FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034705232> ?p ?o ?g. }

• W2034705232 endingPage "31878" @default.
• W2034705232 startingPage "31871" @default.
• W2034705232 abstract "Fibroblasts constitutively express matrix metalloproteinase 2 (MMP-2), which specifically cleaves type IV collagen, a major structural component of basement membranes. The level of MMP-2 expression was not altered by serum withdrawal, suggesting that MMP-2 expression is regulated by a series of steady-state conditions that impinge on the MMP-2 promoter. Expression of a dominant-negative Ras protein significantly inhibited MMP-2 transcription, thereby suggesting a role for steady-state Ras function in the regulation of MMP-2 expression. Kirsten-Ras (K-Ras) knockout fibroblasts express undetectable basal levels of MMP-2, whereas N-Ras knockout fibroblasts expressed constitutive levels of MMP-2 similar to those observed in wild-type control fibroblasts. Using an MMP-2 promoter-luciferase reporter assay, we demonstrated that the transcription of MMP-2 in K-Ras knockout fibroblasts was partially restored by transient expression of c-K(B)-Ras but not c-K(A)-Ras. A phosphoinositide-3 (PI-3) kinase-specific inhibitor (LY294002) decreased the basal level of MMP-2 in wild-type fibroblasts. Blocking PI-3 kinase signaling by overexpression of the regulatory domain of PI-3 kinase (p85) also down-regulated the steady-state MMP-2 levels. Fibroblasts that fail to express AKT1 also expressed decreased amounts of MMP-2 compared with wild-type fibroblasts. These data suggest that steady-state MMP-2 expression is regulated by c-K(B)-Ras through a PI-3 kinase:AKT-dependent signaling pathway. Because the majority of the MMP-2 assays were performed using conditioned media from serum-starved fibroblasts, these data also highlight our previous observations that Ras proteins have functions in the absence of acute mitogenic stimulations. In addition, this is the first demonstration of a specific steady-state function attributable to K(B)-Ras. Fibroblasts constitutively express matrix metalloproteinase 2 (MMP-2), which specifically cleaves type IV collagen, a major structural component of basement membranes. The level of MMP-2 expression was not altered by serum withdrawal, suggesting that MMP-2 expression is regulated by a series of steady-state conditions that impinge on the MMP-2 promoter. Expression of a dominant-negative Ras protein significantly inhibited MMP-2 transcription, thereby suggesting a role for steady-state Ras function in the regulation of MMP-2 expression. Kirsten-Ras (K-Ras) knockout fibroblasts express undetectable basal levels of MMP-2, whereas N-Ras knockout fibroblasts expressed constitutive levels of MMP-2 similar to those observed in wild-type control fibroblasts. Using an MMP-2 promoter-luciferase reporter assay, we demonstrated that the transcription of MMP-2 in K-Ras knockout fibroblasts was partially restored by transient expression of c-K(B)-Ras but not c-K(A)-Ras. A phosphoinositide-3 (PI-3) kinase-specific inhibitor (LY294002) decreased the basal level of MMP-2 in wild-type fibroblasts. Blocking PI-3 kinase signaling by overexpression of the regulatory domain of PI-3 kinase (p85) also down-regulated the steady-state MMP-2 levels. Fibroblasts that fail to express AKT1 also expressed decreased amounts of MMP-2 compared with wild-type fibroblasts. These data suggest that steady-state MMP-2 expression is regulated by c-K(B)-Ras through a PI-3 kinase:AKT-dependent signaling pathway. Because the majority of the MMP-2 assays were performed using conditioned media from serum-starved fibroblasts, these data also highlight our previous observations that Ras proteins have functions in the absence of acute mitogenic stimulations. In addition, this is the first demonstration of a specific steady-state function attributable to K(B)-Ras. MMP-2 1The abbreviations used are: MMP, matrix metalloproteinase; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CMV, cytomegalovirus; PI-3, phosphoinositide-3; MEF, mouse embryonic fibroblast. belongs to an evolutionarily conserved proteinase family that requires Zn2+ for enzymatic activity. MMP-2 is synthesized and secreted as inactive zymogen. The activation of MMP-2 requires the removal of prodomain by the membrane-type MMPs. The activity of MMP-2 is tightly regulated by its natural inhibitor TIMP2 (1Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3239) Google Scholar, 2Curran S. Murray G.I. J. Pathol. 1999; 189: 300-308Crossref PubMed Scopus (611) Google Scholar, 3Westermarck J. Kahari V.M. FASEB J. 1999; 13: 781-792Crossref PubMed Scopus (1398) Google Scholar). The expression of MMP-2 is tissue and developmentally regulated. MMP-2 is expressed in the developing murine lung and kidney, with minimum expression in bone and the central nervous system. In the adult animal, MMP-2 is minimally expressed, primarily confined to cells of mesangial origin (4Reponen P. Sahlberg C. Huhtala P. Hurskainen T. Thesleff I. Tryggvason K. J. Biol. Chem. 1992; 267: 7856-7862Abstract Full Text PDF PubMed Google Scholar). The synthesis of MMP-2 is relatively constitutive. Sequence analysis revealed that the transcription of MMP-2 is under control of a number of cis-acting regulatory elements in the 5′-flanking region, including P53, Y-box, AP2, AP-1, ETS, C/EBP, CREB, PEA3, and Sp1 (5Qin H. Sun Y. Benveniste E.N. J. Biol. Chem. 1999; 274: 29130-29137Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 6Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar, 7Zucker S. Cao J. Chen W.T. Oncogene. 