Inadequate Activation of the GTPase RhoA Contributes to the Lack of Fibronectin Matrix Assembly in von Hippel-Lindau Protein-defective Renal Cancer Cells
2008; Elsevier BV; Volume: 283; Issue: 36 Linguagem: Inglês
10.1074/jbc.m709390200
ISSN1083-351X
AutoresMónica Feijoo‐Cuaresma, Luis Fernando Méndez-López, Alfredo Maqueda, Miguel A. Esteban, Salvador Naranjo-Suárez, María C. Castellanos, Mercedes Hernández del Cerro, Silvia Vázquez, Angeles García‐Pardo, Manuel O. Landázuri, Marı́a J. Calzada,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoThe von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL(+) cells. We have previously reported that VHL(-) cells fail to form β1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells. The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL(+) cells. We have previously reported that VHL(-) cells fail to form β1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells. The von Hippel-Lindau disease is an autosomal dominant hereditary cancer syndrome caused by germ line mutations of the von Hippel-Lindau (VHL) 4The abbreviations used are:VHLvon Hippel-LindauFNfibronectinBSAbovine serum albuminPBSphosphate-buffered salineGSTglutathione S-transferaseGFPgreen fluorescent proteinPKCprotein kinase CAbantibodysiRNAsmall interfering RNARCCrenal cancer cellsHIFhypoxia-inducible transcription factorsFAfibrillar adhesionFCfocal contacts. 4The abbreviations used are:VHLvon Hippel-LindauFNfibronectinBSAbovine serum albuminPBSphosphate-buffered salineGSTglutathione S-transferaseGFPgreen fluorescent proteinPKCprotein kinase CAbantibodysiRNAsmall interfering RNARCCrenal cancer cellsHIFhypoxia-inducible transcription factorsFAfibrillar adhesionFCfocal contacts. tumor suppressor gene (1Kaelin Jr., W.G. Nat. Rev. Cancer. 2002; 2: 673-682Crossref PubMed Scopus (689) Google Scholar). According to the clinical manifestations, the disease has been classified into VHL type 1 and VHL type 2. VHL type 2 disease is further classified into three categories: type 2A, type 2B, and type 2C (1Kaelin Jr., W.G. Nat. Rev. Cancer. 2002; 2: 673-682Crossref PubMed Scopus (689) Google Scholar). VHL protein (pVHL) plays an important role in the oxygen-sensing pathway, and its best known function is to promote the ubiquitination and subsequent elimination by the proteasome of the hypoxia-inducible transcription factors HIF1α and HIF2α (2Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3878) Google Scholar, 3Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4439) Google Scholar). 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Genet. 2007; 3: 3Google Scholar), and in addition, it has been recently shown that it serves as a protein kinase adaptor regulating the activity of NF-κB (24Yang H. Minamishima Y.A. Yan Q. Schlisio S. Ebert B.L. Zhang X. Zhang L. Kim W.Y. Olumi A.F. Kaelin Jr., W.G. Mol. Cell. 2007; 28: 15-27Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Adequate interaction of cells with the surrounding matrix regulates essential aspects of normal cell function, and breakdown of the basement membrane occurs in cancer progression and is often associated with solid tumors (25Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22336) Google Scholar). With regard to RCC tumors, we and others have shown that RCCs lacking VHL fail to organize a normal extracellular fibronectin (FN) matrix (14Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. Garcia-Pardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar, 26Ohh M. Yauch R.L. Lonergan K.M. Whaley J.M. Stemmer-Rachamimov A.O. Louis D.N. Gavin B.J. Kley N. Kaelin Jr., W.G. Iliopoulos O. Mol. Cell. 1998; 1: 959-968Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 27Kurban G. Hudon V. Duplan E. Ohh M. Pause A. Cancer Res. 2006; 66: 1313-1319Crossref PubMed Scopus (105) Google Scholar). Therefore, the lack of a proper cell matrix might be involved in the aggressive behavior of VHL(-) tumors. In addition, highly angiogenic tumors are not only dependent on the pVHL-HIF 2α degradation pathway but are also a consequence of loss of a proper extracellular matrix assembly (28Tang N. Mack F. Haase V.H. Simon M.C. Johnson R.S. Mol. Cell Biol. 2006; 26: 2519-2530Crossref PubMed Scopus (75) Google Scholar). Extracellular matrix assembly is a complex multistep process, which first requires binding of FN to cell surface integrins, mostly α5β1 (29Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9007) Google Scholar). Additionally, other events such as actin stress fiber formation and cell contraction are required for the progressive ordination into detergent-insoluble fibrils (30Wierzbicka-Patynowski I. Schwarzbauer J.E. J. Cell Sci. 2003; 116: 3269-3276Crossref PubMed Scopus (390) Google Scholar, 31Pankov R. Cukierman E. Katz B.Z. Matsumoto K. Lin D.C. Lin S. Hahn C. Yamada K.M. J. Cell Biol. 2000; 148: 1075-1090Crossref PubMed Scopus (384) Google Scholar). These processes are mediated by the small Ras-type GTPase member RhoA (32Zhong C. Chrzanowska-Wodnicka M. Brown J. Shaub A. Belkin A.M. Burridge K. J. Cell Biol. 1998; 141: 539-551Crossref PubMed Scopus (493) Google Scholar, 33Zhang Q. Magnusson M.K. Mosher D.F. Mol. Biol. Cell. 1997; 8: 1415-1425Crossref PubMed Scopus (171) Google Scholar). In this respect, we have reported the inability of VHL(-) cells to form β1 fibrillar adhesions (14Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. Garcia-Pardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar) and intracellular actin stress fibers (16Calzada M.J. Esteban M.A. Feijoo-Cuaresma M. Castellanos M.C. Naranjo-Suarez S. Temes E. Mendez F. Yanez-Mo M. Ohh M. Landazuri M.O. Cancer Res. 2006; 66: 1553-1560Crossref PubMed Scopus (64) Google Scholar), and other authors have shown that these cells also lack the proper assembly of actin and vinculin, which promotes actin stress fiber formation (8Kamada M. Suzuki K. Kato Y. Okuda H. Shuin T. Cancer Res. 2001; 61: 4184-4189PubMed Google Scholar). Although expression of VHL is necessary for proper extracellular matrix assembly, the mechanism by which pVHL mediates this process is not well understood. To approach this, we have evaluated the levels and functionality of the FN produced by VHL(+) and VHL(-) cells. We have also studied the role of the signaling pathway that controls formation of actin stress fibers and cell contraction in the regulation of FN matrix assembly in VHL(+) and VHL(-) cells. Our results demonstrated that FN expression levels in VHL(+) and (-) cells showed no correlation with the ability to assemble a FN matrix and that VHL(-) cells expressed functional FN that was properly assembled by VHL(+) cells. Additionally, we found that the lack of FN matrix in VHL(-) cells was partly due to sustained down-regulation of RhoA activity via p190RhoGAP-increased activation. These results provide an insight into novel mechanisms altered in VHL(-) renal cancer cells. Cell Culture—786-O cells lacking VHL (RC3) and subclones stably producing wild-type VHL (WT8) (34Iliopoulos O. Kibel A. Gray S. Kaelin Jr., W.G. Nat. Med. 1995; 1: 822-826Crossref PubMed Scopus (596) Google Scholar), or different VHL mutants such as L188V (type 2C mutant) (35Ohh M. Takagi Y. Aso T. Stebbins C.E. Pavletich N.P. Zbar B. Conaway R.C. Conaway J.W. Kaelin Jr., W.G. J. Clin. Investig. 1999; 104: 1583-1591Crossref PubMed Scopus (83) Google Scholar), RRR, and KRR (type 2C mutant) (36Stickle N.H. Chung J. Klco J.M. Hill R.P. Kaelin Jr., W.G. Ohh M. Mol. Cell Biol. 2004; 24: 3251-3261Crossref PubMed Scopus (146) Google Scholar), Y112H (type 2A mutant), and C162F (type 2B) mutant. These mutants were provided by Dr. Michael Ohh (University of Toronto, Canada): parental RCC4 and RCC10 and subclones stably producing wild-type VHL (37Krieg M. Haas R. Brauch H. Acker T. Flamme I. Plate K.H. Oncogene. 2000; 19: 5435-5443Crossref PubMed Scopus (323) Google Scholar) and parental UMRC cell lines and their stable VHL transfectants. Cell lines were all maintained as previously described (16Calzada M.J. Esteban M.A. Feijoo-Cuaresma M. Castellanos M.C. Naranjo-Suarez S. Temes E. Mendez F. Yanez-Mo M. Ohh M. Landazuri M.O. Cancer Res. 2006; 66: 1553-1560Crossref PubMed Scopus (64) Google Scholar). Antibodies and Reagents—Polyclonal anti-FN antibody was from Sigma. Anti-RhoA was from Santa Cruz Biotechnology, anti-p190RhoGAP from Upstate, and anti-phosphotyrosine antibody PY20 was from Biomol. Anti-paxillin was from BD Biosciences, and anti-activated β1 integrin (HUTS21) was generously provided by Dr. Carlos Cabañas (CSIC). LPA and 2,3-butanedione-monoxime (BDM) were from Sigma, and the Rho kinase inhibitor Y-27632 was from Calbiochem. Isolation of Deoxycholate-soluble and -insoluble Materials—Cells were cultured in 6-well plates with RPMI 1640 and GLUTAMAX-I (Invitrogen) supplemented with 5% FN-free fetal bovine serum. Deoxycholate-soluble and -insoluble material were obtained as described elsewhere (38Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (131) Google Scholar). A detailed protocol is described under supplemental data. Immunofluorescence Microscopy—Immunofluorescence staining was performed as previously described (14Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. Garcia-Pardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar). A detailed protocol is described under supplemental data. Quantitative Real Time PCR Analysis—Quantitative RNA analysis protocol and primer pairs for β-actin have been described elsewhere (39Naranjo-Suarez S. Castellanos M.C. Alvarez-Tejado M. Vara A. Landazuri M.O. del Peso L. J. Biol. Chem. 2003; 278: 31895-31901Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). FN primers used and a detailed protocol are described under supplemental data. Cell Adhesion Assay—FN purified from VHL(+) or (-) cells was absorbed at 5 μg/ml onto Maxisorb 96-well plates (Costar). The wells were aspirated and blocked with 1% BSA/PBS for 30 min. Cells (20 × 103/well) were resuspended in M199/0.1% BSA and incubated for 1 h at 37 °C in 5% CO2. After washing with PBS, cells were fixed with 1% glutaraldehyde/PBS and stained with 1% crystal violet/PBS. Cells were washed with distilled water, and the absorbance at 590 nm was determined on an ORION-2 plate reader. A negative control consisting of cells attached to BSA was subtracted from each sample. RhoA Activity Assay—RhoA activity was measured using a GST-rhotekin-Rho binding domain (GST-C21), a gift of Dr. John Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Briefly, a total of 7 × 106 cells were lysed in radioimmune precipitation assay lysis buffer. After centrifugation (20,000 × g, 15 min, 4 °C), equal amounts of protein from the supernatant were incubated overnight at 4 °C with beads coated with GST-C21. Beads were washed, and bound protein was eluted by boiling in Laemmli buffer. Total and bound RhoA subjected to electrophoresis and transferred to nitrocellulose membranes were detected using an anti-RhoA mAb. For statistical purposes, gels from active and total RhoA normalized with respect to the loading control were subjected to densitometric analysis and analyzed using the Quantity-One™ program (Bio-Rad). Analysis of p 190RhoGAP Phosphorylation—VHL(+) and (-) RCCs (6 × 106) were lysed in 500 μl of radioimmune precipitation assay buffer. Cell supernatants were incubated overnight at 4 °C with protein G-agarose beads (Pierce) coated with 4 μg of anti-p190RhoGAP mAb (Upstate) or with the IgG1 anti-CD45 T200 antibody (mock). Bound proteins were extracted by boiling in Laemmli buffer, subjected to