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• W2137567423 abstract "Vascular endothelial growth factors (VEGFs) are a highly conserved family of growth factors all angiogenic in vivo with mitogenic and chemotactic activity on endothelial cells. VEGFs are expressed in fibroblasts either in hypoxia or in response to growth factors. Here we report that, differently from the other members of the family,Vegf-D is induced by cell-cell contact. By in situ hybridization we demonstrated that noninteracting fibroblasts express low levels of Vegf-D mRNA, whereas contacting cells express high levels of Vegf-D transcripts. By immunostaining we observed that the surface protein cadherin-11 is localized at the opposite sites of interacting cell surfaces. Ca2+ deprivation from the culture medium determined the loss of cadherin-11 from the cell surfaces and down-regulation ofVegf-D mRNA. Moreover, a cadherin-11 antisense RNA construct inhibited Vegf-D expression in confluent BALB/c fibroblasts, whereas in NIH 3T3 cells, which express low levels of cadherin-11, Vegf-D induction could be obtained by overexpression of cadherin-11. This suggests that cell interaction mediated by cadherin-11 induces the expression of the angiogenic factorVegf-D in fibroblasts. Vascular endothelial growth factors (VEGFs) are a highly conserved family of growth factors all angiogenic in vivo with mitogenic and chemotactic activity on endothelial cells. VEGFs are expressed in fibroblasts either in hypoxia or in response to growth factors. Here we report that, differently from the other members of the family,Vegf-D is induced by cell-cell contact. By in situ hybridization we demonstrated that noninteracting fibroblasts express low levels of Vegf-D mRNA, whereas contacting cells express high levels of Vegf-D transcripts. By immunostaining we observed that the surface protein cadherin-11 is localized at the opposite sites of interacting cell surfaces. Ca2+ deprivation from the culture medium determined the loss of cadherin-11 from the cell surfaces and down-regulation ofVegf-D mRNA. Moreover, a cadherin-11 antisense RNA construct inhibited Vegf-D expression in confluent BALB/c fibroblasts, whereas in NIH 3T3 cells, which express low levels of cadherin-11, Vegf-D induction could be obtained by overexpression of cadherin-11. This suggests that cell interaction mediated by cadherin-11 induces the expression of the angiogenic factorVegf-D in fibroblasts. vascular endothelial growth factor interleukin phosphate-buffered saline poly-2-hydroxyethyl methacrylate The VEGF1 family is composed of several structurally and functionally related growth factors involved in vascular development. This family includes the vascular endothelial growth factor (VEGF), the placental growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E (1Betsholtz C. Johnsson A. Heldin C.-H. Westermark B. Lind P. Urdea M.S. Eddy R. Shows T.B. Philphott K. Mellor A.L. Knott T.J. Scott J. Nature. 1986; 320: 695-699Crossref PubMed Scopus (538) Google Scholar, 2Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connolly D.T. Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1811) Google Scholar, 3Leung D.W. Cachianes G. Kuang W. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar, 4Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar, 5Olofsson B. Pajusola K. Kaipanen A. von Euler G. Joukov V. Saksela O. Orpana A. Petterson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar, 6Grimmond S. Lagerkrantz J. Drinkwater C. Silins G. Towson S. Pollock P. Gotley D. Carson E. Rakar S. Nordenskjold M. Ward L. Hayard N. Weber G. Genome Res. 1996; 6: 124-131Crossref PubMed Scopus (116) Google Scholar, 7Joukov V. Pajusola K. Kaipainen A. Chilov D. Lathinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar, 8Lee J. Gray A. Yan J. Luoh S.-M. Avraha H. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1988-1992Crossref PubMed Scopus (330) Google Scholar, 9Orlandini M. Marconcini L. Ferruzzi R. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11675-11680Crossref PubMed Scopus (265) Google Scholar, 10Nicosia R.F. Am. J. Pathol. 