1 Division of Nephrology, c-Ret, a
protein tyrosine kinase receptor, and its ligand glial-derived
neurotropic factor (GDNF) are critical for early regulation of ureteric
bud development and nephrogenesis. To address whether c-ret directly
initiates epithelial cell morphogenesis, the c-ret receptor was
expressed in murine inner medullary collecting duct cells (mIMCD-3, a
cell line of ureteric bud origin, which has no detectable endogenous
c-ret expression). Stable expression of wild-type c-ret was found to
yield a constitutively tyrosine-phosphorylated receptor, with no change
after the addition of GDNF. Examination of mRNA from these cells
demonstrated the message for endogenous GDNF, suggesting that c-ret was
potentially being constitutively activated by an autocrine mechanism.
When mIMCD-3 cells stably expressing the phosphorylated c-ret receptor
were cultured in a type I collagen matrix, they exhibited little
GDNF-independent or -dependent branching process formation at early
time points compared with the known morphogen hepatocyte growth factor
(HGF) (48 h; control, 0.33 ± 0.33; GDNF, 1.0 ± 0.58, P = nonsignificant; and HGF, 6.33 ± 0.33 processes/20 cell clusters,
P < 0.001), whereas extended culture (7 days) under serum-free conditions revealed a marked
increase in cell survival and the spontaneous development of
rudimentary branching process formation. Extended culture (7 days) of
c-ret-expressing clones in type I collagen with the epithelial morphogens HGF and/or epidermal growth factor (EGF) resulted in the
development of complex three-dimensional spiny cysts, whereas parental
mIMCD-3 cells died under these conditions. We conclude that activated
c-ret appears to mediate epithelial morphogenesis by prolonging cell
survival and, in conjunction with activation of the morphogenic
receptors c-met and the EGF receptor, initiates the events
required for very early branching morphogenesis.
glial-derived neurotropic factor; hepatocyte growth factor; epidermal growth factor; tubulogenesis
THE DEVELOPMENT of the mature kidney involves a
process of reciprocal induction between ureter and mesenchyme
(22). In the murine embryo, once the ureteric bud grows from the
Wolffian duct into the metanephric mesenchyme at embryonic
day
11.5,
the formation of the metanephric kidney is initiated. The collecting
ducts of the kidney arise from the ureteric bud during a complex
process requiring both branching and linear tubulogenesis. Mesenchymal cells near the ureteric bud are induced to undergo epithelial transformation, and the proximal nephron is generated as these cells
undergo linear tubulogenesis. It has recently been demonstrated that
the c-ret tyrosine kinase receptor is essential to the process of
ureteric bud development. Mice lacking either the c-ret tyrosine kinase
receptor or its ligand, glial cell line-derived neurotropic factor
(GDNF), exhibit renal agenesis resulting from failure of ureteric bud
formation (9, 30, 31). To date, the mechanism by which c-ret mediates
ureteric bud development is poorly understood.
c-Ret mRNA encodes multiple transcripts resulting from alternative
splicing of 5' or 3' exons (11, 19, 21, 34). Alternative splicing of the 3' end of the c-ret mRNA results in transcripts encoding c-ret isoforms with different COOH-terminal amino acids. These
transcripts encode c-ret isoforms with distinct 9, 43, or 51 COOH-terminal amino acids. c-Ret protein exists as a short isoform
(1,072 amino acids) or longer isoforms (1,106 and 1,114 amino acids),
respectively. The shortest isoform (RET9 transcript) is the most highly
expressed isoform and is present in the developing and adult kidney
(12). RET51, the transcript encoding the longest isoform, appears to be
developmentally regulated because it is expressed at very low levels
during early gestation and increases later during renal development to
the levels present in the adult kidney (12). The biological function of
the proteins derived from these transcripts is unknown. However, two of
the predicted isoforms encode membrane-spanning receptors with a
truncated extracellular domain. The third is predicted to encode a
soluble, secreted form of the receptor. Two c-ret glycoproteins are
produced from each isoform via posttranslational glycosylation (150 and
170 kDa for the short isoform, 155 and 175 kDa for the longer
isoforms). An important difference between the c-ret receptor and other
protein tyrosine kinase receptors is that activation of c-ret does not occur via direct ligand binding. Instead, a glycosyl
phosphoinositol-linked cell surface receptor binds the required
extracellular ligand, activating the c-ret receptor. Two structurally
related ligands, GDNF and neuroturin (NTN), have been identified, which
activate c-ret through their glycosyl phosphoinositol-linked receptors, GDNFR- The ability of activation of the c-met receptor and the EGF receptor
(EGFR) to initiate morphogenesis in renal epithelial cells (2, 7, 29)
has led us to investigate how c-ret signaling may regulate ureteric bud
development. It has been shown that activation of c-ret in
neuroepithelioma cells could induce the early morphogenetic events of
lamellipodia formation (38), arguing that this ligand/receptor
combination, like hepatocyte growth factor (HGF)/c-met and
EGF/TGF All chemicals were purchased from Sigma unless otherwise noted.