electrophoresis, and transferred into nitrocellulose membranes. Phosphorylated proteins were detected using PY20 mAb followed by a horseradish peroxidase-labeled secondary antibody. Retroviral Infection—Constructs of a constitutively active form (RhoA Q63L) or a dominant negative form (RhoA N19) of RhoA were kindly provided by Dr. Silvio Gutkind (Bethesda, MD). These constructions were subcloned into the BamH1/EcoR1 site of the retroviral vector pLZR IRES-GFP. 786-O cell infection was performed as described previously (16Calzada M.J. Esteban M.A. Feijoo-Cuaresma M. Castellanos M.C. Naranjo-Suarez S. Temes E. Mendez F. Yanez-Mo M. Ohh M. Landazuri M.O. Cancer Res. 2006; 66: 1553-1560Crossref PubMed Scopus (64) Google Scholar). siRNA Transfection—Control small interfering RNA (siRNA) or siRNA directed to human p190RhoGAP (5′GAACAGCGAUUUAAAGCAUTT-3′ and 3′AUGCUUUAAAUCGCUGUUCTT-5′) (Eurogentec) were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. Three days after transfection, cells were used for the experiment. FN mRNA and Protein Expression Levels in VHL(+) and VHL(-) RCC Cells Reveal No Correlation with the Ability to Assemble an FN Matrix—The reduced FN matrix deposition previously observed by immunofluorescence and ELISA studies (14Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. Garcia-Pardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar, 26Ohh M. Yauch R.L. Lonergan K.M. Whaley J.M. Stemmer-Rachamimov A.O. Louis D.N. Gavin B.J. Kley N. Kaelin Jr., W.G. Iliopoulos O. Mol. Cell. 1998; 1: 959-968Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 40Clifford S.C. Cockman M.E. Smallwood A.C. Mole D.R. Woodward E.R. Maxwell P.H. Ratcliffe P.J. Maher E.R. Hum. Mol. Genet. 2001; 10: 1029-1038Crossref PubMed Google Scholar) could be partially explained by an aberrant production or a defective secretion of FN in VHL(-) RCC cells (11Bluyssen H.A. Lolkema M.P. van Beest M. Boone M. Snijckers C.M. Los M. Gebbink M.F. Braam B. Holstege F.C. Giles R.H. Voest E.E. FEBS Lett. 2004; 556: 137-142Crossref PubMed Scopus (42) Google Scholar, 41He Z. Liu S. Guo M. Mao J. Hughson M.D. Cancer Genet. Cytogenet. 2004; 152: 89-94Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). To determine whether this was a general phenomenon in RCCs, we first analyzed the FN mRNA and protein levels in several RCC cell lines and in cells with different pVHL mutations corresponding to types 2A, 2B, and 2C phenotype of the VHL disease. These included the mutants Y112H (type 2A), C162F (type 2B), L188V (type 2C), and a nonneddylateable version of pVHL termed RRR (type 2C), or the control termed KRR (36Stickle N.H. Chung J. Klco J.M. Hill R.P. Kaelin Jr., W.G. Ohh M. Mol. Cell Biol. 2004; 24: 3251-3261Crossref PubMed Scopus (146) Google Scholar), all of them on the RCC 786-O background. Remarkably, FN mRNA levels measured by quantitative RT-PCR were significantly lower in 786-O and UMRC VHL(+) cells than in their corresponding VHL(-) counterparts (Fig. 1A). Similarly, FN mRNA levels in the various pVHL mutants were higher when compared with the corresponding levels in 786-O VHL(+) cells (Fig. 1A). In contrast, in RCC10 cells, FN mRNA expression was always higher in VHL(+) versus VHL(-) cells (Fig. 1A). We also analyzed FN protein levels in cell lysates or deoxycholate-insoluble fractions derived from all these cell types. In agreement with the mRNA data, VHL(-) and mutants derived from 786-O parental cells consistently showed higher FN protein expression than 786-O VHL(+) cells (Fig. 1B). Because VHL(-) cells do not assemble FN, the FN encountered in the deoxycholate-insoluble fraction could be due to FN interacting with other cytoskeletal proteins. In contrast, RCC4, RCC10, and UMRC VHL(-) cells had lower FN protein levels than their VHL(+) counterparts, both in lysates and in deoxycholate-insoluble fractions (Fig. 1B). Therefore, protein levels did not always correspond with mRNA levels, and this might be partially explained by the fact that protein levels were measured in total cell lysates. Therefore, we discarded part of the FN contained in the conditioned medium. Interestingly, we purified FN from the conditioned medium of the different VHL(+) and (-) cell lines and found similar FN-secreted levels (data not shown). To further confirm the above conclusion, we analyzed the assembly of FN matrix in the absence or presence of exogenous FN. We have previously reported that addition of exogenous FN did not re-establish assembly in 786-O VHL(-) cells (14Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. Garcia-Pardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar). Here we analyzed the assembly in three additional cell lines, RCC4, RCC10, and UMRC and in the VHL mutants described above. Our results showed that VHL(-) cells cultured on uncoated or FN-coated coverslips did not form visible FN fibrils, suggesting that FN availability is not exclusively responsible for their incapacity to form a matrix (Fig. 2A). Interestingly, VHL(+) but not VHL(-) UMRC cells, only formed a visible matrix when cultured on exogenous FN (Fig. 2A), indicating that, in this particular case, low secretion of FN (see Fig. 1, A and B) might be responsible for the lack of FN matrix in this VHL-expressing cell line. Additionally, none of the three different mutants, type 2A, 2B, or 2C, were able to assemble FN even when it was exogenously added (Fig. 2B). Altogether, these results indicated a heterogeneous expression of FN on the various VHL(+) and VHL(-) RCCs and most importantly, that lack of matrix assembly by VHL(-) cells could not always be attributed to low FN production in these cells. Fibronectin Produced by VHL(-) Cells Is Functional and Normally Assembled into Fibrils—If FN production and secretion do not always explain the failure to assemble FN by VHL(-) cells, the possibility exists that FN produced by cells lacking VHL is malfolded or malprocessed and hence not functional for its assembly. We therefore studied whether the FN produced by VHL(-) cells was able to bind to its receptor, the α5β1 integrin, and to form fibrils. To this end, we first performed cell adhesion experiments using FN purified from VHL(+) or VHL(-) cell-conditioned medium. As shown in Fig. 3A, all cell lines showed similar levels of adhesion to FN from either source, and this was independent of their VHL status. These results indicated that FN synthesized by VHL(-) cells is able to mediate cell adhesion as efficiently as that synthesized by VHL(+) cells. Moreover, they also indicated that the lack of FN assembly by VHL(-) cells is not due to a decreased α5β1 integrin activity, further extending our previous observations (14Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. Garcia-Pardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar) as well as those of others (13Davidowitz E.J. Schoenfeld A.R. Burk R.D. Mol. Cell Biol. 2001; 21: 865-874Crossref PubMed Scopus (95) Google Scholar), showing that expression and activation of α5β1 on VHL(-) cells are normal. We also compared the ability of FN purified from the conditioned medium of VHL(+) and VHL(-) cells to assemble into fibrils. Our results confirmed that VHL(-) RCCs did not assemble a FN matrix, even in the presence of exogenous FN from VHL(+) cells (Fig. 3B). In contrast, VHL(+) RCCs consistently formed a FN matrix when cultured either on FN purified from their own conditioned medium or from that of VHL(-) cells (Fig. 3B). To discern between assembly of endogenous and exogenous FN, we used UMRC VHL(+) cells, because they barely produce FN, and hence they do not assemble unless we provide them with exogenous FN (see Fig. 2A). As shown in Fig. 3B, UMRC VHL(+) cells showed