1998; 153: 11-16Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 11Ogawa S. Oku A. Sawano A. Yamaguchi S. Yazaki Y. Shibuya M. J. Biol. Chem. 1998; 273: 31273-31282Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). All members of this family are angiogenic in vivo and able to stimulate proliferation of endothelial cells in vitro. Each member of the family recognizes and activates specific receptors on endothelial cells: VEGF recognizes VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1); placental growth factor and VEGF-B recognize VEGFR-1 (12Terman B.I. Khandke L. Dougher-Vermazan M. Maglione D. Lassam N.J. Gosporadowicz D. Persico M.G. Bohlen P. Eisinger M. Growth Factors. 1994; 11: 187-196Crossref PubMed Scopus (82) Google Scholar, 13Sawano A. Takahasci T. Yamaguki S. Aunuma M. Shibuya M. Cell Growth Differ. 1996; 7: 213-221PubMed Google Scholar, 14Olofsson B. Korpelainen E. Pepper M.S. Mandriota S.J. Aase K. Kumar V. Gunji Y. Jeltsch M.M. Shibuya M. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11709-11714Crossref PubMed Scopus (450) Google Scholar); VEGF-C and VEGF-D recognize VEGFR-2 and VEGFR-3 (Flt-4) (7Joukov V. Pajusola K. Kaipainen A. Chilov D. Lathinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar, 15Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar, 16Marconcini L. Marchiò S. Morbidelli L. Cartocci E. Albini A. Ziche M. Bussolino F. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9671-9676Crossref PubMed Scopus (229) Google Scholar). This latter is almost exclusively expressed in lymphatic vessels, suggesting that these factors, beside playing a role in angiogenesis, are also involved in the formation of lymphatic vessels. Due to the similarity of structure and promiscuity of receptor recognition, the specific role of each member of the family has not yet been identified. In differentiating tissues, specific regulation of each factor may be required to determine the correct succession and composition of the appropriated angiogenic factors for vessel formation. VEGF expression has been extensively studied. It responds to low levels of oxygen with induced transcription and increasing mRNA stability (17Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (879) Google Scholar, 18Minchenco A. Bauer T. Salceda S. Caro J. Lab. Invest. 1994; 71: 374-379PubMed Google Scholar, 19Ikeda E. Achen M.G. Breier G. Risau W. J. Biol. Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Moreover, VEGFmRNA expression is up-regulated by epidermal growth factor, transforming growth factor-β, IL-6 in several cell types, and by IL-1β in smooth muscle cells (20Pertovaara L. Kaipainen A. Mustonen T. Orpana A. Ferrara N. Sakesela O. Alitalo K. J. Biol. Chem. 1994; 269: 6271-6274Abstract Full Text PDF PubMed Google Scholar, 21Choen T. Nadhari D. Cerem L.W. Neufeld G. Levi B.Z. J. Biol. Chem. 1996; 271: 736-741Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar, 22Li J. Perrella M.A. Tsai J.C. Yet S.F. Hsieh C.M. Yoshizumi M. Patterson C. Endego W.O. Zhou F. Lee M. J. Biol. Chem. 1995; 270: 308-312Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). VEGF-C expression in cultured fibroblasts is induced by serum, phorbol 12-myristate 13-acetate, and several factors, including IL-1β and tumor necrosis factor α (23Enholm B. Paavonen K. Ristimäki A. Kumar V. Gunji Y. Klefstrom J. Kivinen L. Laiho M. Olofsson B. Jukov V. Eriksson U. Alitalo K. Oncogene. 1997; 14: 2475-2483Crossref PubMed Scopus (387) Google Scholar, 24Ristimaki A. Narko K. Enholm B. Joukov V. Alitalo K. J. Biol. Chem. 1998; 273: 8413-8418Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Vegf-D appeared to be differently regulated, because it was expressed in cells grown in low serum conditions (9Orlandini M. Marconcini L. Ferruzzi R. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11675-11680Crossref PubMed Scopus (265) Google Scholar). Analyzing Vegf-D mRNA expression in mouse fibroblasts we observed that this growth factor, differently from the other members of the VEGF family, was induced by calcium-dependent cell-cell interactions. Cell-cell adhesion is mediated by cadherins, a large family of transmembrane calcium-dependent adhesive glycoproteins that form homotypic binding with their extracellular domain on adjacent cells (25Steinberg M.S. McNutt P.M. Curr. Opin. Cell Biol. 1999; 11: 554-560Crossref PubMed Scopus (248) Google Scholar, 26Marrs J.A. Nelson W.J. Int. Rev. Cytol. 