2000; 19: 6642-6650Crossref PubMed Scopus (501) Google Scholar, 8Mertens P.R. Steinmann K. Alfonso-Jaume M.A. En-Nia A. Sun Y. Lovett D.H. J. Biol. Chem. 2002; 277: 24875-24882Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The high specific activity of MMP-2 toward collagen and its pericellular localization imply that it is a key component in the degradation of basement membranes and cell migration (1Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3239) Google Scholar, 9Brooks P.C. Stromblad S. Sanders L.C. von Schalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Quigley J.P. Cheresh D.A. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar). Increased expression of MMP-2 in tumor cells correlates with invasiveness and metastatic potential (10Azzam H.S. Arand G. Lippman M.E. Thompson E.W. J. Natl. Cancer Inst. 1993; 85: 1758-1764Crossref PubMed Scopus (207) Google Scholar, 11Vaisanen A. Tuominen H. Kallioinen M. Turpeenniemi-Hujanen T. J. Pathol. 1996; 180: 283-289Crossref PubMed Scopus (135) Google Scholar, 12Liabakk N.B. Talbot I. Smith R.A. Wilkinson K. Balkwill F. Cancer Res. 1996; 56: 190-196PubMed Google Scholar, 13Sawaya R.E. Yamamoto M. Gokaslan Z.L. Wang S.W. Mohanam S. Fuller G.N. McCutcheon I.E. Stetler-Stevenson W.G. Nicolson G.L. Rao J.S. Clin. Exp. Metastasis. 1996; 14: 35-42Crossref PubMed Scopus (169) Google Scholar). Tumor cells expressing oncogenic Ras proteins possess higher potential to metastasize, in part because of the up-regulation of MMP-2 expression (14Giunciuglio D. Culty M. Fassina G. Masiello L. Melchiori A. Paglialunga G. Arand G. Ciardiello F. Basolo F. Thompson E.W. Albini A. Int. J. Cancer. 1995; 63: 815-822Crossref PubMed Scopus (73) Google Scholar, 15Arbiser J.L. Moses M.A. Fernandez C.A. Ghiso N. Cao Y. Klauber N. Frank D. Brownlee M. Flynn E. Parangi S. Byers H.R. Folkman J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 861-866Crossref PubMed Scopus (422) Google Scholar, 16Charvat S. Le Griel C. Chignol M.C. Schmitt D. Serres M. Clin. Exp. Metastasis. 1999; 17: 677-685Crossref PubMed Scopus (17) Google Scholar, 17Moon A. Kim M.S. Kim T.G. Kim S.H. Kim H.E. Chen Y.Q. Kim H.R. Int. J. Cancer. 2000; 85: 176-181Crossref PubMed Scopus (112) Google Scholar). Ras proteins are a group of small G proteins. The most common forms of oncogenic Ras have mutations at either codon 12, 13, or 61 (18Bos J. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). These mutations result in a higher proportion of Ras proteins with GTP in their binding site. Introduction of oncogenic H-Ras into fibroblasts results in increased expression of MMP-2 (14Giunciuglio D. Culty M. Fassina G. Masiello L. Melchiori A. Paglialunga G. Arand G. Ciardiello F. Basolo F. Thompson E.W. Albini A. Int. J. Cancer. 1995; 63: 815-822Crossref PubMed Scopus (73) Google Scholar, 17Moon A. Kim M.S. Kim T.G. Kim S.H. Kim H.E. Chen Y.Q. Kim H.R. Int. J. Cancer. 2000; 85: 176-181Crossref PubMed Scopus (112) Google Scholar). Blocking Ras-dependent signaling decreased both expression of MMP-2 and invasion of Src-transformed cells, indicating a role for Ras in regulating MMP-2 expression (19Thant A.A. Sein T.T. Liu E. Machida K. Kikkawa F. Koike T. Seiki M. Matsuda S. Hamaguchi M. Oncogene. 1999; 18: 6555-6563Crossref PubMed Scopus (32) Google Scholar). Three genes encode the four immediate members of the Ras family: H-, N-, K(A)-, and K(B)-Ras. K(A)- and K(B)-Ras proteins arise from alternative splicing of the K-Ras mRNA (20Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-154Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar, 21Ellis C.A. Clark G. Cell Signal. 2000; 12: 425-434Crossref PubMed Scopus (133) Google Scholar). We have generally found c-Ha-Ras protein expression to be detectable only in cells of neuronal origin, whereas c-N-Ras and both K-Ras isoforms are expressed in all tested cell lines. Recent data suggest that different Ras isoforms possess distinct cellular functions. The most compelling evidence is that both the H-Ras knockout and the N-Ras knockout mice are viable, whereas the K-Ras knockout is embryonic lethal. The loss of two K-Ras proteins was not mimicked by the Ha-, N-Ras double knockout mice, which, similar to the single knockouts, were viable (22Johnson L. Greenbaum D. Cichowski K. Mercer K. Murphy E. Schmitt E. Bronson R.T. Umanoff H. Edelmann W. Kucherlapati R. Jacks T. Genes Dev. 1997; 11: 2468-2481Crossref PubMed Scopus (442) Google Scholar, 23Koera K. Nakamura K. Nakao K. Miyoshi J. Toyoshima K. Hatta T. Otani H. Aiba A. Katsuki M. Oncogene. 1997; 15: 1151-1159Crossref PubMed Scopus (288) Google Scholar, 24Esteban L.M. Vicario-Abejon C. Fernandez-Salguero P. FernandezMedarde A. Swaminathan N. Yienger K. Lopez E. Malumbres M. McKay R. Ward J.M. Pellicer A. Santos E. Mol. Cell. Biol. 2001; 21: 1444-1452Crossref PubMed Scopus (250) Google Scholar, 25Umanoff H. Edelmann W. Pellicer A. Kucherlapati R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1709-1713Crossref PubMed Scopus (206) Google Scholar, 26Ise K. Nakamura K. Nakao K. Shimizu S. Harada H. Ichise T. Miyoshi J. Gondo Y. Ishikawa T. Aiba A. Katsuki M. Oncogene. 