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. 2015 Jun 9:13:29.
doi: 10.1186/s12964-015-0105-y.

The importance of claudin-7 palmitoylation on membrane subdomain localization and metastasis-promoting activities

Sarah Heiler  1 , Wei Mu  2 , Margot Zöller  3 , Florian Thuma  4
Affiliations

The importance of claudin-7 palmitoylation on membrane subdomain localization and metastasis-promoting activities

Sarah Heiler et al. Cell Commun Signal. .

Abstract

Background: Claudin-7 (cld7), a tight junction (TJ) component, is also found basolaterally and in the cytoplasm. Basolaterally located cld7 is enriched in glycolipid-enriched membrane domains (GEM), where it associates with EpCAM (EpC). The conditions driving cld7 out of TJ into GEM, which is associated with a striking change in function, were not defined. Thus, we asked whether cld7 serines or palmitoylation affect cld7 location and protein, particularly EpCAM, associations.

Results: HEK cells were transfected with EpCAM and wild type cld7 or cld7, where serine phopsphorylation or the palmitoylation sites (AA184, AA186) (cld7(mPalm)) were mutated. Exchange of individual serine phosphorylation sites did not significantly affect the GEM localization and the EpCAM association. Instead, cld7(mPalm) was poorly recruited into GEM. This has consequences on migration and invasiveness as palmitoylated cld7 facilitates integrin and EpCAM recruitment, associates with cytoskeletal linker proteins and cooperates with MMP14, CD147 and TACE, which support motility, matrix degradation and EpCAM cleavage. On the other hand, only cld7(mPalm) associates with TJ proteins.

Conclusion: Cld7 palmitoylation prohibits TJ integration and fosters GEM recruitment. Via associated molecules, palmitoylated cld7 supports motility and invasion.

