Pleiotrophin disrupts calcium-dependent homophilic cell-cell adhesion and initiates an epithelial-mesenchymal transition.
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.
- [show abstract] [hide abstract]
ABSTRACT: Glioblastoma multiforme is the most common highly aggressive human brain cancer, and receptor tyrosine kinases have been implicated in the progression of this malignancy. We have recently identified anaplastic lymphoma kinase (ALK) as a tyrosine kinase receptor for pleiotrophin, a secreted growth factor that is highly expressed during embryonic brain development and in tumors of the central nervous system. Here we report on the contribution of pleiotrophin-ALK signaling to glioblastoma growth. We found ALK overexpressed in human glioblastoma relative to normal brain and detected ALK mRNA in glioblastoma cell lines. We reduced the endogenous ALK in glioblastoma cells by ribozyme targeting and demonstrated that this prevents pleiotrophin-stimulated phosphorylation of the anti-apoptotic protein Akt. Furthermore, this depletion of ALK reduced tumor growth of xenografts in athymic nude mice and prolonged survival of the animals because of increased apoptosis in the tumors. These findings directly implicate ALK signaling as a rate-limiting factor in the growth of glioblastoma multiforme and suggest potential utility of therapeutic targeting of ALK.Journal of Biological Chemistry 05/2002; 277(16):14153-8. · 4.65 Impact Factor
- Nature reviews. Cancer 07/2002; 2(6):442-54. · 35.00 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Pleiotrophin (PTN) is a platelet-derived growth factor-inducible, 18-kDa heparin-binding cytokine that signals diverse phenotypes in normal and deregulated cellular growth and differentiation. To seek the mechanisms of PTN signaling, we studied the interactions of PTN with the receptor protein tyrosine phosphatase (RPTP) beta/zeta in U373-MG cells. Our results suggest that PTN is a natural ligand for RPTP beta/zeta. PTN signals through "ligand-dependent receptor inactivation" of RPTP beta/zeta and disrupts its normal roles in the regulation of steady-state tyrosine phosphorylation of downstream signaling molecules. We have found that PTN binds to and functionally inactivates the catalytic activity of RPTP beta/zeta. We also have found that an active site-containing domain of RPTP beta/zeta both binds beta-catenin and functionally reduces its levels of tyrosine phosphorylation when added to lysates of pervanidate-treated cells. In contrast, an (inactivating) active-site mutant of RPTP beta/zeta also binds beta-catenin but fails to reduce tyrosine phosphorylation of beta-catenin. Finally, in parallel to its ability to inactivate endogenous RPTP beta/zeta, PTN sharply increases tyrosine phosphorylation of beta-catenin in PTN-treated cells. The results suggest that in unstimulated cells, RPTP beta/zeta is intrinsically active and functions as an important regulator in the reciprocal control of the steady-state tyrosine phosphorylation levels of beta-catenin by tyrosine kinases and phosphatases. The results also suggest that RPTP beta/zeta is a functional receptor for PTN; PTN signals through ligand-dependent receptor inactivation of RPTP beta/zeta to increase levels of tyrosine phosphorylation of beta-catenin to initiate downstream signaling. PTN is the first natural ligand identified for any of the RPTP family; its identification provides a unique tool to pursue the novel signaling pathway activated by PTN and the relationship of PTN signaling with other pathways regulating beta-catenin.Proceedings of the National Academy of Sciences 04/2000; 97(6):2603-8. · 9.74 Impact Factor
Pleiotrophin disrupts calcium-dependent
homophilic cell–cell adhesion and initiates
an epithelial–mesenchymal transition
P. Perez-Pinera*, S. Alcantara*†, T. Dimitrov‡, J. A. Vega§, and T. F. Deuel*¶
*Departments of Molecular and Experimental Medicine and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037;†Unit of Cell Biology,
Department of Experimental Pathology and Therapeutics, School of Medicine, University of Barcelona L’Hospitalet de Llobregat, 08907 Barcelona,
Spain;‡Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and§Departamento
de Ciencias Me ´dicas, Seccio ´n de Anatomia y Embriologı ´a, Facultad de Medicina, Universidad San Pablo CEU, 28668 Madrid, Spain
Communicated by Ernest Beutler, The Scripps Research Institute, La Jolla, CA, August 22, 2006 (received for review March 22, 2006)
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 sys-
tems to initiate complex cellular functions remains a formidable
challenge. The receptor protein tyrosine phosphatase (RPTP)??? is
a transmembrane tyrosine phosphatase whose substrates include
proteins important in intracellular and transmembrane protein-
signaling pathways, cytoskeletal structure, cell–cell adhesion, en-
docytosis, and chromatin remodeling. Pleiotrophin (PTN the pro-
tein and Ptn the gene) is a ligand for RPTP???; PTN inactivates
RPTP???, leaving unchecked the continued endogenous activity of
tyrosine kinases that increase phosphorylation of the substrates of
RPTP??? at sites dephosphorylated by RPTP??? in cells not stimu-
lated by PTN. Thus, through the regulation of the tyrosine phos-
phatase activity of RPTP???, the PTN?RPTP??? 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-depen-
dent homophilic cell–cell adhesion, stimulates ubiquitination 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 RPTP??? 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 devel-
opment and in malignant cells that inappropriately express Ptn.
receptor protein tyrosine phosphatase??? ? glioblastoma ? cadherin ?