2000; 19: 2951-2956Crossref PubMed Scopus (114) Google Scholar). Furthermore, over-expression experiments suggest that the different Ras isoforms might have preferential targets (27Voice J.K. Klemke R.L. Le A. Jackson J.H. J. Biol. Chem. 1999; 274: 17164-17170Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 28Yan J. Roy S. Apolloni A. Lane A. Hancock J.F. J. Biol. Chem. 1998; 273: 24052-24056Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). We have shown in mouse fibroblasts that Raf-1 is the preferential binding partner for plasma membrane-associated c-N-Ras (29Hamilton M. Wolfman A. Oncogene. 1998; 16: 1417-1428Crossref PubMed Scopus (62) Google Scholar, 30Hamilton M. Liao J. Cathcart M.K. Wolfman A. J. Biol. Chem. 2001; 276: 29079-29090Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Using immortalized fibroblasts derived from the N-Ras knockout mice, we demonstrated that c-N-Ras provides a steady-state antiapoptotic function (31Wolfman J.C. Wolfman A. J. Biol. Chem. 2000; 275: 19315-19323Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The N-Ras and K-Ras knockout fibroblasts provide excellent tools to study the role the K- and N-Ras isoforms in regulating the expression of MMP-2. Reagents—Gelatin was obtained from Sigma. MMP-2 cDNA probe was obtained from American Type Culture Collection. PD098059, SB203580, LY294002, genistein, PD153035, and the AKT-specific inhibitor were obtained from Calbiochem. Antibodies—MMP-2 (Ab-3) was obtained from Oncogene. Anti-HSP90 monoclonal antibody was obtained from BD Transduction Laboratories. Anti-AKT1 antibody was obtained from Cell Signaling Technology, Inc. The polyclonal antibody against K(B)-Ras, P85, and the monoclonal antibodies against N-Ras and Ha-Ras were obtained from Santa Cruz Biotchnology, Inc. Cell Culture—All cell lines were maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf serum. For the preparation of conditioned medium for zymography, cells (at ∼80% confluence) were washed three times with serum-free medium. Cells were then cultured for an additional 18 h in serum-free medium before collection of the conditioned medium for MMP assays. When using pharmacological inhibitors, cells were cultured for 24 h in the presence of the inhibitor, washed, and then placed in serum-free medium containing the same concentration of the specific inhibitor for an additional 18 h. Preparation of Cell Lysates—All lysis buffers contained the following phosphatase and protease inhibitors: 30 mm β-glycerophosphate, 5 mm p-nitrophenyl phosphate, 1 mm phosphoserine, 1 mm phosphothreonine, 0.2 mm phosphotyrosine, 100 μm sodium orthovanadate, 25 μg/ml each aprotinin, leupeptin, pepstatin A, and 1 mm phenylmethylsulfonyl fluoride. Cells were scraped into phosphate-buffered saline and pelleted by centrifugation (500 × g for 5 min). The cell pellet was resuspended in p21 buffer (20 mm MOPS, 5 mm MgCl2, 0.1 mm EDTA, 200 mm sucrose, pH 7.4) containing 1% CHAPS (United States Biochemical Corp.). The cells were lysed for 20 min on ice and centrifuged at 13,000 × g for 10 min to remove nuclei and cell debris. The supernatant solution was retained for further experiments. The protein concentration was determined by the method of Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216391) Google Scholar). Zymography of MMP in Conditioned Medium—Conditioned, serum-free medium was prepared as described above. The medium was centrifuged at 15,000 × g to remove cellular debris. The amount of conditioned medium was normalized to the amount of protein in the corresponding parallel cell lysates (prepared as described above). Non-reducing sample buffer was added to the appropriate amount of conditioned medium and the samples were separated by SDS-PAGE using gels made with 1 mg/ml gelatin in the separating gel. The gels were electrophoresed at 25 mA until the bromphenol blue dye front reached the bottom. The gels were rinsed three times for 10 min each with 50 mm Tris, pH 7.4, and 2% Triton X-100 followed by three washes (5 min each) with 50 mm Tris, pH 7.4. The gels were then incubated overnight at 37 °C in developing buffer (50 mm Tris, pH 7.4, 0.2 m NaCl, 1% Triton X-100, 0.02% NaN3, and 5 mm CaCl2). The next day, the gels were rinsed and stained with 0.2% Coomassie blue in 50% methanol and 10% acetic acid for 1 h. Finally, the gels were destained in 20% methanol and 10% acetic acid. MMP activity was observed by the generation of a negative clear band in the blue-gelatin staining background. The gels were quantitated using a Microtek scanner and NIH Image software version 1.60b7 and normalized with respect to untreated controls. Reverse Transcription-PCR—1 μg of total RNA from K+C, K+13, K-2B, and K-6 were used for reverse transcription with Roche TaqMan reverse transcription reagents (poly-dT primer). Reverse transcription was performed at 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. PCR were performed with Roche High-Fi polymerase. The denature temperature was 94 °C, annealing temperature was 60 °C, and elongation temperature was 72 °C. The oligonucleotide primers were synthesized by Sigma. The sequences of primers were: atg gag gca cga gtg gcc tg (MMP-2 