1996; 165: 159-205Crossref PubMed Google Scholar, 27Gumbiner B.M. J. Cell Biol. 2000; 148: 399-403Crossref PubMed Scopus (690) Google Scholar). Although it is generally thought that cadherin expression results in a tight cell association, this is not a general principle and mesenchymal cells, which are loosely associated, express mesenchyme-specific cadherins like cadherin-11 (28Okazaki M. Takeshita S. Kaway S. Kikuno R. Tsujimura A. Kudo A. Amann E. J. Biol. Chem. 1994; 269: 12092-12098Abstract Full Text PDF PubMed Google Scholar, 29Hoffmann I. Balling R. Dev. Biol. 1995; 169: 337-346Crossref PubMed Scopus (118) Google Scholar, 30Kimura Y. Matsunami H. Inoue T. Shimamura K. Uchida N. Ueno T. Miyazaki T. Takeichi M. Dev. Biol. 1995; 169: 347-358Crossref PubMed Scopus (218) Google Scholar, 31Simonneau L. Kitagawa M. Suzuki S. Thiery J.P. Cell. Adhes. Commun. 1995; 3: 115-130Crossref PubMed Scopus (124) Google Scholar). The data presented in this report demonstrate that Vegf-Dmessenger is strongly induced by direct cell-cell contact. This induction can be inhibited by depletion of extracellular Ca2+ from the culture medium. Inhibition of cadherin-11 expression in contacting fibroblasts reduces Vegf-D mRNA induction, whereas cadherin-11 expression in fibroblasts, that do not express cadherin-11, restores Vegf-D induction. These results identify cadherin-11 as a surface molecule involved inVegf-D regulation by cell-cell interaction. The mouse cadherin-11full-length cDNA was amplified from a mouse fibroblast cDNA library (9Orlandini M. Marconcini L. Ferruzzi R. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11675-11680Crossref PubMed Scopus (265) Google Scholar), using the primers 5′-GAGAGGATCCACCACCATGAAGGAGAACTACTG-3′ and 5′-GAGACTCGAGTTAAGAGTCATCATCAAAAGTG-3′. The polymerase chain reaction product was cloned into the plasmid pcDNA3 (Invitrogen Corp.) in the sense (giving MO447) and antisense (giving MO334) orientation under the control of the cytomegalovirus promoter. The oligonucleotide sequences were obtained from the EBI Nucleotide Sequence Data Base under accession numbers D21253 (OB-cadherin) andD31963 (cadherin-11). All constructs were checked by automated sequencing. Mouse embryo fibroblasts were isolated from 14-day CD1 mouse embryos as described previously (32Orlandini M. Semplici F. Ferruzzi R. Meggio F. Pinna L.A. Oliviero S. J. Biol. Chem. 1998; 273: 21291-21297Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Unless otherwise stated, mouse embryo, mouse 3T3-type, NIH 3T3, and BALB/c 3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified, 5% CO2 atmosphere. Stable clones expressing mouse cadherin-11 were obtained from NIH 3T3 cells transfected with thecadherin-11 expression vector MO447 by standard CaPO4 precipitation procedures (32Orlandini M. Semplici F. Ferruzzi R. Meggio F. Pinna L.A. Oliviero S. J. Biol. Chem. 1998; 273: 21291-21297Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Transfectants were selected using 1 mg/ml G418 (Life Technologies, Inc.). Stable clones expressing mouse cadherin-11 in the antisense orientation were obtained from BALB/c 3T3 fibroblasts transfected with the plasmid MO334, and transfectants were selected using 0.4 mg/ml G418. The same empty vector was used to generate mock stable clones. Mouse fibroblasts were plated 14–16 h before day 0 on 10-cm tissue culture dishes at different density, and starting from day 0, culture medium was changed every 2 days. Low, medium, and high cell densities corresponded to cells plated from about 20% to about 70% confluence. The degree of cell confluence was monitored under an inverted microscope. The cell cycle was arrested by adding cyclosporin A (0.95 μg/ml), colchicine (0.11 μg/ml), or tunicamycin (0.5 μg/ml) in the culture medium of subconfluent fibroblasts for 24 h. Conditioned medium was obtained from the culture medium of fibroblasts growing at high cell confluence, diluted 1:1 (v/v) with complete medium, and used to stimulate subconfluent fibroblasts for 33 h. The heparin wash of confluent fibroblasts was performed by using a solution of heparin (100 μg/ml, Sigma-Aldrich) in PBS. After taking off the medium, the heparin solution was left on confluent cells for 2 h at room temperature, collected, centrifuged, diluted in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, and used to stimulate subconfluent fibroblasts for 33 h. As negative control the heparin solution not left on the cells was used. To