?-catenin ? cytoskeleton
tyrosine phosphorylation of key proteins essential for many
important cellular functions. The regulated disruption of this
balance through growth factor and?or cytokine-activated
receptor-transduced signals is an important mechanism of signal
transduction but, when deregulated, is a mechanism frequently
underlying different diseases and a major feature in the patho-
genesis of many human malignancies (1). An important gap,
however, is in understanding the mechanism of how different
pathways and systems are coordinated to initiate the many
different cellular functions required for normal cellular ho-
meostasis, proliferation, and differentiation of cells.
The diverse substrates of the receptor protein tyrosine phos-
phatase (RPTP)??? (2) include ?-catenin (2), ?-adducin (3, 4),
Fyn (5), GIT1?Cat-1 (6), and P190RhoGAP (7), indicating that
RPTP??? is promiscuous in substrate specificity, but through its
activity, is critically positioned to coordinately regulate the
steady-state levels of tyrosine phosphorylation of proteins in
different signaling networks and cellular systems. Pleiotrophin
he balanced activities of tyrosine kinases and tyrosine phos-
phatases dynamically regulate the steady-state levels of
(PTN the protein and Ptn the gene) is a secreted, highly
conserved cytokine (8, 9) that signals through inactivation of
RPTP???; as a consequence, PTN coordinately increases ty-
rosine phosphorylation of the many substrates of RPTP???
through persistent activity of the tyrosine kinases that phosphor-
ylate the same sites that are dephosphorylated by RPTP??? in
cells not stimulated by PTN. The diverse substrates regulated
through the PTN?RPTP??? signaling pathway thus are likely to
account for the diverse functions signaled by PTN in different
cellular systems and in the different malignant cell lines with
inappropriate expression of Ptn (10).
In these studies, we pursued the biochemical and phenotypic
consequences of the PTN-dependent inactivation of RPTP???
in PTN-stimulated U373 cells; the data demonstrate that PTN
stimulates a morphological epithelial–mesenchymal transition
(EMT) in U373 cells and, thus, suggest that the diversity of
responses needed for an EMT are initiated coordinately through
the PTN-dependent increase in tyrosine phosphorylation of
substrates of RPTP??? in different signaling networks.
Pleiotrophin Stimulates Increased Tyrosine Phosphorylation of
?-Catenin. Pleiotrophin stimulates increased tyrosine phosphor-
ylation of ?-catenin through inactivation of RPTP??? (2).
Because tyrosine phosphorylation of ?-catenin is known to
perturb adherent junction protein complexes and homophilic
cell–cell adhesion (11, 12), the increase in tyrosine phosphory-
lation of ?-catenin was compared with the ability of ?-catenin to
associate with N-cadherin in PTN-stimulated U373 cells. U373
cells were stimulated with PTN for 60 min with increasing
concentrations of PTN; ?-catenin was immunoprecipitated with
anti-?-catenin antibodies from lysates of control, nonstimulated
cells and PTN-stimulated cells and analyzed in Western blots
probed with anti-phosphotyrosine antibodies. As found in ref. 2,
PTN induced a rapid increase in tyrosine phosphorylation of
?-catenin when U373 cells were stimulated with PTN at con-
centrations from 0 to 10 ng?ml and slightly higher levels of
tyrosine phosphorylation were seen as the concentration of PTN
was increased to 25 ng?ml. In cells stimulated with 50 and 100
ng?ml PTN, concentrations of PTN previously found to be in
excess of saturating concentrations (2, 3), the levels of tyrosine
phosphorylation of ?-catenin fell somewhat (Fig. 1). The reason
for the decrease in tyrosine phosphorylation of ?-catenin in cells
Author contributions: P.P.-P., S.A., T.D., J.A.V., and T.F.D. designed research; P.P.-P., S.A.,
T.D., and J.A.V. performed research; P.P.-P., S.A., T.D., J.A.V., and T.F.D. analyzed data; and
T.F.D. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: EMT, epithelial–mesenchymal transition; RPTP, receptor protein tyrosine
¶To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
November 21, 2006 ?
vol. 103 ?
no. 47 ?
stimulated with PTN in excess of saturation is unknown. A very
striking increase in tyrosine phosphorylation of ?-catenin also
was seen in U373 cells stimulated with sodium pervanadate (50
?M) (ref. 13; Fig. 1, lane 6), suggesting that more than one
tyrosine phosphatase regulates steady-state tyrosine phosphor-
ylation levels of ?-catenin.
