M.L. Hoffstrom B.G. Otey C.A. J. Cell Biol. 1996; 124: 1383-1390Crossref Scopus (333) Google Scholar). The downstream signals to which pp125FAK has been linked include the caspase family of cell death proteases. Caspases, which are considered the executioners of cell death in apoptosis, are thought to orchestrate cell disintegration through a cascade of caspase activation. This family of aspartate proteases seems to be involved in apoptotic pathways regulated by the ECM, because caspase inhibitors prevent apoptosis triggered by an altered matrix, loss of the ECM, or disruption of pp125FAK function (2Kapila Y.L. Niu J. Johnson P.W. J. Biol. Chem. 1997; 272: 18932-18938Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 3Hungerford J.E. Compton M.T. Matter M.L. Hoffstrom B.G. Otey C.A. J. Cell Biol. 1996; 124: 1383-1390Crossref Scopus (333) Google Scholar, 4Boudreau N. Sympson C.J. Werb Z. Bissell M.J. Science. 1995; 267: 891-893Crossref PubMed Scopus (1116) Google Scholar). Furthermore, caspase 3 has recently been shown to cleave pp125FAK as part of the mechanism that leads to apoptosis (5Gervais F.G. Thornberry N.A. Ruffolo S.C. Nicholson D.W. Roy S. J. Biol. Chem. 1998; 273: 17102-17108Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 6Levkau B. Herren B. Koyama H. Ross R. Raines E.W. J. Exp. Med. 1998; 187: 579-586Crossref PubMed Scopus (228) Google Scholar). Other signals implicated in apoptosis triggered by an altered matrix or disruption of ECM-integrin signaling pathways include the tumor suppressor p53 and the oncogene c-myc, which are known mediators of apoptosis in other pathways (7Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1363) Google Scholar). In general, activation of p53 and c-Myc corresponds to the apoptotic phenotype, and conversely, inactivation of p53 and c-Myc corresponds to cell survival. In the case of apoptosis regulated by the ECM, p53 may either play a direct role (3Hungerford J.E. Compton M.T. Matter M.L. Hoffstrom B.G. Otey C.A. J. Cell Biol. 1996; 124: 1383-1390Crossref Scopus (333) Google Scholar) or only modulate the kinetics of this pathway (8McGill G. Shimamura A. Bates R.C. Savage R.E. Fisher D.E. J. Cell Biol. 1997; 138: 901-911Crossref PubMed Scopus (112) Google Scholar). c-Myc, which in some apoptotic pathways modulates p53 function, is involved in apoptosis triggered by disruption of integrin signals, because during c-Myc-induced apoptosis there is targeted proteolysis of pp125FAK, which can be suppressed by integrin signaling (9Crouch D.H. Fincham V.J. Frame M.C. Oncogene. 1996; 12: 2689-2696PubMed Google Scholar). The protein encoded by the p53 response gene, the inhibitor of cyclin-dependent kinases, p21 (10Clarke A.S. Lotz M.M. Chao C. Mercurio A.M. J. Biol. Chem. 1995; 270: 22673-22676Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), has also been implicated in integrin-ECM-mediated apoptosis. However, we now document an interesting and new alternative pathway of apoptosis that is regulated by the alternatively spliced V region and the heparin-binding domain of fibronectin (FN) and leads to transcriptionally dependent decreases in p53 and c-Myc in primary, nontransformed cells. Furthermore, the decreases in p53 and c-Myc are in part driving this mechanism, because transfection with eitherp53 or c-myc rescues the apoptotic phenotype, and p53-deficient and c-myc antisense conditions trigger a faster rate of apoptosis. Primary cultures of human fibroblasts were obtained, cultured, and tested for apoptosis as previously described (11Kapila Y.L. Wang S. Johnson P.W. J. Biol. Chem. 1999; 274: 30906-30913Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). P53-deficient and wild-type mouse fibroblasts were provided by Dr. Caroline Damsky (1Ilic D. Almeida E.A.C. Schlaepfer D.D. Dazin P. Aizawa S. Damsky C.H. J. Cell Biol. 1998; 143: 547-560Crossref PubMed Scopus (437) Google Scholar) and maintained in culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin/fungizone) prior to experimentation. Four recombinant FN protein fragments were tested in these experiments. These fragments, described elsewhere (2Kapila Y.L. Niu J. Johnson P.W. J. Biol. Chem. 1997; 