chelate Ca2+ in the culture medium, confluent fibroblasts were grown for 24 h in the presence of 2.2 mm EGTA. Poly-HEME (Sigma-Aldrich) was used to inhibit cell adhesion to growth surface in culture dishes. Culture plates were coated with 6 mg/ml poly-HEME in 95% ethanol and allowed to air dry in a sterile environment. Fibroblast cells were seeded at high cell density in plates precoated with poly-HEME and after 48 h cells were collected by centrifugation and RNA was extracted. To block or stimulate Ca2+ flux through calcium channels, fibroblasts were grown either for 18 h at high cell density in the presence of Ca2+ channel antagonists (10 μm diltiazem, 50 μm amiloride, 20 μm nifedipine, 10 μm verapamil, 1 μm ω-conotoxin GVIA) or for 24 h at low cell density in the presence of a Ca2+channel agonist (1.25 μm BAY K-8644). Cyclosporin A, colchicine, tunicamycin, diltiazem, amiloride, and ω-conotoxin GVIA were purchased from BioMol Research Laboratories, and nifedipine, verapamil, BAY K-8644 were from Sigma-Aldrich. Cell synchronization agents and calcium channel modulators were used at concentrations established from the literature to have maximal effects on their targets. Total cellular RNA was extracted from cells by the guanidinium thiocyanate method (33Chomzyski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159PubMed Google Scholar). Total RNA (10 μg) was run on denaturing formaldehyde-agarose gel, transferred onto nylon membranes, and cross-linked by UV irradiation using a Stratalinker (Stratagene). Filters were hybridized with32P-labeled probes, washed as described (9Orlandini M. Marconcini L. Ferruzzi R. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11675-11680Crossref PubMed Scopus (265) Google Scholar), and analyzed by using a PhosphorImager (Molecular Dynamics). Rat glyceraldheyde-3-phosphate dehydrogenase (gapdh) was used as a control for RNA loading. Digoxigenin-labeled Vegf-Dsense and antisense RNA probes were generated from a cDNA fragment corresponding to the complete coding sequence of the mouseVegf-D gene. Mouse fibroblasts were grown on microscopic slides at different degrees of confluence and fixed for 20 min with 4% paraformaldehyde in PBS. In situ hybridization was performed as described previously (34Boom R. Geelen J.L. Sol C. Raap A.K. Minaar R.P. Klavier B.P. Noordaa J.v. d. J. Virol. 1986; 58: 851-859Crossref PubMed Google Scholar) with minor modifications. Briefly, to increase permeability cells were treated for 10 min with 0.2n HCl and for 25 min at 37 °C with 1 μg/ml proteinase K (Sigma-Aldrich) in 50 mm Tris-HCl, pH 8. Then cells were washed in PBS and post-fixed for 10 min with 4% paraformaldehyde in PBS. Cells were hybridized overnight in a humidified chamber at 37 °C with the digoxigenin-labeled probes diluted at 1 μg/ml in hybridization buffer (60% deionized formamide, 2× SSC buffer, 50 mm sodium phosphate, 5% dextran sulfate, 250 μg/ml yeast RNA, and 250 μg/ml salmon sperm DNA). The slides were washed, and the hybridized digoxigenin-conjugated probes were detected by using the fluorescent antibody enhancer set (Roche Diagnostics) according to standard procedures. Slides were counterstained with propidium iodide (Sigma-Aldrich) at 100 ng/ml, mounted in PBS containing 2% 1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich) and 50% glycerol, and examined under a Leica TCS confocal laser-scanning microscope. The sense strand gave no signal. Whole cell extracts were prepared by rinsing cultures with cold buffer (20 mm HEPES, pH 7.4, 130 mm NaCl, 5 mm KCl, 1 mmMgCl2, 2 mm EGTA). Cells were harvested with a rubber policeman, centrifuged, and lysed in 0.5% Nonidet P-40 buffer (20 mm HEPES, pH 7.4, and 2 mm EDTA) containing Complete protease inhibitors (Roche Diagnostic). Protein concentration of cell extracts were determined by using the BCA protein assay reagent (Pierce). The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Equal loading was confirmed by staining in Ponceau S solution (Sigma-Aldrich). The membranes were blocked for 1 h at room temperature in PBS containing 3% dry milk and 0.1% Triton X-100 and incubated with goat polyclonal antibodies against OB-cadherin (Santa Cruz Biotechnology, Inc.) at 0.4 μg/ml for 2 h at room temperature. The blots were washed, incubated with horseradish peroxidase-labeled donkey anti-goat IgG (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature and washed in PBS, and finally the bound antibodies were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). Mouse fibroblasts were seeded onto glass coverslips, cultured overnight, and fixed with 3% paraformaldehyde in PBS for 15 min. Cells were then permeabilized in 0.5% Triton X-100 in PBS for 3 min and blocked for 1 h with 1% bovine serum albumin in PBS. Coverslips were incubated for 1 h at 37 °C with goat polyclonal anti-OB-cadherin antibodies. After washing, the coverslips were incubated for 45 min at 37 °C in the presence of donkey anti-goat IgG labeled with tetramethylrhodamine isothiocyanate (Jackson ImmunoResearch Laboratories). To localize actin filaments, fluorescein isothiocyanate-labeled phalloidin (Sigma-Aldrich) was added along with secondary antibodies at 2 μg/ml. Coverslips were then mounted in Mowiol 4-88 (Calbiochem) and examined under a Leica TCS confocal laser-scanning microscope. UnlikeVEGF-C, whose expression is induced by several growth factors (23Enholm B. Paavonen K. Ristimäki A. Kumar V. Gunji Y. Klefstrom J. Kivinen L. Laiho M. Olofsson B. Jukov V. Eriksson U. Alitalo K. Oncogene. 1997; 14: 2475-2483Crossref PubMed Scopus (387) Google Scholar, 24Ristimaki A. Narko K. Enholm B. Joukov V. Alitalo K. J. Biol. Chem. 1998; 273: 8413-8418Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), Vegf-D is not induced by cell treatment with platelet-derived growth factor, epidermal growth factor, fibroblast growth factor 4, basic fibroblast growth factor, or transforming growth factor β (data not shown). Moreover, we previously observed that in low serum conditions fibroblasts expressed high levels of Vegf-D transcripts (9Orlandini M. Marconcini L. Ferruzzi R. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11675-11680Crossref PubMed Scopus (265) Google Scholar). To test whether Vegf-D mRNA induction may require high cell density, RNA was collected at various time points from fibroblasts plated at different densities. At day 0, when cells were plated at low (20% confluence) or medium (40% confluence) density, the expression of Vegf-D was barely detectable, whereas someVegf-D expression could be detected in cells plated at the highest density (70% confluence) (Fig.1 A, lanes 1–3). Two days later, after the cells reached a higher confluence, we observed a correspondent induction of Vegf-D transcripts. In particular, Vegf-D expression was increased in the cells originally plated at medium and high density (lanes 5 and6). In fact, after 2 days, these cells reached about 90 and 98% confluence, respectively, with elevated cell-cell interactions. At day 2, cells that were originally plated at low density (lane 4) were at about 30% confluence and showed still lowVegf-D expression. Quantitative analysis revealed that cells plated at higher density reached the highest expression ofVegf-D between days 4 and 6 from plating, cells plated at medium density at day 6, and cells plated at low density at about day 8 (Fig. 1 B). Thus, the levels of Vegf-D transcripts and cell density are directly correlated. Next we examined whether cell cycle arrest or soluble autocrine growth factor(s) accumulating in the culture medium, or on the surface of cells growing at high cell density, could be responsible for Vegf-D induction. The treatment of subconfluent cells with the cell cycle inhibitors cyclosporin A, colchicine, or tunicamycin did not lead to Vegf-D induction (Fig.2 A). Neither the treatment of subconfluent cells with conditioned medium nor with a heparin wash from confluent cells could induce Vegf-D expression, suggesting that autocrine-soluble factors were not involved in Vegf-Dinduction (Fig. 2 B, compare lane 1 withlanes 2 and 4). To test whether cell-cell interaction and/or cell-plate contacts would play a role inVegf-D induction, we analyzed Vegf-D mRNA levels in cells growing either in the presence of EGTA or in plates precoated with poly-HEME. In the presence of EGTA, cells grew at a normal rate, acquired a round shape, and lost interactions with each other and with the culture plate. Deprivation of Ca2+ from the culture medium strongly inhibited Vegf-D mRNA accumulation (Fig. 2 C, lane 1). On the contrary, culture plates precoated with poly-HEME, which inhibited cell adhesion to the plate, did not affect