Loss of Association of ?-Catenin with N-Cadherin in PTN-Stimulated
Cells. The levels of N-cadherin that coimmunoprecipitate with
?-catenin from lysates of nonstimulated and PTN-stimulated
cells were compared in Western blots and quantitated by scan-
ning densitometry. An inverse linear relationship between levels
of tyrosine phosphorylation of ?-catenin and levels of N-
cadherin that co-immunoprecipitated with ?-catenin was dem-
onstrated with a coefficient of variation of ?0.98 (see Fig. 8A,
which is published as supporting information on the PNAS web
site). Anti-PTN antibodies (5 ?g?ml) added together with PTN
(50 ng?ml) effectively blocked the PTN-dependent increase in
tyrosine phosphorylation of ?-catenin and loss of its association
with N-cadherin (Fig. 1, compare lanes 1 and 7), establishing the
specificity of PTN as responsible for the increase in tyrosine
with N-cadherin in PTN-stimulated cells.
In recent studies, Wellstein et al. (14–16) reported that the
physiological receptor of PTN is anaplastic lymphoma kinase; in
the present studies, we used a short hairpin RNA to ‘‘knock
down’’ RPTP??? and demonstrated that RPTP??? is required
for the PTN-initiated responses we report (Fig. 9, which is
published as supporting information on the PNAS web site).
Furthermore, by using RT-PCR and Western blot analysis, we
found that anaplastic lymphoma kinase is not expressed in U373
cells as reported in ref. 17. Thus, the data we provide depends
on the PTN?RPTP??? signaling pathway.
Disruption of Adherent Junction Complexes in PTN-Stimulated Cells.
N-cadherin associates also with ?-catenin, P120, and IQGAP-1
known to compete for the same site in the C-terminal region of
the cadherins and through ?-catenin link the cadherins to
filamentous actin to stabilize cell–cell adhesion. Recent evi-
dence, however, suggests that ?-catenin regulates actin filament
assembly and does not associate simultaneously with actin and
P120 binds to the juxtamembrane domain of cadherins and
regulates cadherin turnover at the cell surface (19). IQGAP1
associates more strongly with cadherins and ?-catenin under
conditions in which cells have lost cell–cell adhesion (20).
IQGAP1 in PTN-stimulated U373 cells, lysates of control (non-
stimulated) and PTN-stimulated cells were prepared and immu-
noprecipitated with anti-pan-cadherin antibodies; the immuno-
precipitates were analyzed in Western blots probed with anti-
?-catenin, anti-?-catenin, anti-P120, and anti-IQGAP-1
antibodies (Fig. 2). In the control studies, equal amounts of
N-cadherin were immunoprecipitated with anti-pan-cadherin
antibodies from control (nonstimulated) and PTN-stimulated
cells (Fig. 2). In PTN-stimulated cells, N-cadherin was found to
associate no longer with ?-catenin (Fig. 2) and to decrease the
levels of association with ?-catenin (?84%). A modest reduction
in the levels of association of N-cadherin with P120 (?27%) and
IQGAP-1 (?32%) also was observed in lysates of PTN-
stimulated cells (Fig. 2). An ?2.4-fold increase in tyrosine
phosphorylation of ?-catenin was demonstrated in immunopre-
cipitates from lysates immunoprecipitated with anti-?-catenin
antibodies in comparison with lysates from unstimulated cells;
the increase in tyrosine phosphorylation of ?-catenin was asso-
ciated with a ?59% loss of association of ?-catenin with
?-catenin (Fig. 2).
Loss of Adherent Junction Protein Levels in PTN-Stimulated Cells.
P120 catenin regulates turnover of cadherins at the cell
surface, suggesting the possibility that N-cadherin maybe
subject to proteolytic degradation through the ubiquitin-
proteasome proteolysis system targeted by P120 catenin (19).
To test the possibility that PTN stimulates degradation of
N-cadherin in PTN-stimulated cells, lysates from PTN-
stimulated and control, nontreated cells were prepared and
analyzed in Western blots probed with anti-pan-cadherin
antibodies. The levels of N-cadherin in lysates of U373 cells
stimulated with PTN for 5, 10, and 20 min were reduced to a
level ?50% the levels of N-cadherin in U373 cells before
stimulation (Fig. 3A). The same lysates were analyzed in
Western blots probed with anti-?-catenin and anti-?-catenin
antibodies (Fig. 3A); the levels of ?-catenin decreased to
?80% of the levels in control, nonstimulated cells 10 min after
PTN stimulation and remained essentially constant for the
pervanadate or with anti-PTN antibodies (5 ?g?ml) before incubation with 50
ng?ml PTN. Cell lysates were prepared and immunoprecipitated with anti-?-
probed with anti-phosphotyrosine, anti-pan-cadherin, and anti-?-catenin anti-
bodies. A statistical analysis of the inverse correlation of the levels of the phos-
phorylation of ?-catenin with the levels of the association of ?-catenin with
N-cadherin is shown (Fig. 8A).
Confluent U373 cells were not stimulated or stimulated with 10, 25, 50,
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-?-catenin, anti-?-catenin,
anti-IQGAP1, or anti-pan-cadherin antibodies. Lysates from the same cells
were immunoprecipitated with anti-?-catenin antibodies and analyzed in
Western blots probed with anti-phosphotyrosine, anti-?-catenin, or anti-?-
Western blots were prepared from immunoprecipitates of lysates
www.pnas.org?cgi?doi?10.1073?pnas.0607299103Perez-Pinera et al.
remainder of the experiment. The levels of ?-catenin rapidly
and progressively decreased at 20 min to levels ?25% of the
control, nonstimulated cells. After 60 min, the levels of
?-catenin returned to ?50% of the levels observed in control,
nonstimulated cells, and the levels of each protein returned to
baseline in the subsequent 1–2 h (data not shown).
To test whether N-cadherin is ubiquitinated, Rad-23 conju-
gated to agarose beads was used to ‘‘capture’’ ubiquitinated
proteins from control and PTN-stimulated U373 cells; Rad-23
binds ubiquitin, monoubiquitinated, and polyubiquitinated pro-
teins (21) with high affinity and specificity. Lysates were incu-
bated with Rad-23, and proteins captured by Rad-23 were
analyzed in Western blots probed with either anti-pan-cadherin
antibodies or with antibodies that recognize mono- or polyubiq-
uitin covalently coupled to proteins (see Experimental Proce-
dures). The results demonstrate that levels of ubiquitinated
N-cadherin captured by Rad-23 were increased ?4- and ?5-fold
above the levels of ubiquitinated N-cadherin in control, non-
stimulated cells 30 and 60 min after stimulation with 50 ng?ml
PTN (Fig. 3B, lane 1–3). The Western blots probed with
anti-ubiquitin antibodies confirmed equal loading and that many
ubiquitinated proteins are pulled down by Rad23 in PTN-
stimulated cells. We also preincubated cells with lactacystin (10
?M) before they were stimulated with PTN; lactacystin pre-
vented the degradation of N-cadherin seen in PTN-stimulated
cells that were not incubated with lactacystin (data not shown).
The data suggest that PTN rapidly targets N-cadherin for
proteolysis through the ubiquitin proteasome degradation path-
way in PTN-stimulated cells; however, additional experiments
are needed to fully characterize the basis of N-cadherin degra-
dation in PTN-stimulated cells.
Loss of Homophilic Cell–Cell Adhesion in PTN-Stimulated Cells. To
pursue the possibility that PTN also disrupts homophilic cell–
cell adhesion, the ratio of cells dissociated in calcium-containing
media (NTC) vs. the cells dissociated in calcium-free media (NTE)
(a ‘‘dissociation index,’’ see Experimental Procedures) was mea-
ratio NTC?NTEof U373 cells stimulated with 50 ng?ml PTN in
this assay was 0.85, whereas the ratio NTC?NTEof nonstimulated
decreases calcium-dependent homophilic cell–cell adhesion in
PTN-stimulated U373 cells (see Fig. 8B).
Loss of Adherent Junction Complexes and Cell–Cell Adhesion in
PTN-Stimulated Cells. To directly visualize the structure of cell–
cell adhesion complexes in PTN-stimulated cells, we treated
control and PTN-stimulated confluent U373 cells with anti-?-
catenin and anti-pan-cadherin FITC-tagged antibodies and com-
pared the two preparations by using confocal microscopy at
different planes in the z axis. Fig. 4 is representative of the
different transverse planes of the z axis taken to illustrate points
of cell–cell contact from untreated and PTN-stimulated cells; it
demonstrates that in cells not stimulated with PTN, both ?-cate-
nin and N-cadherin are evenly distributed at juxtamembrane
sites at sites of cell–cell contact (Fig. 4 A and C). In contrast, in
cells stimulated with PTN for 60 min, the intensity of immuno-
staining of both ?-catenin and N-cadherin is greatly decreased
at the sites of cell–cell contact; neither immunoreactive ?-cate-
nin nor N-cadherin were consistently seen at adherent junctions,
and the cell–cell adhesion complexes are disrupted. Further-
more, loss of cell–cell contact is seen at different sites (Fig. 4 B
and D, arrows). The loss of cell–cell adhesion complexes and
homophilic cell–cell contact at different sites occurs in parallel
(A) Lysates of confluent U373 cells stimulated with 50 ng?ml PTN for 0, 5, 10,
(Upper Top), anti-?-catenin (Upper Middle), and anti-?-catenin antibodies
and expressed as percent of intensity compared with control nonstimulated
stimulated with 50 ng?ml PTN for 30 and 60 min probed with anti-pan-
cadherin antibodies and reprobed with anti-ubiquitin antibodies.