272: 18932-18938Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), either included (V+) or excluded (V−) the alternatively spliced V region and contained either an unmutated (H+) or a mutated, nonfunctional, high-affinity, heparin-binding domain (H−). For Western blot analysis, cells were incubated with the V+H− fragment, the V+H+ fragment, or control serum-free medium as indicated for each figure. After incubation, cell lysates were prepared using 100 μl/well TNE buffer (1% Nonidet P-40, 10% glycerol, 150 mm sodium chloride in Tris, pH 7.4, and 1 mmEDTA) containing various protease inhibitors (1 mm sodium orthovanadate, 50 μm sodium molybdate, 25 μg/ml aprotinin, 25 μg/ml leupeptin, 1 mm sodium fluoride, and 1 mm phenylmethylsulfonyl fluoride). Lysates were adjusted for protein concentration using the BCA protein assay kit (Pierce) and then analyzed by standard SDS-PAGE. After electrophoresis, the gels were transferred to nitrocellulose by standard methods. Blots were then probed with primary antibodies and developed using the Enhanced Chemiluminescence-Plus detection system (Amersham Biosciences, Inc.). Primary antibodies included mouse anti-human c-Myc (Ab-1, OP10) and mouse anti-human p53 (Ab-1, OP03), both from Oncogene Research; mouse anti-human p21 (F-5, SC-6246) and mouse anti-human c-Myc (C33, SC42), both from Santa Cruz, Biotechnology; and mouse anti-human Bcl2 (clone 124, M0887) from DAKO. An antisense oligodeoxynucleotide corresponding to the c-mycproto-oncogene was used to inhibit c-Myc protein expression in primary cells prior to treatment with the V+H− protein. Antisense (5′-CACGTTGAGGGGCAT-3′) and non-sense (5′-AGTGGCGGAGACTCT-3′) c-myc oligonucleotides (12Yufang S. Glynn J.M. Guilbert L.J. Cotter T.G. Bissonnette R.P. Green D.R. Science. 1992; 257: 212-214Crossref PubMed Scopus (673) Google Scholar) were synthesized, purified, and analyzed by the University of California San Francisco Biomolecular Resource Center. Cells were transfected for 24 and 48 h with Oligofectin reagent (Invitrogen) and 2 μmoligonucleotides, then treated for various times with the V+H− protein as indicated in the figure legends. For c-Myc and p53 Northern blot analysis, cells were incubated with the V+H− protein, control V+H+ protein, or control medium for various time intervals. After incubation, total RNA was isolated from cells using the QIAshredder and RNeasy mini kit (Qiagen, Valencia, CA). Total RNA was then separated by electrophoresis on 1% agarose gels and transferred to nylon membranes by capillary blotting. The quality of the RNA was verified by ethidium bromide staining before and after transfer. RNA was fixed to the membrane by exposure to UV light for 2 min. Membranes were prehybridized in QuikHyb hybridization solution (Stratagene) at 68 °C for 20 min. They were then hybridized at 68 °C for 1–1.5 h in the same solution containing 100 μg/ml denatured salmon sperm DNA and denatured radiolabeled cDNA probes for human c-myc (a 2.5-kbEcoRI/HindIII fragment) (13Favera R.D. Gelmann E.P. Martinotti S. Franchini G. Papas T.S. Gallo R.C. Staal F.W. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6497-6501Crossref PubMed Scopus (273) Google Scholar) or humanp53 (a 1.8-kb fragment) (14Matlashewski G.P. Lamb D. Pim J. Peacock L. Crawford S. Benchimol S. EMBO J. 1984; 3: 3257-3262Crossref PubMed Scopus (206) Google Scholar). cDNA probes were radiolabeled using a random primer kit (Promega) and [α-32P]dCTP (PerkinElmer Life Sciences), and separated from free nucleotide using NuctrapTM push columns (Stratagene). Specific radioactivities of the individual probes were ∼109 cpm/mg. Hybridized filters were washed in 2× SSC, 0.1% SDS at room temperature for 15 min and then in 0.1× SSC, 0.1% SDS at 60 °C for 30 min and finally exposed to radiographic film with an intensifying screen at −70 °C for 1–2 days. To normalize the blots for differences in RNA loading and/or transfer to the membranes, the blots were stripped and rehybridized with a 1.3-kb glutaraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (15Fort P. Marty L. Piecaczyk M. Sabrouty S.E. Dani C. Jeanteur P. Blanchard J.M. Nucleic Acids Res. 1985; 13: 1431-1442Crossref PubMed Scopus (1972) Google Scholar). Results are expressed as the ratio of the c-Myc or p53 mRNA to the GAPDH mRNA signal. Assays were performed as described previously (16Neumann J.R. Morency C.A. Russian K.O. BioTechniques. 