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Vol. 17, Issue 4, 2046-2056, April 2006

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H-Ras, R-Ras, and TC21 Differentially Regulate Ureteric Bud Cell Branching Morphogenesis

Ambra Pozzi ^*  , Sergio Coffa , Nada Bulus , Wenqin Zhu , Dong Chen , Xiwu Chen , Glenda Mernaugh , Yan Su , Songmin Cai , Amar Singh , Marcela Brissova ^* , and Roy Zent ^*   ||

^* Department of Research Medicine, Veterans Affairs Hospital, Nashville, TN 37232; Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232; Division of Endocrinology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232; Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232; and ^|| Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232

Submitted August 25, 2005; Revised January 3, 2006; Accepted January 26, 2006
Monitoring Editor: Keith Mostov

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The collecting system of the kidney, derived from the ureteric bud (UB), undergoes repetitive bifid branching events during early development followed by a phase of tubular growth and elongation. Although members of the Ras GTPase family control cell growth, differentiation, proliferation, and migration, their role in development of the collecting system of the kidney is unexplored. In this study, we demonstrate that members of the R-Ras family of proteins, R-Ras and TC21, are expressed in the murine collecting system at E13.5, whereas H-Ras is only detected at day E17.5. Using murine UB cells expressing activated H-Ras, R-Ras, and TC21, we demonstrate that R-Ras–expressing cells show increased branching morphogenesis and cell growth, TC21-expressing cells branch excessively but lose their ability to migrate, whereas H-Ras–expressing cells migrated the most and formed long unbranched tubules. These differences in branching morphogenesis are mediated by differential regulation/activation of the Rho family of GTPases and mitogen-activated protein kinases. Because most branching of the UB occurs early in development, it is conceivable that R-Ras and TC-21 play a role in facilitating branching and growth in early UB development, whereas H-Ras might favor cell migration and elongation of tubules, events that occur later in development.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The formation of the kidney starts when the ureteric duct (UB) invades the adjacent metanephric mesenchyme. The UB gives rise to the collecting system of the adult kidney (from the collecting ducts to the trigone of the bladder), whereas the metanephric mesenchyme gives rise to the nephron. During development, the UB undergoes repetitive dichotomous branching events, leading to the formation of UB tips available for interactions with the metanephric mesenchyme and consequent formation of additional nephrons (Davies and Davey, 1999; Pohl et al., 2000; Davies and Fisher, 2002). These events are mediated by several soluble factors such as transforming growth factor (TGF)-; Wnt family members; fibroblast growth factor; glial cell line-derived neurotrophic factor; hepatocyte growth factor (HGF); and epidermal growth factor (EGF) as well as cell–extracellular matrix interactions, which act in a cooperative manner either as pro- or antitubulogenic factors (Piscione and Rosenblum, 2002; Karihaloo et al., 2005). Cells respond to these stimuli by initiating signaling pathways that regulate cell proliferation, migration, and morphogenesis.

Members of the Ras superfamily modulate many signaling pathways activated by cell surface receptors involved in tubulogenesis and branching morphogenesis, including growth factor receptors and integrins. Ras superfamily proteins are able to bind/activate downstream effectors only when bound to guanosine triphosphate (GTP), and GTP hydrolysis leads to the release of the effectors and attenuation of downstream signaling. The Ras superfamily members, Ras oncoproteins (H-Ras, N-Ras, and K-Ras), and the Related to Ras (R-Ras and TC21) share a high level of amino acid similarity. R-Ras has an overall amino acid identity with H-Ras of 55% (Lowe et al., 1987), and TC21shows 70% homology to R-Ras (Lowe and Goeddel, 1987). TC21 and H-Ras bind/activate many of the same effectors, including phosphatidylinositol 3-kinase (PI3-K), RalGDS, and Raf; however, each of these Ras family members mediates highly divergent cellular functions.

The role of the Ras proteins is virtually unexplored in kidney development. The only study that examines the expression of these family members in the adult kidney showed strong K-Ras staining in the collecting ducts but weak staining of H- and N-Ras (Kocher et al., 2003). In vitro studies with Madin-Darby canine kidney (MDCK) cells demonstrated that overexpression of H-Ras inhibits HGF-induced tubulogenesis because of excessive activation of Raf and its downstream effector extracellular signal-regulated kinase (ERK). In contrast, expression of activated R-Ras is per se sufficient to promote tubulogenesis because of the ability of R-Ras to activate PI3-K but not Raf (Khwaja et al., 1998). These observations raised the possibility that the R-Ras family of proteins, rather than H-Ras, may be key regulators of renal cell tubulogenesis.

We therefore investigated the role of the R-Ras family members in the development of the renal collecting system. We demonstrate that both R-Ras and TC21 are expressed at early stages of the collecting system development (i.e., E13.5), whereas H-Ras is only expressed at E17.5. Using murine UB cells expressing activated forms of H-Ras, R-Ras, and TC21, we show that H-Ras inhibits the ability of UB cells to branch in three-dimensional (3D) cultures, but it does not alter their ability to form tubular structures. In contrast, R-Ras increases branching morphogenesis, whereas TC21 induces epithelial-mesenchymal transition, which induces branching but prevents tubule elongation. Thus H- and R-Ras proteins differentially modulate UB cell branching morphogenesis, and differences in their temporal expression could alter the pattern of branching morphogenesis of the developing collecting system in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cell Culture
Phoenix 293 cells obtained from Dr. Albert Reynolds (Vanderbilt University, Nashville, TN) with the permission of Dr. Gary Nolan (Stanford University, Stanford, CA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Murine UB cells were cultured in minimal essential medium (MEM) (Mediatech, Herndon, VA) supplemented with 10% FBS as described previously (Sakurai et al., 1997; Sakurai and Nigam, 1997).

