(Received for publication, January 3, 1996)
From the
Contact-induced growth inhibition is a characteristic feature of
normal cells grown in monolayer. The importance of reversible tyrosine
phosphorylation in mitogenic signaling, together with earlier reports
of increased levels of protein-tyrosine phosphatases (PTPs) in densely
cultured cells, has led to the proposal that PTPs may be involved in
mediating contact inhibition of cell growth. We have compared net
levels of ligand-induced tyrosine phosphorylation of the epidermal
growth factor (EGF) receptor in mink lung epithelial cells cultured
under sparse or dense conditions. The levels of net tyrosine
phosphorylation of the stimulated EGF receptor was found to be more
than 4-fold higher in sparse cultures. This difference was greatly
reduced when cells were pretreated with the PTP inhibitor phenyl arsine
oxide. Monitoring of dephosphorylation rates in vivo of the
stimulated EGF receptors revealed increased EGF receptor-directed PTP
activity in dense cultures. The platelet-derived growth factor
-receptor, expressed in stably transfected porcine aortic
endothelial cells, also displayed lower levels of ligand induced net
tyrosine phosphorylation in cells from dense cultures. This
density-dependent difference in tyrosine phosphorylation was reduced by
pretreatment of cultures with the PTP inhibitor orthovanadate. A
PTP-mediated decrease of the in vivo net levels of ligand
induced tyrosine phosphorylation of EGF and platelet-derived growth
factor receptors in cells at high density have thus been demonstrated.
Loss of this previously unnoticed regulatory pathway may be involved in
cellular transformation.
Contact-induced growth inhibition is a characteristic feature of
normal cells grown in monolayer. This negative growth regulatory
mechanism is lost in many tumor cells. The molecular events underlying
contact inhibition remain largely unknown. The importance of reversible
tyrosine phosphorylation in growth factor-induced mitogenic signaling,
together with the potential of protein-tyrosine phosphatases (PTPs) ()to antagonize tyrosine kinase signaling, has led to the
proposal that PTPs may be involved in contact inhibition (for a recent
review see (1) ). The finding that treatment of normal rat
kidney cells with the PTP inhibitor orthovanadate relieved cells from
contact inhibition of cell growth provided early experimental support
for this hypothesis(2) . During recent years additional data
have been obtained that support this idea.
Increased PTP activity in
membrane fractions or cell lysates from cells harvested at high cell
densities has been reported(3, 4, 5) . Cell
density-dependent up-regulation of the receptor-like PTPs
density-enhanced phosphatase-1 and PTP-µ at the protein level has
been demonstrated(6, 7) . Comparison of mRNA levels of
PTPs in growing and contact-inhibited cells also revealed higher mRNA
levels for PTP- in dense cells(8) . The demonstration of
specific homophilic interactions of the extracellular domains of the
transmembrane PTP-µ and -
also points toward a role in cell
contact-induced signaling(9, 10, 11) .
Finally, the recent demonstration of physical association between
PTP-µ and cadherins is compatible with a regulatory role for
PTP-µ in signaling triggered by cell contact (12) .
The ability of PTPs to reduce receptor tyrosine phosphorylation in vivo has been directly demonstrated in co-transfection experiments of transiently expressed tyrosine kinase receptors and PTPs (13) . In addition, signaling through the PDGF receptor was shown to be attenuated in cells stably expressing transfected CD45(14, 15) . Also, antisense mediated blocking of endogenous leukocyte antigen-related phosphatase increased ligand-induced tyrosine phosphorylation of the insulin-receptor(16) . Together, these results demonstrate that the net level of tyrosine phosphorylation of tyrosine kinase receptors in vivo will depend on the combined action of tyrosine kinases themselves and counteracting PTPs.
In this study we have compared
net levels of growth factor-induced receptor tyrosine phosphorylation
in cells cultured under sparse or dense conditions. A density-dependent
reduction in ligand-induced tyrosine phosphorylation of EGF receptors
in mink lung epithelial cells, as well as of transfected PDGF
-receptors in endothelial cells, is demonstrated. In both cases
the density-dependent decrease in growth factor receptor tyrosine
phosphorylation was reduced if cells were pretreated with PTP
inhibitors. Also, monitoring of the in vivo dephosphorylation
of the EGF receptor revealed an increased rate of dephosphorylation in
cells from dense cultures.
Figure 1:
Characterization of
EGF-induced DNA synthesis and EGF receptor expression in sparse and
dense Mv1Lu cultures. A, microphotographs of sparse (1.3
10
cells/cm
) and dense (1.3
10
cells/cm
) cultures. B,
[
H]thymidine incorporation of serum-starved
sparse and dense cultures after stimulation with different
concentrations of EGF. C, Scatchard analysis of EGF binding to
sparse and dense cultures. D, EGF receptor (EGFR)
immunoblotting of WGA-Sepharose fractions from equal number of cells
from sparse and dense cultures.
For further
comparison of ligand-induced tyrosine phosphorylation of the EGF
receptor in sparse and dense cultures, it was of importance to compare
the number of EGF receptors on sparsely and densely cultured cells as
well as the receptor affinities. To that end EGF binding experiments
were performed with I-EGF, and results were subjected to
Scatchard analysis. Fig. 1C shows the results from one
such experiment. Both sparse and dense cells were found to express
approximately 10,000 receptors/cell, and K
values
for the receptors in sparse and dense cells were determined to be 0.8
nM and 0.9 nM, respectively. Thus, neither with
regard to receptor number per cell nor K
were any
major difference observed between sparse and dense cultures. That
sparsely and densely cultured cells express equal amounts of EGF
receptors was also confirmed by immunoblot analysis. As shown in Fig. 1D, no difference in the amounts of EGF receptor
was found between cells harvested at sparse or dense culture
conditions.