1987; 5: 444-446Google Scholar). In brief, the constructs/reporter plasmids pCBp53-CAT (17Schroeder M. Mass M.J. Biochem. Biophys. Res. Commun. 1997; 235: 403-406Crossref PubMed Scopus (74) Google Scholar), 1.6Bgl myc-CAT (18Frazier M.W. He X. Wang J.L. Gu Z. Cleveland J.L. Zambetti G.P. Mol. Cell. Biol. 1998; 18: 3735-3743Crossref PubMed Scopus (176) Google Scholar), and Rous sarcoma virus (pRSV-CAT (19Gorman C.M Merlino G.T. Willingham M.C. Pastan I. Howard B.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6777-6781Crossref PubMed Scopus (881) Google Scholar)) were transfected into cells by electroporation with a Bio-Rad Gene Pulser (250 V at a capacity setting of 960 microfarads). To normalize for different transfection efficiencies, a plasmid containing the β-galactosidase reporter gene driven by the actin promoter was cotransfected into the cells. After electroporation, cells were treated with the recombinant FN proteins V+H− and V+H+ or with control medium for different times. Cells were washed with phosphate-buffered saline and lysed with lysis buffer (250 mm Tris-HCl, pH 7.5, 0.1% Triton X-100). CAT assays were performed as described previously (16Neumann J.R. Morency C.A. Russian K.O. BioTechniques. 1987; 5: 444-446Google Scholar). β-Galactosidase activity was measured by the Galacto-Light Plus chemiluminescent assay (Tropix, Bedford, MA) using a luminometer (Analytical Luminescence Laboratory, model 2010). CAT activity was normalized by β-galactosidase activity and compared for all treatments from triplicate experiments. Cells that were 60–80% confluent in 96-well tissue culture plates were transiently transfected with 0.1 μg of DNA (as indicated in each figure legend) in 50 μl of serum-free medium or with vector control, using the LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. Transfection efficiency in these primary cells is ∼30–35% as measured using a green fluorescent protein vector (pEGFP, CLONTECH). Transfected cells were then rinsed, fresh serum-free medium plus the FN protein (V+H−) was added to the test wells, and apoptosis was assessed. pCMV-jun (as described previously (20Rauscher III, F.J. Voulalas P.J. Franza Jr., B.R. Curran T. Genes Dev. 1988; 12B: 1687-1699Crossref Scopus (348) Google Scholar)) was used to construct cmv-c-myc, c-jun was removed, and human c-myc (2.5 kb) was inserted withHindIII. The apoptotic population was assessed in primary human fibroblasts transfected with c-myc, p53, c-myc antisense, c-myc non-sense, p21, C-p21, or vector control and in p53−/− and wild-type mouse fibroblasts using a flow cytometric assay (21Hamel W. Dazin P. Israel M. Cytometry. 1996; 25: 173-181Crossref PubMed Scopus (43) Google Scholar). Treated and control cells were resuspended in 1 ml of ice-cold phosphate-buffered saline containing 2% fetal calf serum, 3% enzyme-free phosphate-buffered saline-based cell dissociation buffer (Invitrogen), and 1 μg/ml propidium iodide (Sigma). Cells were kept on ice until 10–15 min before the addition of Hoechst 33342 stain. After equilibration to room temperature, 5 μg/ml Hoechst 33342 was added to the cell suspension. Detection of Hoechst 33342 staining was done after 6 min using a dual laser Triple Vantage S.E. cell sorter (Becton Dickinson, San Jose, CA). In primary human fibroblasts (11Kapila Y.L. Wang S. Johnson P.W. J. Biol. Chem. 1999; 274: 30906-30913Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), we initially observed that a recombinant fragment of FN containing the alternatively spliced V region and a mutation in the high-affinity heparin-binding domain (2Kapila Y.L. Niu J. Johnson P.W. J. Biol. Chem. 1997; 272: 18932-18938Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) induced apoptosis in these cells (Fig. 1,a–c). This apoptotic pathway is proteoglycan- and caspase-mediated and is associated with changes in pp125FAKphosphorylation (11Kapila Y.L. Wang S. Johnson P.W. J. Biol. Chem. 1999; 274: 