Correspondence to: Rik Derynck, Department of Growth and Development, University of California at San Francisco, San Francisco, CA 94143-0640. Tel:(415) 476-7322 Fax:(415) 476-1499 E-mail:derynck{at}itsa.ucsf.edu.
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Abstract |
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Transforming growth factor- (TGF-
) is a member of the EGF growth factor family. Both transmembrane TGF-
and the proteolytically released soluble TGF-
can bind to the EGF/TGF-
tyrosine kinase receptor (EGFR) and activate the EGFR-induced signaling pathways. We now demonstrate that transmembrane TGF-
physically interacts with CD9, a protein with four membrane spanning domains that is frequently coexpressed with TGF-
in carcinomas. This interaction was mediated through the extracellular domain of transmembrane TGF-
. CD9 expression strongly decreased the growth factor and PMA- induced proteolytic conversions of transmembrane to soluble TGF-
and strongly enhanced the TGF-
induced EGFR activation, presumably in conjunction with increased expression of transmembrane TGF-
. In juxtacrine assays, the CD9-induced EGFR hyperactivation by transmembrane TGF-
resulted in increased proliferation. In contrast, CD9 coexpression with transmembrane TGF-
decreased the autocrine growth stimulatory effect of TGF-
in epithelial cells. This decrease was associated with increased expression of the cdk inhibitor, p21CIP1. These data reveal that the association of CD9 with transmembrane TGF-
regulates ligand-induced activation of the EGFR, and results in altered cell proliferation.
Key Words:
transforming growth factor-, CD9, epidermal growth factor receptor, ectodomain, shedding
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Introduction |
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Transforming growth factor- (TGF-
)1 is a member of a family of structurally related growth factors that includes EGF, heparin-binding EGF-like growth factor (HB-EGF), and amphiregulin (
is synthesized as a transmembrane protein that can undergo a regulated cleavage of the ectodomain to release a soluble form of TGF-
(
/EGF family (
/EGF family also activate the EGFR or structurally closely related tyrosine kinase receptors (
TGF- is expressed by a variety of cell types from neuroectodermal origin, most commonly by epithelial cells including keratinocytes. In these cells, the transmembrane TGF-
formed at the cell surface seems to be the predominant form of TGF-
. In vivo, transmembrane TGF-
is likely to interact in a juxtacrine or paracrine manner with the EGFR on neighboring cells, or in an autocrine manner on the same cells. TGF-
is also expressed in a large diversity of tumors, not only in tumors of neuroectodermal origin, but also in mesenchymally derived tumors (
. Although no controlled comparisons have been done, the expression of TGF-
is generally upregulated when an epithelial cell progresses into a carcinoma cell. In addition, the proteolytic cleavage of transmembrane TGF-
to release soluble TGF-
seems to be also induced or enhanced concomitantly with this transition. Since carcinomas also express EGFR, often at increased levels when compared with normal cells (
and EGFR activation are likely to regulate tumor cell proliferation and tumor development, a notion that is strongly supported by cell culture (
In contrast to the well characterized ability of the activated EGFR to activate several signaling pathways, little is known about how the ligands are presented to the EGFR. Although transmembrane TGF- potently activates the EGFR, its presentation at the cell surface and its interaction with the EGFR may be regulated by accessory proteins. Several putative transmembrane proteins have genetically been implicated in the activation of the EGFR by TGF-
related proteins in Drosophila (
related factors in vertebrates. Transmembrane TGF-
has been shown to interact with another transmembrane protein, p106, but its identity has not been reported (
has been reported.
CD9 is a 2427-kD cell surface protein with four predicted transmembrane domains that belong to the tetraspanin family. Originally identified as a surface antigen on lymphohematopoietic cells, CD9 is expressed by epidermal, basophil, pre-B cells, activated T cells, platelets, as well as neural cell lines ( and EGFR (
or a potential role for CD9 in carcinoma development. However, an inverse correlation has been found between CD9 expression in carcinomas and the prognosis for the behavior, invasiveness, and prognosis of the cancer (
In this report, we demonstrate that transmembrane TGF- and CD9 interact with each other, and that this interaction is mediated through the extracellular sequence of TGF-
. Increased CD9 expression decreases the growth factorinduced release of the TGF-
ectodomain and enhances the transmembrane TGF-
induced EGFR stimulation. The increased EGFR activity in epithelial cells results in a high level of expression of the cdk inhibitor p21CIP1, thus, decreasing the TGF-
induced cell proliferation.