sense primer), tca gca gcc cag cca gtc tg (MMP-2 antisense primer), tcg gcg tgaa cgg att tgg ccg ta (glyceraldehyde-3-phosphate dehydrogenase sense primer), and tgg cat gga ccg tgg tca tga gtc (glyceraldehyde-3-phosphate dehydrogenase antisense primer). Immunoblotting—Lysates containing equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Hybond P; Amersham Biosciences). The membrane was blocked with blocker casein in phosphate-buffered saline (Pierce Chemical Co.) containing 10% newborn calf serum (Invitrogen). The washed blots were incubated with primary antibodies (1:1000) for 2 to 3 h at room temperature or overnight at 4 °C. After washing in Tris-buffered saline/0.1% Tween 20, the blots were incubated with secondary antibodies (1:1000) for 1 h at room temperature. After washing, the signals were detected using standard enhanced chemiluminescence techniques. The signals were quantitated using a Microtek scanner and NIH Image software version 1.60b7 and normalized to untreated controls. Luciferase Reporter Assay—1 × 105 cells per well were seeded into 12-well dishes. The MMP-2 promoter luciferase reporter and pRL-CMV were transiently introduced into fibroblasts in combination with other indicated constructs using LipofectAMINE/Plus Reagent (Invitrogen). The amount of total DNA was kept constant using the appropriate empty vectors. The luciferase activity of the MMP-2 reporter was analyzed with the dual-luciferase reporter assay system (Promega) and normalized to the activity of the Renilla reniformis luciferase. The basal level of luciferase activity was analyzed by transfection with pGL2-Basic plasmid and pRL-CMV. The relative luciferase activity is reported as the fold increase of the MMP-2 reporter luciferase activity compared with the basal level of luciferase activity. Steady-state Expression of MMP-2 in Mouse Fibroblasts—We first tested whether the production of MMP-2 maintains its tissue specificity in cultured cells. Conditioned medium from each cell line was analyzed either by gelatin zymography (Fig. 1A, top) or by immunoblotting the conditioned media for MMP-2 (Fig. 1A, bottom). Both murine fibroblast cell lines, NIH3T3 and C3H10T1/2, secrete easily detectable amounts of MMP-2. Epithelial cells (IEC-6 and COS-7), however, did not secrete detectable levels of MMP-2 (Fig. 1A). We next tested whether the generation of MMP-2 was a constitutive, steady-state phenomenon or might be regulated through short-term signal transduction mechanisms. In Fig. 1B, the expression of MMP-2 activity in conditioned medium was monitored through a serum-deprivation time course. Cells were serum-starved for a 24-hour period. After this initial starvation period, cells were incubated with fresh serum-free medium. Aliquots of this medium were taken every 6 h and analyzed for the presence of MMP-2 by zymography. The data suggest that MMP-2 production continue at a linear rate (Fig. 1B, right), even after this prolonged period of serum withdrawal. These data suggest that the generation of MMP-2 in the extracellular medium is not regulated through a serum-dependent mechanism. The converse experiment, examining whether MMP-2 expression was altered upon serum addition, confirmed the steady-state nature of MMP-2 expression (Fig. 1C). Challenging quiescent cultures with serum did not induce any further expression or secretion of MMP-2. Interestingly, however, we did observe a reproducible increase in MMP-9 activity 24 h after serum addition. These data suggest that MMP-2 expression and secretion are regulated through a steady-state mechanism, rather than an inducible mechanism that might be dependent on acute signal transduction. K-Ras Knockout Fibroblasts Fail to Secrete MMP-2—The constitutive production of MMP-2 by mouse fibroblasts allowed us to use N-Ras and K-Ras negative cell lines to examine the role of each Ras isoform in the maintenance of MMP-2 synthesis and secretion. To avoid potential artifacts arising from cell line divergence, we generated multiple immortalized cell lines from the K-Ras knockout and control MEF populations. These are represented by K-1C, K-2B, K-3, K-4, and K-6 for the K-Ras negative cell lines and K+C, K+2, K+13, and K+14 for the control cell lines. N–/– and N+/+ indicate, for these experiments, a single N-Ras negative and control immortalized fibroblast population. We verified, by immunoblotting, the presence and absence of the different Ras isoforms in this spectrum of cell lines (Fig. 2A). As expected, the K-Ras negative cell lines fail to express K(B)-Ras and K(A)-Ras, whereas the N-Ras negative cells express both K(B) and K(A)-Ras but not N-Ras. There was no significant difference in the level of c-N-Ras expressed by the control K-Ras-expressing cells and the K-Ras negative cells (expressing 9 ng of c-N-Ras per 30 μg of cell lysate). Consistent with our previous experience using mouse fibroblasts, c-Ha-Ras was not detected. In this case, a lysate generated from C3H10T1/2 cells transformed by the minimal expression of an oncogenic G12V-Ha-Ras (11A) and PC12 cell lysate were used as positive controls for Ha-Ras expression. As has been our experience with cultured cell lines, c-Ha-Ras is only detectable in cell lines of neuronal origin (with a 2-ng sensitivity). The five K-Ras negative fibroblast cell lines