QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02777

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ogawa, K. Articles by Tsuyama, S. Search for Related Content PubMed PubMed Citation Articles by Ogawa, K. Articles by Tsuyama, S. Social Bookmarking                         What's this?

EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases

¹ Department of Veterinary Anatomy, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
² The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
³ Department of Molecular and Cell Biology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan

View larger version (21K): [in a new window]   Fig. 1. Expression of B-class Eph receptors and ephrins in the adult mouse kidney. (A,B) Amplification of ephrin-B and EphB mRNAs by RT-PCR. (A) Substantial levels of endogenous ephrin-B1, EphB2 and EphB6 are amplified from adult mouse kidney. Bands were not detected in controls in which the RT reaction was omitted (not shown). (B) Amplification of ephrin-B1, EphB2 and EphB6 from the cortex and the medulla of the kidney. (C) Densitometric quantification of the amplification levels of ephrin-B1, EphB2 and EphB6 mRNA in the cortex and medulla of the kidney. Data from three independent experiments, normalized to the levels of the GAPDH amplification products, are shown as mean ± s.d. Comparisons were performed using unpaired t-test. EphB2 expression in the medulla is significantly higher than in the cortex (P=0.002), whereas expression levels of ephrin-B1 and EphB6 are not significantly different between the two (P=0.407 and 0.692, respectively). (D) EphB2 tyrosine phosphorylation in adult mouse kidney. Lysates from the whole kidney were immunoprecipitated with anti-EphB2 antibodies. Immunoprecipitates were separated by SDS-PAGE and probed by immunoblotting with antibodies as indicated at the right.

View larger version (127K): [in a new window]   Fig. 2. Localization of ephrin-B1, EphB2, EphB4 and EphB6 in the adult mouse kidney. Immunoperoxidase labeling of frozen sections with anti-ephrin-B1 (A), anti-EphB2 (B), anti-EphB4 (C) and anti-EphB6 (D) antibodies. (A) Ephrin-B1 immunoreactivity is broadly expressed in the nephron. In the proximal tubules (PT) of cortex, ephrin-B1 is localized in the basolateral membranes. Upper panel, anti-ephrin-B1p antibody; lower panels, anti-ephrin-B1e antibody. (B) EphB2 immunoreactivity is distinctly restricted to the medulla and absent from the cortex. EphB2 is localized in medullary tubules, including the distal straight tubules (DST) and the thin limb of the loop of Henle (TL). In the distal straight tubules, EphB2 is concentrated in the basal infolds. Upper panel, anti-EphB2p antibody; lower panel, anti-EphB2e antibody. (C) EphB4 is primarily localized in capillaries of the medulla (arrows in the lower panels). Faint immunoreaction of EphB4 is present in collecting duct (CD) in the cortex. (D) EphB6 immunoreactivity is primarily localized in tubules of the cortex and the outer medulla, including proximal tubules and distal tubules. In the distal straight tubules (DST) EphB6 is concentrated in the basal infolds and in the proximal tubules (PT) EphB6 is concentrated in granular structures of the cytoplasm. (E) There is no immunoreaction in the control without the primary antibodies. C, cortex; IM, inner medulla; OM, outer medulla; CD, collecting duct; DT, distal tubule; DST, distal straight tubule; PT, proximal tubule; TL, thin limb of the loop of Henle. Sections were developed with NiCl2 (upper panel in A and B) or without NiCl2 (lower panels in A and B, and all panels in C, D and E) and the sections in the lower panels except for the lower left of C were counterstained with hematoxylin. (F) Schematic drawings showing the expression patterns of ephrin-B1 (yellow), EphB2 (green), and EphB6 (blue) and predicted EphB2 tyrosine phosphorylation levels (dark green, high; light green, low) in the nephron. CD, collecting duct; CT, connecting tubule; DCT, distal convoluted tubule; DST, distal straight tubule (also known as thick ascending limb of loop of Henle); PCT, proximal convoluted tubule; PST, proximal straight tubule; TL, the thin limb of loop of Henle.