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Materials and Methods |
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Plasmids
The mammalian expression plasmids pRK5 and pRK7 were previously described (
The expression plasmids for full size TGF- or the cytoplasmically truncated version (pRK7-
c) were previously described (
coding sequence was modified to encode the derivatives TGF-
-myc or TGF-
E. TGF-
-myc corresponds to the full size transmembrane TGF-
with the GGEQKLISEEDLGG sequence, i.e., the myc epitope tag flanked by diglycines, inserted between amino acids 93 and 94. This location is immediately downstream the cleavage site for mature TGF-
and before the transmembrane segment. TGF-
E corresponds to the transmembrane TGF-
-myc from which the 50amino acid mature TGF-
was removed. The TGF-
-myc and TGF-
E coding sequences were inserted into the EcoRI-HindIII sites of the pRK5 expression plasmid. pRK5-EGFR was generated by excising the XbaI-HindIII fragment from pXER (a gift from Dr. G. Gill, University of California, San Diego, CA) containing 3.5 kbp of EGFR cDNA, and inserting it into the corresponding sites of pRK5.
Cell Culture and Transfections
CHO cells were cultured in Ham's F12 medium supplemented with 10% FBS and 10 µg/ml penicillin/streptomycin. 293 cells were cultured in DME medium, supplemented with 4.5 g glucose per liter, 10% FBS, and 10 µg/ml penicillin/streptomycin. MDCK cells were grown in DME medium, supplemented with 4.5 g glucose per liter, 10% FBS, and 10 µg/ml penicillin/streptomycin. WEHI-3B cells were cultured in RPMI 1640 medium supplemented with 15% FBS, 10 µg penicillin/streptomycin. 32D cells and 32D-EGFR (EP170.7) cells (
Stable transfected MDCK cells expressing TGF- were obtained from Dr. R. Coffey (Vanderbilt University Medical School, Nashville, TN) (
Metabolic Labeling, Coimmunoprecipitation, and Immunoblot Analysis
Metabolic labeling and coimmunoprecipitations were carried out essentially as described ( mAb
1 (5 µg/ml) (
, cells were first incubated with the
1 mAb (
For Western blotting, the immunoprecipitated protein membranes separated by SDS-PAGE, were electrotransferred onto nitrocellulose using a Mini Transblot Apparatus (Bio-Rad Laboratories). The immunoblots were pretreated with 5% BSA (for antiphosphotyrosine Western blots) or 5% dry milk (for the use of other antibodies) in TBST (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween) for 1 h at room temperature. After three washes with TBST, antibody was added at a concentration of 110 µg/ml in TBST, and the incubation proceeded overnight at 4°C. The blot was washed again three times and incubated with a 1:2,500-fold diluted HRP-conjugated goat antimouse IgG (Amersham Inc.) for 1 h at room temperature. The blots were washed again and developed using ECL substrates (Pierce Chemical Co.). AntiTGF- antibody was purchased from Calbiochem-Novabiochem, antiCD9 and antip21 antibodies from PharMingen, and antiphosphotyrosine antibody from Transduction Laboratories.
Measurement of Transmembrane TGF- Cleavage
The release of soluble TGF- from transmembrane TGF-
was measured in transiently transfected CHO cells, as described previously (
with or without 1 µg pRK7-CD9-hygro or the control plasmid pRK7-hygro. Cells were switched into F12 medium, supplemented with 10% FBS, cultured for another 5 h, and then serum-starved overnight. To analyze the levels of cell-associated TGF-
, cells were pulse-labeled with [35S]methionine/cysteine in F12 medium for 20 min, rinsed with medium, and incubated for 1 h at 37°C in F12/DME (1:1) medium, 0.1% BSA with or without 10% heat-inactivated FBS, or 10 nM PMA. Cells were again rinsed with cold medium and lysed in TBS containing 1% NP-40. Transmembrane TGF-
was immunoprecipitated from the cell extracts using the mAb
1 (
were quantitated by ImageQuant software (Molecular Dynamics). To measure the levels of soluble TGF-
in the medium, [35S]methionine/cysteine labeling was performed for 2 h. Soluble TGF-
, which was released into the medium, was analyzed by immunoprecipitation followed by gel analysis or liquid scintillation counting.