a similar assembly pattern when cultured on FN from VHL(+) or VHL(-) cells. Therefore, these results indicated that FN produced by VHL(-) cells does not interfere with normal FN function in VHL(+) cells. Additionally, they clearly established that FN secreted by VHL(-) cells is fully functional and able to be assembled by VHL(+) cells. VHL(-) Cells Are Unable to Trigger the FN Stretching Necessary for Its Assembly—We have previously shown that VHL(-) cells fail to assemble proper β1 fibrillar adhesions (FAs), a cell matrix adhesion structure essential for FN assembly (14Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. Garcia-Pardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar). Therefore, the lack of proper formation of actin stress fibers and cell contraction could explain the defective assembly of FN by VHL(-) cells, because these events are required for the exposure of cryptic sites in FN and its progressive ordination into detergent-insoluble fibrils (33Zhang Q. Magnusson M.K. Mosher D.F. Mol. Biol. Cell. 1997; 8: 1415-1425Crossref PubMed Scopus (171) Google Scholar). To further analyze this process, we repeated our previous studies but now in the presence of exogenous FN to determine if VHL(-) cells were able to initialize the first steps of FN stretching required for its assembly into fibrils. After 4 h in culture, we analyzed by confocal microscopy the formation of focal contacts (FCs) by staining with paxillin, a major component of these structures, and with a FN antibody to see how FN was being stretched from these structures. Our results showed that VHL(+) cells arranged proper FCs, and the initial steps of fibril formation with short fibrils appearing at the edge of the FCs were very evident (Fig. 4A, panels c and d, white arrow-heads). In contrast, VHL(-) cells, although forming proper FCs, were not as efficient as VHL(+) cells, and the number of cells forming proper FCs were lower compared with VHL(+) cells (Fig. 4, A, panels f-h and B). These results strongly support our findings in that the impaired or defective formation of focal contacts may affect the proper formation of fibrillar adhesions required for FN assembly into fibrils. After 24 h, these structures became more mature and organized elongated FAs, which colocalized with FN fibrils in VHL(+) cells (Fig. 4, panels j-l, see magnified area). On the contrary, and even in the presence of exogenous FN, VHL(-) cells failed to form FA structures as well as arranging FN into fibrils, (Fig. 4, panels n-p). These results and our previous observation of a diminished organization of actin stress fibers in VHL(-) cells (16Calzada M.J. Esteban M.A. Feijoo-Cuaresma M. Castellanos M.C. Naranjo-Suarez S. Temes E. Mendez F. Yanez-Mo M. Ohh M. Landazuri M.O. Cancer Res. 2006; 66: 1553-1560Crossref PubMed Scopus (64) Google Scholar) support the hypothesis that a decreased FN stretching could contribute to the lack of FN assembly in these cells. Activation of the Small GTPase RhoA Is Defective in VHL(-) Cells—It is well established that the GTP-binding protein RhoA mediates the formation of actin stress fibers and regulates cellular functions such as cell adhesion and contractility (42Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). Furthermore, inhibitors of RhoA-mediated cellular contractility prevent FN assembly into fibrils (32Zhong C. Chrzanowska-Wodnicka M. Brown J. Shaub A. Belkin A.M. Burridge K. J. Cell Biol. 1998; 141: 539-551Crossref PubMed Scopus (493) Google Scholar), and a constitutively active form of RhoA restores the assembly of FN into fibrils in cells lacking active β1 integrins (43Cali G. Mazzarella C. Chiacchio M. Negri R. Retta S.F. Zannini M. Gentile F. Tarone G. Nitsch L. Garbi C. J. Cell Sci. 1999; 112: 957-965PubMed Google Scholar). According
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