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. 2006 Nov 21;103(47):17795-800.
doi: 10.1073/pnas.0607299103. Epub 2006 Nov 10.

Pleiotrophin Disrupts Calcium-Dependent Homophilic Cell-Cell Adhesion and Initiates an Epithelial-Mesenchymal Transition

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Pleiotrophin Disrupts Calcium-Dependent Homophilic Cell-Cell Adhesion and Initiates an Epithelial-Mesenchymal Transition

P Perez-Pinera et al. Proc Natl Acad Sci U S A. .
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Abstract

Regulation of the levels of tyrosine phosphorylation is essential to maintain the functions of proteins in different signaling pathways and other cellular systems, but how the steady-state levels of tyrosine phosphorylation are coordinated in different cellular systems to initiate complex cellular functions remains a formidable challenge. The receptor protein tyrosine phosphatase (RPTP)beta/zeta is a transmembrane tyrosine phosphatase whose substrates include proteins important in intracellular and transmembrane protein-signaling pathways, cytoskeletal structure, cell-cell adhesion, endocytosis, and chromatin remodeling. Pleiotrophin (PTN the protein and Ptn the gene) is a ligand for RPTPbeta/zeta; PTN inactivates RPTPbeta/zeta, leaving unchecked the continued endogenous activity of tyrosine kinases that increase phosphorylation of the substrates of RPTPbeta/zeta at sites dephosphorylated by RPTPbeta/zeta in cells not stimulated by PTN. Thus, through the regulation of the tyrosine phosphatase activity of RPTPbeta/zeta, the PTN/RPTPbeta/zeta signaling pathway coordinately regulates the levels of tyrosine phosphorylation of proteins in many cellular systems. We now demonstrate that PTN disrupts cytoskeletal protein complexes, ablates calcium-dependent homophilic cell-cell adhesion, stimulates ubiquitination and degradation of N-cadherin, reorganizes the actin cytoskeleton, and induces a morphological epithelial-mesenchymal transition (EMT) in PTN-stimulated U373 cells. The data suggest that increased tyrosine phosphorylation of the different substrates of RPTPbeta/zeta in PTN-stimulated cells alone is sufficient to coordinately stimulate the different functions needed for an EMT; it is possible that PTN initiates an EMT in cells at sites where PTN is expressed in development and in malignant cells that inappropriately express Ptn.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Confluent U373 cells were not stimulated or stimulated with 10, 25, 50, or 100 ng of PTN/ml for 60 min. Confluent U373 cells also were preincubated with pervanadate or with anti-PTN antibodies (5 μg/ml) before incubation with 50 ng/ml PTN. Cell lysates were prepared and immunoprecipitated with anti-β-catenin antibodies, and the immunoprecipitates were analyzed in Western blots probed with anti-phosphotyrosine, anti-pan-cadherin, and anti-β-catenin antibodies. A statistical analysis of the inverse correlation of the levels of the phosphorylation of β-catenin with the levels of the association of β-catenin with N-cadherin is shown (Fig. 8A).
Fig. 2.
Fig. 2.
Western blots were prepared from immunoprecipitates of lysates incubated with anti-pan-cadherin antibodies of confluent nonstimulated control U373 cells or of U373 cells that were stimulated with 50 ng/ml PTN for 60 min. The blots were probed with anti-P120, anti-β-catenin, anti-γ-catenin, anti-IQGAP1, or anti-pan-cadherin antibodies. Lysates from the same cells were immunoprecipitated with anti-β-catenin antibodies and analyzed in Western blots probed with anti-phosphotyrosine, anti-α-catenin, or anti-β-catenin antibodies.
Fig. 3.
Fig. 3.
Degradation of adherent junction proteins in PTN-stimulated cells. (A) Lysates of confluent U373 cells stimulated with 50 ng/ml PTN for 0, 5, 10, 20, and 60 min were analyzed in Western blots probed with anti-pan-cadherin (Upper Top), anti-α-catenin (Upper Middle), and anti-β-catenin antibodies (Upper Bottom). (Lower) The results also were analyzed by using densitometry and expressed as percent of intensity compared with control nonstimulated cells. (B) Western blots of proteins captured by Rad23 from lysates of U373 cells stimulated with 50 ng/ml PTN for 30 and 60 min probed with anti-pan-cadherin antibodies and reprobed with anti-ubiquitin antibodies.
Fig. 4.
Fig. 4.
Confocal microscopy analysis of confluent U373 cells not stimulated (A and C) or stimulated (B and D) with 50 ng/ml PTN for 60 min were stained by using FITC-tagged anti-β-catenin (A and B) and anti-pan-cadherin antibodies (C and D) and analyzed by using confocal microscopy. The section is from a single plane in the z axis and is representative of the entire z axis from both control and PTN-stimulated cells. Loss of cell–cell contact is seen at different sites (arrows).
Fig. 5.
Fig. 5.
U373 cells not stimulated (A) or stimulated (B) with 50 ng/ml PTN for 60 min were stained by using FITC-tagged anti-pan-cadherin antibodies and Texas red-conjugated anti-LAMP1 antibodies to visualize lysosomes and observed by using confocal microscopy. The sections are from a single plane in the z axis and are representative of the entire z axis (see Fig. 10) from both PTN-stimulated and control cells.
Fig. 6.
Fig. 6.
U373 cells were seeded in culture plates and incubated for 1 h with serum-free media (AC) or media containing 50 ng/ml PTN (DF). The cells were stained by using FITC-tagged anti-tubulin antibodies (B and E), Texas red-conjugated phalloidin to visualize F-actin (A and D), and DAPI to visualize nuclei and observed by using confocal microscopy. Overlay images are shown in C and F.
Fig. 7.
Fig. 7.
U373 cells not stimulated (A and C) or stimulated with 50 ng/ml PTN for 60 min (B and D) were stained by using anti-β-catenin antibodies and visualized by using confocal microscopy (A and B). In separate experiments, U373 cells not stimulated or stimulated with 50 ng/ml PTN for 60 min were visualized by using scanning electron microscopy (C and D). Cytoplasmic extensions used for cells to initiate cell–cell contact (A and C, arrows) are blunted or lost (B, arrows) in PTN-stimulated cells.

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