Immunofluorescence
Cells were grown in 4-well chamber slides (Falcon Plastics) and fixed with 3.7% formaldehyde in PBS for ~10 min. They were rinsed twice with PBS and permeabilized with 0.1% Triton-X 100, 1% BSA in PBS for 7 min, washed twice with PBS with a 5-min interval, and treated with 3% BSA in PBS for 30 min. After washing with PBS, the cells were incubated with the 1 mAb (4 µg/ml) in 2% horse serum in PBS for 1 h at room temperature. The unbound antibody was removed by repeated washes in PBS. The cells were incubated with rhodamine (TRITC)-conjugated affinity-purified donkey antimouse IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc.) (4 µg/ml) for 3060 min. After washing the cells thoroughly with PBS, the slides were mounted with Gel-mount (Sigma Chemical Co.). All procedures were performed at room temperature. The fluorescence staining was examined with a Nikon fluorescence microscope.
Measurements of DNA Synthesis
Cells were plated at 2 x 104 cells/well in 12-well plates in normal culture medium and incubated overnight. 300 µl of serum-free medium containing 4 µCi/ml of [3H]thymidine was added and incubation proceeded for 4 h at 37°C. After removal of the medium, the cells were washed twice with ice-cold 5% TCA and three times with ice-cold water. The cells were lysed with 250 µl 0.5 M NaOH, and the incorporated [3H]thymidine in the cell lysates was determined using liquid scintillation counting.
Determination of Cell Numbers
MDCK cells were plated at 40% confluency in multiple 60-mm diam plates. At daily intervals after plating, one plate of cells was trypsinized and the cells were resuspended in 5 ml medium. The cells in a 10-µl sample were counted, and the total cell number was calculated as a function of time to determine the growth rate.
Measurement of Juxtacrine Growth Stimulation
CHO cells, which either express transmembrane TGF- or not, were tested for their ability to stimulate proliferation of 32D-EGFR cells through cellcell contact. The method was modified from S.
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Results |
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Physical Interaction of CD9 with Transmembrane TGF-
It has been reported that CD9 interacts with pro-HB-EGF, but not transmembrane TGF-. Since transmembrane TGF-
and pro-HB-EGF both have a structurally related extracellular domain sequence with six conserved cysteines, we evaluated whether CD9 physically associates with transmembrane TGF-
. As shown in Fig 1a and Fig b, CD9 coimmunoprecipitated with transmembrane TGF-
. No interaction was seen with another transmembrane protein, such as the type II TGF-ß receptor (data not shown). In addition, CD9 did not interact with the EGFR, the receptor for TGF-
(Fig 1 C).
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To characterize the structural basis of this interaction, we evaluated the ability of CD9 to associate with several derivatives of transmembrane TGF-. One of these, TGF-
C, lacked the cytoplasmic domain but still associated with CD9, indicating that the cytoplasmic domain of transmembrane TGF-
is not required for this interaction (Fig 2 A). To assess whether the extracellular domain of transmembrane TGF-
plays a role in the association with CD9, we also examined the interaction with a derivative of the transmembrane TGF-
, TGF-
E. In TGF-
E, the 50amino acid core sequence of fully processed TGF-
was replaced with a Myc epitope tag. CD9 was unable to associate with this derivative of transmembrane TGF-
(Fig 2 A), suggesting that the 50amino acid core TGF-
sequence is required for this interaction. In control experiments, CD9 still coprecipitated with a derivative of transmembrane TGF-
in which the 50amino acid TGF-
core was followed by the Myc epitope sequence (data not shown), strongly suggesting that the Myc antibody does not interfere with the association with CD9. Finally, expression of HB-EGF decreased the level of CD9 that associated with transmembrane TGF-
(Fig 2 B), strongly suggesting that transmembrane HB-EGF and TGF-
compete with each other for interaction with CD9. This observation further supports the notion that the 50amino acid core TGF-
sequence is essential for association with CD9, since the sequence similarity of transmembrane HB-EGF and TGF-
is only apparent in this core segment.
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CD9 Coexpression Inhibits Growth Factor and PMA-induced Transmembrane TGF- Cleavage
The interaction of CD9 with the extracellular domain of transmembrane TGF- raises the possibility that CD9 association regulates the cleavage of the ectodomain of transmembrane TGF-
. This cleavage occurs immediately downstream from the 50amino acid core sequence through the action of a cell surfaceassociated metalloprotease (
expressing cells undergo a basal level of soluble TGF-
release, which is controlled by the p38 MAP kinase signaling cascade, whereas growth factor receptorstimulated TGF-
ectodomain release (e.g., in response to EGFR activation) is mediated through the Erk/MAP kinase pathway (
. As shown in Fig 3, the serum- and PMA-induced release of soluble TGF-
, as assessed by the levels of both the soluble TGF-
in the medium and the cell surfaceassociated transmembrane TGF-
, were strongly inhibited by CD9. These results suggest that the interaction of CD9 with transmembrane TGF-
strongly decreases its susceptibility to ectodomain cleavage.
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Increased EGFR Activation in Cells Coexpressing Transmembrane TGF- and CD9
To assess the physiological effects of CD9 on the activity of transmembrane TGF-, we expressed CD9, TGF-
, or both in CHO cells, which lack endogenous EGFR, and in MDCK cells, which express EGFR endogenously. Stably transfected CHO cells were tested for their ability to stimulate EGF receptors on transfected 32D cells, a hematopoietic cell line that lacks endogenous EGFR and the related HER-2, -3, and - 4 receptors, but was transfected to express EGFR (
and 32D-EGFR cells allowed transmembrane TGF-
to stimulate the tyrosine autophosphorylation of the EGFR, a measure of juxtacrine receptor activation induced by TGF-
. In contrast, cells expressing only CD9 did not stimulate EGFR autophosphorylation. Coexpression of CD9 with transmembrane TGF-
, however, resulted in a higher level of EGFR receptor activation than TGF-
alone (Fig 4 A). This was observed using both live CHO cells (Fig 4 A) or fixed CHO cells, which do not have the ability to release soluble TGF-
(data not shown), as donor cells.
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We also generated stably transfected MDCK cells, which express TGF-, CD9, or both. Like most epithelial cells, MDCK cells make low levels of endogenous TGF-
and EGFR, and the autocrine stimulation of the EGFR by TGF-
is thought to be important for epithelial cell proliferation (
and CD9 were considerably lower than in the transfected CHO cells (data not shown). Using these stable cell lines, we evaluated the level of EGFR tyrosine phosphorylation as a measure of autocrine, TGF-
induced receptor activation. The MDCK cells that coexpressed TGF-
and CD9 showed a higher level of EGFR activation than the cells expressing only TGF-
(Fig 4 B). This result is consistent with the higher level of juxtacrine EGFR stimulation of 32D cells by TGF-
/CD9 coexpressing CHO cells, when compared with TGF-
expressing cells (Fig 4 A). The low level of EGFR phosphorylation in the control-transfected MDCK cells or the MDCK cells expressing CD9 alone is most likely due to stimulation by endogenous TGF-
.
We next evaluated whether the increased EGFR stimulation was due to an increased ability of transmembrane TGF- to activate the receptor and/or an increased number of transmembrane TGF-
proteins at the cell surface. As shown in Fig 5 A, coexpression of CD9 resulted in increased expression of transmembrane TGF-
both at the cell surface of stably transfected CHO cells (Fig 5 A) and in total cell lysates of the MDCK cells (data not shown). This increased expression of transmembrane TGF-
, when CD9 was coexpressed, was consistently seen in multiple stably transfected CHO (data not shown) and MDCK cell clones (Fig 5 B). Immunofluorescence staining of permeabilized MDCK cells also demonstrated increased transmembrane TGF-
not only at the cell surface, but also in the perinuclear region (Fig 5 C). The increase in juxtacrine and autocrine EGFR activation in 32D and MDCK cells, respectively, is therefore not necessarily a direct consequence of the interaction of CD9 with transmembrane TGF-
per se, but may result from the increased transmembrane TGF-
levels at the surface of cells that coexpress CD9.
