Expression of c-ret promotes morphogenesis and cell survival in mIMCD-3 cells

Dawn A. O'Rourke1, Hiroyuki Sakurai2, Katherine Spokes1, Crystal Kjelsberg1, Masahide Takahashi3, Sanjay Nigam2, and Lloyd Cantley1

1 Division of Nephrology, Beth Israel Deaconess Medical Center, and 2 Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215; and 3 Department of Pathology, Nagoya University School of Medicine, Nagoya 466, Japan


    ABSTRACT
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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


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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-alpha and NTNR-alpha , 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.

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/TGFalpha -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.


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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-alpha 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'.

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 beta -mercaptoethanol and was separated by SDS-polyacrylamide gel electrophoresis as above.

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.


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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-alpha 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-alpha , we used RT-PCR to amplify part of the GDNFR-alpha 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-alpha but fail to express the c-ret receptor. This finding is consistent with the observations of others (13, 14) that GDNFR-alpha is expressed in the kidney and suggests that expression of c-ret in these cells could reconstitute a GDNF-responsive signaling pathway.


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Fig. 1.   Glial cell line-derived neurotropic factor receptor (GDNFR)- alpha 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-alpha . M, Phi X174 RF DNA markers; C, control PCR without mRNA; N2A, Neuro2 A cells (positive control known to express GDNFR-alpha ).

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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Fig. 2.   Stable transfection of c-ret in mIMCD-3 cells. Six clones that stably express transfected long isoform of c-ret receptor were incubated in absence (-) or presence (+) of GDNF (100 ng/ml GDNF for 10'), immunoprecipitated with a polyclonal anti-c-ret antibody, isolated by SDS-PAGE, and immunoblotted with anti-c-ret (top) or anti-phosphotyrosine (anti-pY; bottom). Lanes 1 and 2, parental mIMCD-3; lanes 3 and 4, Ret1; lanes 5 and 6, Ret2; lanes 7 and 8, Ret3; lanes 9 and 10, Ret4; lanes 11 and 12, Ret5; lanes 13 and 14, Ret6.

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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Fig. 3.   Endogenous GDNF expression. mRNA from mIMCD-3, Ret clone, and embryonic kidney (mouse and rat) was isolated, reverse transcribed, and PCR amplified with primers to the mouse GDNF sequence. Lane 1, 100-bp ladder; lane 2, no cDNA; lane 3, no RT; lane 4, mIMCD-3; lane 5, Ret clone 2; lane 6, mouse embryonic day 13.5; lane 7, rat embryonic day 19; lane 8, Phi X174 RF DNA markers.

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).

                              
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Table 1.   Results of early branching morphogenesis assay

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).


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Fig. 4.   Activated c-ret promotes cell survival and branching morphogenesis. mIMCD-3 cells and Ret3 clones were incubated 7 days in the absence of serum (A-D) or in GDNF (100 ng/ml, E and F). Ret3 cells show process formation in the absence of serum (B and D, magnification, ×10 and ×40, respectively). mIMCD-3 cells under the same conditions die and disintegrate (A and C, magnification, ×10 and ×40, respectively). GDNF did not have any effect on branching morphogenesis of c-ret-3 clone (F vs. D, magnification, ×40).

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.


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Fig. 5.   Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) stimulate formation of spiny cysts in c-ret 3 clone. mIMCD-3 cells and Ret3 clones were incubated 7 days in HGF (40 ng/ml, A and B) or EGF (20 ng/ml, C and D). Addition of HGF or EGF induced the formation of multicellular spiny cysts (B and D) in c-ret-3 cells. Parental mIMCD-3 cells die and disintegrate under similar conditions (A and C). Magnification, ×40.

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.