Production of Stable Cell Populations
The LZRS-GFP vector, modified from the original LZRS vector (Kinsella and Nolan, 1996) to allow bicistronic expression of the protein of interest and green fluorescent protein (GFP), was a generous gift from Dr. A. Reynolds (Ireton et al., 2002). This vector was used to generate stable UB cell populations overexpressing H-Ras, R-Ras, and TC21 mutants.

PCR with appropriate primers was performed on pcDNA3-H-Ras (G12V), pcDNA3-H-Ras (G38V) (generous gift from Dr. T. Sethi, University of Edinburgh Medical School, Edinburgh, Scotland), and pRSV-TC21 (Q72L) (a gift from Dr. M. Hansen, University of Copenhagen, Copenhagen, Denmark). The PCR products were subcloned into Bluescript plasmids, sequenced, and transferred into the LZRS-GFP vector.

The LZRS-GFP empty vector or carrying the different Ras family members was transfected into the packaging cell line Phoenix 293 using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN). Transfection of 80% of cells is usually achieved 48 h after transfection as assessed by the percentage of GFP-positive Phoenix cells. UB cells were subsequently infected with the retrovirus daily for 2 wk. Stable cell populations of UB cells expressing equal GFP levels were selected using an FACStar Plus cell sorter (BD Biosciences, Franklin Lakes, NJ). Gates were set to collect cells that expressed GFP in the lower, middle, or top one-third of the GFP range. Polyclonal cell populations from the top one-third of gated cells were used in experiments presented in this article. Cell populations were used to avoid some of the pitfalls associated with isolation of monoclonal cell lines.

Three-dimensional Cell Culture
Tubulogenesis of UB cells were performed in 3D gels as described previously (Cantley et al., 1994; Sakurai et al., 1997; Chen et al., 2004). Briefly, for the collagen I/Matrigel (MG) gels, a collagen I solution (BD Biosciences) composed of 0.1 mg/ml CI in Dulbecco's minimal essential media containing 20 mM HEPES, pH 7.2, was initially made. This collagen solution was then mixed with growth factor-reduced MG (BD Biosciences) in a 1:1 ratio (Sakurai et al., 1997). Then, 100-µl gels (containing 1.5 x 10³ UB cells) were placed into 96-well plates. Media (100 µl) supplemented with 10% fetal calf serum (FCS) was added to the gels after they had solidified. Cells were allowed to grow for 6 d before quantification analysis was performed by counting the number of branch points in each tubular structure. Branching structures (defined as more than 1 branch) were counted in five random high-power fields. Three independent experiments were performed in triplicate.

To determine the signaling molecules involved in UB branching morphogenesis, cells were cultured as indicated above in the presence or absence of 1 µM Y27632 (a Rho kinase inhibitor), 10 µg/ml C3 (a Rho inhibitor), 1 µM LY294002 (a PI3-K inhibitor), 20 nM wortmannin (a PI3-K inhibitor), 5 µM PD98059 (an ERK inhibitor), and 0.5 µM PD169316 (a p38 inhibitor) (Calbiochem, San Diego, CA), which were added to the medium at the start of the cultures. These concentrations were used after assessing the toxic doses of the inhibitors for UB cells.

Cell Migration
Cell migration was assayed in transwells consisting of polyvinylpyrolidone-free polycarbonate filters with 8-µm pores as described previously (Cai et al., 2005). The bottom of the filters were coated with MG (1 µg/ml) in phosphate-buffered saline (PBS) overnight at 4°C and subsequently incubated with 1% bovine serum albumin (BSA) in PBS for 1 h at 37°C. Then, 100 µl of a cell suspension (1 x 10⁶ cells/ml) in serum-free medium containing 0.1% BSA was added to the top wells, and cells were allowed to migrate into the bottom wells for 4 h at 37°C. Cells on the top of the filter were removed by wiping, and the filter was fixed in 4% formaldehyde in PBS. Migrating cells were stained with 1% crystal violet, and nine randomly chosen fields were counted at 200x magnification. Three independent experiments were performed in triplicate in the presence and absence of 10 µM Y27632, 5 µM LY294002, 50 nM wortmannin, 50 µM PD98059, and 50 µM PD169316.

Cell Proliferation
UB cell proliferation was evaluated as described previously (Chen et al., 2004). Briefly, UB cells (5 x 10³) were embedded in the 3D collagen I/MG gels (100-µl final volume) in 96-well plates as described above and incubated in 100 µl of media supplemented with 10% FCS. After 48 h in culture, cells were pulsed for an additional 24 h with [³H]thymidine (1 µCi/well). The gels were removed from the plates and dialyzed against PBS for 24 h to remove free [³H]thymidine. The gels were lysed in 10% SDS (100-µl final volume), and the lysates were measured with a beta counter. Three independent experiments were performed in triplicate in the presence and absence of specific inhibitors of Rho, Rho kinase, mitogen-activated protein kinase kinase (MEK), PI3-K, and p38 mitogen-activated protein kinase (MAPK) at the same concentrations indicated above.

Immunohistochemistry
Paraffin sections of embryonic and adult mouse kidneys were stained with rabbit anti-mouse H-Ras (SC-520), R-Ras (SC-523), and TC21 (SC-883) (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies followed by incubation with goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) and Sigma Fast DAB chromogenic tablets (Sigma-Aldrich, St. Louis, MO).