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Coexpression of CD9 Enhances the Juxtacrine, but Decreases the Autocrine Mitogenic Activity of Transmembrane TGF-
We next assessed the effect of CD9 expression on the ability of transmembrane TGF- to stimulate the EGFR-induced DNA synthesis and cell proliferation. Coculture of cells expressing transmembrane TGF-
with EGFR-expressing 32D cells allowed us to measure the juxtacrine activity of transmembrane TGF-
. CHO cells, which lack endogenous TGF-
and EGFR did not induce [3H]dT incorporation in the 32D-EGFR cells, nor did TGF-
expressing CHO cells stimulate the DNA synthesis in parental 32D cells, which lack EGFR expression. In addition, expression of CD9 alone did not affect the DNA synthesis in EGFR-expressing 32D cells either. In contrast, TGF-
expressing CHO cells stimulated DNA synthesis in EGFR-expressing 32D cells, and this stimulation was substantially increased when CD9 was coexpressed with transmembrane TGF-
. This was demonstrated using both live (Fig 6 A) or fixed donor CHO cells (Fig 6 B). The basis for the higher level of DNA synthesis in control- or CD9-stimulated 32D-EGFR cells, when compared with the parental 32D cells (Fig 6 A), is unclear but may be related to a background activity of the EGFR in the absence of exogenous stimulation.
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Consistent with the ability of transmembrane TGF- to activate the EGFR in 32D cells, the TGF-
expressing MDCK cells had a higher rate of DNA synthesis and cell proliferation than control-transfected MDCK cells (Fig 7A and Fig B). This observation is also consistent with the growth stimulatory activity of soluble TGF-
on these cells (data not shown) and many other cell lines. The rate of DNA synthesis and cell proliferation of CD9-expressing cells was comparable to the control cells. In contrast to the TGF-
expressing MDCK cells, coexpression of CD9 and transmembrane TGF-
resulted in a lower rate of DNA synthesis (Fig 7 A) and cell proliferation (Fig 7 B). This effect of CD9 was remarkable since the expression level of transmembrane TGF-
and the level of EGFR activation were considerably higher in these cells than in the TGF-
expressing MDCK cells. Such a growth inhibitory activity could not be mimicked by addition of high levels of soluble TGF-
to these cells (data not shown). This result is also in contrast with the ability of coexpressed CD9 to enhance the juxtacrine, TGF-
induced stimulation of DNA synthesis in 32D cells (Fig 6 A). Furthermore, the expression level of transmembrane TGF-
in the CHO cells that were used in the juxtacrine stimulation assays, was considerably higher than in MDCK cells used in the autocrine stimulation (data not shown).
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These results indicate that CD9 coexpression with transmembrane TGF- results in increased transmembrane TGF-
levels at the cell surface and increased activation of the EGFR both in a juxtacrine (32D cells) and autocrine (MDCK cells) manner. Cells expressing both CD9 and TGF-
induced an enhanced juxtacrine stimulation of DNA synthesis and proliferation of 32D-EGFR cells. However, the strong increase in EGFR activation in MDCK cells, expressing both CD9 and TGF-
, resulted in growth inhibition, when compared with MDCK cells expressing only transmembrane TGF-
.
The CD9-induced Growth Inhibition in Transmembrane TGF-Expressing Cells Correlates with Increased p21CIP1 Expression
Although moderate activation of Raf, an effector of Ras signaling and stimulator of MAP kinase activation, results in stimulation of cell proliferation, a high level activation of Raf (.