Degradation of adherent junction proteins in PTN-stimulated cells.
(A and C) or stimulated (B and D) with 50 ng?ml PTN for 60 min were stained
ies (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
Confocal microscopy analysis of confluent U373 cells not stimulated
Perez-Pinera et al.
November 21, 2006 ?
vol. 103 ?
no. 47 ?
with loss of association of N-cadherin and ?-catenin in PTN-
stimulated cells illustrated above.
Pan-cadherin immunoreactive proteins are also seen scattered
within the cytosolic compartment of PTN-stimulated cells in Fig.
4 B and D. The pan-cadherin immunoreactive proteins appear to
be in vesicles and likely are degradation products of N-cadherin
targeted for proteolysis through the ubiquitin-proteasome pro-
teolysis pathway. However, endocytosis through the lysosomal
vesicles recently has emerged as a regulatory mechanism to
modulate the levels of cadherin cell surface expression in
epithelial cells (22, 23), raising the possibility that loss of
‘‘uncap’’ N-cadherin in PTN-stimulated cells and target N-
cadherin for degradation within the lysosome compartments
(24). U373 cells therefore were stimulated with PTN for 60 min,
and cells stained with anti-N-cadherin antibodies and anti-
LAMP1 antibodies to mark lysosomal compartments were an-
alyzed with confocal microscopy at different z axis planes
through the cell. The data in a representative focal plane (Fig.
5) failed to demonstrate colocalization of N-cadherin and the
lysosomal protein LAMP1 in nonstimulated or in PTN-
The images from the different z axis planes then were used to
quantitatively analyze the height of PTN-stimulated U373 cells
compared with nonstimulated cells (see Fig. 10, which is pub-
lished as supporting information on the PNAS web site). The
distance between the highest and the lowest focal planes in
PTN-stimulated cells measured in 10 separate microscopic fields
in three different slides from independent experiments was 5.31
?m, whereas in cells that were not stimulated, the average height
was 3.97 ?m; this difference in cell height furthermore demon-
strates that PTN induces striking changes in the cytoskeletal
architecture of PTN-stimulated cells (see Fig. 8C).
Pleiotrophin Stimulates a Reorganization of the Actin Cytoskeleton
and Initiates an Epithelial–Mesenchymal-Like Transition. To pursue
further the ability of PTN to alter the cytoskeletal structure of
PTN-stimulated cells, PTN-stimulated and control, nonstimu-
lated cells were stained with FITC-tagged anti-tubulin anti-
bodies and Texas red-tagged phalloidin to visualize F-actin and
observed with confocal microscopy. U373 cells characteristi-
cally are rounded but relatively flat; they have a large cytosolic
compartment and a centrally located nucleus. In nonstimu-
lated cells, F-actin was localized in sites immediately beneath
the plasma membrane and in stress fibers throughout the
cytoplasm (Fig. 6 A–C). In contrast, PTN-stimulated U373
cells for 1 h were elongated; they had a fibroblast-like shape
and had numerous evenly distributed, nonpolarized filopodia
and lamelipodia protruding from the cell membrane. F-actin
was localized clearly in those protrusions and also in the
cortical regions. The PTN-stimulated cells appear to have a
markedly reduced cytosolic compartmental volume (Fig. 6
D–F). It was found that the numbers of cells with the mesen-
chymal phenotype increased ?40% within 1 h in PTN-
stimulated cell cultures. These data and the data presented
above are consistent with the conclusion that PTN stimulates
U373 cells treated with PTN for 60 min and control non-
treated cells were compared with scanning electron microscopy.
U373 cells usually are round and flat-shaped with numerous
cytoplasmic extensions (Fig. 7C, arrows). The PTN-stimulated
U373 cells again acquired an elongated morphology consistent
with mesenchymal phenotype and enhanced motility, and the
small cytoplasmic extensions used for cells to initiate cell–cell
contact were blunted or lost (Fig. 7D); this observation was
confirmed when the cells were stained by using anti-?-catenin
antibodies and observed with confocal microscopy (Fig. 7 A and
B). The data thus confirm the morphological transition of U373
cells treated with PTN for 60 min from an epithelial-like to
fibroblast-like phenotype and, thus, the transition to the mes-
serum-free media (A–C) or media containing 50 ng?ml PTN (D–F). 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.
U373 cells were seeded in culture plates and incubated for 1 h with
for 60 min (B and D) were stained by using anti-?-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.