Cell lines. Immortalized murine inner
medullary collecting duct (mIMCD-3; Ref. 26), Madin-Darby canine kidney
(MDCK), and ureteric bud (28) cells were grown in DMEM-Ham's F-12
(DMEM-F12) media supplemented with 10% fetal calf serum. The Neuro-2A
mouse neuroblastoma cell line (16) was grown in minimal essential medium supplemented with 10% fetal calf serum. The human c-ret expression plasmid, Rc/CMV-Ret (from M. Takahashi), was
transfected into mIMCD-3 cells with Lipofectin (GIBCO) and was selected
with G418 to generate stable subclones as previously described (6, 15).
Transient transfections were performed similarly, but cells were
utilized 48 h after the transfection.
RT-PCR.
Poly(A)+ mRNA was isolated with a
quick prep micro mRNA protocol (Pharmacia). Reverse transcription was
performed with Superscript II (GIBCO). The following sense and
antisense primer sequences, respectively, were used to PCR amplify
GDNFR- Protein analysis. For whole cell lysates, subconfluent cells
were washed twice with PBS and refed with DMEM-F12 medium in the
absence of fetal calf serum. After 24 h of serum deprivation, cells
were treated with either GDNF (100 ng/ml in PBS + 0.01% bovine serum
albumin) or vehicle control for 10 min, washed twice with ice-cold PBS,
and lysed in lysis buffer (137 mM NaCl, 20 mM Trizma base, 1 mM
MgCl2, 1 mM
CaCl2, 1 mM sodium orthovanadate, 10% glycerol, 0.5% Igepal CA-630, 1 mM phenylmethylsulfonyl fluoride) at 40°C. Cell lysates were vortexed vigorously and centrifuged for
10 min at 12,000 g, and solubilized
proteins were resolved by SDS-polyacrylamide gel electrophoresis (6%)
and transferred to Immobilon (Millipore) as previously described (6,
15). For immunoprecipitation experiments, protein concentrations of the
resulting supernatants were determined by the Bradford assay and 500 µg of protein were immunoprecipitated for 3 h at 4°C with either
c-ret antibody (from M. Takahashi) or anti-phosphotyrosine (Upstate Biotechnology) with protein A-Sepharose beads (Sigma). Beads
were washed three times in PBS, 1% Nonidet P-40, and 200 µM sodium
vanadate, pH 7.5, and two times in 10 mM Tris, 100 mM NaCl, 1 mM EDTA,
and 200 µM sodium vanadate, pH 7.5. The final pellet was boiled for 5 min in the presence of Immunoblots. Proteins were
electrophoretically transferred to Immobilon-P membranes (Millipore).
Blots were probed with either the antibody to c-ret 1:1,000 (35),
anti-phosphotyrosine 1:3,000 (Upstate Biotechnology), anti-phospho-MAPK
1:1,000 (NEB), or anti-p42/44 1:1,000 (NEB). Proteins were detected
with a chemiluminescence system (ECL, Amersham International).
Tubulogenesis. mIMCD-3 cells were
trypsinized and resuspended in type I collagen and cultured in the
presence or absence of the desired growth factor as previously
described (7). After a 48-h period of incubation at 37°C, cells
were examined for the presence or absence of branching tubular
processes. Quantification of the early events of tubulogenesis was
performed by counting the number of processes per cell cluster for 20 randomly selected single to multicellular cell clusters and comparing
with control cells grown in the absence of the morphogen. Cells were
then allowed to incubate for 7 days, with fresh addition of growth
factor every other day. Cysts and tubules were photographed at
day 7 through a ×10 or ×40 objective with a Nikon Diaphot
inverted microscope with a Hoffman modulator for phase contrast.