did not possess detectable MMP-2 activity. In contrast, all the wild-type, control fibroblast cell lines expressed MMP-2 activity comparable with that of other mouse fibroblast cell lines (Fig. 2B, top). Both N-Ras knockout and wild-type, N+/+, control fibroblasts expressed levels of MMP-2 activity consistent with that observed for both the NIH3T3 and C3H10T1/2 cell lines. These data were confirmed by Western analysis of the conditioned media (Fig. 2B, bottom). These data indicate that the steady-state level of MMP-2 in mouse fibroblasts requires the function(s) of a c-K-Ras gene product rather than that of c-N-Ras. The failure to detect c-Ha-Ras protein in any of these cell lines rules out its involvement in the regulation of steady-state MMP-2 expression. K-Ras Regulates MMP-2 Levels through a Transcriptional Mechanism—We first tested whether the K-Ras negative cells were producing MMP-2 but failing to secrete the protein into the extracellular milieu. Similar to our observations with conditioned medium, whole-cell extracts generated from the K-Ras knockout fibroblasts possessed significantly less (an 85% decrease) MMP-2 protein compared with control cells (Fig. 3A). MMP-2 mRNA, as detected by reverse transcription-PCR analysis, suggested that K-Ras regulated the steady-state transcription of the MMP-2 gene (Fig. 3B). In contrast, there was a lack of MMP-2 mRNA amplification in the K-Ras negative cell lines suggesting the absence of MMP-2 mRNA. The 519-bp glyceraldehyde-3-phosphate dehydrogenase fragment was amplified in all samples as a loading control. Similar results were obtained by Northern analysis in that we did not detect significant levels of MMP-2 mRNA in the K-Ras-negative cell lines. MMP-2 mRNA was detected in both K+/+ cell lines and the positive control, HT1080, a human fibrosarcoma transformed by an activated N-Ras allele (data not shown). We confirmed this observation using a transient, luciferase reporter assay (Fig. 3C). MMP-2 transcription levels from K-Ras knockout and wild-type fibroblasts were measured by transfection of a rat MMP-2 promoter luciferase reporter construct and pRL-CMV. The luciferase activity of the MMP-2 reporter construct was analyzed with the dual-luciferase reporter assay system (Promega) and normalized to the activity of R. reniformis luciferase. The relative luciferase activity indicates the fold increase of MMP-2 reporter luciferase activity related to the basal level luciferase activity. Only minimal reporter activity was detected in the K-Ras negative cell lines, whereas 10–30-fold higher reporter activity was detected in the K-Ras-expressing control fibroblasts. These data are consistent with the hypothesis that K-Ras function is required for the transcription of the MMP-2 gene. Ras Signaling Is Required for Transcription of MMP-2—To explore the possible role of Ras signaling in the regulation of steady-state MMP-2 expression, a dominant-negative Ha-Ras-N17 vector was co-transfected with the previously described Rat MMP-2 promoter luciferase reporter construct. Ras-N-17 blocks Ras activity be tying up Ras exchange factors into a non-productive cycle (32Schweighoffer F. Cai H. Chevallier-Multon M.C. Fath I. Cooper G. Tocque B. Mol. Cell. Biol. 1993; 13: 39-43Crossref PubMed Scopus (49) Google Scholar). The luciferase activity of the MMP-2 reporter construct was analyzed as described in Fig. 3C. Expression of the dominant-negative Ha-Ras-N17 inhibited MMP-2 promoter activity by ∼80% in the K-Ras-expressing control fibroblasts (Fig. 4A). We next used expression plasmids encoding the separate K-Ras isoforms in an attempt to modulate MMP-2 reporter activity (Fig. 4B). Only c-K(B)-Ras restored MMP-2 reporter activity in the transient transfection assay described in Fig. 4A (p < 0.05). In this instance, we used phorbol 12-myristate 13-acetate addition as a positive control. In separate experiments, we found that phorbol ester addition increased the basal expression of MMP-2 protein by 2-fold (data not shown). Therefore, the level of transcriptional activation by K(B)-Ras, though only 50% above the vector control, is consistent with the level of transcriptional activation by the positive control. Combining both K(A)- and K(B)-Ras encoding plasmids did not give rise to a synergistic activation of MMP-2 luciferase activity. These results suggest that constitutive expression of MMP-2 is controlled through the function(s) of only c-K(B)-Ras and not c-K(A) or c-N-Ras. PI-3 Kinase/AKT Signaling Pathway Is Required for the Steady-state MMP-2 Expression in Fibroblasts—Ras proteins are key molecular switches that mediate transmembrane signaling through the activation of multiple downstream pathways, which include the MAPK and PI-3 kinase pathways (33Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (920) Google Scholar). A series of pharmacological inhibitors were used to test the potential contribution of one or more Ras-dependent signaling pathways that might regulate the steady-state transcription of the MMP-2 gene. Secretion of MMP-2 into the medium of K-Ras-expressing control fibroblasts was analyzed by immunoblotting for MMP-2 after a 48-h treatment with one of the following signaling inhibitors: PD098059 (mitogen-activated protein kinase kinase-specific inhibitor, 37 μm), SB203580 (p38-specific inhibitor, 10 μm), LY294002 (PI-3 kinase-specific inhibitor, 20 μm), genistein (tyrosine kinase inhibitor, 100 μm) or PD153035 (epidermal growth factor receptor tyrosine kinase inhibitor, 10 μm). LY294002 treatment dramatically inhibited the secretion of MMP-2 in wild-type fibroblasts (Fig. 5A). There was also a less pronounced, but reproducible, effect using the general tyrosine kinase inhibitor genistein. Others have also reported the inhibition of MMP-2 expression by genistein (34Alhasan S.A. Aranha O. Sarkar F.H. Clin. Cancer Res. 2001; 7: 4174-4181PubMed Google Scholar). The PI-3 kinase-dependent, constitutive expression of MMP-2 was mimicked upon examination of the protein levels in whole-cell lysates (Fig. 5B). The specific PI-3 kinase inhibitor LY294002 was effective in blocking the steady-state accumulation of MMP-2 in whole-cell lysates, again suggesting that the expression of MMP-2 is regulated through a K-Ras-dependent pathway that flows through PI-3 kinase. We used overexpression of the regulatory p85 subunit of PI-3 kinase as a dominant negative to block basal signaling through receptor-mediated tyrosine kinases. Overexpression of the p85 regulatory subunit inhibited MMP-2 promoter activity in both K-Ras-expressing control cell lines (Fig. 5C). The K+C control cell line was more susceptible to this inhibition, which is consistent with this cell line's increased sensitivity of MMP-2 promoter activity to the tyrosine kinase inhibitor, genistein (Fig. 5B). These data all support the hypothesis that constitutive MMP-2 expression is regulated through a steady-state signaling event passing through c-K(B)-Ras and PI-3 kinase. The lipid products of PI-3 kinase activity result in the activation of a downstream serine kinase, AKT. Using a similar strategy, we examined the levels of MMP-2 secretion in cells that were wild-type, heterozygous, or homozygous knockouts for AKT1. In complete agreement with our previous observations and the documented relationship between PI-3 kinase lipid products and AKT activation, there was a significant decrease in constitutive MMP-2 expression in the AKT1-negative MEF populations (Fig. 6A, top). It is interesting that the decreased MMP-2 expression was similar between the AKT1+/– and the AKT1–/– MEFs, suggesting a possible threshold effect for AKT expression. We have observed a similar phenomenon in N-Ras heterozygous MEFs in their heightened response to apoptotic agents; N-Ras+/– cells responded in a manner more similar to the N–/– cells than the N+/+ cells. 2J. C. Wolfman and A. Wolfman, unpublished observations. Western analysis of AKT1 verified that the AKT1+/– cells expressed AKT1 at about 50% of the level observed in the AKT1+/+ MEFS. To verify the threshold nature of the MMP-2 expression of AKT levels, we quantitated the amount of AKT in the different MEF populations (Fig. 6A, bottom). To confirm the role of AKT signaling in mediating MMP-2 expression, we used an AKT-specific inhibitor (20 μm) to block AKT-dependent gene expression in control K-Ras-expressing fibroblasts. Similar reductions in the steady-state levels of MMP-2 in whole-cell lysates were observed in the presence of either the PI-3 kinase inhibitor LY294002 or the specific AKT inhibitor (Fig. 6B). These data support the hypothesis that constitutive MMP-2 expression is regulated through the steady-state functions of c-K(B)-Ras, PI-3 kinase and AKT. The data presented in this manuscript support the newly established consensus that the different immediate Ras isoforms do not have completely overlapping functions (20Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-154Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar, 21Ellis C.A. Clark G. Cell Signal. 2000; 12: 425-434Crossref PubMed Scopus (133) Google Scholar). Several groups have shown that the Ras isoforms possess different potencies in their respective abilities to activate Raf-1 and PI-3 kinase (27Voice J.K. Klemke R.L. Le A. Jackson J.H. J. Biol. Chem. 1999; 274: 17164-17170Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 28Yan J. Roy S. Apolloni A. Lane A. Hancock J.F. J. Biol. Chem. 1998; 273: 24052-24056Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). We have also shown, in mouse fibroblasts, that plasma membrane associated c-N-Ras is the preferential binding partner for Raf-1 (29Hamilton M. Wolfman A. Oncogene. 1998; 16: 1417-1428Crossref PubMed Scopus (62) Google Scholar, 30Hamilton M. Liao J. Cathcart M.K. Wolfman A. J. Biol. Chem. 2001; 276: 29079-29090Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Here, using immortalized N-Ras and K-Ras knockout fibroblasts, we have shown that, in the absence of short-term mitogenic stimulation, the expression of MMP-2 is regulated through a c-K(B)-Ras dependent process. Every K-Ras-negative cell line tested was devoid of MMP-2 secretion, whereas the N-Ras-negative cells expressed levels of MMP-2 consistent with that observed in control fibroblasts. The decreased expression of MMP-2 in K-Ras-negative cells was mimicked by incubation of control cells with either a PI-3 kinase or AKT inhibitor, by expression of a dominant-negative PI-3 kinase, or by the failure to express AKT-1, suggesting the involvement of these signaling intermediates in the regulation of MMP-2 levels. Indeed, cells