U373 cells not stimulated (A and C) or stimulated with 50 ng?ml PTN
60 min were stained by using FITC-tagged anti-pan-cadherin antibodies and
Texas red-conjugated anti-LAMP1 antibodies to visualize lysosomes and ob-
served 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.
U373 cells not stimulated (A) or stimulated (B) with 50 ng?ml PTN for
www.pnas.org?cgi?doi?10.1073?pnas.0607299103Perez-Pinera et al.
These studies support the conclusion that PTN stimulates an
EMT in PTN-stimulated U373 cells; these features of an EMT
include loss of cell–cell adhesion, increased motility and inva-
siveness, and the morphological phenotype needed for cells to
exit early developmental sites for subsequent differentiation in
the process of morphogenesis and organ development (25). To
initiate an EMT, many different properties of the cell need to be
altered coordinately for the cell to assume the mesenchymal
phenotype; in this context, for PTN to stimulate an EMT, PTN
needs to coordinate different regulatory pathways and cellular
systems through initiation of a single signaling mechanism. Many
malignant cells with inappropriate expression of Ptn also exhibit
these features of an EMT needed for the malignant cells to
the malignant cells constitutively express Ptn, the malignant cells
have a ‘‘stable EMT’’ or perhaps more appropriately, an ‘‘ar-
Pleiotrophin signals through inactivation of the endogenous
protein tyrosine phosphatase activity of RPTP???; it increases
tyrosine phosphorylation of the different substrates of
RPTP??? through the persistent activity of tyrosine kinases
acting at the same sites as RPTP??? (2–5). Pleiotrophin was
the first natural ligand to be discovered for this class of
receptor-type transmembrane tyrosine phosphatases (2) and
the PTN?RPTP??? signaling pathway triggered by PTN is
unique. ?-catenin was identified as the first substrate of
RPTP??? (2), and, subsequently, ?-adducin (3, 4), Fyn (5),
GIT-1 (6), and P190RhoGAP (7) have been identified as
substrates of RPTP??? and downstream targets of the PTN?
RPTP???-signaling pathway. Through regulation of tyrosine
phosphorylation of these different substrates of RPTP???,
PTN thus regulates proteins involved in cytoskeletal stability
and function, endocytosis, chromatin remodeling, and both
intracellular and transmembrane receptor-type tyrosine ki-
nase signaling pathways. RPTP??? thus is centrally positioned
to coordinately regulate steady-state levels of tyrosine phos-
phorylation of different proteins in diverse pathways depend-
ing on the relative affinity and levels of each for RPTP???.
Pleiotrophin thus is positioned as the single mediator to
up-regulate tyrosine phosphorylation of each of these proteins
through its ability to inactivate the tyrosine phosphatase
activity of RPTP??? (26). Because these studies were under-
taken within the first hour of stimulation by PTN, it is
reasonable to conclude that responses initiated by transcrip-
tion activation are minimal, but it is the PTN?RPTP???
signaling pathway that directly initiates the profound changes
that underlie the EMT through the coordinated increase of
tyrosine phosphorylation of the different substrates of
Phosphorylation of tyrosines 86, 142, and 654 in ?-catenin is
known to result in loss of affinity and, thus, loss of association
of ?-catenin with cadherins (27), and increased tyrosine phos-
phorylation of ?-catenin is known to destabilize cytoskeletal
structures and to ablate homophilic cell–cell adhesion (11)
through decreased affinity of phosphorylated ?-catenin for
cadherins (27). ?-adducin also was identified as a target of the
PTN?RPTP???-signaling pathway (3, 4), and PTN disrupts
?-adducin–actin–spectrin complexes supporting cystoskeletal
Finally, N-cadherin was found to be degraded in PTN-
stimulated cells. Whereas the data suggest that degradation of
N-cadherin is modulated through the ubiquitin-proteasome pro-
teolytic pathway, a feature of PTN-stimulated cells likely to
further destabilize the cytoskeleton, further experiments are
needed to better determine the molecular mechanisms of N-
cadherin degradation in PTN-stimulated cells. As cited in ref. 1,
degradation of selected cytoskeletal and adhesion proteins, and
particularly E-cadherin and N-cadherin, are necessary early
steps in differentiation and in EMT. One candidate that may
mediate degradation of N-cadherin in this study is the Cbl-like
ubiquitin ligase Hakai, previously discovered to target cadherins
for degradation upon dissociation of the cadherins from their
binding partners (28).