Thymidine incorporation. mIMCD-3 cells
or c-ret clones were plated (50,000 cells/well) on 24-well plates.
Cells were serum starved for 24 h before the addition of either GDNF
(100 ng/ml) or 10% fetal calf serum. After 12 h of stimulation, cells
were treated with a second dose of the appropriate stimulus for an additional 12 h.
[3H]thymidine (1 µCi) was then added to each well and incubated for 12 h. Incorporated
thymidine was precipitated with cold 5% trichloroacetic acid.
Precipitates were washed with MeOH and dissolved in 0.1 M NaOH and
dried overnight at room temperature. The resulting precipitate was
resuspended in liquid scintillation fluid (10 ml) and counted on a beta
scintillation counter. Protein determination on lysates was performed
by Bradford assay and used to normalized the results. Each well
represents an n of 1.
Chemotaxis. Chemotaxis assays were
performed with a modified Boyden chamber assay as previously described
(7, 32). Briefly, a 48-well bottom plate (Neuro Probe, Cabin John, MD)
was filled with DMEM-F12 media containing the appropriate
chemoattractant or vehicle control. The well was overlaid with a rat
tail collagen type I (Collaborative Biomedical) coated polycarbonate
filter (Nucleopore). The top compartment was connected, and 1.5 × 104 cells suspended in DMEM-F12
were added to the top of each well. After 4 h of incubation at
37°C, the filters were removed and stained with Diff-Quik (Baxter
Healthcare), and the cells remaining on the top of the membrane were
mechanically scraped off. Cells that had passed through the pores were
counted to determine the number of cells per millimeter squared of
membrane. Each well represents an n of 1.
Expression of c-ret in renal epithelial
cells. To investigate GDNF signaling in renal
epithelial cells, we first examined several cell lines for the presence
of the c-ret receptor and the GDNFR-
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
and NTNR-
, respectively (13, 17). Activation of c-ret has
been found to initiate both the phosphatidylinositol 3-kinase (PI
3-kinase) pathway (38) and the mitogen-activating protein kinase
(MAPK)-signaling cascade (40). Shc-Grb2 adaptor proteins bind to
tyrosine-1062 in the carboxy terminus of c-ret, thus activating MAPK
(23). Although it has been postulated that other SH2-signaling molecules, including PI 3-kinase, are activated by c-ret through tyrosine-1062 as well (1), this remains to be proven.
-EGFR (3, 29), may directly induce epithelial cell
morphogenesis. To address this, we explored the ability of wild-type
c-ret to induce cell migration and branching morphogenesis in
immortalized cells of ureteric bud origin.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
cDNA: 5'
579TGAAGAAAGAGAAGAATTGTCTG-3' and 5'
1221AGGCTGCTGGAGTCTAGTG-3' (13). For GDNF cDNA
amplification, the sense and antisense primer sequences, respectively,
were 5' 267CGCTGACCAGTGACTCCAATATGC-3' and
5' 615GTTAGCCTTCTACTCCGAGACAGG-3'.
-mercaptoethanol and was separated by
SDS-polyacrylamide gel electrophoresis as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
coreceptor. mIMCD-3, MDCK, and
ureteric bud cells were screened for the presence of the c-ret receptor
by western analysis with anti-c-ret and RT-PCR. None of these three
cell lines had detectable expression of the c-ret protein by Western analysis, although the c-ret message was detectable in the ureteric bud
cells by RT-PCR (29). To detect GDNFR-
, we used RT-PCR to amplify
part of the GDNFR-
message from mIMCD-3 and ureteric bud cells (Fig.
1). The expected 642-bp band was
successfully amplified from each cell type. Thus mIMCD-3 cells express
the message for the GDNF coreceptor GDNFR-
but fail to
express the c-ret receptor. This finding is consistent with the
observations of others (13, 14) that GDNFR-
is expressed in the
kidney and suggests that expression of c-ret in these cells could
reconstitute a GDNF-responsive signaling pathway.
View larger version (39K):
[in a new window]
Fig. 1.