Vegf-D induction (Fig.2 C, lane 2), suggesting that cell-matrix interactions are not involved in Vegf-D expression. Because Ca2+ depletion from the culture medium might affect calcium influx into the cells, we tested the expression of Vegf-DmRNA in cells treated with different Ca2+ channel blockers. The treatment of confluent cells with the calcium channel antagonists nifedipine, verapamil, amiloride, diltiazem, or ω-conotoxin GVIA did not inhibit Vegf-D mRNA expression, and the treatment of sparse cells with the calcium channel agonist BAY K-8644 did not induce Vegf-D expression (Fig.2 D). Therefore, this excluded that calcium influx plays a role in the Vegf-D up-regulation. To directly observe Vegf-D mRNA expression in contacting cells we performed in situexperiments with cultured fibroblasts plated at various degrees of confluence. Hybridization with Vegf-D antisense probe showed that single cells expressed very poor levels of Vegf-DmRNA, whereas contacting fibroblasts did express high levels ofVegf-D messenger. This could be observed even at the level of two interacting cells (Fig. 3,A and B). Taken together, the above experiments demonstrated that both cell-cell interaction and extracellular calcium ions are required forVegf-D up-regulation in mouse fibroblasts. Homophilic calcium-dependent cell-cell interactions are mediated by cadherins (25Steinberg M.S. McNutt P.M. Curr. Opin. Cell Biol. 1999; 11: 554-560Crossref PubMed Scopus (248) Google Scholar). We therefore tested the hypothesis that direct interaction between contacting fibroblasts, mediated by cadherins, could be responsible for Vegf-D up-regulation. First we examined by Northern blot analysis the expression of cadherins in sparse and confluent fibroblasts with different cadherin probes. We observed that, in fibroblasts, grown at different degrees of confluence, cadherin-11, a mesenchymal-specific cadherin, was strongly induced by cell-cell contact (Fig.4). Importantly, cadherin-11mRNA induction was preceding Vegf-D mRNA of 8–12 h, suggesting that cadherin-11 could be involved in Vegf-Dregulation. Immunostaining of contacting fibroblasts using anti-cadherin-11 antibodies revealed a positive staining at the cell surfaces (Fig.5). In sparse cells cadherin-11 was localized at intercellular contacts, whereas it was mostly absent from surfaces free of cell contact (Fig. 5 A). At confluence cadherin-11 staining was observed at the level of the whole cell membrane (Fig. 5 B). To examine whether the localization of cadherin in fibroblasts depends on Ca2+, contacting cells were treated with EGTA. As expected, within the first hour cadherin-11 signal disappeared from the cell surface and became mostly cytoplasmic (Fig. 5 C). Addition of Ca2+ to the media restored cell-cell contacts, with reappearance of cadherin-11 at the intercellular contacts and induction of Vegf-D mRNA in the cells (not shown). In our study, mouse 3T3 type fibroblasts, derived from mice strain 129/SvJ × C57BL/6J (129-B6) (35Hu E. Mueller E. Oliviero S. Papaioannou V.E. Johnson R. Spiegelman B.M. EMBO J. 1994; 13: 3094-3103Crossref PubMed Scopus (169) Google Scholar), were used for comparative analysis of gene expression, because Vegf-D was strongly expressed in these cells. We tested whether other fibroblasts showed the same Vegf-D mRNA regulation. Primary embryo fibroblasts obtained from CD1 mice and BALB/c 3T3 fibroblasts revealed a Vegf-D mRNA strong induction that correlated withcadherin-11 high expression in confluent cells. Instead, NIH 3T3 fibroblasts expressed barely detectable levels of bothcadherin-11 and Vegf-D mRNAs (Fig.6). Thus, extending the correlation between Vegf-D mRNA induction and cadherin 11expression in the same cells. To directly evidence that cadherin-11 is required for Vegf-Dexpression, we generated, from BALB/c 3T3 cells, stable cell lines overexpressing cadherin-11 in the antisense orientation. Analysis of several stable clones revealed a variable level of cadherin-11 measured by Western blot of cell lysates. Two clones expressing low levels of cadherin-11 were analyzed forVegf-D expression (Fig.7 A). By Northern blot analysis of the cadherin-11 antisense clones using Vegf-Dprobe, we observed that inhibition of cadherin-11 resulted in a strong reduction of Vegf-D expression (Fig. 7, compare Aand B). The converse experiment