Article |
Address correspondence to Andrew P. Kowalczyk, Dept. of Dermatology, Woodruff Memorial Building, Rm. 5007, Emory University School of Medicine, 1639 Pierce Dr., Atlanta, GA 30322. Tel.: (404) 727-8517. Fax: (404) 727-5878. email: akowalc{at}emory.edu
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Abstract |
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Key Words: adhesion; cytoskeleton; endocytosis; cadherin; catenin
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Introduction |
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Similar to other cadherins (Cowin, 1994; Pokutta and Weis, 2002), the VE-cadherin cytoplasmic domain interacts with several binding partners that couple the adhesion molecule to actin (Navarro et al., 1995) and vimentin cytoskeletal networks (Kowalczyk et al., 1998; Shasby et al., 2002; Calkins et al., 2003). Two distinct regions of the cytoplasmic domain of the classical cadherins have been identified, and these domains bind to different subfamilies of armadillo proteins (Anastasiadis and Reynolds, 2000). The membrane distal region of the cadherins interacts with ß-catenin and plakoglobin (Cowin and Burke, 1996; Angst et al., 2001; Hatsell and Cowin, 2001). ß-Catenin links cadherins to the actin cytoskeleton through direct and indirect interactions with actin binding proteins, such as -catenin and
-actinin (Knudsen and Wheelock, 1992; Aberle et al., 1994; Jou et al., 1995; Knudsen et al., 1995). Plakoglobin also links cadherins to the actin cytoskeleton, but in addition, plakoglobin interacts with desmoplakin (Kowalczyk et al., 1999; Green and Gaudry, 2000), an intermediate filament binding protein that is important for vascular organization during mammalian development (Gallicano et al., 2001).
In addition to ß-catenin and plakoglobin, cadherins interact with a second subfamily of armadillo proteins through a highly conserved domain on the cytoplasmic side of the cadherin membrane spanning domain (Anastasiadis and Reynolds, 2000). This juxtamembrane domain is thought to play an important role in cadherin clustering and in strengthening of cadherin adhesive interactions (Yap et al., 1998). The major binding partner for the cadherin juxtamembrane domain is an armadillo protein termed p120 catenin (p120ctn; Thoreson et al., 2000). p120ctn is part of a group of related armadillo proteins that includes ARVCF, -catenin, and p0071 (Hatzfeld, 1999). The juxtamembrane domain of VE-cadherin is known to bind directly to both p120ctn and p0071 (Calkins et al., 2003). Several studies indicate that p120ctn functions to promote cadherin clustering and to strengthen adhesion (Thoreson et al., 2000; Pettitt et al., 2003), but other studies have suggested that p120ctn may function as a negative regulator of cadherin function (Aono et al., 1999; Ohkubo and Ozawa, 1999). Recently, the absence of p120ctn in a colon carcinoma cell line was found to cause a corresponding loss of E-cadherin metabolic stability, indicating an important role for p120ctn in the maintenance of E-cadherin expression in differentiated epithelial cells (Ireton et al., 2002). However, recent studies in Drosophila melanogaster indicate that p120ctn is not an essential component of adherens junctions in these organisms (Myster et al., 2003; Pacquelet et al., 2003), underscoring the elusive nature of p120ctn contributions to cadherin function.
Here, we examined whether VE-cadherin internalization and degradation are regulated by armadillo family proteins that bind to the cadherin cytoplasmic tail. Morphological analysis indicated that neither p120ctn nor ß-catenin colocalized with VE-cadherin that had entered an endocytic pathway, suggesting that the disruption of catenin binding to the cadherin cytoplasmic tail might be associated with cadherin endocytosis. Consistent with this possibility, expression of cadherin mutants that compete for catenin binding caused the disruption of intercellular junctions and a dramatic down-regulation of endogenous VE-cadherin. Interestingly, competition for p120ctn binding, but not ß-catenin binding, was found to be critical for the induction of VE-cadherin degradation. Similarly, siRNA knockdown experiments revealed that the loss of p120ctn resulted in a corresponding loss of VE-cadherin. Finally, overexpression of p120ctn inhibited VE-cadherin entry into endocytic compartments, and caused a corresponding increase in cell surface levels of VE-cadherin. These findings indicate that p120ctn levels function as a set point for cadherin expression levels, and demonstrate for the first time that p120ctn regulates cadherin cell surface presentation by preventing cadherin degradation via an endosomallysosomal pathway.