Figure 2: Comparison of EGF receptor tyrosine phosphorylation in sparse and dense cultures. Phosphotyrosine immunoblotting of 20 and 80% of WGA-Sepharose fractions from equal numbers of cells from unstimulated or stimulated sparse and dense cultures. EGF-R, EGF receptor.
Figure 3: Effect of PAO treatment on EGF receptor phosphorylation in sparse and dense cultures. Phosphotyrosine immunoblotting of WGA-Sepharose fractions from equal number of cells from unstimulated or stimulated sparse and dense cultures, preincubated with or without PAO. EGF-R, EGF receptor.
This method was used to compare the rate of EGF receptor dephosphorylation in sparse and dense cultures (Fig. 4). After stimulation with EGF, cells were transferred to ice and exposed to either vehicle alone or the EGF receptor-specific tyrphostin AG1517. After indicated time points cells were lysed, and EGF receptor tyrosine phosphorylation was determined as above. In sparse cultures (Fig. 4, left panel) tyrosine phosphorylated receptors were easily demonstrated after 10 min of incubation with or without the kinase inhibitor, indicating very low receptor directed PTP activity in these cells. In contrast, in samples from dense cultures (Fig. 4, right panel), a decrease in the net levels of tyrosine phosphorylation of the EGF receptor is seen over time in dense cultures treated with vehicle only, indicating a net dephosphorylation under these conditions. Furthermore, this rate is dramatically increased in cells exposed to AG1517. Already after 5 min of incubation with kinase inhibitor, the levels of phosphotyrosine had decreased below the sensitivity of this assay. Thus, these experiments directly demonstrate increased EGF receptor-directed PTP activity in vivo in dense as compared with sparse cultures.
Figure 4: Comparison of in vivo EGF receptor dephosphorylation in sparse and dense cultures. Sparse and dense cultures were stimulated with EGF and then exposed to vehicle alone or the EGF receptor kinase blocker AG1517 for the indicated length of time. WGA-Sepharose fractions from equal number of cells were then analyzed by phosphotyrosine immunoblotting. EGF-R, EGF receptor.
Figure 5:
Comparison of PDGF -receptor tyrosine
phosphorylation in sparse and dense PDGF
R-PAE cultures. A, [
H]thymidine incorporation in
serum-starved sparse and dense cultures after stimulation with
different concentrations of PDGF-BB. B, phosphotyrosine
immunoblotting of WGA-Sepharose fractions from an equal number of cells
from unstimulated and stimulated sparse and dense PDGF-
R-PAE
cultures, preincubated with or without orthovanadate
(Vd).
Both endogenous EGF receptors in Mv1Lu cells and transfected
PDGF -receptors in porcine aortic endothelial cells were found to
show decreased ligand-induced tyrosine phosphorylation in cells of
dense cultures. The density-dependent difference in receptor tyrosine
phosphorylation was reduced by pretreatment of cells with PTP
inhibitors. We therefore conclude that the difference in net tyrosine
phosphorylation of PDGF
- and EGF receptors between sparse and
dense cultures is caused by increased receptor-directed PTP activity in
dense cultures. This was also directly demonstrated for the EGF
receptor.
Earlier studies have demonstrated increased total PTP activity in lysates or membrane fractions derived from high density cultures, as well as increased levels of different transmembrane PTPs(3, 4, 5, 6, 7, 8) . However, no density-dependent differences in the net levels of tyrosine phosphorylation in vivo of key signaling molecules have been demonstrated. Our experiments show that one group of in vivo targets for the increased PTP activity in high cell density cultures are growth factor receptor tyrosine kinases. It is therefore possible that contact-induced growth inhibition is at least partially caused by PTP-mediated attenuation of growth factor signaling already at the level of receptor tyrosine phosphorylation. Loss of this previously unnoticed regulatory mechanism may be involved in cellular transformation.
Both EGF and PDGF -receptors are known to be
phosphorylated on a number of tyrosine
residues(22, 23) . Various tyrosine phosphorylated
sites will activate different signaling pathways by recruiting
different SH-2 domain containing proteins (reviewed in (24) ).
Preliminary analysis of the substrate specificity of PTPs have revealed
some sequence specificity. For example, when a dodecapeptide derived
from the insulin receptor containing three phosphorylated tyrosine
residues was used as substrate, both CD45 as well as leukocyte
antigen-related phosphatase preferentially dephosphorylated the
tyrosine corresponding to amino acid 1146 of the insulin
receptor(25, 26) . It is therefore possible that the
observed density-dependent difference in receptor phosphorylation
reflects specific phosphorylation of some sites, rather than complete
dephosphorylation of a subset of receptors. This is a question that
should be addressed in future studies.
Identification of the PTPs
responsible for the increased dephosphorylation of EGF- and PDGF
-receptors in dense cultures of Mv1Lu cells and PDGF-
R-PAE
cells is another important subject for future studies. When identified,
they should be interesting candidates for comparative analysis
regarding expression in normal cells and cancer cells. Finally,
targeting of these PTPs with antisense or knock-out techniques should
make it possible to formally test if those are true mediators of
contact inhibition.