30906-30913Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). On examining p53 and c-Myc function, we found surprising results, namely that p53 and c-Myc protein (Fig.1 d) and RNA levels (Fig.2 a) were down-regulated as part of this apoptotic mechanism. However, levels of Bcl-2, an antiapoptotic protein, remained unchanged under the same conditions (Fig. 1 d), illustrating the specificity of the p53 and c-Myc response. In addition, when cells were treated with the control FN protein V+H+ or control medium, no apoptosis was triggered, and p53 and c-Myc levels remained unchanged (Figs. 1 and2). Time course experiments demonstrated that the drop in c-Myc levels preceded that of p53, because the c-Myc signal began its downward trend at 30 min, whereas the p53 signal started to decline at 45 min (data not shown).Figure 2Correspondence between down-regulation of c-Myc and p53 proteins and RNA levels by the V+H− FN fragment. a, RNA levels for c-Myc and p53 were analyzed by Northern blots at 3 and 7 h using standard methods. Blots were probed with denatured radiolabeled cDNA probes for human c-myc or humanp53. Blots were normalized for differences in RNA loading and/or transfer to the membranes by stripping and rehybridizing with a 1.3-kb GAPDH cDNA probe. Results are expressed as the ratio of the c-Myc or p53 mRNA to the GAPDH mRNA signal. b, CAT reporter assays were used to assess the activity of the p53, c-myc, and control RSV promoters. Representative experiments are presented for both Northern blots and promoter assays. Values represent means and S.D. for three experiments. Data were evaluated statistically using one-way analysis-of-variance with the Newman-Keuls test for significance by GraphPad InStat software. *, p< 0.05, and **, p < 0.01, versuscontrol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether the decrease in p53 and c-Myc levels was a transcriptionally regulated event, primary human fibroblasts were first transfected with a p53promoter/reporter-CAT construct, a c-mycpromoter/reporter-CAT construct, or a control RSV-CAT promoter/reporter construct and then treated with the apoptosis-inducing FN fragment, V+H−. After transfection with thep53 or c-myc promoters, promoter activity declined (Fig. 2 b) mirroring the decline in the corresponding protein and RNA species and illustrating the transcriptional down-regulation of p53 and c-Myc in this pathway. However, RSV promoter activity was not down-regulated under the same conditions, further illustrating the specificity of the p53 and c-Myc response. Suspecting that the decrease in p53 and c-Myc was driving the FN fragment-mediated apoptotic pathway, we transfected primary human fibroblasts with p53, c-myc, or vector controls to determine whether ectopic expression of p53 or c-Myc could rescue the apoptotic phenotype. Indeed, transfection with eitherp53 or c-myc, but not the control vector, rescued cells from FN fragment-mediated apoptosis (Fig.3, a–c). Cell shape changes for the c-myc- and p53-transfected cells demonstrated a more well spread phenotype, characteristic of live cells, whereas the vector-transfected cells demonstrated cell membrane blebbing and a rounded phenotype associated with apoptosis (Fig.3 a). The c-myc-transfected cell population showed a 10.8% base-line level of apoptosis, which increased to 14% upon treatment with the V+H− protein. The p53-transfected cell population showed a 9.5% base-line level of apoptosis, which increased to 10.8% upon treatment with the V+H− protein. The vector-transfected control cell population showed a 5.1% base-line level of apoptosis, which increased to 15% upon treatment with the V+H−protein. Thus, the c-myc- and p53-transfected cells showed a 1.3-fold (14/10.8%) and 1.1-fold (10.8/9.5%) increase in apoptosis, respectively, compared with the 2.9-fold (15/5.1%) increase in apoptosis in the vector- transfected cells (Fig.3 b). This is approximately a 2.5-fold difference. Although, the rescue from apoptosis was not complete with c-myc andp53 transfection (and with other treatments; Fig.Figure 4, Figure 5, Figure 6), this likely represents an incompletely transfected cell population; transfection efficiency