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Results |
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The first approach undertaken was to express various cadherin mutants to compete with endogenous VE-cadherin for binding to p120ctn and/or ß-catenin. In previous studies, the expression of an IL-2 receptor-VE-cadherin chimera comprising the interleukin-2 receptor (IL-2R) extracellular domain and the VE-cadherin cytoplasmic tail (IL-2R-VE-cadcyto) was shown to cause the down-regulation of endogenous VE-cadherin (Xiao et al., 2003). Two additional mutants were constructed for the present work (Fig. 3). The IL-2R-VE-cadCBD mutant lacks the ß-catenin binding site and the IL-2R-VE-cadJMD-AAA mutant contains a triple alanine substitution at amino acids 562564 (EMD-AAA), which abrogates p120ctn and p0071 binding to classical cadherins (Thoreson et al., 2000; Calkins et al., 2003). To determine the localization of endogenous VE-cadherin in MEC expressing the various VE-cadherin mutants, immunofluorescence analysis was performed after infection with adenovirus carrying the empty virus or the various mutants (Fig. 3). An antibody directed against the VE-cadherin extracellular domain was used to specifically identify endogenous VE-cadherin, and antibodies directed against the myc epitope were used to detect the VE-cadherin mutants. In control cells expressing empty virus, extensive VE-cadherin staining was observed at MEC cell borders (Fig. 3 A). In striking contrast, in MEC cultures expressing the IL-2R-VE-cadcyto mutant, endogenous VE-cadherin was distributed in a punctate cytoplasmic distribution (Fig. 3 B). MEC expressing the IL-2R-VE-cad
CBD mutant also exhibited disrupted junctions, as evidenced by the thinning of VE-cadherin staining at intercellular junctions (Fig. 3 C). However, overall, the IL-2R-VE-cad
CBD mutant had somewhat less dramatic effects than the IL-2R-VE-cadcyto. Interestingly, the IL-2R-VE-cadJMD-AAA mutant had only minor effects on MEC intercellular junctions (Fig. 3 D), suggesting that competition for p120ctn was required for the rapid disruption of MEC junctions in cells expressing mutant cadherins.
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If the depletion of cytoplasmic p120ctn by cadherin mutants increases VE-cadherin internalization, then increased levels of p120ctn in the cytosol should inhibit cadherin internalization. To test this possibility, exogenous p120ctn was expressed in MEC and the amount of VE-cadherin internalization was monitored. For these experiments, MEC were surface labeled at 4°C with VE-cadherin antibody and switched to 37°C for 6 h in the presence of chloroquine to allow for extensive VE-cadherin internalization. Under these conditions, numerous cytoplasmic vesicles containing cell surface-derived VE-cadherin were visualized (Fig. 7 A). Very little p120ctn (Fig. 7 B) or ß-catenin (not depicted) colocalized with the internalized cadherin. Expression of exogenous ß-catenin had no discernible impact on the amount of VE-cadherin internalization (Fig. 7, DF). In contrast, expression of exogenous p120ctn dramatically inhibited entry of cell surface VE-cadherin into cytoplasmic vesicles (Fig. 7, GI). These results suggested that p120ctn expression was inhibiting constitutive internalization and degradation of VE-cadherin. Therefore, the accumulation of an intracellular pool of VE-cadherin in chloroquine-treated cells was monitored by Western blot analysis in MEC expressing p120ctn or ß-catenin. As discussed above (Fig. 6 J), chloroquine treatment results in the accumulation of an intracellular 100-kD processed form of VE-cadherin. As shown in Fig. 7 K, the accumulation of this intracellular pool of VE-cadherin was prevented by the expression of exogenous p120ctn but not ß-catenin. These data are consistent with the inhibition of VE-cadherin internalization observed in MEC expressing exogenous p120ctn (Fig. 7, GI). Collectively, these findings indicate that cytoplasmic availability of p120ctn regulates the delivery of cell surface VE-cadherin into endocytic compartments for endosomallysosomal degradation.