Glial cell line-derived neurotropic factor receptor (GDNFR)- 1
expression in murine inner medullary collecting duct (mIMCD-3) and
ureteric bud (UB) cells. mRNA from mIMCD-3 and UB cells was isolated,
reverse transcribed, and PCR amplified with primers designed to amplify
a 642-bp product of GDNFR-
. M,
X174 RF DNA markers; C, control
PCR without mRNA; N2A, Neuro2 A cells (positive control known to
express GDNFR-
).
To examine the role of GDNF and c-ret in kidney epithelial cell
morphogenesis, the long isoform of wild-type c-ret was transfected into
mIMCD-3 cells. Six clones that stably expressed c-ret were identified
by immunoblotting with anti-c-ret (Fig. 2,
top), with clones
1, 2,
3, and
4 expressing both the 155- and 175-kDa
glycosylated products of the long c-ret isoform.
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Constitutive phosphorylation of c-ret in mIMCD-3 cells. The ability of GDNF to trigger c-ret activation was next investigated by examining the tyrosine phosphorylation state of the transfected receptor in the presence and absence of GDNF. Unexpectedly, both the 155- and 175-kDa isoforms of c-ret were found to be constitutively phosphorylated in each of the stable c-ret clones, with no increase after GDNF stimulation (Fig. 2, bottom). To determine whether expression of c-ret in a different cell type of ureteric bud origin would result in GDNF-regulated receptor phosphorylation, we transiently transfected c-ret into ureteric bud and MDCK cells. Transient expression of either the short or long c-ret isoforms in these cells again resulted in a constitutively phosphorylated receptor, with little change after GDNF addition (data not shown). Sequence analysis of the carboxy terminus of the c-ret cDNA, including the tyrosine kinase domain and those regions where known activating mutations occur in multiple endocrine neoplasia (1), was performed, and no mutations were found in the transfected cDNA.
RT-PCR with primers designed to amplify GDNF indicated that both
parental mIMCD-3 cells and c-ret clones express endogenous GDNF (Fig.
3). Thus continuous expression of c-ret in
these immortalized renal epithelial cells results in a constitutively
phosphorylated receptor either because of autocrine stimulation by
endogenously expressed GDNF or potentially because of
ligand-independent receptor homodimerization. Consistent with this
result is the observation that mIMCD-3 cells expressing the
phosphorylated c-ret receptor exhibited modest constitutive
phosphorylation of extracellular signal-related kinase (ERK)1 and ERK2
compared with parental mIMCD-3 cells, with no further ERK1-ERK2
activation after stimulation with exogenous GDNF (data not shown).
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Clones expressing c-ret demonstrate enhanced survival
and morphogenesis in a collagen matrix. To determine if
c-ret could directly mediate epithelial cell tubulogenesis in vitro,
mIMCD-3 cells expressing c-ret were utilized to examine both the early events of branching process formation and the later events of complex
morphogenesis. Cells were grown suspended in type I collagen ± GDNF
in the absence of serum. Quantification of the early events in tubule
formation after 24 and 48 h revealed that the c-ret 3 clone did not
exhibit significant early process formation either spontaneously or in
response to GDNF (Table 1), although these cells exhibited normal branching morphogenesis in response to HGF and
EGF. These results were reproduced with examination of the c-ret 1 clone (data not shown).
|
Culturing under serum-free conditions for 7 days in a type I collagen
matrix, however, resulted in a marked difference between cells
expressing c-ret and the parental mIMCD-3 cells. Typically, mIMCD-3
cells maintained in a collagen matrix in the absence of serum die and
disintegrate within 4 or 5 days, even in the presence of growth factors
such as HGF or EGF. However, when c-ret-3 cells were maintained for
7-10 days in collagen matrix, they exhibited enhanced survival
under serum-free conditions (Fig. 4,
B and
D vs.
A and
C). In addition, these cells tended
to display rudimentary branching process formation (lamellipodia-like
structures) independent of the addition of GDNF (Fig. 4,
B and
D), with no effect of GDNF on this
phenotype (Fig. 4, F vs.
