Mitogen-Activated Protein Kinase (MAPK) Pathway Regulates Branching by Remodeling Epithelial Cell Adhesion

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Abstract

Although the growth factor (GF) signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK) pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA) protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions. © 2014 Ihermann-Hella et al.

Figures

  • Figure 1. Localization of MAPK pathway activity in developing kidney. (A–A9) Representative cross sections of wild type E11.5 kidneys stained with anti-phospho-ERK1/2 (red) demonstrate MAPK pathway activity both in the ureteric (UB) bud, visualized by calbindin staining (green), and metanephric mesenchyme (arrow). (B–B9) In E13.5 wild type kidneys, pERK1/2 localization in the ureteric bud epithelium is concentrated to UB tips where the staining is unevenly distributed among the epithelial cells (arrowheads), which all express UB marker calbindin (green). Additional pERK1/2 staining is detected in nephron primordia (arrows). (C) Chromogenic pERK1/2 staining on E14.5 wild type kidneys shows strong but heterogeneous MAPK activity in UB tips with lack of activity in sporadic cells (red arrowheads). (D) Ret expression in the ureteric bud tips of E13.5 wild type kidney as detected by in situ hybridization of mRNA on vibratome sections. In A and B, nuclei are labeled with Hoechst. Scale bars: A–B 50 mm, C–D 500 mm. doi:10.1371/journal.pgen.1004193.g001
  • Figure 2. MAPK co-localizes with G1/S-phase markers in the ureteric bud epithelial cells. (A–A9) Confocal images of the wild type Wolffian duct at E10.5, including the region that will give rise to the UB, demonstrate MAPK activity (red) only on the side where kidney mesenchyme is located (m). No pERK1/2 staining was detected in the mesenchyme at this stage. The presumptive UB shows a typical appearance of pseudo-stratified epithelium where cell nuclei (blue) are located at several levels of epithelium stained with E-cadherin (green). Scattered MAPK activity (red) localizes to both cytoplasm and nuclei, where it is detected at different levels (arrows) of epithelium (E-cadherin, green). Mitotic, pHH3-positive (yellow) nuclei (arrowhead) located in apical surface of the epithelium shows no pERK1/2. (B–B9) MAPK pathway is active (red) during and after DNA replication as seen by pERK1/2 staining in some UB epithelial cells that have incorporated EdU (white in B, arrowheads) during an hour pulse. E-cadherin (green) marks cell borders and Hoechst (blue) visualizes nuclei while B9 shows only pERK1/2. (C) MAPK pathway activity (red) and pHH3 (white) in UB tips of E13.5 wild type kidneys. Arrowheads mark the mitotic epithelial cells with pHH3 label, which lack MAPK activity as seen in C9. Arrows indicate UB tips. (D–F9) Confocal stack through E13.5 wild type UB tip shows pERK1/2 (red) in cytoplasm and nuclei of tip cells but lacking in mitotic, pHH3+ (white, arrowheads in C, D9, E9 and F9) and some other cells (asterisks). Scale bars: A&C 50 mm, B, D–F 20 mm. doi:10.1371/journal.pgen.1004193.g002
  • Figure 3. Genetic ablation of MAPK pathway specifically in ureteric bud epithelium results in severe renal hypodysplasia. (A) Newborn kidneys with Mek1 deleted specifically in ureteric bud (UB) derivatives are comparable to wild type kidneys. (B) Absence of three out of four Mek1 and Mek2 alleles is enough to support normal kidney development as shown in newborn Hoxb7CreGFP;Mek1F/+;Mek2-/- kidneys. (C) Simultaneous lack of Mek1 and Mek2 in UB results in very small kidneys with hydroureters (arrow). Histology of newborn (D) control and (E) dko kidneys demonstrates huge cysts (yellow arrowheads) and reduced nephron epithelium in double knockout newborns. Calbindin staining visualizes numerous UBs and collecting ducts in (F) newborn control kidney whereas (G) the amount of collecting duct epithelia is minimal and dilated in dko kidneys. Asterisks mark glomeruli; a, adrenal gland; b, bladder; CD, collecting duct; k, kidney. Scale bars: A–C 1 mm, D–G 100 mm. doi:10.1371/journal.pgen.1004193.g003