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Discussion |
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The findings presented here reveal a remarkable reciprocity in the regulation of cadherin and catenin expression levels. Previous studies demonstrated that cytoplasmic pools of ß-catenin and plakoglobin are metabolically unstable, and that cadherin binding to ß-catenin or plakoglobin rescues these armadillo family proteins from degradation (Kowalczyk et al., 1994; Aberle et al., 1997). Because of the important functions of ß-catenin in the regulation of gene expression, the rapid turnover of cytosolic ß-catenin is central to the regulation of ß-catenin entry into the nucleus and in the modulation of cell proliferation and migration (Willert and Nusse, 1998; Polakis, 1999; Gottardi and Gumbiner, 2001). As shown here, p120ctn rescues VE-cadherin from entry into a degradative pathway. VE-Cadherin is degraded in an endosomallysosomal compartment when p120ctn is removed from the cadherin tail, either by competition with cadherin mutants or by siRNA knock down approaches. Because cadherins antagonize ß-catenin signaling (Heasman et al., 1994; Fagotto et al., 1996; Sadot et al., 1998), the regulation of cadherin expression by p120ctn may control ß-catenin availability to signal transduction pathways. Thus, interactions between p120ctn and the cadherin cytoplasmic domain may function as a global regulator of cadherin and catenin signaling.
The role of p120ctn in junction assembly and cadherin function has been difficult to establish and the results obtained through the use of various model systems have been difficult to reconcile (Aono et al., 1999; Ohkubo and Ozawa, 1999; Thoreson et al., 2000). The Reynolds laboratory recently demonstrated in a colon carcinoma cell line that the loss of p120ctn expression due to gene mutation resulted in a corresponding loss of E-cadherin (Ireton et al., 2002). This was an important finding because it provided a potential explanation for the widespread down-regulation of E-cadherin expression that is observed in tumor cells, even when mutations in the E-cadherin gene are not apparent (Thoreson and Reynolds, 2002). However, recent studies in Drosophila indicate that p120ctn is not required for adherens junction assembly or for DE-cadherin expression (Myster et al., 2003; Pacquelet et al., 2003). It is possible that certain tumor cell lines might harbor additional genetic anomalies that would render E-cadherin vulnerable to the loss of p120ctn. However, our current results using siRNA knock down approaches in primary cultures of MEC indicate clearly that p120ctn is required for cadherin expression in normal human cells. The reason for this apparent discrepancy between flies and mammalian systems is not clear. However, it is interesting that vertebrates express not only p120ctn, but also several other related armadillo family members, including ARVCF, -catenin, and p0071 (Hatzfeld, 1999; Anastasiadis and Reynolds, 2000). Thus, the appearance of multiple p120ctn family members in vertebrates may reflect the evolution of distinct cadherin regulatory mechanisms that are required for tissue patterning or integrity in higher organisms.
The present work, as well as our previous work (Xiao et al., 2003), indicates that cadherin mutants trigger endocytosis and degradation of endogenous cadherins. Elimination of the p120ctn binding site on the mutant cadherin severely compromised the ability of the mutant to trigger VE-cadherin internalization (Fig. 6). In contrast, deletion of the ß-catenin binding domain had very little effect on this process, at least over the relatively short time courses that were examined. Thus, we conclude that VE-cadherin mutants trigger degradation of endogenous VE-cadherin by competing for p120ctn binding. This interpretation is based on the fact that overexpression of p120ctn could prevent the down-regulation of endogenous VE-cadherin, whereas ß-catenin overexpression could not. Furthermore, knock down of p120ctn levels using siRNA resulted in a corresponding decrease in VE-cadherin expression. Interestingly, overexpression of N-cadherin also caused the down-regulation of VE-cadherin in this model system (unpublished data). These data are consistent with previous studies suggesting that cells possess mechanisms that function as sensors for cadherin levels (Troxell et al., 1999). The results presented here indicate that p120ctn is the central component of this sensing mechanism.
Recently, endocytosis has emerged as a regulatory mechanism that modulates cadherin cell surface levels in epithelial cells (Le et al., 1999; Akhtar and Hotchin, 2001; Palacios et al., 2002). E-Cadherin is internalized and recycled back to the plasma membrane (Le et al., 1999), and this process is modulated by PKC (Le et al., 2002). It is formally possible that p120ctn does not regulate the initial cadherin internalization event, but rather that p120ctn regulates subsequent sorting decisions. The juxtamembrane domain of E-cadherin binds to Hakai, an E3 ubiquitin ligase that targets E-cadherin for internalization and degradation in epithelial cells (Fujita et al., 2002). Although Hakai binds to sequences unique to E-cadherin, these findings suggest that the cadherin juxtamembrane domain, and proteins that bind this region of cadherins, are critical in the control of cadherin expression levels. Consistent with this possibility, we found that p0071, which also binds to the VE-cadherin juxtamembrane domain (Calkins et al., 2003), also regulates VE-cadherin expression levels (unpublished data). Reynolds and colleagues (Ireton et al., 2002) found that p120ctn mutants that are unable to bind to E-cadherin are also unable to rescue E-cadherin expression in a p120ctn null background. These data suggest that p120ctn family proteins bind to the cadherin juxtamembrane domain and function as a "cap" that prevents cadherin internalization and degradation. Such a model is consistent with the competitive association of Hakai and p120ctn for the cadherin juxtamembrane domain (Fujita et al., 2002) and with the apparent dissociation of p120ctn from the cadherin tail (Fig. 2) during endocytosis. Regardless of the precise mechanism, this work reveals a key role for p120ctn in the regulation of cadherin cell surface levels by modulating cadherin delivery to degradative endocytic pathways.