As shown in Fig 7 C, the level of p21CIP1 expression was considerably higher in MDCK cells, which coexpress transmembrane TGF- and CD9, when compared with parental MDCK cells or the TGF-
expressing MDCK cells. Cells expressing TGF-
alone also had enhanced p21CIP1 expression, albeit to a considerably lower level than in cells coexpressing CD9 and TGF-
. Thus, the level of p21CIP1 expression correlated with the level of EGFR activation (Fig 4 B). Furthermore, the high level of p21CIP1 and its role as an inducer of growth arrest also correlated with the lower level of proliferation of the TGF-
/CD9 coexpressing cells, when compared with cells expressing TGF-
alone. The levels of p21CIP1 in these MDCK cell lines correlated with the transcription levels from the p21CIP1 promoter, as assessed using a p21CIP1 promoter-luciferase reporter, transfected into the MDCK stable cells (data not shown). These results show that the high level of EGFR activation, induced by coexpression of transmembrane TGF-
and CD9, results in a high level of p21CIP1 expression in MDCK cells. This increase in p21CIP1 expression, a CDK inhibitor that can induce growth arrest when expressed beyond a certain threshold, may explain why cells that coexpress TGF-
and CD9 proliferate at a lower level than cells expressing TGF-
alone, even though they have a higher level of EGFR activation.
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Discussion |
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TGF- and related growth factors are expressed as transmembrane proteins and bind to and activate the EGFR, both as transmembrane proteins and as proteolytically released, soluble ligands (
activates the EGFR (
.
CD9 can associate with the transmembrane forms of HB-EGF and amphiregulin, and enhances their juxtacrine proliferative effect (, and no effect of CD9 on TGF-
stimulated DNA synthesis was seen in EGFR-expressing cells (
, which lacks such a domain (
, and that this interaction requires the extracellular 50amino acid core sequence of TGF-
. Accordingly, CD9 does not interact with the
E mutant that lacks the TGF-
core sequence. In addition, transmembrane HB-EGF and TGF-
, which have sequence similarity only in their EGF/TGF-
like core sequence, compete with each other for association with CD9. The interaction of CD9 with TGF-
is most likely mediated through the second and largest extracellular loop of CD9, which has been implicated in the association with HB-EGF.
CD9 and several other tetraspanins can associate with several types of integrins (3ß1 integrin dimer have been observed (
and, through the formation of oligomeric complexes with integrins, direct its localization to sites of cell contact or cell adhesion. In this way, CD9 could facilitate and increase the efficiency of the TGF-
induced stimulation of the EGFR, which has also been localized at these sites. Tetraspanins, such as the CD9-related CD151, CD63, and CD81, can also regulate other types of intracellular signaling. CD63, CD81, and CD151 recruit phosphatidylinositol 4-kinase toward the integrintetraspanin complex (
with CD9 may mediate other signaling events, which await further characterization.
The interaction of CD9 with transmembrane TGF- strongly decreased the serum- or PMA-induced ectodomain cleavage of TGF-
and consequent release of soluble TGF-
. This observation may have considerable significance. EGFR stimulation by e.g., TGF-
itself normally induces the release of soluble TGF-
, thus, decreasing the level of transmembrane TGF-
(
induces internalization of the ligandreceptor complex and consequent attenuation of the response, binding of transmembrane TGF-
to EGFR is unlikely to do so, and is expected to result in a high level, sustained activation of EGFR. This would result in a much stronger EGFR response, as in internalization-defective EGFR mutants (
ectodomain cleavage and consequent attenuation of EGFR signaling, and allow transmembrane TGF-
to sustain signaling through the EGFR.
The much stronger stimulation of the EGFR induced by coexpressing CD9 and TGF-, when compared with TGF-
expression alone, was evident from its increased tyrosine phosphorylation. This was observed both in an autocrine system, whereby TGF-
and CD9 were coexpressed in the same MDCK cells as the EGFR, and in juxtacrine assays, whereby the CD9/TGF-
expressing cells were in contact with the EGFR on adjacent cells. The increased EGFR activation could be explained by inhibition of the TGF-
ectodomain cleavage and consequent sustained signaling by transmembrane TGF-
, combined with the higher levels of uncleaved TGF-
at the cell surface. In addition, CD9 coexpression may also increase the stability and half-life of transmembrane TGF-
, and increased EGFR stimulation may enhance the expression of transmembrane TGF-
, as described previously (
levels in the presence of CD9 fully accounts for the increased EGFR activation is unclear, and additional effects of CD9 on the presentation of transmembrane TGF-
to the EGFR (e.g., by increasing its affinity or concentrating it at the cell surface in clusters) may be envisioned.