View larger version (24K): [in a new window]   Fig. 3. Expression of ephrin-B1, EphB2, EphB6 and the cytoplasmic adaptor protein Dishevelled in cortical and medullary tubule cells. (A) RT-PCR amplification of mRNA prepared from cultured cortical and medullary tubule cells shows that the cells in culture retain ephrin-B1, EphB2 and EphB6 expression patterns similar to those in the regions of the kidney from which the cells are derived (see Fig. 1B,C). (B) Densitometric quantification of ephrin-B1, EphB2 and EphB6 amplification products from cortical and medullary tubule cells. Data from three independent experiments, normalized to the levels of the GAPDH amplification products, are shown as mean ± s.d. Comparisons were performed using unpaired Student's t-test. The levels of the EphB2 and EphB6 amplification products are significantly higher in medullary than in cortical tubule cells (P=0.0001 and P=0.009, respectively), whereas the expression level of ephrin-B1 is not significantly different between the two (P=0.367).

View larger version (67K): [in a new window]   Fig. 4. Treatment with ephrin-B1–Fc induces retraction of medullary tubule cells plated on Matrigel-coated surfaces. (A) Cells were stimulated with 1 µg/ml ephrin-B1–Fc or 1 µg/ml human Fc for the indicated time periods. Cell lysates were immunoprecipitated with anti-EphB2 antibody. The immunoprecipitates were separated by SDS-PAGE, probed by immunoblotting with phosphotyrosine (PY) antibodies, and reprobed for EphB2. (B,C) Phase-contrast time-lapse microscopy pictures at a low (B) and high (C) magnification. Cells were stimulated with 1 µg/ml ephrin-B1–Fc and a series of phase-contrast images of the same field were obtained at the indicated times. Treatment with ephrin-B1–Fc leads to dramatic changes in cell morphology. Cell retraction is particularly prominent at the free edges of the cells (arrowheads in B) and partial separation between the adjacent cell surfaces of neighboring cells is also evident (arrow in C). (D) Morphological changes are induced by ephrin-B1–Fc but not EphB2-Fc treatment. Cells were stimulated with ephrin-B1–Fc (1 µg/ml) or EphB2-Fc (1 µg/ml) with or without crosslinking with anti-human Fc antibody (0.25 µg/ml). 1 µg/ml Fc with or without anti-human Fc antibody, or vehicle only, were used as controls. Phase-contrast images of the same field were obtained at 0 minute and 15 minutes after beginning the stimulation and the fraction of retracting cells at the 15 minutes time point was calculated. Only cells at the periphery of the epithelial sheets were quantitated for statistical analysis. Data from three independent experiments are shown as mean ± s.d. Comparisons were performed using one-way factorial ANOVA.

View larger version (16K): [in a new window]   Fig. 5. EphB2 activation promotes cell attachment to Matrigel-coated surfaces. Medullary tubule cells were plated on wells coated with Matrigel alone or together with ephrin-B1–Fc (0.3 µg/cm2) and allowed to adhere for 40 minutes. Ephrin-B1–Fc (2.0 µg/ml) or human Fc (2.0 µg/ml) with or without crosslinking with anti-human Fc antibodies (0.5 µg/ml) was also added in soluble form at the time of plating. Cell attachment measurements from three independent experiments are shown as mean ± s.d. Values were normalized to the control (stimulation with human Fc). Comparisons were performed using one-way factorial ANOVA. Exogenous ephrin-B1–Fc significantly stimulates cell attachment (P=0.004).

View larger version (109K): [in a new window]   Fig. 6. Rearrangement of focal adhesions in medullary tubule cells is induced by treatment with ephrin-B1–Fc but not EphB2-Fc. Cells plated on Matrigel were stimulated with 1 µg/ml ephrin-B1–Fc, EphB2-Fc or human Fc for 15 minutes and stained with an anti-vinculin antibody. Focal adhesions revealed by vinculin staining are much more prominent in the cells stimulated with ephrin-B1–Fc, but remain unchanged in the cells stimulated with EphB2-Fc. Left panels, lower magnification; right panels, higher magnification.