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Fig. 6.   Expression of c-ret does not cause enhanced mitogenesis in mIMCD-3 cells. mIMCD-3 and Ret3 clones were serum deprived for 48 h. Cells were then incubated for 24 h either serum free, with GDNF (100 ng/ml), or with FCS before labeling with [3H]thymidine (n = 6 for each condition). [3H]thymidine uptake was then determined by counting on a beta scintillation counter and normalized to protein content. NS, nonsignificant. P < 0.001 for FCS vs. control.

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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Fig. 7.   Expression of constitutively active c-ret augments HGF-dependent cell migration. c-Ret clones 1 (n = 6) and 3 (n = 11) were examined for cell migration in response to GDNF, compared with known chemoattractant HGF (40 ng/ml). Ret clones exhibited basal cell migration rates similar to parental mIMCD-3 cells (n = 11) and did not chemotax in response to GDNF, whereas migration in response to HGF was nearly twice that observed for parental cells. P < 0.05 for Ret1 + HGF vs. mIMCD-3 + HGF; P = NS for Ret3 + HGF vs. mIMCD-3 + HGF.


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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-alpha . 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-alpha . The role of GDNFR-alpha expression independent of c-ret is unclear (the extracellular cadherin-like domain raises the possibility that GDNFR-alpha 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-alpha 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-alpha 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-alpha . 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-alpha is required for the complete development of the renal collecting duct tubule.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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[Abstract/Free Full Text].

2.   Barros, E. J., O. F. Santos, K. Matsumoto, T. Nakamura, and S. K. Nigam. Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development. Proc. Natl. Acad. Sci. USA 92: 4412-4416, 1995[Abstract].

3.   Cantley, L. G., E. J. G. Barros, M. Gandhi, M. Rauchman, and S. K. Nigam. Regulation of mitogenesis, motogenesis, and tubulogenesis by hepatocyte growth factor in renal collecting duct cells. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F271-F280, 1994[Abstract/Free Full Text].

4.   Cantley, L. G. Growth factors and the kidney: regulation of epithelial cell movement and morphogenesis. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F1103-F1113, 1996[Abstract/Free Full Text].

5.   Clarkson, E. D., W. M. Zawada, and C. R. Freed. GDNF improves survival and reduces apoptosis in human embryonic dopaminergic neurons in vitro. Cell Tissue Res. 289: 207-210, 1997[Medline].

6.   Derman, M. P., J. Y. Chen, K. C. Spokes, Z. Songyang, and L. G. Cantley. An 11-amino acid sequence from c-met initiates epithelial chemotaxis via phosphatidylinositol 3-kinase and phospholipase C. J. Biol. Chem. 271: 4251-4255, 1996[Abstract/Free Full Text].

7.   Derman, M. P., M. J. Cunha, E. J. G. Barros, S. Nigam, and L. G. Cantley. HGF-mediated chemotaxis and tubulogenesis require activation of the phosphatidylinositol 3-kinase. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F1211-F1217, 1995[Abstract/Free Full Text].

8.   Derman, M. P., A. Toker, J. H. Hartwig, K. Spokes, J. R. Falck, C.-S. Chen, L. C. Cantley, and L. G. Cantley. The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C. J. Biol. Chem. 272: 6465-6470, 1997[Abstract/Free Full Text].

9.   Durbec, P., C. V. Marcos-Gutierrez, C. Kilkenny, M. Grigoriou, K. Wartiowaara, P. Suvanto, D. Smith, B. Ponder, F. Costantini, M. Saarma, H. Sariola, and V. Pachnis. GDNF signalling through the Ret receptor tyrosine kinase. Nature 381: 789-793, 1996[Medline].

10.   Holgado-Madruga, M., D. R. Emlet, D. K. Moscatello, A. K. Godwin, and A. J. Wong. A Grb2-associated docking protein in EGF- and insulin-receptor signalling. Nature 379: 560-564, 1996[Medline].

11.   Ivanchuk, S. M., C. Eng, W. K. Cavenee, and L. M. Mulligan. The expression of RET and its multiple splice forms in developing human kidney. Oncogene 14: 1811-1818, 1997[Medline].

12.   Ivanchuk, S., S. Myers, and L. Mulligan. Expression of RET 3' splicing variants during human kidney development. Oncogene 16: 991-996, 1998[Medline].

13.   Jing, S., D. Wen, Y. Yu, P. L. Holst, Y. Luo, M. Fang, R. Tamir, L. Antonio, Z. Hu, R. Cupples, J. C. Louis, S. Hu, B. W. Altrock, and G. M. Fox. GDNF-induced activation of the Ret protein tyrosine kinase is mediated by GDNFR-alpha , a novel receptor for GDNF. Cell 85: 1113-1124, 1996[Medline].

14.   Jing, S., Y. Yu, M. Fang, Z. Hu, P. L. Holst, T. Boone, J. Delaney, H. Schultz, R. Zhou, and G. M. Fox. GFRalpha -2 and GFRalpha -3 are two new receptors for ligands of the GDNF family. J. Biol. Chem. 272: 33111-33117, 1997[Abstract/Free Full Text].

15.   Kjelsberg, C., H. Sakurai, K. Spokes, C. Birchmeier, I. Drummond, S. Nigam, and L. G. Cantley. Met -/- kidneys express epithelial cells that chemotax and form tubules in response to EGF receptor ligands. Am. J. Physiol. 272 (Renal Physiol. 41): F222-F228, 1997[Abstract/Free Full Text].

16.   Klebe, R. J., T. Chen, and F. H. Ruddle. Controlled production of proliferating somatic cell hybrids. J. Cell Biol. 45: 74-82, 1970[Abstract/Free Full Text].

17.   Klein, R. D., D. Sherman, W. Ho, D. Stone, G. L. Bennett, B. Moffat, R. Vandlen, L. Simmons, Q. Gu, J. Hongo, B. Devaux, K. Paulsen, M. Armanini, C. Nozaki, N. Asai, A. Goddard, H. Phillips, C. E. Henderson, M. Takahashi, and A. Rosenthal. A GPI-linked protein that interacts with Ret to form a candidate neuroturnin receptor. Nature 387: 717-724, 1997[Medline].

18.   Lin, L., D. H. Doherty, J. D. Lile, S. Bektesh, and F. Collins. GDNF: a glial cell-line derived neurotrophic factor for midbrain dopamingergic neurons. Science 260: 1130-1132, 1993[Medline].

19.   Lorenzo, M., C. Eng, L. Mulligan, T. Stonehouse, C. Healey, B. Ponder, and D. Smith. Multiple mRNA isoforms of the human RET proto-oncogene generated by alternate splicing. Oncogene 10: 1377-1383, 1995[Medline].

20.   Montesano, R., K. Matsumoto, T. Nakamura, and L. Orci. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67: 901-908, 1991[Medline].

21.   Myers, S. M., C. Eng, B. A. J. Ponder, and L. M. Mulligan. Characrization of RET proto-oncogene 3' splicing variants and polyadenylation sites: a novel C-terminus for RET. Oncogene 11: 2039-2045, 1995[Medline].

22.   Nigam, S. K., A. C. Aperia, and B. M. Brenner. Development and maturation of the kidney. In: The Kidney (5th ed.), edited by B. M. Brenner. Philadelphia: Saunders, 1996, p. 72-98.

23.   Ohiwa, M., H. Murakami, T. Iwashita, N. Asai, Y. Iwata, T. Imai, H. Funahashi, H. Takagi, and M. Takahashi. Characterization of Ret-Shc-Grb2 complex induced by GDNF, MEN 2A, and MEN 2B mutations. Biochem. Biophys. Res. Commun. 237: 747-751, 1997[Medline].

24.   Pachnis, V., B. Mankoo, and F. Costantini. Expression of the c-ret proto-oncogene during mouse embryogenesis. Development 119: 1005-1017, 1993[Abstract/Free Full Text].