D). Likewise, the c-ret
clone 1 was cultured under similar
conditions to investigate whether lower level expression of c-ret
altered this phenotype. Again, the expression of c-ret resulted in
enhanced survival in a type I collagen matrix with rudimentary
branching process formation (data not shown).
|
When cultured for 7 days in the presence of HGF or EGF, c-ret-3 cells
again exhibited enhanced serum-free survival and formed multicellular
cystic structures with multiple extra-cystic spiny processes (Fig.
5, B and
D, respectively). Parental mIMCD-3
cells cultured under these conditions failed to form cysts and were judged to be dead by light microscopy (Fig. 5,
A and
C). These morphological changes in
the c-ret clones were observed in three independent experiments.
|
To determine whether the enhanced viability of c-ret clones in
serum-free conditions was due to c-ret-mediated cellular proliferation, we looked at
[3H]thymidine uptake
in c-ret clones ± GDNF. Cells expressing c-ret exhibit a marked
proliferative response to serum but did not demonstrate a significant
increase in basal
[3H]thymidine uptake
compared with parental mIMCD-3 cells (Fig. 6). In addition, there was no increase in
[3H]thymidine uptake
after GDNF stimulation.
|
mIMCD-3 cells expressing constitutively phosphorylated
c-ret do not demonstrate basal enhanced cell migration but upregulate migration in response to HGF. The ability of c-ret
expression to alter epithelial cell chemotaxis was determined by
examining the migratory response in the presence or absence of GDNF of
c-ret clones
1 and
3 compared with parental mIMCD-3
cells. Neither clone exhibited enhanced basal chemotaxis, nor did a
significant number of cells move toward a gradient of GDNF (mIMCD-3,
control 15.2 ± 1.6 cells/mm2;
GDNF, 8.4 ± 1.1 cells/mm2;
Ret1, control 10.2 ± 1.4 cells/mm2; GDNF, 5.0 ± 1 cells/mm2; Ret3, control 16.8 ± 2.0 cells/mm2; GDNF, 13.2 ± 1.0 cells/mm2; Fig.
7). Of note, expression of c-ret did alter
the normal mIMCD-3 cell migratory response toward a gradient of HGF. In
both c-ret-expressing clones, the migratory response to HGF was nearly
double that observed with parental mIMCD-3 cells, although this only
reached statistical significance for the Ret1 clone (mIMCD-3 + HGF, 168 ± 24.3 cells/mm2; Ret1 + HGF,
293 ± 25 cells/mm2,
P < 0.05; Ret3 + HGF, 288 ± 50 cells/mm2).
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DISCUSSION |
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Gene knockout data in mice have demonstrated that the GDNF-c-ret
signaling pathway is critical for ureteric bud development and early
nephrogenesis (9, 30, 31), yet the mechanism by which c-ret mediates
these events in the kidney is unknown. In the present study, we
demonstrate that stable expression of the long isoform of the c-ret
receptor in a cell line derived from the ureteric bud, mIMCD-3 cells,
results in a constitutively phosphorylated receptor that mediates
enhanced serum-free cell survival when those cells are cultured in a
type I collagen matrix. The surviving cells also demonstrate
rudimentary branching process formation (an early phase of in vitro
tubulogenesis) but do not undergo complex three-dimensional structure
formation (i.e., spiny cysts) unless cultured in the presence of known
epithelial morphogens such as HGF, EGF, or TGF-. These results
suggest that activation of c-ret in the ureteric bud may play a dual
role of preventing apoptosis and initiating early morphogenesis
necessary for ureteric bud outgrowth and branching.
Before transfection with c-ret, we examined mIMCD-3 cells for the
expression of endogenous c-ret. In none of the cells examined could we
detect either the c-ret mRNA or 150- or 170-kDa c-ret proteins. The
absence of c-ret in these cells was not entirely unexpected because
they are derived from the fully formed inner medullary collecting duct
of embryonic mice, whereas c-ret appears to be selectively expressed
only at the very tip of the invading ureteric bud (24, 27).