failing to express AKT1 were also deficient in the expression of MMP-2. The data presented in this manuscript are the first to document a specific signaling pathway endpoint regulated solely by K(B)-Ras. It is likely that other Ras isoforms regulate PI-3 kinase and AKT. In previous work, we have shown that the N-Ras-negative cell lines also have depressed steady-state levels of activated AKT (31Wolfman J.C. Wolfman A. J. Biol. Chem. 2000; 275: 19315-19323Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 35Wolfman J.C. Palmby T. Der C.J. Wolfman A. Mol. Cell. Biol. 2002; 22: 1589-1606Crossref PubMed Scopus (62) Google Scholar), although this does not apparently affect their ability to regulate MMP-2 expression. The mechanism that renders the PI-3 kinase/AKT-dependent regulation of MMP-2 expression specific for K(B)-Ras is unclear. It is possible that the subcellular localization of the specific K(B)- Ras, PI-3 kinase and AKT molecules that function to control the MMP-2 promoter significantly contribute toward specificity between c-K(B)-Ras, AKT and MMP-2 expression. Based on previous results and those reported in this communication, regulation of a specific downstream target is not necessarily sufficient to obtain an identical phenotype. Both c-N-Ras and c-K(B)-Ras can regulate steady-state AKT activity, but only c-K(B)-Ras does so in a manner that results in the corresponding regulation of MMP-2 expression. These subtle differences highlight the importance of performing experiments at endogenous levels of protein expression to avoid upsetting the delicate balance between these unique steady-state signaling pathways. Signal transduction has traditionally been examined by short-term stimulation of cells with agents that induce mitogenesis, differentiation, or apoptosis (36Crespo P. Leon J. Cell Mol. Life Sci. 2000; 57: 1613-1636Crossref PubMed Scopus (153) Google Scholar, 37Kerkhoff, E., and Rapp, U. R. (1998) 17, 1457–1462Google Scholar, 38Rebollo A. Martinez A.C. Blood. 1999; 94: 2971-2980Crossref PubMed Google Scholar). We have found, however, that maintenance of basal cell function also requires “steady-state” signal transduction. In this specific instance, the steady-state production of a single matrix metalloproteinase, MMP-2, is regulated through the steady-state functions of c-K(B)-Ras, PI-3 kinase, and AKT. Our previous work using N-Ras-negative cells suggested that short-term signaling pathways readily adapt to the absence of essential signal transduction components (31Wolfman J.C. Wolfman A. J. Biol. Chem. 2000; 275: 19315-19323Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 35Wolfman J.C. Palmby T. Der C.J. Wolfman A. Mol. Cell. Biol. 2002; 22: 1589-1606Crossref PubMed Scopus (62) Google Scholar). In contrast, we have found that cellular steady-state functions do not adapt as rapidly. Using immortalized N-Ras- and K-Ras-negative cell lines, we have identified a novel steady-state function for c-K(B)-Ras. The K-Ras-negative cell lines expressed detectable levels of only c-N-Ras. Consistent with our previous observations, we have not observed detectable levels of c-Ha-Ras in mouse fibroblasts, even as a compensatory mechanism for the loss of either the N-ras or K-ras genes. The K-Ras-negative cells failed to synthesize detectable levels of MMP-2, one of the matrix metalloproteinases involved in cell detachment, migration, and metastasis of invasive tumors. Failure to express the c-N-Ras gene product did not alter the levels of MMP-2 generation, suggesting a direct relationship between one of the K-Ras gene products and the steady-state production of MMP-2. Transient assays confirmed that only one of the K-Ras gene products, c-K(B)-Ras, is responsible for controlling the transcriptional activity of the MMP-2 gene. This observation is supported by the inhibition of basal MMP-2 production by transient expression of a dominant-negative Ras construct, N17-Ha-Ras, which functions through the sequestering of Ras exchange factors. The identification of steady-state Ras dependent functions opens novel possibilities regarding the role of each oncogenic Ras isoform in specific cancers. The common hypothesis of the involvement of Ras in the development of tumors has always stemmed from their role in short-term signal transduction. Given the data presented in this manuscript, we could now predict that tumors containing an activating K(B)-Ras rather than N-Ras should expressed elevated levels of MMP-2. This should result in a higher metastatic potential and probably a poorer overall prognosis. Therefore, although much of the transforming potential of each Ras isoform might arise from “continuous short-term signaling,” there is probably a secondary component that arises from changes in Ras isoform-dependent “steady-state” signal transduction that has been overlooked. We thank Dr. Tyler Jacks for providing K-Ras knockout and wild-type MEFs, Dr. Raju Kucherlapati for providing N-Ras knockout and wild-type MEFs, Dr. Nissim Hay for providing AKT1 Knockout and wild-type MEFs, and Dr. David Lovett for providing rat MMP-2 promoter luciferase reporter. We thank Drs. Thomas Patterson and Sarah Planchon for their helpful suggestions." @default.
• W2034705232 created "2016-06-24" @default.
• W2034705232 creator A5031842352 @default.
• W2034705232 creator A5055322707 @default.
• W2034705232 creator A5086656920 @default.
• W2034705232 date "2003-08-01" @default.
• W2034705232 modified "2023-09-26" @default.
• W2034705232 title "K-Ras Regulates the Steady-state Expression of Matrix Metalloproteinase 2 in Fibroblasts" @default.
• W2034705232 cites W1502644042 @default.
• W2034705232 cites W1517339521 @default.
• W2034705232 cites W1580959101 @default.
• W2034705232 cites W1800142641 @default.
• W2034705232 cites W1965972240 @default.
• W2034705232 cites W1983609115 @default.
• W2034705232 cites W1989510858 @default.
• W2034705232 cites W1993163893 @default.
• W2034705232 cites W1999441881 @default.
• W2034705232 cites W2013256513 @default.
• W2034705232 cites W2026440079 @default.
• W2034705232 cites W2032707394 @default.
• W2034705232 cites W2038982544 @default.
• W2034705232 cites W2043250294 @default.
• W2034705232 cites W2052660171 @default.
• W2034705232 cites W2057534797 @default.
• W2034705232 cites W2072103796 @default.
• W2034705232 cites W2076743657 @default.
• W2034705232 cites W2078459253 @default.
• W2034705232 cites W2079389107 @default.
• W2034705232 cites W2081516439 @default.
• W2034705232 cites W2093965961 @default.
• W2034705232 cites W2101160383 @default.
• W2034705232 cites W2102884576 @default.
• W2034705232 cites W2122374538 @default.
• W2034705232 cites W2123004850 @default.
• W2034705232 cites W2128049766 @default.
• W2034705232 cites W2135242575 @default.
• W2034705232 cites W2146651717 @default.
• W2034705232 cites W2316297264 @default.
• W2034705232 cites W2324319461 @default.
• W2034705232 cites W2325577633 @default.
• W2034705232 cites W2333923319 @default.
• W2034705232 cites W4254861860 @default.
• W2034705232 cites W4293247451 @default.
• W2034705232 doi "https://doi.org/10.1074/jbc.m301931200" @default.
• W2034705232 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12805379" @default.
• W2034705232 hasPublicationYear "2003" @default.
• W2034705232 type Work @default.
• W2034705232 sameAs 2034705232 @default.
• W2034705232 citedByCount "59" @default.
• W2034705232 countsByYear W20347052322012 @default.
• W2034705232 countsByYear W20347052322013 @default.
• W2034705232 countsByYear W20347052322014 @default.
• W2034705232 countsByYear W20347052322015 @default.
• W2034705232 countsByYear W20347052322016 @default.
• W2034705232 countsByYear W20347052322017 @default.
• W2034705232 countsByYear W20347052322018 @default.
• W2034705232 countsByYear W20347052322020 @default.
• W2034705232 countsByYear W20347052322021 @default.
• W2034705232 countsByYear W20347052322022 @default.
• W2034705232 countsByYear W20347052322023 @default.
• W2034705232 crossrefType "journal-article" @default.
• W2034705232 hasAuthorship W2034705232A5031842352 @default.
• W2034705232 hasAuthorship W2034705232A5055322707 @default.
• W2034705232 hasAuthorship W2034705232A5086656920 @default.
• W2034705232 hasBestOaLocation W20347052321 @default.
• W2034705232 hasConcept C106487976 @default.
• W2034705232 hasConcept C109523444 @default.
• W2034705232 hasConcept C147789679 @default.
• W2034705232 hasConcept C185592680 @default.
• W2034705232 hasConcept C202751555 @default.
• W2034705232 hasConcept C2780381497 @default.
• W2034705232 hasConcept C43617362 @default.
• W2034705232 hasConcept C55493867 @default.
• W2034705232 hasConcept C55728118 @default.
• W2034705232 hasConcept C8171440 @default.
• W2034705232 hasConcept C86803240 @default.
• W2034705232 hasConcept C95444343 @default.
• W2034705232 hasConceptScore W2034705232C106487976 @default.
• W2034705232 hasConceptScore W2034705232C109523444 @default.
• W2034705232 hasConceptScore W2034705232C147789679 @default.
• W2034705232 hasConceptScore W2034705232C185592680 @default.
• W2034705232 hasConceptScore W2034705232C202751555 @default.
• W2034705232 hasConceptScore W2034705232C2780381497 @default.
• W2034705232 hasConceptScore W2034705232C43617362 @default.
• W2034705232 hasConceptScore W2034705232C55493867 @default.
• W2034705232 hasConceptScore W2034705232C55728118 @default.
• W2034705232 hasConceptScore W2034705232C8171440 @default.
• W2034705232 hasConceptScore W2034705232C86803240 @default.
• W2034705232 hasConceptScore W2034705232C95444343 @default.
• W2034705232 hasIssue "34" @default.
• W2034705232 hasLocation W20347052321 @default.
• W2034705232 hasOpenAccess W2034705232 @default.
• W2034705232 hasPrimaryLocation W20347052321 @default.
• W2034705232 hasRelatedWork W1437588494 @default.
• W2034705232 hasRelatedWork W2040997967 @default.
• W2034705232 hasRelatedWork W2043473655 @default.
• W2034705232 hasRelatedWork W2081311681 @default.
• W2034705232 hasRelatedWork W2104905716 @default.

• next