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Figures

Figure 1
Figure 1
Claudin-7 palmitoylation: Impact on membrane subdomain localization and protein association (a-e) HEK cells were transfected with EpC and/or wt cld7 or EpC with a mutation of (mAG) in the transmembrane region or with cld7, with a palmitoylation site mutation. (a) WB of non-transfected and transfected HEK with anti-EpC, anti-cld7 and anti-actin (loading control); an example of flow cytometry analysis of EpC and cld7 in transfected HEK cells: single fluorescence overlays of negative controls and anti-EpC or anti-cld7 staining; (b) HEK-EpC-cld7 and HEK-EpCmAG-cld7 were lysed in the presence of N-ethylmaleimide to irreversibly block unmodified thiol groups. After incubation with HAM buffer for unmasking palmitoylated cysteine thiol groups, samples where incubated in biotin-BMCC for selective labeling palmitoylated cysteines. Samples were blotted with streptavidin-HRP and after stripping with anti-cld7. (c) WB of sucrose density fractions of untreated and 2-BP-treated HEK-EpC-cld7 and HEK-EpC-cld7mPalm with anti-cld7 and anti-EpC; (d) IP of HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm lysates with control IgG, anti-EpC and anti-cld7 and WB with anti-EpC and anti-cld7; (e) WB of non-mutated and mutated EpC and cld7 in light and heavy density fractions after density gradient centrifugation; constitutively GEM-located CD81 served as control; relative protein band intensity and the protein ratio in light to heavy fractions is indicated; (f) immunoprecipitation with control IgG, anti-EpC and anti-cld7 of lysates of non-mutated and mutated EpC and cld7 in light and heavy density fractions and WB with anti-EpC and anti-cld7; (g-i) SDS-PAGE, Coomassie-blue staining, in vitro kinase assay of whole cell and membrane lysates and WB with anti-p-tyrosine of anti-cld7 immunoprecipitates of HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm lysates. Co-transfection of HEK-EpC with cld7 shifts EpC into GEM. Instead, recovery of EpCmAG in GEM and co-immunoprecipitation with cld7 is strikingly reduced. Palmitoylation site mutated cld7 is hardly recovered in GEM and only few, mostly non-palmitoylated molecules associate with cld7mPalm.
Figure 2
Figure 2
Claudin7 palmitoylation and TJ formation (a) Flow cytometry analysis of claudin and ZO-1 expression in HEK cells; the mean fluorescence index (% stained cells x mean intensity of staining ± SD; three assays) and representative examples are shown; (b) colocalization of cld7 with cld3 and ZO-1 and of cld4 with ZO-1 (scale bar: 10 μm); (c) immunoprecipitation with control IgG, anti-cld7 and anti-ZO-1 and co-immunoprecipitation of cld7, EpC, cld3, cld4 and ZO-1 in HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm lysates. Palmitoylated cld7 poorly colocalizes and co-immunoprecipitates with TJ proteins.
Figure 3
Figure 3
Claudin7 palmitoylation and homophilic EpC cell-cell adhesion (a,b) CFSE-labeled transfected HEK cells were seeded on a monolayer of HEK-EpC-cld7 cells with/without mutations of EpC or cld7. Where indicated, cells were cultured in the presence of anti-EpC (10 μg / ml) or TAPI (50 μM / ml). The percent of adherent cells (mean ± SD; triplicates) is shown; significant differences between HEK-EpC-cld7 and HEK-EpCmAG-cld7 or HEK-EpC-cld7mPam: *, significant differences by anti-EpC: s; significant differences in the presence of TAPI: +. (c) Supernatant of confluent PMA stimulated HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm cells was collected after 48 h of culture in the absence of FCS. Where indicated cultures contained 50 μM / ml TAPI. Cell debris- and vesicle-depleted supernatant were adjusted to 100 μg / ml protein, were separated by SDS-PAGE and after transfer were blotted with anti-EpC or ELISA plates were coated with the concentrated supernatant. After blocking, EpEx was detected by anti-EpC binding. The OD at 595 nm (mean ± SD; triplicates) is shown; significant differences between HEK-EpC-cld7 and HEK-EpCmAG-cld7 or HEK-EpC-cld7mPalm: *, significant differences in the presence of TAPI: +. (d) Confocal microscopy of HEK, HEK-EpC, HEK-cld7, HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm cells stained with anti-TACE and anti-EpC or anti-cld7. Green and red fluorescence and overlays of red and green fluorescence are shown (scale bar: 10 μm). (e) Lysates of cells as above were separated by sucrose gradient centrifugation and blotted with anti-TACE. (f) Lysates of the cells as above were precipitated with anti-TACE. After sucrose gradient centrifugation of the precipitates, SDS-PAGE and transfer, membranes were blotted with anti-EpC, anti-cld7 and anti-TACE. Palmitoylated cld7 interferes with homophilic EpC adhesion, which is partly due to the association of palmitoylated cld7 with TACE in GEM, which promotes EpEx cleavage.
Figure 4
Figure 4
Palmitoylated claudin7 promotes cell motility (a) Cells were seeded in FN-coated 24-well plates. Subconfluent cultures were scratched with a pipette tip. Wound healing was followed by light microscopy. Examples and mean ± SD (triplicates) of wound closure are shown (scale bar: 250 μm). (b) Cells suspended in RPMI / 1%BSA were seeded in the upper chamber of a Boyden chamber. The lower chamber contained RPMI/20%FCS. After 16 h of incubation, cells at the lower membrane site were stained with crystal violet and OD 595 nm was determined after lysis; mean ± SD of triplicates. (c) Cells were seeded on BSA-coated 24-well plates. Cell migration was followed for 12 h by videomicroscopy taking pictures every 20 min; examples and the mean ± SD migration of 20 individual cells. (a-c) Significant differences between HEK, HEK-EpC, HEK-EpC-cld7, HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm: *. Palmitoylated cld7 significantly strengthens HEK cell migration.
Figure 5
Figure 5
Cooperativity of palmitoylated claudin-7 with integrins (a) Confocal microscopy of F-actin staining and co-localization (overlay of green and red fluorescence) with EpC and cld7 (scale bar: 10 μm). (b, c) Flow cytometry analysis of adhesion molecules and cytoskeletal linker proteins in untransfected and transfected HEK cells; the mean fluorescence index (% stained cells x mean intensity of staining ± SD; three assays) and representative examples are shown. (d) Cld7 co-localization with integrins and CD166 (confocal microscopy, green and red fluorescence and overlays, scale bar: 10 μm). Cld7 expression allows for actin filament formation in HEK cells, which likely is supported by the association of cld7 with integrins. Cld7 palmitoylation also facilitates colocalization with CD166.
Figure 6
Figure 6
Cooperativity of palmitoylated claudin-7 with the cytoskeleton (a) Cld7 co-localization with ezrin and src (confocal microscopy, green and red fluorescence and overlays, scale bar: 10 μm) and (b) control IgG and cld7 co-immunoprecipitation with ezrin and p-ezrin in the absence or presence of a palmitoylation inhibitor (2-BP, 15 μM) and (c) control IgG, cld7 and ezrin co-immunoprecipitation with cld7, EpC, CD104 and ezrin; EpC, CD49d and CD166 co-immunoprecipitation with cld7, ezrin and EpC; (d) cld7 co-immunoprecipitation with src, FAK, paxillin and cld7 in transfected HEK cells. Cld7 associates with the cytoskeletal linker protein ezrin. Cld7 palmitoylation facilitates ezrin binding (and F-actin formation). This also accounts for the association with CD49e, CD166, src, FAK and paxillin. Recruitment of the latter two towards cld7 does not essentially depend on, but becomes strengthened by cld7 palmitoylation.
Figure 7
Figure 7
Palmitoylated claudin-7 supports the generation of EpIC (a) Confocal microscopy of HEK-EpC-cld7 and HEK-EpC-cld7mPalm cells stained with anti-EpIC and -cld7; single fluorescence staining and overlays are shown (scale bar: 10 μm) and WB of lysates of PMA-stimulated transfected HEK cells with anti-EpIC; EpIC and EpC bands are indicated. (b) Confocal microscopy of HEK-EpC-cld7 and HEK-EpC-cld7mPalm cells stained with anti-presenilin2 and -cld7 (single fluorescence and overlays, scale bar: 10 μm); sucrose gradient fractions of lysates of PMA-stimulated HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm cells were immunoprecipitated with anti-presenilin2, precipitates were blotted with anti-EpC, anti-cld7 and anti-presenilin2. (c) WB of presenilin2 and β-catenin in PMA-stimulated transfected HEK cells. (d) Flow cytometry analysis of mesenchymal proteins in PMA-stimulated transfected HEK cells; the mean fluorescence index (% stained cells x mean intensity of staining ± SD; three assays) and representative examples are shown (mean % stained ± SD, mean intensity ± SD; triplicates and representative examples); significant differences between HEK-cld7 versus HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm cells: *. (e) Representative examples of FGF, vimentin and N-cadherin co-localization with cld7 (confocal microscopy, scale bar: 10 μm). Palmitoylated cld7 facilitates the generation of EpIC by associating with presenilin-2 in GEM. EpIC generation is accompanied by a slight upregulation of mesenchymal protein expression.
Figure 8
Figure 8
Palmitoylated claudin-7 and invasion (a) HEK cells were seeded in matrigel-coated transwell plates, matrigel invasion (recovery within the matrigel) and penetration (recovery at the lower site of the insert) was evaluated after 16 h; mean number ± SD (triplicates) of invading and penetrating cells and representative examples (scale bar: 250 μm); significant differences between HEK-EpC-cld7 and HEK-EpCmAG-cld7 or HEK-EpC-cld7mPalm: *. (b) Flow cytometry of MMP and CD147 expression in untransfected and transfected HEK cells; the mean fluorescence index (% stained cells x mean intensity of staining ± SD; three assays) and representative examples are shown; significant differences in transfected versus non-transfected HEK cells: *. (c) Confocal microscopy of transfected HEK cells stained with anti-cld7 and anti-MMP3 or -MMP14; single fluorescence and overlays of representative examples (scale bar: 10 μm). Despite slight downregulation of MMP3, MMP7 and MMP9 by cld7, palmitoylated cld7 promotes invasiveness.
Figure 9
Figure 9
Palmitoylated claudin-7, MMP activity and CD147 (a) Zymography of HEK, HEK-EpC, HEK-cld7, HEK-EpC-cld7 and HEK-EpCmAG-cld7 or HEK-EpC-cld7mPalm supernatants. (b) HEK cells were seeded in matrigel-coated transwell plates in the presence of anti-CD147 (10 μg / ml) (a representative example is included in Figure 8a); invasion and penetration was evaluated as above; significant inhibition by anti-CD147: s. (c) Confocal microscopy of HEK-cld7, HEK-EpC-cld7 and HEK-EpCmAG-cld7 or HEK-EpC-cld7mPalm cells stained with anti-cld7 and anti-CD147 (single fluorescence and overlays, scale bar: 10 μm). (d) HEK-cld7, HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm lysates were precipitated with control IgG or anti-CD147; after SDS-PAGE and transfer precipitates were blotted with anti-EpC, anti-cld7 and anti-CD147. Palmitoylated cld7-promoted invasiveness likely is supported by associated MMP14 and CD147, which can strengthen MMP2 and MMP9 activation and expression.
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References

    1. Tsukita S, Furuse M. Overcoming barriers in the study of tight junction functions: from occludin to claudin. Genes Cells. 1998;3:569–73. doi: 10.1046/j.1365-2443.1998.00212.x. - DOI - PubMed
    1. Schneeberger EE, Lynch RD. The tight junction: a multifunctional complex. Am J Physiol Cell Physiol. 2004;286:C1213–28. doi: 10.1152/ajpcell.00558.2003. - DOI - PubMed
    1. Singh AB, Sharma A, Dhawan P. Claudin family of proteins and cancer: an overview. J Oncol. 2010;2010:541957. doi: 10.1155/2010/541957. - DOI - PMC - PubMed
    1. Schulzke JD, Günzel D, John LJ, Fromm M. Perspectives on tight junction research. Ann NY Acad Sci. 2012;1257:1–19. doi: 10.1111/j.1749-6632.2012.06485.x. - DOI - PubMed
    1. Kwon MJ. Emerging roles of claudins in human cancer. Int J Mol Sci. 2013;14:18148–80. doi: 10.3390/ijms140918148. - DOI - PMC - PubMed

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