Interestingly, mIMCD-3 cells do express the message for the c-ret
coreceptor GDNFR-. The role of GDNFR-
expression independent of
c-ret is unclear (the extracellular cadherin-like domain raises the
possibility that GDNFR-
may play a role in cell adhesion) but
suggested to us that coexpression of c-ret in these cells should
reconstitute a GDNF-responsive pathway. Instead, transfection with
wild-type c-ret in mIMCD-3 cells resulted in a constitutively
tyrosine-phosphorylated receptor. Recently, Tang et al. (36)
demonstrated that c-ret expressed in MDCK cells was activated in a
ligand-independent manner by simply overexpressing c-ret protein,
similar to our result. They found that the addition of GDNF and soluble
GDNFR-
could regulate this activation. On the basis of our detection
of the message for GDNF in mIMCD-3 cells, we postulate that the
constitutive activation of c-ret in these cells may be due to autocrine
activation. However, spontaneous, ligand-independent c-ret dimerization
due to overexpression in excess of GDNFR-
might occur in these cells as well. Further attempts to prove whether c-ret phosphorylation in
mIMCD-3 cells is due to autocrine activation of c-ret via endogenous GDNF expression or c-ret dimerization via overexpression is beyond the
scope of the present study.
The presence of GDNF has been shown to prevent apoptosis in neuronal cells and is felt to be important for normal brain development, at least in part, due to this effect (5, 14, 18). In a system utilizing hanging-drop primary ureteric bud cultures, the addition of GDNF was found to decrease overall apoptosis and increase cell adhesiveness, presumably by activation of the endogenous c-ret receptor (27). It has been our consistent observation that activation of either of the known mitogenic-morphogenic receptors c-met and/or the EGFR does not prevent mIMCD-3 cell death when these cells are cultured in a collagen matrix under serum-free conditions. In the present study, we demonstrate the novel finding that selective expression of the phosphorylated c-ret receptor can convey enhanced serum-free survival of renal tubular epithelial cells (Fig. 5, compare A and C vs. B and D). Taken together, these results demonstrate that the local expression of GDNF and subsequent activation of c-ret may play a critical role in preventing apoptosis of the developing ureteric bud, whereas activation of the c-met and/or EGFR do not appear capable of mediating this increase in cell survival. Interestingly, Towers et al. (37) have recently found that GDNF increased cell numbers in a primary ureteric bud culture system when the cells were cultured on laminin or fibronectin. This effect was solely due to enhanced cell survival when the cells were cultured on fibronectin, whereas GDNF enhanced both cell division and survival when the culture was performed on a laminin substratum.
In contrast to the effects on cell survival, expression of the constitutively phosphorylated c-ret receptor did not result in enhanced mitogenesis in our experiments. Prior studies addressing the mitogenic properties of GDNF in ureteric bud cultures are conflicting, with Sainio et al. (27) reporting no effect of GDNF on mitogenesis, whereas Pepicelli et al. (25) in whole organ explants did detect an increase in mitogenesis with GDNF. As noted above, Towers et al. (37) indicated that GDNF induced proliferation of cells grown on laminin but not those grown on fibronectin. In the present study, we found no effect of activated c-ret on mIMCD-3 cell mitogenesis in either the presence or absence of exogenous GDNF when the cells were cultured in a monolayer on standard tissue culture plates. However, in the presence of activated c-ret, the addition of either HGF or EGF did result in apparent cell proliferation when cultured under serum-free conditions in a collagen matrix (compare Fig. 5, B and D vs. Fig. 4, D and F). Thus the present results lead us to believe that under the conditions of constitutive phosphorylation, c-ret is not mitogenic itself but rather mediates ureteric bud cell survival and thereby allows other growth factors to trigger actual cell division.
Our finding that activated c-ret mediates the very early events of branching process formation, but not the more complex events of tubulogenesis, argues that although this receptor may not be a true morphogenic receptor in these cells like the c-met and EGF receptors, activated c-ret can trigger at least the initial events in actin skeletal reassembly. This is consistent with the observations of van Weering and Bos (38), who found that activation of c-ret in neuroepithelioma cells results in lamellipodia formation (38), one of the earliest events in cell migration (33). In agreement with our finding, they demonstrated that transfection of wild-type c-ret into neuroepithelioma cells resulted in the expression of both the 150- and 170-kDa c-ret glycosylation products but found tyrosine phosphorylation only after stimulation with GDNF. The addition of GDNF to the c-ret-expressing neuroepithelioma cells resulted in actin filament reorganization and lamellipodia formation, an event that was prevented by the PI 3-kinase inhibitors wortmannin and LY-294002.