The effect of CD9 on the cell surface levels of the transmembrane growth factor may also explain why CD9 expression enhanced the juxtacrine mitogenic stimulation of transmembrane HB-EGF (
Consistent with the increased EGFR stimulation, CD9 enhanced the DNA synthesis in 32D cells, when compared with the effect of transmembrane TGF- alone. This higher juxtacrine mitogenic response resembled the effect of CD9 on the activity of HB-EGF in a similar assay (
in MDCK cells was decreased in the presence of CD9, even though the level of EGFR activation (and TGF-
expression) was much higher than in cells expressing TGF-
alone. EGFR-induced growth inhibition has previously only been shown in squamous carcinoma cells, such as A431 cells, with a strongly amplified level of EGFR expression. In A431 cells, very low EGF levels induce a moderate proliferative response, and the more commonly used EGF concentrations induce growth inhibition (
.
The autocrine growth inhibitory effect of CD9 in TGF-expressing MDCK cells is in contrast with its stimulatory effect in juxtacrine assays using 32D cells. This difference in response does not correlate with higher EGFR levels in MDCK versus 32D cells, as could have been invoked based on the findings in A431 cells. Instead, the endogenous EGFR levels in MDCK cells are physiological and lower than in EGFR-transfected 32D cells, and the TGF-
levels in the transfected MDCK cells are lower than in the transfected CHO cells, which were used in the juxtacrine assays (data not shown). The growth inhibitory effect of TGF-
/CD9 in an autocrine context versus the stimulatory effect in juxtacrine assays may reflect a qualitative difference between these two modes of presentation of TGF-
/CD9 to the EGFR. Alternatively or in addition, this difference may also be due to cell type differences between the 32D cells of hematopoietic origin and the epithelial MDCK cells.
The autocrine growth inhibitory effect of CD9 in TGF-expressing MDCK cells was associated with a high expression level of p21CIP1. p21CIP1 binds to cdk4 and cdk2 complexes, and increased p21CIP1 expression inhibits cyclin D and cyclin Edependent kinase activities and induces growth inhibition (
expressing MDCK cells is, therefore, likely to be a direct consequence of the induction of p21Cip1. Accordingly, the growth inhibitory effect of EGF on A431 cells with their extremely high EGFR levels is also paralleled by an induction of p21CIP1 (
. Induction of p21CIP1 as a result of EGFR stimulation may also explain the decreased proliferation of cells expressing transmembrane HB-EGF, when compared with cells expressing soluble HB-EGF (
Finally, the association of CD9 with transmembrane TGF- and its effect on EGFR signaling may play a role in the development of carcinoma tumors. When compared with normal epithelial cells, carcinomas, especially squamous carcinomas, frequently show increased TGF-
(
levels and autocrine EGFR activation are known to contribute to carcinoma formation and proliferation in mice (
and EGFR numbers may contribute to carcinoma development in humans. The frequent expression of CD9 in carcinomas (
signaling at normal EGFR levels (i.e., without the need for increased EGFR levels) and, in this way, regulate the behavior of carcinomas in vivo. A few studies have correlated the expression of CD9 with the clinical prognosis of carcinomas (
induced activation of EGFR signaling and, consequently, cell proliferation.
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Footnotes |
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The present address for L. Shum is Craniofacial Development Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892-2745.
1 Abbreviations used in this paper: EGFR, EGF/TGF- tyrosine kinase receptor; HB-EGF, heparin-binding EGF-like growth factor; MAP, mitogen-activated protein; TGF-
, transforming growth factor-
.
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Acknowledgements |
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We are grateful to Dr. E. Mekada and Dr. S. Higashiyama for providing the original CD9 and HB-EGF expression plasmids. We thank Dr. Xin-Hua Feng for helpful discussion and critical comments, K.W. Tzung for constructing the TGF--myc and TGF-
E plasmids, and T. Ko for help with the densitometric scanning of the autoradiograms.
This work is supported by the National Institutes of Health grant CA54826 to R. Derynck and postdoctoral fellowships from the American Lung Association to W. Shi, the Tobacco Related Disease Research Program to W. Shi and H. Fan, and the Leukemia Society of America to L. Shum.
Submitted: 12 July 1999
Revised: 15 December 1999
Accepted: 5 January 2000
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References |
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