Immunofluorescence
To determine the localization of zonula occludens-1 (ZO-1) and E-cadherin, UB cells were plated at low density (1 x 10³) on chamber slides in MEM containing 10% FCS and cultured at 37°C until cell patches formed (usually 72 h). Cells were then fixed in cold methanol for 10 min, blocked with 3% BSA for 1 h, and incubated with anti-ZO-1 (1:100; Zymed Laboratories, South San Francisco, CA) or anti-E-cadherin (1:100; BD Transduction Laboratories, Lexington, KY) antibodies at 4°C for 12 h. The cells were washed and incubated with the appropriate rhodamine-conjugated secondary antibody. Chamber slides were then processed as indicated above.

To examine cell morphology, serum-starved UB cells were plated in serum-free medium onto chamber slides coated with 10 µg/ml MG. After 4 h, the cells were fixed in 4% paraformaldehyde in PBS for 15 min and permeabilized in 0.1% Triton X-100 in PBS for 10 min. Cells were subsequently blocked with 3% BSA for 1 h and incubated with rhodamine-phalloidin (1:1000; Molecular Probes, Carlsbad, CA) for 1 h. Chamber slides were mounted with ProLong Antifade kit (Molecular Probes), and cells were imaged on a Zeiss Axiophot microscope and an RT Slider Spot digital camera.

To examine cell morphology of UB cells grown in 3D collagen I/MG gels (1500 cells/gel), the gels were fixed as described above at days 2, 4, and 6. Fixed gels were incubated with rhodamine-phalloidin (1:1000; Molecular Probes) in PBS for 3 h and then washed in PBS for 12 h. To determine the presence of lumens within the tubules, gels were fixed at day 6 in 4% paraformaldehyde in PBS for 20 min, washed with PBS for 1 h, and subsequently incubated in 30% sucrose in PBS for 12 h at 4°C. The gels were embedded in OCT, and 5-µm frozen sections were incubated with rhodamine-phalloidin as described above. All the samples were optically sectioned using a Zeiss LSM510 META confocal laser scanning microscope.

Immunoblotting
Twenty micrograms of total UB cell lysates were run onto a 10% SDS gel and subsequently transferred to nitrocellulose membranes. Membranes were then immunoblotted with polyclonal antibodies to mouse H-Ras, R-Ras, and TC21 (all 1:250; Santa Cruz Biotechnology) to determine the expression of endogenous and activated forms of these small G proteins; anti-rabbit FSP-1 antibody (1:100; a gift from Dr. E. G. Neilson, Vanderbilt University) (Strutz et al., 1995) to determine epithelial-mesenchymal transition; and anti-mouse E-cadherin antibody (1:250; BD Transduction Laboratories) to determine cell adhesion proteins. To determine the compartmentalization of the E-cadherin, the cells were fractionated into Triton X-100–soluble and –insoluble fractions, as described previously (Singh and Harris, 2004). Briefly, cells were scraped in cell lysis buffer (10 mM HEPES, pH 7.2, 1% Triton X-100, 100 mM NaCl, 2 mM EDTA, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/ml leupeptin, and soybean and lima bean trypsin inhibitors) and incubated at 4°C for 20 min, followed by centrifugation at 10,000 x g for 20 min. The resulting supernatant was considered the Triton X-100/detergent-soluble fraction. For the insoluble fraction, the pellet was dissolved by pipetting in pellet solubilization buffer (10 mM HEPES, pH 7.2, 1% SDS, 100 mM NaCl, 2 mM EDTA, 1 mM benzamidine, 1 mM PMSF, 10 µg/ml leupeptin, and soybean and lima bean trypsin inhibitors), and the proteins were released by sonication. The resulting preparation was incubated at 4°C for 20 min, followed by centrifugation at 10,000 x g for 20 min. Then, 20 µg of total cell lysates was analyzed by Western blot using anti-E-cadherin antibodies, as indicated above. To ensure equal loading, membranes were stained with Ponceau red.

To determine downstream signaling, 24-h serum-starved UB cells were removed from plates with trypsin followed by addition of 1 mg/ml trypsin inhibitor (Sigma-Aldrich). Cells were then centrifuged and resuspended in serum-free medium. Cells were kept in suspension for 30 min and replated on 10 µg/ml MG for 0, 30, 60, and 120 min (Hanks et al., 1992). Cells were washed twice with PBS and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail, 1 mM Na₃VO₄, and 1 mM NaF). Then, 20 µg of total proteins was run onto a 10% SDS gel and subsequently transferred to nitrocellulose membranes. Membranes were incubated with anti-phospho-ERK, anti-phospho-p38, anti-phospho-AKT, anti-ERK, anti-p38, or anti-AKT antibodies (all from Cell Signaling Technology, Beverly, MA) followed by the appropriate HRP-conjugated secondary antibodies. Immunoreactive bands were identified using enhanced chemiluminescence according to the manufacturer's instructions.

PAK1-PBD Pull Down Assay
PAK1-PBD-conjugated glutathione-Sepharose beads were prepared as described previously (del Pozo et al., 2000). We scraped 24-h serum-starved cells in 1ysis buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 200 mM NaCl, 1% NP-40, 5% glycerol, 0.05% Tween 20, 1 mM NaF, 1 mM Na₃VO₄, and proteinase inhibitor (Roche Diagnostics). Then, 800 µg of total cell lysates was added to 30 µl of PAK1-PBD–conjugated glutathione-Sepharose beads and incubated at 4°C for 1 h with gentle rocking. After four washes with washing buffer (25 mM Tris-HCl, pH 7.6, 30 mM MgCl₂, 40 mM NaCl, 1 mM dithiothreitol, and 1% NP-40), the beads were resuspended in 20 µl of 2x Laemmli's sample buffer, boiled for 5 min, and run on 15% SDS-PAGE. The gels were then transferred to a nitrocellulose membrane and incubated with anti-Rac1 or anti-Cdc42 (BD Transduction Laboratories) antibodies in 3% BSA at 4°C overnight. Immunoreactive proteins were visualized using a peroxidase-conjugated goat anti-mouse antibody and an ECL kit according to manufacture's protocol. Densitometry was performed using a UVP bioimaging system (UVP, Upland, CA).

Rho Kinase Assay
Rho kinase assay was performed using the Rho Kinase kit of Upstate Technology (Lake Placid, NY) following manufacturer's instructions. Briefly, 24-h serum-starved cells were scraped in a magnesium lysis buffer after which a glutathione S-transferase–tagged fusion protein, corresponding to residues 7-89 of mouse Rhotekin Rho binding domain was added to 800 µg of total cell lysates. The beads were then washed, reduced with 1 M dithiothreitol, and the supernatant and beads were subjected to SDS-PAGE. Immunoblotting was performed with an anti-Rho antibody (BD Transduction Laboratories). Densitometry was performed using a UVP bioimaging system.

Embryonic Kidney Culture
E12.5 kidneys were isolated from CD1 mouse embryos, where the day of the vaginal plug was designated as E0.5. The isolated kidneys were placed on 0.4-µm transwell inserts in 24-well plates. DMEM supplemented with 10% FCS was added to the top and bottom of the transwells. For the inhibitor experiments, 20 µM Y27632, 50 µM LY294002, 100 µM PD98059, and 100 µM PD169316 were added to the medium. These were the lowest inhibitor concentrations that induced an effect on UB branching morphogenesis and were not toxic to the cultures. The kidneys were grown for 72 h after which they were fixed in methanol for 10 min and washed twice in PBS containing 0.1% Tween 20 (PBST) for 10 min. Kidneys were subsequently incubated with anti-E-cadherin antibodies (1:100 in 2% goat serum in PBST) for 4 h at 4°C. The kidneys were then washed in PBST four times for 1 h and incubated with a fluorescein isothiocyanate-conjugated secondary antibody (1:100 in 2% goat serum in PBST) for 3 h at room temperature. The kidneys were then washed in PBST, placed on a slide with ProLong Antifade kit (Molecular Probes), and imaged using a fluorescence microscope (Nikon, Tokyo, Japan). The number of branches in each structure was counted, and the size of the UB was calculated by from the measured length and breadth.

Statistical Analysis
The Student's t test was used for comparisons between two groups, and analysis of variance using Sigma Stat software was used for statistical differences between multiple groups. A p 0.05 was considered statistically significant.

View larger version (169K): [in this window] [in a new window]   Figure 1. Localization of H-Ras, R-Ras, and TC21 during renal development. Mouse kidneys at different stages of development were stained with antibodies to H-Ras, R-Ras, and TC21. The arrowheads indicate UB-derived structures in the embryonic kidneys. Original magnification, 200x.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

Expression of Activated Forms of H-Ras and TC21, but Not R-Ras, in UB Cells Promotes Epithelial–Mesenchymal Transition
Based on the observations that 1) overexpression of H-Ras in MDCK inhibits tubulogenesis, whereas expression of activated R-Ras promotes tubulogenesis (Khwaja et al., 1998), and 2) there is a different temporal expression of the R-Ras family of proteins and H-Ras in the developing kidney (Figure 1), we developed murine UB cell populations expressing activated forms of H-Ras, R-Ras, and TC21 to determine their effects on branching morphogenesis.

Populations of UB cells expressing equal levels of H-Ras, R-Ras, and TC21 were generated using the LZRS-GFP vector and sorted by GFP expression by using flow cytometry (our unpublished data). Western blot analysis revealed that endogenous H-Ras, R-Ras, and TC21 are present in UB cells (Figure 2A, left) and that approximately 5 times the endogenous level of protein was present in the overexpressing cell populations (Figure 2A, right). Cell populations expressing more or less of the activated forms had similar effects on cell behavior to those shown in this study (our unpublished data).

View larger version (109K): [in this window] [in a new window]   Figure 2. Generation and characterization of UB cell populations expressing H-Ras, R-Ras, and TC21. (A) UB cells were infected with a retrovirus carrying either the empty vector (LZRS) or activated H-Ras, R-Ras, and TC21 as described in Materials and Methods. Expression of endogenous H-Ras, R-Ras, and TC21 in vector-transfected (LZRS) cells is shown in the left panel, whereas the right panel shows over expression of H-Ras, R-Ras, and TC21 in the different cell populations. Twenty micrograms of total UB cell lysate was used for Western blot analysis as described in Materials and Methods. (B) Typical morphology of the different UB cell populations cultured on plastic for 3 d. (C) The different UB cell populations indicated were grown on chamber slides for 3 d to allow clustering and subsequently stained with antibodies to E-cadherin or ZO-1. (D) Total cell lysates (20 µg) were immunoblotted for E-cadherin (E-cad) and GAPDH as described in Materials and Methods. Thirty micrograms of protein from Triton X-100–soluble (TS) and –insoluble (TI) fractions, prepared as described in Materials and Methods, was used to determine E-cadherin localization. (E) The levels of FSP-1 were analyzed in 20 µg of total UB cell lysates by Western Blot analysis as described in Materials and Methods. Membranes were incubated with anti-AKT antibody to confirm equal loading.