was performed in NIH 3T3 fibroblasts, because these cells expressed low levels of cadherin-11. From NIH 3T3 we generated stable clones expressing, under the control of a constitutive promoter, cadherin-11 and analyzed Vegf-D mRNA expression levels in contacting cells. Two clones expressing higher levels of cadherin-11 were chosen for Vegf-D expression analysis (Fig. 7 C). In these cells the ectopic expression of cadherin-11 induced a significant increase of Vegf-Dtranscripts (Fig. 7, compare C and D). In multicellular organisms, intercellular interactions and inductive signals play a major role in cell fate during development. Cell-cell adhesion, dictated by homophilic surface molecules like cadherins, determine cell patterning establishing the tissue architecture; however, secreted growth factors, which act at a few cell diameters, modify the expression pattern of neighboring target cells. Here we demonstrate that in cultured fibroblasts direct cell-cell interaction, mediated by the mesenchyme-specific cadherin-11, triggersVegf-D mRNA induction, suggesting cross-talk occurs between cell adhesion and growth factor signaling. Several lines of evidence support the model that Vegf-Dexpression is regulated by direct cell-cell interactions via cadherin-11. First, Vegf-D is not induced in subconfluent cells under diverse culture conditions, whereas its expression is dramatically increased in cells that reach confluence. Second, the addition of conditioned media from cells highly expressingVegf-D does not induce its expression in cells grown at low density, excluding the possibility that autocrine-diffusible factors are instrumental in this activation. Third, depletion of extracellular calcium ions, but not inhibition of cells to signal through calcium flux, blocks Vegf-D expression, demonstrating that direct calcium-dependent cell-cell interactions are required. Fourth, Vegf-D expression directly correlates with cadherin-11 localization on the cell-interacting surfaces. Fifth, down-modulation or overexpression of cadherin-11 in fibroblasts affectsVegf-D expression in a negative and positive manner, respectively. Thus, the experiments described in this report provide evidence that cadherin-11 mediates a cell interaction signaling that leads to the regulation of Vegf-D in contacting fibroblasts. Cadherins play an important role in cell recognition and sorting during development (36, 37; and references therein). Their function has been perceived to link and stabilize connections between cells through interaction with the cytoskeleton. However, cytoplasmic domains of cadherins are highly diversified, and examples of cadherins have been found associated with signal transduction molecules and/or able to induce intracellular messengers, suggesting that cadherins mediate signal transduction pathways upon ligand binding (for review see Ref.38Yagi T. Takeichi M. Genes Dev. 2000; 14: 1169-1180PubMed Google Scholar). During development Vegf-D mRNA expression appears to be restricted to cadherin-11-positive mesenchymal cells. In the developing mouse embryo cadherin-11 is expressed in migratory cells derived from neural crest cells and in cells involved in mesenchymal condensation (30Kimura Y. Matsunami H. Inoue T. Shimamura K. Uchida N. Ueno T. Miyazaki T. Takeichi M. Dev. 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Vegf-D regulation by cell-cell contact is consistent with a model in which aggregating fibroblasts switch on the expression of a factor that is chemotactic to endothelial cells and thus could recruit them to the forming tissue. The cell-cell contact mediated by cadherin-11 in vitro may mimic, in part, the angiogenic process and suggests that Vegf-D expression might be involved in the initial steps of the angiogenic process. The neovascularization and/or formation of lymphatic vessels in solid tumors is induced by tumor cells that express inductive angiogenic factors, which promote vessels sprouting with the recruitment of endothelial cells to the growing tumor mass (10Nicosia R.F. Am. J. Pathol. 1998; 153: 11-16Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 41Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 42Fukumura D. Xavier R. Sugiura T. Chen Y. Park E.C. Lu N. Selig M. Nielsen G. Taksir T. Jain R.K. Seed B. 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