  • Figure 4. Normal ureteric bud outgrowth in dko kidneys is followed by severely compromised branching morphogenesis. (A–F) Time lapse imaging of kidneys cultured for 48 h, and ureteric buds visualized by the green fluorescent protein tag encoded by the Hoxb7CreGFP construct. (A) In control kidney, ureteric bud is formed and swollen at E11.5, followed by branching (arrow) (B) 24 h later. (C) At 48 h, several generations of branches (average UB tip number: 10.5, n = 6) have been generated in the control kidney. (D) The UB formation in dko kidney at E11.5 is indistinguishable from the control kidney. (E) At 24 h, branching morphogenesis is progressing very slowly in the dko kidney as indicated by deepening of the cleft (arrowhead) on top of the developing T-bud, which however fails to complete and further generate secondary branches (arrows, average tip number: 3.8, n = 5) even at 48 h (F). (G–H) E13.5 ureteric epithelium visualized by calbindin (green) staining, followed by 3- dimensional reconstruction from confocal optical sections, shows the typical shape and distribution of UB tips at the surface of control kidney (G). Note that the control UBs are distributed over the entire surface area of the kidney, while in (H) dko kidney UB tips are very infrequent due to defective branch formation, leaving large areas of the kidney surface devoid of UB epithelium. (I–J) Calbindin staining on E13.5 kidneys shows several UB tips and branches (brown) in (I) control but only few in (J) dko. Scale bar: Scale bars: A–F 500 mm, G–H 50 mm, I–J 250 mm. doi:10.1371/journal.pgen.1004193.g004
  • Figure 5. Relationship of intracellular MAPK pathway to RET receptor tyrosine kinase signaling. In situ hybridization of the RET signaling target Cxcr4 in E13.5 (A) control kidney shows expression in UB tips (arrow) as well as in kidney mesenchyme, while expression is lost in (B) UB tip (arrow) of dko kidney. (C–D) Another RET signaling target Dusp6, which is a negative regulator of MAPK activity, shows no changes (arrows) in the absence of MAPK pathway activity at E13.5. (E) Table summarizing the expression results of RET signaling targets in UB of dko kidneys. Scale bar: A–D 500 mm. doi:10.1371/journal.pgen.1004193.g005
  • Figure 7. Lack of MEK1/2 function in UB cells intensifies cell surface localization of FA proteins and E-cadherin. (A) Baseline MAPK activity as measured by phosphorylation of ERK1/2 is dose-dependently inhibited by UO126 in 2 h. (B) Paxillin phosphorylation on serine 83 is slightly increased by addition of FBS but greatly reduced by inhibition of MEK1/2 function. (C–D) Control and (E–F) UO126-treated UB cells (15 mM for 4 h) were double-stained with E-cadherin (green), and pPaxillin (white). (C) and (E) show overlay of E-cadherin and nuclei (Hoechst, blue), while pPaxillin is separately shown in (D) and (F). Addition of UO126 increased membrane-associated E-cadherin and FA proteins (arrowheads). (G–H) Co-staining of phalloidin (green) with vinculin (red) demonstrated increase in vinculin at cell-cell contacts upon 4 h of MAPK inhibition. G9 and H9 show only vinculin. (I–N) Primary UB cells originating from E11.5 UBs isolated from (I–K) control and (L–N) dko kidneys. E-cadherin in (I) control cells appears in thick sheet like structures while it is fuzzy and spiky in (L) dko cells. Both paxillin (J, M) and vinculin (K, N) are intensified in FAs of dko cells (M–N) while are more diffusely distributed in control cells (J–K). Additionally to amplified FA localization, vinculin is also intensively distributed to cell surfaces (arrowheads). Scale bar: 20 mm. doi:10.1371/journal.pgen.1004193.g007
  • Figure 8. MAPK pathway activity is required for normal E-cadherin localization and epithelial cell adhesion. (A) Merged montage of confocal image through E13.5 control and (B) dko UB stained with calbindin (green) and E-cadherin (red) to show the cell-cell contacts. (C–D) Maximal intensity Z projections of stacks shown in A and B, respectively. Arrowheads point to the baso-lateral points, which show only occasional weak Ecadherin in control UB (A, C), but strong and widespread localization in the UB lacking MAPK activity (B, D). Asterisk indicates stronger intensity at lateral membranes in dko epithelium. (E) Electron microscopy image of E12.5 control UB shows continuous cell-cell contacts (arrows) while (F) in dko UB, the connections are disrupted at several sites (arrows), but the sites where they are maintained (arrowhead) appear electron dense. Also extracellular space (asterisk) between UB epithelial cells and metanephric mesenchymal (MM) cells is enlarged in dko kidneys. Scale bars: C–D 10 mm, E–F 2 mm. doi:10.1371/journal.pgen.1004193.g008

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Ihermann-Hella, A., Lume, M., Miinalainen, I. J., Pirttiniemi, A., Gui, Y., Peränen, J., … Kuure, S. (2014). Mitogen-Activated Protein Kinase (MAPK) Pathway Regulates Branching by Remodeling Epithelial Cell Adhesion. PLoS Genetics, 10(3). https://doi.org/10.1371/journal.pgen.1004193

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