View larger version (84K): [in this window] [in a new window]   Figure 3. Increased branching morphogenesis in R-Ras and TC21 UB cells. UB cells (1.5 x 103) were plated in collagen I/MG gels as described in Materials and Methods. Six days later, the cells were imaged (A), and the number of branches was counted and evaluated (B). Values are means ± SD of three experiments performed in triplicate and are expressed as the percentage of branching relative to control. Differences between LZRS cells and H-Ras, R-Ras, or TC21 cells were significant (*p < 0.05). Bar, 50 µm.

H-Ras-, R-Ras-, and TC21-expressing UB Cells Undergo Alterations in Branching Morphogenesis as a Result of Changes in Cell Spreading, Migration, and Proliferation
UB cells undergo branching morphogenesis in 3D collagen I/MG gels after serum stimulation (Chen et al., 2004). To determine the effects of overexpression of activated H-Ras, R-Ras, and TC21 on UB cell branching morphogenesis, the cells were placed in collagen I/MG gels, stimulated with serum, and allowed to grow for 6 d before the number of branches was evaluated. There were markedly different phenotypes among the R-Ras, H-Ras, and TC21 cell populations. The H-Ras cells formed long, minimally branched tubules, the R-Ras cells formed large multibranched structures, whereas the TC21 cells branched significantly from a focal point, but the branches were short (Figure 3, A and B).

Based on the finding that activation of H-Ras and R-Ras plays a role in cell adhesion (Zhang et al., 1996), proliferation (Cox et al., 1994), and migration (Keely et al., 1999; Jeong et al., 2005), we investigated whether alterations of any of these specific cell functions account for the different branching morphogenic patterns shown in Figure 3. Interestingly, no differences in cell adhesion on MG were observed between the cell populations (our unpublished data). In addition, expression of the activated Ras constructs did not alter the levels of 1, 2, 3, 5, 6, 1, and 4 integrin expression (our unpublished data). The different UB cell populations did, however, demonstrate distinct morphologies when plated onto MG for 4 h. As shown in Figure 4A, UB cells transfected with LZRS vector only, spread on MG with pronounced rings of cortical actin present. In contrast, UB cells expressing activated H-Ras exhibited well defined actin stress fibers and developed lamellopodia, a phenotype compatible with their mesenchymal phenotype. R-Ras cells also exhibited an increase in stress fiber formation and marked ruffling at the cell edges. Despite the fact that the TC21 cells had features of EMT and showed increased FSP-1 expression (Figure 2E), these cells had a marked spreading defect.

View larger version (16K): [in this window] [in a new window]   Figure 4. Cell morphology, proliferation, and migration of H-Ras, R-Ras, and TC21 UB cell populations. (A) The different UB cell populations indicated were serum starved for 24 h and subsequently plated on 10 µg/ml MG-coated dishes. After 4 h, the cells were fixed and stained with rhodamine-phalloidin to visualize the cytoskeleton. Bar, 50 µm. (B) The different UB cell populations indicated were plated in transwell dishes coated with 1 µg/ml MG, and migration was evaluated 4 h after plating as described in Materials and Methods. The values indicate the mean ± SD of three independent experiments performed in triplicate. (C) The different UB cell populations indicated were plated within 3D collagen I/MG gels. After 2 d, cells were treated with [3H]thymidine (1 µCi/well) and incubated for a further 2 d. [3H]Thymidine incorporation was then determined as described in Materials and Methods. Values are means ± SD of three experiments performed in triplicates and are expressed as the percentage of proliferation relative to LZRS vector control. Asterisk (*) indicates statistically significant differences (p < 0.05) between the Ras cell populations and UB cells transfected with LZRS control vector.

To determine how the alterations in cell spreading, migration, and proliferation resulted in the different branching morphogenesis patterns, cells were grown in 3D gels and stained with rhodamine-phalloidin 2, 4, and 6 d after plating. As shown in Figure 5, striking differences between the cell populations was already evident at day 2, with increased cell number and branching in the R-Ras and TC21 cells compared with UB cells transfected with LZRS vector only or H-Ras cells. In addition, similar to the morphology shown in Figure 3A, cortical actin was present in the H-Ras cells compared with LZRS-transfected cells, whereas both the R-Ras and TC21 cells had a more rounded phenotype. By day 4, branched LZRS tubules with single parallel branched multicellular cords on either side of the lumen were seen. Similarly, H-Ras cells developed well defined but less well branched tubules with lumens, and the cortical actin was easily visualized. The R-Ras and TC21 cells were more rounded and showed increased branching with no dominant tubule at this time point compared with LZRS and H-Ras cells. Because of the presence of multiple multicellular cords on either side of the lumen, the lumens were difficult to distinguish in the R-Ras and TC21 cells. The phenotypes observed at day 4 were still evident, but more pronounced, by day 6. Staining of cross-sections of gels at day 6 revealed the presence of lumens with distinct morphologies in all the UB cell populations (Figure 5). The H-Ras cells formed well defined tubules and had a prominent cortical actin cytoskeleton, whereas the R-Ras and particularly the TC21 cells were less well spread and developed poorly formed lumens compared with LZRS-transfected UB cells.

View larger version (107K): [in this window] [in a new window]   Figure 5. Temporal changes in UB cell branching morphogenesis. UB cells (1.5 x 103) were plated in collagen I/MG gels as described in Materials and Methods. In the top three panels, the gels were fixed 2, 4, and 6 d after plating, stained with rhodamine-phalloidin, and subjected to confocal microscopy as described in Materials and Methods. In the lowest panel, frozen sections of gels kept in culture for 6 d were stained with rhodamine-phalloidin and subsequently analyzed by confocal microscopy. Arrows in the top three panels and asterisks in the bottom panel show the lumen of the tubules.

Together, these results suggest that the well-formed long tubules with less branching in the H-Ras cells are likely due to the alterations in the actin cytoskeleton and increased cell migration. In contrast, the short multibranched structures observed in TC21 cells are consistent with the inability of these cells to migrate, most likely because of abnormalities of the actin cytoskeleton. The multicellularity of the R-Ras and TC21 cells correlates well with the increased proliferation of these cells in 3D gels (Figure 4C).

H-Ras-, R-Ras-, and TC21-expressing UB Cells Differentially Activate Small GTPase Family Members and MAPKs
Rho-GTPases play a significant role in modulating the actin cytoskeleton in many cell types. Activation of RhoA promotes actin stress fiber assembly and focal adhesions, whereas activation of Rac1 and Cdc42 induces lamellapodia and filopodia, respectively (Millan and Ridley, 2005). Because of the major differences in the actin cytoskeleton in the various UB cell populations (Figures 4A and 5), we determined the levels of activated Rho-GTPases. As shown in Figure 6, increased levels of activated Rac1, Cdc42, and RhoA were detected in H-Ras cells compared with LZRS-transfected cells. In the R-Ras cells, an even greater increase in activated Rac1 and Cdc42, but not RhoA, was evident compared with H-Ras cells. The most striking feature of TC21 cells was the detection of extremely high levels of activated RhoA and less, but still significant, activation of Cdc42 compared with LZRS cells.

View larger version (49K): [in this window] [in a new window]   Figure 6. Activation of small GTPases by H-Ras, R-Ras, and TC21 UB cell populations. (A) The different UB cell populations indicated were serum starved for 24 h, and 800 µg of total cell lysates was used to detect levels of activated Rac, Cdc42, or Rho as indicated in Materials and Methods, whereas 20 µg of total cell lysates were used to detect the levels of total Rac, Cdc42, and Rho. (B) The bands shown in A were scanned, and their intensity was determined by densitometric analysis and expressed as active GTPases/total GTPase. A representative experiment is shown (a total of three experiments was performed).

View larger version (56K): [in this window] [in a new window]   Figure 7. H-Ras, R-Ras, and TC21 activate specific downstream signaling molecules in UB cells. The different serum-starved UB cell populations indicated were detached from plates and subsequently replated on 10 µg/ml MG for 0, 30, 60, and 120 min. Levels of phosphorylated as well as total Akt, ERK, and p38 were determined on total cell lysates (20 µg/lane) by Western blot analysis as described in Materials and Methods.

Branching in R-Ras and TC21 UB Cells Is Mediated by Activation of ERK and p38
Activation of the Rho GTPases (Maru et al., 1998; Rogers et al., 2003; Wozniak et al., 2003), PI3-K (Lavenburg et al., 2003), and the MAPK family members (Khwaja et al., 1998; Ishibe et al., 2003, 2004; O'Brien et al., 2004) has been shown to play a role in branching morphogenesis. Because these molecules are differentially regulated in the various UB cell populations (Figures 6 and 7), we investigated the role of these signaling molecules in branching morphogenesis. To do this, UB cell populations were grown in 3D collagen I/MG gels with or without Rho, ROCK, PI3-K, ERK, and p38 inhibitors in concentrations that were nontoxic to cells. The inhibition of the Rho downstream effector ROCK by Y27632 (as well as direct inhibition of Rho with C3; our unpublished data) completely inhibited the ability of all the UB cell population to undergo branching morphogenesis (Figure 8). Moreover, ROCK inhibition resulted in a mesenchymal phenotype and loss of cell–cell adhesion (Figure 8A). Treatment with the PI3-K inhibitor LY294002 (as well as wortmannin; our unpublished data) led to decreased length of tubules in both H-Ras and LZRS vector cells, whereas no effect was observed in R-Ras and TC21 cells (Figure 8A). Overall, the amount of branching was not affected by inhibition of PI3-K in any of the cell populations analyzed (Figure 8B). Inhibition of MEK by PD98059 resulted in decreased length and number of branches in vector control cells (Figure 8, A and B). In contrast, PD98059-treated H-Ras cells showed increased number, but reduced length of branches, thus resembling the phenotype observed in untreated vector control cells. MEK inhibition also decreased the length and number of branches in R-Ras and TC21 cells, although this effect was significantly more evident in TC21 cells. The greatest effect on branching morphogenesis was seen with the p38 inhibitor PD169316, because this inhibitor profoundly decreased the number of branches in all the cell populations analyzed with the exception of H-Ras cells (Figure 8, A and B).

View larger version (85K): [in this window] [in a new window]   Figure 8. Effect of kinase inhibitors on UB cell branching morphogenesis. (A) The different UB cell populations indicated were plated within 3D collagen I/MG gels in the presence or absence of 10 µM Y27632, 5 µM LY294002, 50 µM PD98059, or 50 µM PD169316. Representative images of tubelike structures taken 6 d after plating are shown. (B) The number of branching was quantified as described in Materials and Methods. Values are the means ± SD of three experiments performed in triplicate. Differences in branch number between LZRS cells and H-Ras, R-Ras, or TC21 cells in the presence or absence of inhibitors were significant (*p < 0.05).

View larger version (42K): [in this window] [in a new window]   Figure 9. Effect of kinase inhibitors on UB cell proliferation and migration. Cell migration (A) and proliferation (B) of the different UB cell populations indicates was evaluated as described in Figure 4 with the difference that cells were incubated with or without the various kinase inhibitors indicated. Values are the means ± SD of three experiments performed in triplicate. Differences in migration or proliferation between LZRS cells and H-Ras, R-Ras, or TC21 cells in the presence or absence of inhibitors were significant (*p < 0.05).