25.   Pepicelli, C. V., A. Kispert, D. H. Rowitch, and A. P. McMahon. GDNF induces branching and increased cell proliferation in the ureter of the mouse. Dev. Biol. 192: 193-198, 1997[Medline].

26.   Rauchman, M., S. Nigam, E. Delpire, and S. Gullans. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F416-F424, 1993[Abstract/Free Full Text].

27.   Sainio, K., P. Suvanto, J. Davies, J. Wartiovaara, K. Wartiovaara, M. Saarma, U. Arumae, X. Meng, M. Lindahl, V. Pachnis, and H. Sariola. Glial-cell-line-derived neurotrophic factor is required for bud initiation from ureteric epithelium. Development 124: 4077-4087, 1997[Abstract/Free Full Text].

28.   Sakurai, H., E. J. Barros, T. Tsukamoto, J. Barasch, and S. K. Nigam. An in vitro tubulogenesis system using cell lines derived from the embryonic kidney shows dependence on multiple soluble growth factors. Proc. Natl. Acad. Sci. USA 94: 6279-6284, 1997[Abstract/Free Full Text].

29.   Sakurai, H., T. Tsukamoto, C. A. Kjelsberg, L. G. Cantley, and S. K. Nigam. EGF receptor ligands are a large fraction of in vitro branching morphogens secreted by embryonic kidney. Am. J. Physiol. 273 (Renal Physiol. 42): F463-F472, 1997[Abstract/Free Full Text].

30.   Sanchez, M. P., I. Silos-Santiago, J. Frisen, B. He, S. A. Lira, and M. Barbacid. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382: 70-73, 1996[Medline].

31.   Schuchardt, A., V. d'Agati, L. Larsson-Blomberg, F. Costantini, and V. Pachnis. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367: 380-383, 1994[Medline].

32.   Stoker, M. Effect of scatter factor on motility of epithelial cells and fibroblasts. J. Cell. Physiol. 139: 565-569, 1989[Medline].

33.   Stossel, T. P. On the crawling of animal cells. Science 260: 1086-1094, 1993[Medline].

34.   Tahira, T., Y. Ishizaka, F. Itoh, T. Sugimura, and M. Nagao. Characterization of ret proto-oncogene mRNAs encoding two isoforms of the protein product in a human neuroblastoma cell line. Oncogene 5: 97-102, 1990[Medline].

35.   Takahashi, M., Y. Buma, and M. Taniguchi. Identifiction of the ret proto-oncogene products in neuroblastoma and leukemia cells. Oncogene 6: 297-301, 1991[Medline].

36.   Tang, M., D. Worley, M. Sanicola, and G. Dressler. The RET-glial cell-derived neurotrophic factor (GDNF) pathway stimulates migration and chemoattraction of epithelial cells. J. Cell Sci. 142: 1337-1345, 1998.

37.   Towers, P., A. Woolf, and P. Hardman. Glial cell line-derived neurotrophic factor stimulates ureteric bud outgrowth and enhances survival of ureteric bud cells in vitro. Exp. Nephrol. 6: 337-351, 1998[Medline].

38.   Van Weering, D. H., and J. L. Bos. Glial cell line-derived neurotrophic factor induces Ret-mediated lamellipodia formation. J. Biol. Chem. 272: 249-254, 1997[Abstract/Free Full Text].

39.   Weidner, K. M., S. Di Cesare, M. Sachs, V. Brinkmann, J. Behrens, and W. Birchmeier. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature 384: 173-176, 1996[Medline].

40.   Worby, C. A., Q. C. Vega, Y. Zhao, H. H. J. Chao, A. F. Seasholtz, and J. E. Dixon. Glial cell line-derived neurotrophic factor signals through the RET receptor and activates mitogen-activated protein kinase. J. Biol. Chem. 271: 23619-23622, 1996[Abstract/Free Full Text].


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