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Materials and methods |
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cDNA constructs
A cDNA clone encoding full-length human VE-cadherin was provided by E. Dejana (FIRC Institute of Molecular Oncology, Milan, Italy; Navarro et al., 1995), and an expression construct encoding the extracellular and transmembrane domains of the IL-2R was provided by S. LaFlamme (Albany Medical College, Albany, NY; LaFlamme et al., 1994). This IL-2R construct was used to generate a chimeric cDNA with the IL-2R extracellular domain, the entire VE-cadherin cytoplasmic domain, and a carboxyl-terminal c-myc epitope tag, as described previously (Venkiteswaran et al., 2002). A deletion mutant of the VE-cadherin cytoplasmic tail lacking the catenin binding domain of VE-cadherin was constructed based on a previous report in which the catenin binding domain of VE-cadherin was mapped (Navarro et al., 1995). This catenin binding domain deletion construct encodes VE-cadherin amino acid positions 621702 followed by a carboxyl-terminal c-myc epitope tag. To generate this construct, a VE-cadherin cDNA was used as a template for PCR using the 5' primer 5'ATGGAAGCTTCGGCGGCGGCTCCGGAAGCAGGCC3', which includes a HindIII site and the 3' primer 5'ACGTCTCGAGCTACAAGTCCTCTTCAGAAATGAGCTTTTGCTCCACGGGCCCTCCGTGTGC3', which includes a c-myc tag, stop codon, and a XhoI site. The resulting PCR product was ligated in frame to the IL-2R extracellular domain using the HindIII and XhoI restriction sites. An additional VE-cadherin cytoplasmic mutant lacking the p120ctn binding domain was generated as described elsewhere (Calkins et al., 2003). This mutant encodes the VE-cadherin cytoplasmic tail with mutations altering the sequence EMD-AAA at amino acid positions 562564, which abrogates binding of the VE-cadherin cytoplasmic tail to p120ctn as determined by yeast two hybrid analysis (Calkins et al., 2003). This mutant lacking the p120ctn binding site was ligated to the IL-2R extracellular domain as described above to generate the IL-2R-VE-cadJMD-AAA mutant. p120ctn 1A cDNA was provided by A.B. Reynolds (Vanderbilt University School of Medicine, Nashville, TN) and a human, myc tagged ß-catenin cDNA was obtained from P. McCrea (University of Texas M.D. Anderson Cancer Center, Houston, TX).
Adenovirus production
The VE-cadherin mutants, ß-catenin, and p120ctn 1A were subcloned into the pAd-Track vector, which coexpresses GFP with the cDNA of interest (He et al., 1998). Adenoviruses carrying the VE-cadherin constructs, p120ctn, and ß-catenin were produced using the pAdeasy adenovirus-packaging system as described previously (Xiao et al., 2003). For most experiments, infection rates of 8090% were used as monitored by GFP expression.
Immunofluorescence
MEC cultured on gelatin-coated glass coverslips were fixed in methanol or 3.7% PFA followed by extraction in 0.5% Triton X-100. Endogenous VE-cadherin was detected using mouse mAbs cad-5 (Transduction Laboratories), BV6 (Research Diagnostics Inc.), or a goat polyclonal VE-cadherin antibody (Santa Cruz Biotechnology, Inc.). VE-Cadherin mutants were followed using a rabbit antibody directed against the c-myc epitope tag (Bethyl Laboratories, Inc.). The localization of p120ctn and ß-catenin was determined using rabbit polyclonal antibodies against p120ctn (Santa Cruz Biotechnology, Inc.) or ß-catenin (Neo Markers), respectively. An mAb H5C6 directed against CD63 (Developmental Studies Hybridoma Bank at the University of Iowa) was used to detect late endosomes/lysosomes. Secondary antibodies conjugated to various Alexa Fluors (Molecular Probes) were used for dual label immunofluorescence. The mouse BV6 and CD63 antibodies were distinguished using secondary antibodies specific for IgG2a and IgG1 subtypes. Microscopy was performed using a fluorescence microscope (model DMR-E; Leica) equipped with narrow band pass filters and a camera (model Orca; Hamamatsu). Images were captured, pseudo colored, and processed using Open Lab software (Improvision Inc.).