Together, these results suggest that activation of PI3-K plays a minor role in UB cell branching morphogenesis in wild-type and mutant cells, because it only mediates length of LZRS and H-Ras tubules. In contrast, ERK and p38 play a significant role in determining tubule length and branching of both wild-type and Ras-expressing UB cells. The enhanced basal activity of ERK and increased activity of the p38 pathway accounts for the increased branching of TC21 tubules compared with R-Ras.

UB Branching in Kidney Cultures Is Mediated by Signaling Pathways Similar to Those Identified in UB Cell Populations
To identify whether the same pathways controlling UB cell branching morphogenesis in vitro might also mediate UB branching morphogenesis in the developing kidney, E12.5 embryonic kidneys were grown on transwells with or without the ROCK, PI3-K, ERK, and p38 inhibitors described above. Similar to UB cells, the inhibitors had differential effects on UB growth and branching morphogenesis (Figure 10). The ROCK inhibitor Y27632 did not alter the UB size (Figure 10, A and B), but it significantly decreased UB branching (Figure 10C) compared with the untreated UB. Although 70% of UB branching was inhibited by Y27632, branching was completely inhibited in the UB cell system (Figure 8B). Inhibition of PI3-K by LY294002 resulted in a significantly smaller UB, compared with control (Figure 10, A and B). However, no significant changes in the branching was observed (Figure 10C), which parallels the results with the UB cell culture system (Figure 8B). Inhibition of ERK by PD98059 and p38 by PD169316 significantly diminished branching of the UB (Figure 10C), whereas it did not affect the UB size (Figure 10, A and B). This finding parallels the results obtained with the UB cell culture system (Figure 8B). Together, these results suggest that the signaling that mediates UB cell branching morphogenesis is similar to that of the UB in whole kidney cultures. Thus, the results extrapolated from the UB cell culture system may be relevant to UB branching morphogenesis in vivo.

View larger version (36K): [in this window] [in a new window]   Figure 10. Effect of kinase inhibitors on UB branching morphogenesis in embryonic kidney cultures. (A) E12.5 mouse kidneys were isolated and grown on transwells in DMEM and 10% FBS in the presence of no inhibitor (control), 20 µM Y27632, 50 µM LY294002, 100 µM PD98059, or 100 µM PD169316. After 3 d, the kidneys were fixed and stained with anti-E-cadherin antibodies as described in Materials and Methods. The relative area (B) and number of branches (C) of the treated UB compared with the untreated control were evaluated. Values are the mean ± SD of three experiments performed in triplicate. Asterisk (*) indicates significant differences (p < 0.05) between untreated and inhibitor treated kidney cultures.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The present study investigated the role of H-Ras, R-Ras, and TC21 in the developing collecting system of the kidney. R-Ras and TC21 are expressed from day E13.5, when most branching occurs, whereas H-Ras is only expressed from E17.5 when the already branched collecting system is primarily elongating (Cebrian et al., 2004). Using murine UB cells, we demonstrated that overexpression of activated H-Ras resulted in the formation of long unbranched tubules, whereas overexpression of activated R-Ras and TC21 resulted in increased UB cell branching morphogenesis, especially evident in the TC21 cells. Because most UB branching occurs early in development, it is conceivable that R-Ras and TC21 play a role in facilitating early branching and growth, whereas H-Ras might favor cell migration and tubule elongation that occurs later in development.

Little is known about expression of Ras family members in the developing and adult kidney. Our finding that H-Ras is detected in the adult collecting system confirms a report demonstrating expression of H-, K- and N-Ras in human collecting ducts (Kocher et al., 2003). To the best of our knowledge, this is the first data suggesting that temporal expression of Ras proteins may influence renal development, particularly UB branching morphogenesis.

One of the interesting findings in this study is that H-Ras, to some extent, and TC21 cells to a greater degree showed evidence of EMT, yet they displayed very different characteristics with respect to cell migration, F-actin distribution, and tubulogenesis. Both cell types demonstrated an increase in ERK and P38 activity, which might have accounted for the EMT. In this context, it has been shown that H-Ras–transformed mammary epithelial cells (Janda et al., 2002) and rat bladder carcinoma cells (Edme et al., 2002) undergo EMT because of increased ERK activation. Furthermore, p38 activation plays a role in TGF-–induced EMT in breast epithelial cells (Bhowmick et al., 2001; Bakin et al., 2002). Although TC21 cells revealed more signs of EMT than H-Ras cells, they failed to spread and migrate, which could be due to markedly increased levels of Rho A and significantly less Rac activation compared with H-Ras cells. Thus, although H-Ras and TC21 cells might activate similar effectors resulting in ERK and/or p38-dependent EMT, their differential activation of the Rho GTPases might explain the differences in the actin cytoskeleton and the inability of these cells to spread, migrate, and form elongated tubules. Unfortunately, it is difficult to identify a clear role for activated RhoA in UB cell branching morphogenesis in general and TC21 cells in particular because blocking Rho and ROCK activation resulted in complete inhibition of tubulogenesis in all the UB cells, including vector controls.