In summary, this study supports the conclusion that through
the regulation of the intrinsic tyrosine phosphatase activity of
RPTP???, PTN regulates the activity of different systems that
are important in cytoskeletal stability and the strength of
cell–cell adhesion, in stimulating degradation of cytoskeletal
proteins, and potentially many more systems to initiate a mor-
phological EMT that is similar to that in development and during
Cell Lines. U373 cells (human glioblastoma) (American Type
Culture Collection, Manassas, VA) were grown in DMEM
supplemented with 10% FBS and 1% penicillin?streptomycin at
37°C with 5% CO2. U373 cells express high levels of RPTP???
and fail to express anaplastic lymphoma kinase as detected by
using RT-PCR or Western blots (17). Cells were serum starved
for 24 h before stimulation with PTN. PTN-Fc was prepared in
the laboratory; PTN was purchased from R & D Systems
anti-LAMP1, and anti-mouse IgG FITC-conjugated antibodies
were obtained from Sigma–Aldrich (St. Louis, MO). Anti-
anti-phosphotyrosine antibodies were obtained from BD Bio-
sciences (San Diego, CA). Anti-mouse IgG HRP-conjugated and
anti-rabbit IgG HRP-conjugated antibodies were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-Pan-Cadherin Antibodies. In preliminary studies, N-cadherin
was estimated to constitute ?95% of the total cadherins in U373
cells. Anti-pan-cadherin antibodies were more effective in im-
munoprecipitation than specific anti-N-cadherin antibodies
tested (data not shown) and, thus, used for immunoprecipitation
and Western blots. The protein immunoprecipitated with anti-
pan-cadherin antibodies is referred as N-cadherin unless other-
Cell–Cell Aggregation Assays. Confluent cells in 60-mm dishes
were treated with 0.01% trypsin with either 1 mM calcium
chloride or 1 mM EDTA for 10 min at 37°C, scraped, and
dissociated by pipetting 10 times. Individual cells were counted
by using a hemacytometer and the ratio NTC?NTE, the number
of cells dissociated in presence of calcium (NTC) divided by the
number of cells dissociated in presence of EDTA (NTE), the
‘‘dissociation index,’’ was determined as described in ref. 29.
Ubiquitination Assay. Lysates of U373 cells in 50 mM Tris, pH
7.5?150 mM NaCl?1% Nonidet P-40?0.25% sodium deoxy-
cholate?0.1% SDS?Complete EDTA-free Protease Inhibitor
Mixture (Roche, Indianapolis, IN)?2 mM sodium orthovanadate
were incubated overnight with Rad23 agarose-beads (Calbio-
chem, La Jolla, CA). Rad23 recognizes ubiquitin, monoubiq-
uitinated, and polyubiquitinated proteins (21). The beads were
washed four times in lysis buffer, boiled in loading buffer, and
analyzed in Western blots.
Immunoprecipitation. The samples were incubated with 1 ?g?ml
of either ?-catenin or cadherin antibodies at 4°C overnight and
subsequently with 50 ?l of protein G conjugated to magnetic
Perez-Pinera et al.
November 21, 2006 ?
vol. 103 ?
no. 47 ?
beads (Miltenyi, Auburn, CA) for 1 h at 4°C. The lysate was
passed through a 1-ml column placed in a magnetic stand and
washed five times with PBS. Bound proteins were eluted by using
Western Blots. Protein concentrations in cell lysates were measured
by using the BCA Protein Assay Kit (Pierce, Rockford, IL). Cell
lysates were mixed with loading buffer, boiled for 5 min, and equal
amounts of protein were run in polyacrylamide gels, transferred to
nitrocellulose membranes blocked with 50 mM Tris?150 mM
NaCl?0.1% Tween-20 (TBS-T)?5% nonfat milk for 1 h, and
incubated with the primary antibodies at the dilutions indicated
membranes were incubated for 1 h with donkey anti-mouse sec-
ondary antibodies conjugated with horseradish peroxidase (Santa
washed three times in TBS-T, and visualized by using the ECL
Enhanced Method (Amersham, San Francisco, CA).
Immunostaining. Cells grown on coverslips were fixed with 4%
paraformaldehyde in 0.12 M phosphate buffer, pH 7.2, for 30
min, and washed three times with PBS. Nonspecific antibody
binding was reduced by incubation in PBS with 1% BSA. The
cells were permeabilized in PBS with 0.5% Triton X-100 for 1 h
at room temperature, incubated overnight with primary anti-
bodies at an appropriate dilution in PBS with 1% BSA and 0.5%
Triton X-100, washed three times for 5 min, and incubated with
the secondary antibodies conjugated with fluorescein diluted
1:100 in PBS with 1% BSA and 0.5% Triton X-100. The slides
were washed in PBS, stained with Texas red-conjugated phal-
loidin (Molecular Probes, Eugene, OR), diluted 1:100 in PBS for
40 min, washed again in PBS, and mounted by using ProLong
Antifade Kit (Molecular Probes), according to manufacturer’s
instructions and observed in a Nikon TE2000U microscope
coupled with a confocal cell imaging CARV system.