is ∼30–35% in these primary cells (data not shown). The fold changes in apoptosis following transfection with c-myc, p53, and vector control were computed for an average of 3 experiments and graphically illustrated (Fig. 3 c). Confirmatory Western immunoblots indicate the higher expression levels for p53 and c-Myc achieved in the respective transfected cells (Fig. 3 d). The relatively unchanged levels of p53 protein in the c-myc-transfected cells suggest that c-Myc may not be directly upstream of p53 or in its direct regulatory pathway. To determine whether it is the down-regulation of the p53 signal that is required to mediate this mechanism, p53-deficient mouse fibroblasts (p53−/−) and wild-type (p53+/+) controls were treated with the apoptosis-inducing FN fragment and examined for their rate of apoptosis. The p53-deficient cells underwent a faster rate of apoptosis than their p53+/+ counterpart controls (Fig. 4,a, c, and d). Cell shape changes for the primary mouse fibroblasts demonstrated a more well spread phenotype, characteristic of live cells, whereas the p53−/−fibroblasts demonstrated cell membrane blebbing and a rounded phenotype, associated with apoptosis (Fig. 4 a). The wild-type fibroblasts showed a 3.3% base-line level of apoptosis, which increased to 5% at 1.5 h and 5.8% at 3 h upon treatment with the V+H− protein. The p53−/− fibroblasts showed a 3.6% base-line level of apoptosis, which increased to 12% at 1.5 h and 14.2% at 3 h upon treatment with the V+H−, a 3.3- and 3.9-fold increase in apoptosis at 1.5 h and 3 h, respectively, compared with the 1.5- and 1.8-fold increase in apoptosis in the wild-type fibroblasts at the same respective time points (Fig.4 c). This is approximately a 2-fold difference. The fold changes in apoptosis for the p53−/− and wild-type fibroblasts were computed for an average of three experiments and illustrated graphically (Fig. 4 d). Western immunoblots confirmed the absence of p53 in the p53−/− cells and the decrease in p53 and c-Myc proteins in the normal mouse fibroblasts after treatment with the V+H− fragment (Fig.4 b). In p53−/− cells, c-Myc protein levels did not change appreciably or were only slightly decreased in response to the V+H− treatment, suggesting that although c-Myc may not be in a direct regulatory pathway with p53, it may be influenced by p53 status in this apoptotic mechanism. The requirement for down-regulation of c-Myc in this apoptotic pathway was also investigated by using antisense strategies. In this case, c-myc antisense-treated human fibroblasts underwent a more rapid rate of apoptosis than control non-sense oligonucleotide-treated or control-transfected cells upon addition of the V+H− fragment (Fig.5, a, c, and d), further confirming that it is in part the depressed levels of c-Myc that mediate this mechanism. Cell shape changes for the non-sense-treated cells demonstrated a more well spread phenotype, characteristic of live cells, whereas the c-mycantisense-treated cells demonstrated cell membrane blebbing and a rounded phenotype (Fig. 5 a). The antisense-treated cells showed a 1.4% base-line level of apoptosis, which increased to 3.9% at 1 h and 7.9% at 3 h upon treatment with the V+H− protein. The non-sense-treated cells showed a 2% base-line level of apoptosis, which increased to 2.8% at 1 h and 5.9% at 3 h upon treatment with the V+H− protein. The control-transfected cells showed a 3.3% base-line level of apoptosis, which increased to 5.4% at 1 h and 8% at 3 h upon treatment with the V+H− protein. Thus, the antisense-treated cells showed a 2.8- and 5.6-fold increase in apoptosis at 1 and 3 h, respectively, compared with the 1.4- and 2.95-fold increase in apoptosis in the non-sense-treated cells at the same respective time points. Similarly, the transfected controls showed a 1.6- and 2.4-fold increase in apoptosis. Therefore, both of the controls showed an ∼2-fold lower level of apoptosis than the antisense-treated cells (Fig. 5 c). The fold changes in apoptosis for antisense-, non-sense-, and control-transfected cells were computed for an average of three experiments and illustrated graphically (Fig. 5 d). Fig. 5 b illustrates the effect of the oligonucleotides on c-Myc expression. Taken together, these data suggest that down-regulation of either p53 or c-Myc may be sufficient for induction of apoptosis by the FN fragment because loss of either p53 or c-Myc function was sufficient to induce a faster rate of apoptosis. Consistent with the changes in p53, levels of a downstream signal, cyclin-dependent kinase p21, were depressed in tandem (Fig. 1 d). Ectopic expression of full-length p21, but not its C-terminal form (C-p21) (22Luo Y. Hurwitz J. Massague J. Nature. 