VE-Cadherin internalization assay
VE-cadherin internalization assays were performed using procedures adapted from Paterson et al. (2003). An mAb directed against the VE-cadherin extracellular domain (BV6) was dialyzed into MCDB 131 medium containing 20 mM Hepes and 3% BSA. The dialyzed antibody was incubated with MEC cultures at 4°C for 1 h. Unbound antibody was removed by rinsing cells in ice-cold MCDB 131. Cells were incubated at 4°C or transferred to 37°C for various amounts of time (36 h) in the presence of 100150 µM chloroquine. To remove cell surface bound antibody while retaining internalized antibody, cells were washed for 15 min in PBS, pH 2.7, containing 25 mM glycine and 3% BSA. The cells were rinsed, fixed, and processed for dual label immunofluorescence as described in the previous paragraph. For experiments to monitor internalization in response to the IL-2R-VE-cadherin mutants, MEC were infected with adenovirus for 6 h to allow time for infection and expression of the mutants. The cells were surface labeled at 4°C and transferred to 37°C for 3 h. To determine if p120ctn inhibits internalization, cells were infected with p120ctn or ß-catenin overnight to allow expression of the proteins. Cells were labeled at 4°C and transferred to 37°C for 6 h to allow time for significant levels of internalization in control cells. In each case, the amount of vesicular VE-cadherin present was quantified by a blinded observer by counting VE-cadherin vesicles/cell.
Western blot analysis
MEC were harvested in Laemmli gel sample buffer (Bio-Rad laboratories) and analyzed by SDS-PAGE and immunoblot using antibodies directed against the extracellular domain of VE-cadherin (cad-5; Transduction Laboratories), the myc epitope tag (Bethyl Laboratories), p120ctn (Santa Cruz Biotechnology, Inc.), ß-catenin (Transduction Laboratories or Neo Marker), PECAM-1 (Santa Cruz Biotechnology, Inc.), or vimentin (V9; Sigma-Aldrich). HRP-conjugated secondary antibodies (Bio-Rad Laboratories) were used at 1:3,000 dilution and detected using ECL (Amersham Biosciences). In some experiments, MEC were rinsed and incubated in trypsin/EDTA at 37°C for 2 min to proteolytically remove cell surface VE-cadherin before Western blot analysis (Xiao et al., 2003). Trypsin was inactivated using normal growth medium and cells recovered by centrifugation. Cell pellets were dissolved in SDS-PAGE sample buffer for Western blot analysis. Control cells were harvested in SDS-PAGE sample buffer without trypsinization.
siRNA
Inhibition of p120ctn expression in MEC was performed using p120ctn-directed siRNA reagents. A human p120ctn-specific 21-nt siRNA (5'-AACGAGGTTATCGCTGAGAAC-3') was constructed using the SilencerTM siRNA Construction Kit (Ambion). MEC were transfected with the 21-nt duplexes using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Ambion's SilencerTM negative control siRNAs were purchased and used as controls. Cells were rinsed and harvested for Western blot analysis 4872 h after transfection.
ELISA
ELISA was performed to measure cell surface VE-cadherin levels. MEC were seeded into a 96-well plate overnight and infected by empty virus or adenoviruses carrying p120ctn or ß-catenin for 18 h. Cells were rinsed and fixed in 1% formaldehyde at room temperature for 10 min. Cells were blocked in HBSS supplemented with 1 mM Ca2+ and 10% FBS for 45 min, and incubated in a mouse VE-cadherin antibody directed against the cadherin extracellular domain (BD Biosciences). Antibodies directed against vimentin were used as negative controls to verify that only cell surface antigens were being detected in the assay. HRP-conjugated goat antimouse secondary antibodies (Bio-Rad Laboratories) were used at 1:500 and detected using TMB One Step Substrate System (DakoCytomation). The reaction was stopped using 8 N sulfuric acid and the results were determined by Microplate Autoreader.
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Acknowledgments |
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This work was supported by the American Cancer Society (RPG CSM-100348), American Heart Association (AHA) (0355293B), and by National Institutes of Health grants (1R01AR048266 and P30AR042687). K. Xiao was supported by an AHA fellowship.
Submitted: 2 June 2003
Accepted: 19 September 2003
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References |
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