We have made a similar observation that activation of the PI 3-kinase is sufficient to initiate membrane ruffling in either an epithelial cell line expressing a hybrid platelet-derived growth factor receptor/c-met receptor (6) or by direct addition of PtdIns(3,4,5)P3 (the lipid product of the PI 3-kinase) (8) but have found that this in itself was insufficient to initiate the more complex events required for tubulogenesis (H. Sakurai, unpublished observations). Interestingly, the two receptors that trigger in vitro tubulogenesis in cell culture, c-met and the EGFR, have both been found to associate with the intracellular docking protein Gab-1 (10, 39), whereas c-ret was not found to associate with this signaling protein (39). Thus the ability of c-ret to trigger signaling pathways such as the PI 3-kinase, but not associate with Gab-1, may explain the development of simple process formation but not more complex morphogenesis by clones expressing activated c-ret.
In addition to c-ret mediating epithelial cell survival and branching process formation, the expression of this receptor unexpectedly altered the phenotype induced by the known renal epithelial morphogens HGF and EGF. As noted previously, when mIMCD-3 cells are cultured in a type I collagen matrix under serum-free conditions but in the presence of HGF or EGF, they die within 4 to 5 days. However, when cultured with either HGF or EGF in the presence of low concentrations of serum (or with high concentrations of serum alone), mIMCD-3 cells divide and form complex branching tubular structures (2-4). In the present study, mIMCD-3 cells expressing c-ret survived in the absence of serum, allowing us for the first time to examine the effects of addition of HGF or EGF on in vitro morphogenesis in the absence of serum over an extended period of time. Under these conditions, c-ret-expressing mIMCD-3 cells underwent cyst development rather than branching tubule formation when cultured in the presence of either HGF or EGF. Furthermore, the cysts displayed spiny process formation or spikes, a phenotype reminiscent of MDCK cells cultured sequentially in the presence of serum and HGF (20). Whether the development of "spiny cysts" in mIMCD-3 cells is due to the presence of the activated c-ret receptor or the absence of a unique morphogen (such as ureteric bud lumen-forming factor, Ref. 28) normally provided in serum is at present unknown.
Despite the ability of constitutively phosphorylated c-ret to mediate
early branching process formation, we were unable to detect an increase
in basal cell motility in these cells compared with parental mIMCD-3
cells. However, cell migration induced by the known chemoattractant HGF
was nearly doubled in cells expressing constitutively phosphorylated
c-ret. Similar to the results in the tubulogenesis assay, these effects
on cell migration suggest a complex interplay between the downstream
signaling events activated by c-ret and the morphogenic receptors c-met
and the EGFR. Tang et al. (36) observed increased basal and
GDNF-induced cell motility in MDCK cells expressing c-ret when
supplemented with soluble GDNFR-. Our inability to detect an
increase in cell migration in the c-ret-expressing mIMCD-3 cells might
be because constitutive phosphorylation of the receptor results in
downregulation of critical signaling pathways that are typically
activated only transiently after receptor stimulation.
In conclusion, when c-ret is expressed in cultured inner medullary
collecting duct cells, it becomes constitutively phosphorylated and
mediates an increase in both cell survival and very early morphogenic
events but does not trigger the more complex events required for
three-dimensional tubule formation in cell culture. Thus we postulate
that the expression of c-ret in the ureteric bud is required to prevent
cell death and begin the early events of morphogenesis, whereas
coexpression in this region of other morphogens such as HGF and/or
EGF/TGF- is required for the complete development of the renal
collecting duct tubule.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-48871 to L. Cantley and by NIDDK DK-49517 Grant to S. Nigam. S. Nigam is an established investigator of the American Heart Association. H. Sakurai is supported by a fellowship grant from Uehara Memorial Life Science Foundation.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. G. Cantley, Dana 517, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail: lcantley{at}caregroup.harvard.edu).
Received 9 March 1998; accepted in final form 21 December 1998.
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REFERENCES |
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1.
Asai, N.,
H. Murakami,
T. Iwashita,
and
M. Takahashi.
A mutation at tyrosine 1062 in MEN2A-Ret and MEN2B-Ret impairs their transforming activity and association with shc adaptor proteins.
J. Biol. Chem.
271:
17644-17649,
1996
2.
Barros, E. J.,
O. F. Santos,
K. Matsumoto,
T. Nakamura,
and
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