1995; 375: 159-161Crossref PubMed Scopus (516) Google Scholar), rescued the apoptotic phenotype induced by the V+H− fragment (Fig.6, a–c). Thep21-transfected cells demonstrated the more well spread phenotype characteristic of live cells, whereas the C-p21-transfected and untransfected control cells demonstrated a rounded and cell membrane blebbing phenotype (Fig.6 a). The p21-transfected cell population showed a 2.4% base-line level of apoptosis, which increased to 4.8% upon treatment with the V+H− protein. The C-p21-transfected cell population showed a 2.2% base-line level of apoptosis, which increased to 9.7% upon treatment with the V+H− protein. The untransfected cell population showed a 2.7% base-line level of apoptosis, which increased to 11.3% upon treatment with the V+H−protein. Thus, the p21-transfected cells showed only a 2-fold increase in apoptosis, compared with the 4.2- and 4.4-fold increase in the untransfected control and C-p21-transfected cells, respectively. Therefore, the p21-transfected cells showed approximately a 2-fold lower level of apoptosis than the controls (Fig. 6 b). The fold changes in apoptosis forp21- and C-p21-transfected cells, and untransfected cells were computed for an average of three experiments and illustrated graphically (Fig. 6 c). Western immunoblot results confirmed the increased expression levels of p21 in thep21-transfected cells and the more gradual decline of p21 protein in these same cells upon treatment with the FN fragment (Fig.6 d). The decline in p21 was not surprising given that p53 acts as a transcriptional activator of p21. In addition, p53 protein levels remained unchanged after p21 transfection, again suggesting that p53 lies upstream of p21 in this pathway. Although a great deal is known about how the p53 protein interacts with other proteins to control transcription, little is known about the factors that control p53 transcription itself. However, our data shed light on how p53 transcription can be regulated by ECM-generated signals and specifically by the heparin-binding domain and alternatively spliced V region of FN. Our combined data suggest an alternative regulatory pathway for p53, c-Myc, and p21 as part of an apoptotic pathway initiated by the interactions of an altered ECM ligand with cell surface receptors. Proteoglycan and integrin receptors (11Kapila Y.L. Wang S. Johnson P.W. J. Biol. Chem. 1999; 274: 30906-30913Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) 2Y. Kapila, S. Wang, and P. Dazin, manuscript in preparation. then trigger an apoptotic signaling pathway that leads to decreases in pp125FAK phosphorylation, activation of a caspase cascade, and depression of downstream signals that include c-Myc, p53, and p21. Others (23Wu M. Arsura M. Bellas B. Fitzgerald M.J. Lee H. Schauer S.L. Sherr D.H. Sonenshein G.E. Mol. Cell. Biol. 1996; 16: 5015-5025Crossref PubMed Scopus (125) Google Scholar) have reported that a drop in c-Myc levels leads to apoptosis of B lymphoma cells. We report on a unique mechanism of regulation of c-Myc, p53, and p21 in human primary cells by specific domains of FN in the context of apoptosis. The impact of the present findings, when viewed in the context of our other in vivo, in vitro, and clinical data, affords a greater level of significance. In other ongoing studies in our laboratory, we have focused on a 40-kDa proteolytic FN fragment (Invitrogen) that is comparable with the V+H−recombinant FN protein examined here because it contains the heparin-binding domain and part of the V region (CS-1 site). Thus, at least one of the two FN regions, the heparin-binding domain or V region, is compromised in these two fragments. To further explain, the similarities in the 40-kDa fragment and the V+H− protein lie in the fact that they are both missing some part of either the heparin-binding domain or the V region of FN. The importance of this fact relates to our findings suggesting that at least two receptors, the α4β1 integrin and a chondroitin sulfate proteoglycan, are involved in regulating apoptosis by interacting with these two FN domains. Our hypothesis is that if one of these domains is partially missing or nonfunctional, then these two receptors can no longer engage these domains normally and necessary survival cues are no longer initiated; this, in turn, activates a cell death pathway or apoptosis. Furthermore, we have discovered that this 40-kDa fragment not only is found in vivo and associated with inflammation 3Q. Huynh, S. Wang, E. Tafolla, S. Gansky, S, Kapila, G.C. Armitage, and Y. L. Kapila, manuscript submitted for publication. but also triggers apoptosis in these same human primary fibroblasts, specifically and dose-dependently. Furthermore, this 40-kDa FN fragment, identified in inflammatory fluids from periodontally diseased sites, was found to be highly associated with the disease status of the sites. Prevotella intermedia, a known periodontal pathogen, produces a chymotrypsin-like proteinase that can cleave FN (24Homer K.A. Manji F. Beighton D. J. Clin. Periodontol. 1992; 19: 305-310Crossref PubMed Scopus (37) Google Scholar) and thus generate this same chymotryptic 40-kDa fragment. Thus, one possibility is that FN fragments signal cells that their ECM has undergone dissolution secondary to inflammation. These signals may lead to apoptosis of the cellular components of inflamed tissues and thus contribute to the overall degradation of tissues under these conditions. Indeed, other studies have confirmed the presence of higher levels of FN fragmentation in the more advanced stages of inflammatory diseases such as arthritis and periodontal disease (25Carsons S. Lavietes B.B. Diamond H.S. Kinney S.G. Arthritis Rheum. 1985; 28: 601-612Crossref PubMed Scopus (50) Google Scholar, 26Clemmensen I. Andersen R.B. Arthritis Rheum. 1982; 25: 25-31Crossref PubMed Scopus (76) Google Scholar, 27Griffiths A.M. Herbert K.E. Perret D. Scott D.L. Clin. Chim. Acta. 1989; 184: 133-146Crossref PubMed Scopus (35) Google Scholar, 28Talonpoika J.J. Heino J. Larjava H. Hakkinen L. Paunio K. Scand. J. Dent. Res. 1989; 97: 415-421PubMed Google Scholar, 29Talonpoika J. Paunio K. Soderling E. Scand. J. Dent. Res. 1993; 101: 375-381PubMed Google Scholar, 30Xie D-L. Meyers R. Homandberg G.A. J. Rheumatol. 1992; 19: 1448-1452PubMed Google Scholar), and some of these FN fragments can themselves produce tissue destruction in vitro (31Homandberg G.A. Meyers R. Xie D.-L. J. Biol. Chem. 1992; 267: 3597-3604Abstract Full Text PDF PubMed Google Scholar) and in vivo (32Homandberg G.A. Meyer R. Williams J.M. J. Rheumatol. 1993; 20: 1378-1382PubMed Google Scholar). Furthermore, recent evidence suggests that p53 may be important in the regulation of inflammatory pathways (33Tan M. Wang Y. Guan K. Sun Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 109-114Crossref PubMed Scopus (233) Google Scholar) and not just in apoptotic, survival, and cell cycle pathways. Future studies will be needed to decipher the potential intersection of ECM/integrin/proteoglycan/pp125FAK signals with p53/c-Myc/p21 signals and to directly demonstrate the role of these FN fragments as proapoptotic molecules within in vivo model systems. We thank Drs. Bert Vogelstein, Gerard Zambetti, and John Cleveland for their helpful discussions and gifts of reagents. We thank many colleagues for their gifts of reagents, including Dr. Joan Massague for the p21 and C-p21 constructs and Dr. Caroline Damsky for the p53-deficient and wild-type mouse cells. We thank Evangeline Leash for editorial advice." @default.
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• W2140433265 title "The Heparin-binding Domain and V Region of Fibronectin Regulate Apoptosis by Suppression of p53 and c-myc in Human Primary Cells" @default.
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