Based on the finding that activated forms of H-Ras, R-Ras, and TC21 can alter integrin affinity, cell adhesion, and migration (Cox et al., 1994; Zhang et al., 1996; Hughes et al., 1997, 2002; Sethi et al., 1999), major differences in integrin-dependent functions were anticipated in our UB cell populations. Surprisingly, no observable difference in cell adhesion among the UB cell populations was evident. That H-Ras–expressing UB cells show increased cell migration contrasts with the finding that overexpression of H-Ras decreases integrin affinity in Chinese hamster ovary cells (Hughes et al., 1997). In addition, the observation that R-Ras UB cells migrate as much as control cells and TC21 UB cells migrate significantly less, contrasts with data showing that activated mutants of R-Ras increase integrin affinity (Hughes et al., 1997), and both activated R-Ras- and TC21-transfected breast epithelial cells have increased cell migration and invasion (Keely et al., 1999). Interestingly, R-Ras- and TC21-induced cell migration and invasion are mediated by activation of integrin 21 (Keely et al., 1999), and UB cells do not express this integrin and interact with MG in an integrin 3- and 6-dependent manner (Chen et al., 2004). Thus, the effects of R-Ras and TC21 might be integrin and/or cell type specific.

By performing temporal imaging of the tubulogenesis assays, it was clear that lumen formation in UB tubules occurs at early time points (days 2–4) and is not dependent on apoptosis of cells, as seen in mammary acini (Debnath et al., 2002). In addition, UB cell tubulogenesis does not require cyst formation seen with HGF-induced tubulogenesis in MDCK cells (O'Brien et al., 2004), suggesting that mechanisms of UB and MDCK tubulogenesis are different. This discrepancy might explain why the H-Ras UB cells developed long, unbranched, well-defined tubules, whereas MDCK-overexpressing activated H-Ras cells developed diffusely spread clusters lacking organized structures when grown in 3D-collagen gels (Khwaja et al., 1998). Although the H-Ras mutant phenotypes differed between UB and MDCK cells, the rapid growth and increased branching induced by R-Ras cells was similar in both cell types (Khwaja et al., 1998). The effect of activated mutants of TC21 on MDCK cells is not reported. However, G38V R-Ras and 72L TC21 mutants in a T47D breast cell population resulted in disruption of cell differentiation and polarity when grown in collagen gels (Keely et al., 1999). Thus, the phenotypes of epithelial cells expressing activated mutants of Ras proteins are cell type specific.

We were surprised to find that the activated Ras mutants did not alter the levels of activated PI3-K and that inhibition of PI3-K had little effect on tubule branching, because expression of all three Ras mutants resulted in PI3-K activation in other epithelial cells (Khwaja et al., 1998; Berrier et al., 2000; Murphy et al., 2002; Rincon-Arano et al., 2003), and activation of PI3-K is required for HGF-induced tubulogenesis in MCDK cells (Khwaja et al., 1998). Similar to MDCK cells, ERK activity was required for UB cells to undergo branching morphogenesis in 3D gels (Khwaja et al., 1998; O'Brien et al., 2004; Hellman et al., 2005). The primary difference in ERK activation between the three Ras mutants was the increase in its basal activity in the H-Ras and TC21 cells. Sustained activation of Raf has been shown to be sufficient for the induction of EMT in MDCK cell (Lehmann et al., 2000; O'Brien et al., 2004), suggesting that constitutive activation of the ERK pathway might play a major role in the EMT induction observed in the H-Ras and TC21 cells.

Our observation that p38 activation is required for UB branching is consistent with the fact that BMP 7 induced IMCD cell tubulogenesis is a p38-mediated event (Hu et al., 2004). p38 activity was increased in all the UB cells expressing Ras mutants, and the potent effect of p38 inhibition on both R-Ras and TC21 UB cell branching suggested this signaling pathway is the major mediator of the excessive branching in these cells. Thus, regulation of p38 by Ras proteins seems to play an even more a critical role than ERK in UB branching morphogenesis in vitro.

In both the whole kidney and UB culture systems, inhibition of ROCK had the most dramatic effects on branching morphogenesis. The significantly diminished branching of UB cell and mouse organ cultures by the p38 inhibitor contrasts with the results of a day 15 rat organ culture system, where p38 inhibition decreased metanephros growth, but did not change UB branching (Hida et al., 2002). Diminution of branching by the MEK inhibitor in both culture systems agreed with other studies where ERK was shown to be required for UB branching morphogenesis (Fisher et al., 2001). Although PI3-K inhibition did not significantly alter branching morphogenesis in either the UB cell or organ culture system, inhibiting this pathway did decrease UB cell migration and UB size in organ culture. PI3-K has been shown to be essential for the initial outgrowth of the UB and branching morphogenesis in mice at the E11.5 stage, which is earlier than the experiments we performed (Ostrom et al., 2000). Because the effects of the inhibitors on the developing UB in whole organ were similar, but not identical, to UB cell branching morphogenesis, we propose that the UB cell culture is a reasonable model to predict the possible effects of Ras expression in UB development in vivo.

In summary, during renal development members of the R-Ras family, R-Ras and TC21, are expressed when maximal branching of the UB occurs, whereas H-Ras is expressed at later stages when elongation of the tubules takes place (Cebrian et al., 2004). The in vitro evidence that activated mutants of R-Ras and TC21 induced a significant increase in UB branching morphogenesis, whereas activated H-Ras decreased branching, but induced tubule elongation, suggests that the specific temporal expression of Ras proteins might play a role in regulating branching morphogenesis of the developing UB in vivo.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Cancer Institute/National Institutes of Health Grant R01-CA94849 (to A. P.), National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK074359 (to A. P.), National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK 69921 (to R. Z.), and an Advanced Career Development and Merit Award from the Department of Veterans Affairs (to R. Z.).

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-08-0800) on February 8, 2006.

Address correspondence to: Roy Zent (roy.zent{at}vanderbilt.edu).

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Kinsella, T. M., and Nol