Scanning Electron Microscopy. U373 cells grown on culture cover-
slips were fixed with 2.5% glutaraldehide in 0.1 M phosphate
buffer, pH 7.4, for 4 h at 4°C and washed with 0.1 M PBS, pH
7.2. Samples were dehydrated by using ethanol, treated with CO2
by using a Balzers CPD Critical Point Dryer 030, and covered
with gold by using a Balzers BAL-TEC SCD 050. Cells were
observed and photographed by using a scanning electron mi-
croscope Cambridge Stereoscan 240.
This work was supported by National Institutes of Health Grants
CA88440 and CA66029. P.P.-P. was supported by National Institutes of
Health Grant 2 T32 DK007022-26. This is manuscript no. 17827-MEM
from the Scripps Research Institute.
1. Thiery JP (2002) Nat Rev Cancer 2:442–454.
2. Meng K, Rodriguez-Pena A, Dimitrov T, Chen W, Yamin M, Noda M, Deuel
TF (2000) Proc Natl Acad Sci USA 97:2603–2608.
3. Pariser H, Herradon G, Ezquerra L, Perez-Pinera P, Deuel TF (2005)Proc Natl
Acad Sci USA 102:12407–12412.
4. Pariser H, Perez-Pinera P, Ezquerra L, Herradon G, Deuel TF (2005) Biochem
Biophys Res Commun 335:232–239.
5. Pariser H, Ezquerra L, Herradon G, Perez-Pinera P, Deuel TF (2005) Biochem
Biophys Res Commun 332:664–669.
6. Kawachi H, Fujikawa A, Maeda N, Noda M (2001) Proc Natl Acad Sci USA
7. Tamura H, Fukada M, Fujikawa A, Noda M (2006) Neurosci Lett 399:33–38.
8. Li YS, Milner PG, Chauhan AK, Watson MA, Hoffman RM, Kodner CM,
Milbrandt J, Deuel TF (1990) Science 250:1690–1694.
9. Milner PG, Li YS, Hoffman RM, Kodner CM, Siegel NR, Deuel TF (1989)
Biochem Biophys Res Commun 165:1096–1103.
10. Deuel TF, Zhang N, Yeh HJ, Silos-Santiago I, Wang ZY (2002) Arch Biochem
11. Gottardi CJ, Wong E, Gumbiner BM (2001) J Cell Biol 153:1049–1060.
12. Gooding JM, Yap KL, Ikura M (2004) BioEssays 26:497–511.
13. Dewang PM, Hsu NM, Peng SZ, Li WR (2005) Curr Med Chem 12:1–22.
14. Stoica GE, Kuo A, Aigner A, Sunitha I, Souttou B, Malerczyk C, Caughey DJ, Wen
D, Karavanov A, Riegel AT, Wellstein A (2001) J Biol Chem 276:16772–16779.
15. Bowden ET, Stoica GE, Wellstein A (2002) J Biol Chem 277:35862–35868.
16. Powers C, Aigner A, Stoica GE, McDonnell K, Wellstein A (2002) J Biol Chem
17. Lu KV, Jong KA, Kim GY, Singh J, Dia EQ, Yoshimoto K, Wang MY,
Cloughesy TF, Nelson SF, Mischel PS (2005) J Biol Chem 280:26953–26964.
18. Drees F, Pokutta S, Yamada S, Nelson WJ, Weis WI (2005) Cell 123:903–915.
19. Kowalczyk AP, Reynolds AB (2004) Curr Opin Cell Biol 16:522–527.
20. Briggs MW, Sacks DB (2003) EMBO Rep 4:571–574.
21. Chen L, Madura K (2002) Mol Cell Biol 22:4902–4913.
22. Le TL, Yap AS, Stow JL (1999) J Cell Biol 146:219–232.
23. Palacios F, Schweitzer JK, Boshans RL, D’Souza-Schorey C (2002) Nat Cell
24. Xiao K, Allison DF, Buckley KM, Kottke MD, Vincent PA, Faundez V,
Kowalczyk AP (2003) J Cell Biol 163:535–545.
25. Thiery JP (2003) Curr Opin Cell Biol 15:740–746.
26. Bilwes AM, den Hertog J, Hunter T, Noel JP (1996) Nature 382:555–559.
27. Piedra J, Martinez D, Castano J, Miravet S, Dunach M, de Herreros AG (2001)
J Biol Chem 276:20436–20443.
28. Fujita Y, Krause G, Scheffner M, Zechner D, Leddy HE, Behrens J, Sommer
T, Birchmeier W (2002) Nat Cell Biol 4:222–231.
KR, Hummingbird DK, Reynolds AB (2000) J Cell Biol 148:189–202.
www.pnas.org?cgi?doi?10.1073?pnas.0607299103 Perez-Pinera et al.