Abstract

Artilce18 August 2005free access A molecular role for lysyl oxidase-like 2 enzyme in Snail regulation and tumor progression Héctor Peinado Héctor Peinado Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author Maria del Carmen Iglesias-de la Cruz Maria del Carmen Iglesias-de la Cruz Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author David Olmeda David Olmeda Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author Katalin Csiszar Katalin Csiszar Cardiovascular Research Center, John A Burns School of Medicine, University of Hawaii, Honolulu, HI, USA Search for more papers by this author Keith SK Fong Keith SK Fong Cardiovascular Research Center, John A Burns School of Medicine, University of Hawaii, Honolulu, HI, USA Search for more papers by this author Sonia Vega Sonia Vega Instituto Cajal, Avenida Doctor Arce, Madrid, SpainPresent address: Instituto de Neurociencias, Apartado de Correos, 18, 03550 San Juan, Alicante, Spain Search for more papers by this author Maria Angela Nieto Maria Angela Nieto Instituto Cajal, Avenida Doctor Arce, Madrid, SpainPresent address: Instituto de Neurociencias, Apartado de Correos, 18, 03550 San Juan, Alicante, Spain Search for more papers by this author Amparo Cano Corresponding Author Amparo Cano Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author Francisco Portillo Corresponding Author Francisco Portillo Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author Héctor Peinado Héctor Peinado Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author Maria del Carmen Iglesias-de la Cruz Maria del Carmen Iglesias-de la Cruz Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author David Olmeda David Olmeda Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author Katalin Csiszar Katalin Csiszar Cardiovascular Research Center, John A Burns School of Medicine, University of Hawaii, Honolulu, HI, USA Search for more papers by this author Keith SK Fong Keith SK Fong Cardiovascular Research Center, John A Burns School of Medicine, University of Hawaii, Honolulu, HI, USA Search for more papers by this author Sonia Vega Sonia Vega Instituto Cajal, Avenida Doctor Arce, Madrid, SpainPresent address: Instituto de Neurociencias, Apartado de Correos, 18, 03550 San Juan, Alicante, Spain Search for more papers by this author Maria Angela Nieto Maria Angela Nieto Instituto Cajal, Avenida Doctor Arce, Madrid, SpainPresent address: Instituto de Neurociencias, Apartado de Correos, 18, 03550 San Juan, Alicante, Spain Search for more papers by this author Amparo Cano Corresponding Author Amparo Cano Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author Francisco Portillo Corresponding Author Francisco Portillo Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain Search for more papers by this author Author Information Héctor Peinado1,‡, Maria del Carmen Iglesias-de la Cruz1,‡, David Olmeda1, Katalin Csiszar2, Keith SK Fong2, Sonia Vega3, Maria Angela Nieto3, Amparo Cano 1 and Francisco Portillo 1 1Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, Madrid, Spain 2Cardiovascular Research Center, John A Burns School of Medicine, University of Hawaii, Honolulu, HI, USA 3Instituto Cajal, Avenida Doctor Arce, Madrid, Spain ‡These authors contributed equally to this work *Corresponding authors: Instituto de Investigaciones Biomédicas ‘Alberto Sols’, CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain. Tel.: +34 91 585 4411; Fax: +34 91 585 4401; E-mail: [email protected] or Tel.: +34 91 585 4457; Fax: +34 91 585 4401; E-mail: [email protected] The EMBO Journal (2005)24:3446-3458https://doi.org/10.1038/sj.emboj.7600781 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The transcription factor Snail controls epithelial–mesenchymal transitions (EMT) by repressing E-cadherin expression and other epithelial genes. However, the mechanisms involved in the regulation of Snail function are not fully understood. Here we show that lysyl-oxidase-like 2 and 3 (LOXL2 and LOXL3), two members of the lysyl-oxidase gene family, interact and cooperate with Snail to downregulate E-cadherin expression. Snail's lysine residues 98 and 137 are essential for Snail stability, functional cooperation with LOXL2/3 and induction of EMT. Overexpression of LOXL2 or LOXL3 in epithelial cells induces an EMT process, supporting their implication in tumor progression. The biological importance of LOXL2 is further supported by RNA interference of LOXL2 in Snail-expressing metastatic carcinoma cells, which led to a strong decrease of tumor growth associated to increased apoptosis and reduced expression of mesenchymal and invasive/angiogenic markers. Taken together, these results establish a direct link between LOXL2 and Snail in carcinoma progression. Introduction Epithelial tumors are thought to metastasize by initially invading the adjacent tissues, a process involving the loss of their cell–cell adhesions and the acquisition of migratory capabilities. These processes include phenotypical changes associated with epithelial–mesenchymal transitions (EMT), similar to those that take place during certain steps of embryonic development (Thiery, 2002). The invasive and metastatic phenotype is associated with downregulation of E-cadherin expression (Birchmeier and Behrens, 1994). Several mechanisms have been implicated in the regulation of E-cadherin expression during tumor progression, including genetic, epigenetic and transcriptional changes (Christofori and Semb, 1999; Peinado et al, 2004c). Snail transcription factor has been described as a direct repressor of E-cadherin expression in epithelial cells; the expression of Snail induces a full EMT and increases migration/invasion in different physiological and pathological situations (Batlle et al, 2000; Cano et al, 2000; Peinado et al, 2004b). Moreover, Snail expression has been detected in different invasive carcinoma and melanoma cell lines and, importantly, in invasive regions of squamous cell carcinomas and dedifferentiated ductal breast carcinomas and hepatocarcinomas (reviewed in Nieto, 2002; Peinado et al, 2004c). Recently, we have described the recruitment of the mSin3A corepressor complex with histone deacetylases (HDACs) by Snail, through the Snail and Gfi (SNAG) domain, to repress E-cadherin expression (Peinado et al, 2004a). In order to identify additional proteins that might interact with Snail to regulate E-cadherin expression, we carried out a yeast two-hybrid screen. Using Snail as bait, we found members of the lysyl oxidase (LOX) gene family to be potential interacting partners. Five LOX family genes have been identified so far in mammalian genomes encoding the prototypic LOX and four different LOX-like proteins (LOXL1, LOXL2, LOXL3 and LOXL4) (Kagan and Li, 2003; Molnar et al, 2003). LOX and LOX-like proteins are copper-containing enzymes that catalyze the oxidative deamination of the ε-amino group in certain peptidyl lysine residues promoting covalent protein crosslinkages (Kagan and Li, 2003; Molnar et al, 2003). All members of the LOX family show a highly conserved C-terminus region that contains the catalytic domain. The N-terminus of the LOX isoforms is less conserved among the different members and it is thought to determine the individual role and tissue distribution of each isoenzyme (Maki et al, 2001). The prototypic LOX plays a key role in the biogenesis of the connective tissue catalyzing crosslinkage formation in collagen and elastin components (Kagan and Li, 2003) and, recently, it has been shown that LOXL1 is required for proper elastic fiber homeostasis (Liu et al, 2004). The individual function of the remaining members of the family remains unclear, although recent evidences suggest the involvement of LOX, LOXL2 or LOXL4 in breast and head and neck squamous cell carcinoma progression (Kirschmann et al, 2002; Akiri et al, 2003; Holtmeier et al, 2003). In the present report, we show that LOXL2 and LOXL3 collaborate in vivo with Snail to repress E-cadherin transcription. Snail–LOXL2/3 physical interaction depends on the SNAG domain and Snail's lysine residues K98 and K137 are critical for Snail stability and functional cooperation with LOXL2/3. We also present evidence for a role of LOXL2 in tumor growth and progression. Results LOXL2 and LOXL3 interact with Snail in vivo To identify new proteins involved in Snail functionality, we performed a yeast two-hybrid screen. Using the N-terminus part of Snail (amino acids 1–150; Figure 1A) as bait, we identified the catalytic domain of LOX and LOXL1 enzymes as positive clones in the screen (Figure 1B). Since the C-terminus is a region of high conservation among all LOX family members, one or more of the LOX isoforms could be potential Snail interacting partner(s). Thus, we analyzed by reverse transcriptase–polymerase chain reaction (RT–PCR) the expression of the endogenous LOX gene family in a panel of both mouse epidermal keratinocyte (MCA3D, CarB and HaCa4) (Figure 2A) and human melanoma and carcinoma cell lines (MCF7, MDA-MB231 MDA-MB435 and A375P) (Figure 2B). The analysis included from poorly invasive/nonmetastatic cell lines with normal levels of E-cadherin expression and undetectable levels of Snail transcripts (MCA3D and MCF7) to cell lines that show high levels of Snail expression, E-cadherin loss and a highly invasive/metastatic phenotype (CarB, HaCa4, MDA-MB231, MDA-MB435 and A375P) (Cano et al, 2000). We detected expression of LOXL2, LOXL3 and/or LOXL4 in cell lines that were highly invasive and metastatic but not of LOX and LOXL1 mRNAs (Figure 2A and B). Interestingly, we observed a direct correlation between the expression of at least one of the LOXL2, LOXL3 and LOXL4 genes, and the presence of Snail and the loss of E-cadherin transcripts. This result was further confirmed for LOXL2 and LOXL3 proteins by immunoblotting analysis using specific antibodies (Figure 2C and D). These results led us to pursue LOXL2, LOXL3 or LOXL4 as potential Snail's partners for collaborating in EMT. Figure 1.Snail interacts with LOX and LOXL1 in the two-hybrid screen. (A) Diagrammatic representation of the main functional domains of Snail and LOX-like proteins. (Left) Snail organization: N-half part (N-t) used as bait in the two-hybrid screen, containing the N-terminal SNAG domain, the destruction box (DB) and the NES domain. (Right) LOX-like proteins organization: N-terminal region specific to each family member and C-terminal catalytic region common to LOX and LOXL enzymes. (B) Specificity of interactions between Snail (N-t) and LOX and LOXL1 (catalytic domain) in the two-hybrid system. The isolated cDNAs from LOX and LOXL1 isoforms were tested for interaction with Snail in complete medium (middle) or in the absence of adenine and histidine and in the presence of X-αGal (right) at three serial dilutions. Interactions in the absence of bait and prey cDNAs and those between Snf1 and Snf4 cDNAs were tested in parallel as negative and positive controls, respectively. Download figure Download PowerPoint Figure 2.Expression of LOX and LOXL isoforms in mouse and human carcinoma cells. (A, B) The expression of LOX, the indicated LOXL isoforms and Snail was analyzed by RT–PCR in the indicated mouse (A) and (B) human cell lines. GAPDH mRNA levels were analyzed in parallel as a control of the amount of cDNAs. (C, D) LOXL2 and LOXL3 expression was analyzed by Western blot in the indicated mouse (C) and human (D) cell lines; α-tubulin levels were analyzed in parallel as a loading control. Download figure Download PowerPoint To confirm the molecular interaction suggested by the two-hybrid screen, we carried out co-immunoprecipitation analyses in HEK293T cells transiently transfected with tagged versions of Snail and LOXL2, LOXL3 or LOXL4 isoforms. Co-immunoprecipitation of LOXL2 and LOXL3, but not LOXL4, by Snail (Figure 3A, left panels) indicated an in vivo interaction between Snail and either LOXL2 or LOXL3. Furthermore, inverse co-immunoprecipitation analysis reinforced this notion (Figure 3A, right panels). Additional co-immunoprecipitation experiments carried out with several versions of Snail-HA containing different functional domains showed that LOXL2 interacts with the full-length Snail protein, but not with mutants lacking the N-terminal region (ΔNt) or just the first 9 amino acids (ΔSNAG) (Figure 3B), indicating that Snail interaction with LOXL2 requires the repressor SNAG domain (Peinado et al, 2004a). Similar results were obtained in Madin Darby canine kidney (MDCK) cells and in pulldown assays with LOXL2 or LOXL3 (data not shown). Unfortunately, Snail-HA lacking the C-terminal domain (ΔZn-HA) was highly unstable (Figure 3B, left panel) precluding confirmation of the interaction between LOXL2/3 and Snail N-terminal domain detected in the two-hybrid screen. On the other hand, confocal analysis of MDCK cells transiently transfected with tagged versions of the corresponding genes showed that LOXL2/3 and Snail colocalize in the perinuclear compartment (Figure 3C). The perinuclear localization has also been recently observed for LOXL1 in cell cultures (Liu et al, 2004). Taken together, these results show that LOXL2 and LOXL3 interact with Snail through the SNAG domain. Figure 3.Snail interacts with LOXL2 and LOXL3 isoforms. (A) HA-tagged Snail-wt (wild type) or Snail-K98R/K137R constructs were transiently coexpressed with LOXL2-, LOXL3- or LOXL4-flag isoforms in HEK293T cells. (Left panel) Snail immunoprecipitation with anti-HA and detection of LOXL isoforms by Western with anti-flag antibodies. Control IgG immunoprecipitation is also shown. Reversal immunoprecipitation (right panel) with anti-flag and detection of Snail-wt or Snail-K98R-K137R with anti-HA antibodies was performed. IgGs were used to confirm equal immunoprecipitation. The expression of Snail and LOXL isoforms was detected by Western blot in 5% of cell lysates (upper panel). (B) (Right) Co-immunoprecipitation analyses performed after transfection of LOXL2-flag and Snail-HA, or the indicated Snail deletion mutants, with anti-HA and detection of associated LOXL2 with anti-flag antibodies. (Left) Input fractions showing Snail-HA and mutants levels; α-tubulin was used as a loading control. Note the low levels of ΔZn-HA expression precluding its analyses in co-immunoprecipitation. (C) Confocal analyses of MDCK cells transiently transfected with Snail-HA (a, e) and either LOXL2- (b) or LOXL3-flag (f), showing the colocalization of Snail with LOXL2/3 in the perinuclear region (merge images, c, g; and d and h). Snail nuclear localization was confirmed by DAPI staining (i, j). Bar, 5 μm. Download figure Download PowerPoint LOXL2 and LOXL3 collaborate with Snail in E-cadherin repression To get an insight into the functionality of the identified Snail–LOXL2/3 interactions, we next analyzed the effect of human LOXL2 and LOXL3 on E-cadherin promoter activity in MDCK cells in the absence or presence of Snail. To observe a potential cooperation, Snail was transfected under partial repression conditions (50 ng) (Peinado et al, 2004a) (Figure 4A, lane 2). Transfection of human LOXL2 or LOXL3 cDNAs (300 ng) induced a partial repression of the E-cadherin promoter (Figure 4A, lanes 3 and 5) and cotransfection of Snail with either LOXL2 or LOXL3 led to a significant increase, up to 70%, in the repression activity (Figure 4A, lanes 4 and 6), indicating that LOXL2/3 proteins collaborate with Snail in E-cadherin promoter repression. Cotransfection of the ΔSNAG mutant indicated the requirement of the N-terminal SNAG domain for Snail repression and functional collaboration with LOXL2/3 (Figure 4A, lanes 7–9). Figure 4.K98 and K137 residues of Snail are required for its collaboration with LOXL2/3 in E-cadherin promoter repression. (A) E-cadherin promoter was transiently transfected in MDCK cells and the activity was measured in the presence of wild-type Snail or the ΔSNAG mutant (50 ng) in the absence or presence of LOXL2 or LOXL3 cDNAs (300 ng). (B) E-cadherin promoter activity was measured as above in MDCK-EGFPshRNA or MDCK-SnailshRNA stable cell lines in the absence or presence of Snail, LOXL2 or LOXL3. (C) Schematic diagrams of Snail and Slug proteins showing the localization of lysine residues K9, K16, K98 and K137; note the absence of K98 and the location of K137 in the first zing-finger domain of Slug. (D) The activity of the E-cadherin promoter in MDCK cells was measured as above in the absence or presence of the indicated individual mutant forms of Snail (50 ng) (lanes 3–6) or double mutants in the absence or presence of LOXL2 and LOXL3 cDNAs (300 ng), as indicated. The effect of Slug (100 ng) in the absence or presence of LOXL2/3 isoforms (300 ng) was also tested (lanes 11–13). (E) RT–PCR analyses for detection of endogenous E-cadherin transcripts after transient transfection of the indicated plasmids as above. Levels of GAPDH transcript were used as a control of cDNA loading; densitometry of E-cadherin/GAPDH ratio of two independent experiments is shown. Download figure Download PowerPoint To confirm if the moderate E-cadherin promoter repression triggered by LOXL2 or LOXL3 might be caused by cooperation with the endogenous Snail (Peinado et al, 2003), E-cadherin promoter activity was assayed in MDCK cells stably transfected with either SnailshRNA or control EGFPshRNA. Expression of LOXL2 or LOXL3 in MDCK-SnailshRNA cells had no effect on E-cadherin promoter activity (Figure 4B, lanes 6 and 7, compare with lanes 2 and 3). Analysis of E-cadherin promoter in Snail-deficient MCA3D cells showed a very low repressive effect of LOXL2/3 (Supplementary data S2a). Together, these data suggest that LOXL2/3 enzymes can functionally cooperate with Snail in E-cadherin repression as a consequence of their physical interaction through the SNAG domain. Snail Lys98 and Lys137 residues are essential for E-cadherin silencing, induction of EMT and Snail stability Since LOXL enzymes exert their function by modification of specific peptidyl lysine residues, we analyzed the conserved lysine residues in the Snail subfamily of repressors (Sefton et al, 1998) and found that four of them (K9, K16, K98 and K137) are located within the N-terminus fragment used as bait in the protein interaction screen (Figure 4B). To determine if Snail's lysine residues could be required for collaboration with LOXL enzymes, we carried out site-directed mutagenesis of K9, K16, K98 and K137 residues that were replaced by arginine and the mutants were used in E-cadherin promoter assays. None of the individual mutations affected E-cadherin promoter repression mediated by Snail (Figure 4D, lanes 3–6, compare with lane 2) or the collaboration with LOXL2/3 (Supplementary data S1). Next, we analyzed the consequence of the double mutations K9R/K16R and K98R/K137R on Snail repressor activity. The K9R/K16R mutant exhibited a behavior similar to that of the wild-type Snail (Figure 4D, lane 7) and partly relieved the cooperation with LOXL2/3 (Supplementary data S1), probably because it altered the ability to recruit corepressor complexes. In contrast, the double mutant K98R/K137R, although with a conserved intact SNAG domain, was unable to repress the E-cadherin promoter activity (Figure 4D, lane 8) and failed to collaborate with LOXL2/3 (Figure 4D, lanes 9 and 10). Analysis of the effect on endogenous E-cadherin mRNA levels confirmed the collaboration of Snail and LOXL2/3 and the strict requirement of Snail's K98 and K137 residues for E-cadherin repression (Figure 4E). The unsuccessful collaboration of Snail K98R/K137R mutant with LOXL2/3 is not due to a lack of interaction, since the double mutant maintains interaction with either LOXL2 or LOXL3 (Figure 3A). However, the Snail K98R/K137R mutant has impaired ability to recruit corepressor complexes; decreased interaction with mSin3A and HDAC1/2 components has been detected (Supplementary data S2b, and data not shown). Altogether, these results indicate that K98 and K137 residues are essential for Snail to achieve its full repressor capability and suggest that these residues could be the substrates of LOXL2/3 enzymes. Since both Snail and Slug members of the Snail superfamily have been described as repressors of E-cadherin, it is possible that Slug could also be modified by LOXL2/3. Interestingly, K9 and K16 residues are fully conserved in the Snail superfamily, but K98 is replaced by arginine in the Slug subfamily and K137, although conserved, is located in a very different sequence context being embedded in the first zinc-finger domain of Slug (Sefton et al, 1998) (Figure 4C), suggesting that Slug members would not collaborate with LOXL2/3 in silencing E-cadherin promoter. To confirm this assumption, we carried out E-cadherin promoter assays with mouse Slug in the absence and presence of LOXL2/3. Transfection of Slug led to a moderate level of E-cadherin promoter repression in MDCK cells even when used at higher doses (100 ng) than Snail (50 ng) (Figure 4D, compare lanes 11 and 2), in agreement with previous observations (Bolos et al, 2003). No collaboration of LOX2/3 with Slug could be detected over a range of Slug concentration (50–250 ng) (Figure 4C, lanes 11–13, and unpublished data), supporting that, in contrast to Snail, Slug would not require interaction/modification by LOXL2/3 to be active. These data indicate that Snail and Slug use different mechanisms to repress E-cadherin transcription, unveiling the existence of functional differences between the Snail and Slug subfamilies. To further explore whether the Snail K98R/K137R mutation has any in vivo consequence, we evaluated the competence of the mutant Snail to achieve EMT. To this end, MDCK cells were stably transfected with HA-tagged variants of Snail and Snail-K98R/K137R. MDCK cells expressing Snail-HA suffered EMT with complete loss of E-cadherin (Figure 5A and B), while cells expressing the double mutant exhibited an unaltered epithelial phenotype (95% of the clones) similar to that of the mock-control cells (Figure 5A, compare panelse and f with i and j) and maintained the expression of E-cadherin (Figure 5B) organized in cell–cell junctions (Figure 5A, compare panels g and h with k and l). These results reinforce the requirement of intact K98 and K137 residues for Snail-mediated EMT. Figure 5.Lysine residues K98 and K137 of Snail are required for EMT induction. (A) MDCK transfectants obtained after stable expression of Snail-HA (upper panels), Snail-K98R/K137R-HA (middle panels) or pcDNA3-HA control vector (lower panels) were characterized by phase contrast of subconfluent cultures (left panels) and immunofluorescence of E-cadherin (right panels). Two independent clones, out of 20, from each transfection are shown. Bars, 40 μm. (B) Western blot analyses performed on whole cell extracts for the expression of E-cadherin and Snail-HA proteins in the indicated MDCK clones. Detection of α-tubulin was used as a loading control. Download figure Download PowerPoint The K98 and K137 residues are flanking the Snail NES domain (Dominguez et al, 2003) and the K98 residue (K99 in human Snail) is located inside the conserved destruction box (DSGKSS) recently reported to be required for GSK3β-dependent phosphorylation and proteasome degradation of Snail (Zhou et al, 2004). We, therefore, analyzed the stability of wild-type and variant K98R/K137R Snail proteins after transient transfection in HEK293T cells. The mutant K98R/K137R protein exhibits a slightly lower stability than wild-type Snail (Figure 6A and C), which is in agreement with recent reports (Yook et al, 2005). Strikingly, coexpression of LOXL2 led to an increased stability of wild-type Snail while it strongly decreased the stability of the mutant K98R/K137R Snail (Figure 6B and D), an effect that can be prevented by pretreatment with GSK3β and proteasome inhibitors (data not shown). We next evaluated the interaction of wild-type Snail and mutant K98R/K137R protein with GSK3β and their ubiquitination degree. The K98R/K137R mutant protein exhibited a higher degree of interaction with GSK33β and ubiquitination than the wild-type Snail (Figure 6E), in agreement with its highest instability. These data indicate that K98 and K137 residues are crucial for Snail stability and suggest that interaction/modification with LOXL2/3 might prevent its degradation and/or nuclear export, therefore increasing its functional transcription activity. Figure 6.Lysine residues K98 and 137 are crucial for Snail protein stability. HEK293T cells transiently transfected with wild-type or variant K98R/K137R Snail in the absence (A) or presence (B) of LOXL2 were treated with 20 μM cycloheximide for the indicated time intervals and whole cell extracts analyzed by Western blotting. (C, D) Densitometric analysis of blots shown in panels A and B, respectively. Results show the mean±s.d. of two independent experiments. (E) (Right) Immunoprecipitation analysis of Snail wild type or K98R/K137R-HA performed with anti-HA and detection of associated GSK3β or the ubiquitination level by Western blot. IgGs were used as a control to confirm equal immunoprecipitation. The expression level of Snail and GSK3β proteins was detected in 5% of cell lysates (left panel). Download figure Download PowerPoint LOXL2 and LOXL3 induce EMT To further analyze the role of LOXL2 and LOXL3 in E-cadherin downregulation in vivo, we examined the phenotype of MDCK cells stably expressing each of the human enzymes. As a control, we analyzed MDCK cells either transfected with the empty vector (CMV) (Cano et al, 2000) or expressing the human LOXL4 that exhibit an unaltered epithelial phenotype, maintaining growth in an epithelial monolayer (Figure 7Ac) and the expression of E-cadherin (Figure 7B and C) in organized cell–cell junctions (Figure 7Af). No expression of the mesenchymal marker fibronectin was observed in MDCK-hLOXL4 cells (Figure 7B) and vimentin exhibited a distribution (Figure 7Al) typical of control MDCK cells in culture (Cano et al, 2000). In striking contrast, stable expression of hLOXL2 or hLOXL3 in MDCK cells induced a conversion to a fibroblastic/spindle phenotype (Figure 7Aa and b) and vimentin exhibited an organization typical of mesenchymal cells (Figure 7Aj and k). Although both hLOXL2 and hLOXL3 showed a similar expression pattern (Figure 7Ag and h), the EMT effect seems to be stronger in hLOXL2-transfected cells than in hLOXL3-transfected cells. While MDCK-hLOXL2 cells do not express E-cadherin (Figure 7Ad, B and C) and show an induction of fibronectin (Figure 7B), MDCK-hLOXL3 cells still express E-cadherin mRNA and protein, although at reduced levels (Figure 7B and C) and with a disorganized distribution (Figure 7Ae), and do not express fibronectin (Figure 7B). No changes in the expression level of endogenous Snail transcripts were observed in the MDCK-LOX2/3 transfectants (data not shown). To discard a post-transcriptional regulation of E-cadherin by LOXL2/3, we investigated the effect of proteasome inhibition in the different cell lines (Figure 7D) finding no significant differences in the E-cadherin protein levels. Figure 7.Stable expression of LOXL2 or LOXL3 in MDCK cells induces EMT. (A) Characterization of MDCK transfectants obtained after stable expression of hLOXL2 (left panels), hLOXL3 (middle panels) and LOXL4 (right panels) by phase contrast (a–c), and immunofluorescence for the expression of E-cadherin (d–f), ectopically expressed LOXL isoform (g–i) and vimentin (j–l) in the indicated cell clones. Bars, 40 μm. (B) Western blot analyses performed on whole cell extracts for the expression of E-cadherin, fibronectin and ec