View larger version (89K): [in a new window]   Fig. 7. Ephrin-B1–Fc-induced retraction is antagonized by a peptide that selectively binds to EphB2 and blocks ephrin binding. (A) Representative examples of cell morphologies are shown. Upon treatment with 1.5 µg/ml ephrin-B1–Fc, medullary tubule cells retracted at the periphery, leaving behind spike-like protrusions (arrows). Preincubation of cells with the SNEW peptide inhibited cell retraction induced by ephrin-B1–Fc, whereas a control peptide that does not bind any Eph receptor did not inhibit cell retraction. (B) Quantitation of the percent of cells with spike-like protrusions with or without ephrin-B1–Fc and in the presence or absence of 360 µM SNEW peptide is shown. Data from four independent experiments are shown as mean ± s.d.

View larger version (98K): [in a new window]   Fig. 8. EphB activation by ephrin-B1–Fc increases RhoA activity and decreases Rac1 activity, and the RhoA kinase inhibitor Y27632 antagonizes the effects of ephrin-B1–Fc in primary cultures of medullary tubule cells. (A) In cells stimulated for 15 minutes with 1 µg/ml ephrin-B1–Fc, or 1 µg/ml Fc as a control, activated RhoA was isolated by pull-down with a GST fusion protein of the Rho-binding domain of Rhotekin (GST-RBD). Proteins bound to GST-Rhotekin were separated by SDS-PAGE and probed by immunoblotting with an anti-RhoA antibody. The levels of active RhoA in ephrin-B1–Fc-treated cells were normalized to those in Fc control-treated cells. Data from three independent experiments are shown as mean ± s.d. Ephrin-B1 Fc treatment increased the level of activated RhoA by 1.7 fold (P=0.002). (B,C) Phase-contrast time-lapse microscopy photographs. Cells were stimulated with 1 µg/ml ephrin-B1–Fc in the presence of 10 µM Y27632 and a series of phase-contrast images of the same field were obtained at the indicated times. Y27632 was added 10 minutes before (B) and 20 minutes after (C) the addition of ephrin-B1–Fc. Pretreatment with the Y27632 inhibitor essentially abolished the effects of ephrin-B1–Fc (B). Treatment with the inhibitor after cell retraction has begun reverses the effects within 5 minutes (B, arrowheads). (D) Quantification of the percentage of retracting cells stimulated with 1 µg/ml ephrin-B1–Fc under the presence of 10 µM Y27632. Y27632 and ephrin-B1–Fc are added at the same time courses as B and C. Phase-contrast images of the same field were obtained: for the Y27632 addition before the ephrin-B1–Fc stimulation at 0 minute, 10 minutes (after the addition of Y27632) and 30 minutes (20 minutes after the stimulation with ephrin-B1–Fc); for the Y27632 addition after the stimulation at 0 minute, 20 minutes (after the stimulation with ephrin-B1–Fc), and 30 minutes (10 minutes after the addition of Y27632). The fraction of retracting cells at the 10 and 30 minutes time point (open column), and 20 and 30 minutes (dotted column) was calculated, respectively. Only cells at the periphery of the epithelial sheets were quantitated for statistical analysis. Data from three independent experiments are shown as mean ± s.d. (E) Focal adhesion rearrangements induced by treatment with ephrin-B1–Fc are blocked by pretreatment with the Y27632 Rho kinase inhibitor. Medullary tubule cells plated on Matrigel were preincubated with 10 µM Y27632 for 5 minutes and then stimulated for 15 minutes with 1 µg/ml ephrin-B1–Fc, or human Fc as a control. The cells were then stained with an anti-vinculin antibody. (F) In cells stimulated for 5 minutes and 15 minutes with 1 µg/ml ephrin-B1–Fc, or for 15 minutes with 1 µg/ml Fc as a control, activated Rac1 was isolated by pull-down with a GST fusion protein of the Rho-binding domain of PAK1 (GST-PBD). Proteins bound to GST-PAK1 were separated by SDS-PAGE and probed by immunoblotting with an anti-Rac1 antibody. The levels of active Rac1 in ephrin-B1–Fc-treated cells were normalized to those in Fc control-treated cells. Data from three independent experiments are shown as mean ± s.d.

View larger version (26K): [in a new window]   Fig. 9. Schematic drawings illustrating possible effects of EphB2 signaling in medullary kidney epithelial cells. On the basis of the effects of ephrin-B1–Fc treatment in cultured medullary tubule cells, we propose that, in the kidney, EphB2 might regulate the geometry of the basal infolds and the gaps between cells through membrane retraction and remodeling of cell-matrix adhesion sites.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter