Integrin-linked kinase mediates bone morphogenetic protein 7-dependent renal epithelial cell morphogenesis

Abstract

Bone morphogenetic protein 7 (BMP7) stimulates renal branching morphogenesis via p38 mitogen-activated protein kinase (p38(MAPK)) and activating transcription factor 2 (ATF-2) (M. C. Hu, D. Wasserman, S. Hartwig, and N. D. Rosenblum, J. Biol. Chem. 279:12051-12059, 2004). Here, we demonstrate a novel role for integrin-linked kinase (ILK) in mediating renal epithelial cell morphogenesis in embryonic kidney explants and identify p38(MAPK) as a target of ILK signaling in a cell culture model of renal epithelial morphogenesis. The spatial and temporal expression of ILK in embryonic mouse kidney cells suggested a role in branching morphogenesis. Adenovirus-mediated expression of ILK stimulated and expression of a dominant negative ILK mutant inhibited ureteric bud branching in embryonic mouse kidney explants. BMP7 increased ILK kinase activity in inner medullary collecting duct 3 (IMCD-3) cells, and adenovirus-mediated expression of ILK increased IMCD-3 cell morphogenesis in a three-dimensional culture model. In contrast, treatment with a small molecule ILK inhibitor or expression of a dominant negative-acting ILK (ILK(E359K)) inhibited epithelial cell morphogenesis. Further, expression of ILK(E359K) abrogated BMP7-dependent stimulation. To investigate the role of ILK in BMP7 signaling, we showed that ILK overexpression increased basal and BMP7-induced levels of phospho-p38(MAPK) and phospho-ATF-2. Consistent with its inhibitory effects on IMCD-3 cell morphogenesis, expression of ILK(E359K) blocked BMP7-dependent increases in phospho-p38(MAPK) and phospho-ATF-2. Inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, failed to inhibit BMP7-dependent stimulation of ILK activity, suggesting that ILK functions upstream of p38(MAPK) during BMP7 signaling. We conclude that ILK functions in a BMP7/p38(MAPK)/ATF-2 signaling pathway and stimulates epithelial cell morphogenesis.

FIG. 1.

ILK is expressed in the UB and metanephric mesenchyme of E13.5 kidneys and in the collecting duct system at E17.5. E13.5 and E17.5 mouse kidneys were sectioned and stained with nonimmune immunoglobulin G (control) or ILK antibody (anti-ILK). Sections demonstrate ureteric bud (UB), glomerular progenitors (GP), cortex (Ctx), medulla (Med), collecting duct (CD), and glomeruli (G). E13.5 fields shown are magnifications of ×400, and E17.5 fields are magnifications of ×100.

FIG. 2.

ILK controls ureteric bud morphogenesis in embryonic kidney explant cultures. (A) Embryonic (E13.5) mouse kidneys were cultured as explants as described in Materials and Methods. Adenoviruses biscistronically expressing GFP and ILK, dominant negative ILK (GFP-dnILK), or control GFP virus were added to explant cultures at a concentration of 10⁵ PFU/ml. Fluorescent microscopic analysis of GFP expression in whole-mount preparations at 5 days postinfection indicated efficient infection by each virus. (B) Embryonic explants were infected at increasing doses with ILK adenoviruses, as in panel A. At 5 days postinfection, explants were fixed and stained with DBA for specific visualization of ureteric bud branches. Images in both panels are representative of 20 explants examined for each viral infection group.

FIG. 3.

BMP7 and EGF induce morphogenesis and ILK activity.(A) IMCD-3 cells were cultured in three-dimensional collagen gels containing 5% fetal calf serum, 0.25 nM BMP7, or 20 ng of EGF/ml. After 48 h, tubule progenitors were quantitated as described in Materials and Methods. The mean numbers of progenitors (± standard deviation) per microscope field are as follows: serum stimulated, 55 ± 5; BMP7, 75 ± 7; and EGF, 115 ± 9. The statistical significance of BMP7 and EGF stimulations is indicated, as determined by a two-tailed Student's t test. B. IMCD-3 cells were grown in standard two-dimensional cultures and serum starved for 18 h prior to stimulation for 15 min with 0.25 nM BMP7 or 20 ng of EGF/ml. ILK activity was assayed by ILK immune complex kinase assays with exogenous MBP substrate. Relative quantitation was by densitometry of bands. Results are from three independent determinations, and error bars indicate standard errors of the mean. Inset is a representative blot of ³²P incorporation into MBP in the kinase assays.

FIG. 4.

ILK activity induces IMCD-3 tubule progenitors. IMCD-3 cells were infected with Ad-ILK^WT, dominant negative Ad-ILK^E359K, or “empty” Ad^CONTROL viruses (multiplicity of infection [MOI] = 5). At 24 h postinfection, cells were cultured in collagen gels containing 5% serum, and formation of tubule progenitors was quantified 48 h after seeding. Bars represent data from five independent determinations. White arrows indicate linear structures that were enumerated as tubule progenitors. The inset shows a Western blot with expression of myc-tagged ILK constructs at different MOI values. Significant differences between Ad-ILK^WT cultures and control or E359K-infected cultures were determined by a two-tailed Student's t test.

FIG. 5.

Dominant negative ILK mutant blocks BMP7-induced IMCD-3 morphogenesis. (A) Cells were cultured in collagen gels containing 5% serum, 0.25 nM BMP7, or 20 ng of EGF/ml, and induction of morphogenesis was quantitated after 48 h. BMP7-induced morphogenesis in Ad^CONTROL-infected cells was 121 ± 6 per microscope field, decreasing to 21 ± 2 tubule progenitors per field in Ad-ILK^E359K-infected cells. EGF-stimulated morphogenesis was similarly decreased, from 143 ± 5 to 52 ± 8 per field, by Ad-ILK^E359K infection. The statistical significance (n = 6; P < 0.05) between Ad^CONTROL and Ad-ILK^E359K in all treatment cases was determined by a two-tailed Student's t test. (B) Cells were seeded into collagen gels in the presence of 5% serum, with or without 5 μM KP-392 ILK inhibitor. After 48 h, morphogenesis was visually quantified (Fig. 4B). Serum-stimulated morphogenesis was inhibited from 28 ± 2 to 12 ± 4 progenitors/field, and EGF stimulation was inhibited from 45 ± 6 to 10 ± 1 progenitors/field by the KP-392 ILK inhibitor. The dimethyl sulfoxide vehicle controls were at the same dilution as in the inhibitor-treated gels. The significant difference in EGF stimulation between vehicle and KP-392 treated cultures is indicated, calculated by a Student's t test as described in Materials and Methods.

FIG. 6.

BMP7 induction of morphogenesis and ILK activation are PI 3-K dependent. (A) IMCD-3 cells were seeded in collagen gels treated with BMP7 and EGF as shown in Fig. 4, with and without 10 μM LY294002. Tubule progenitors were quantified after 48 h. The statistical significance level between vehicle and LY294002-treated cultures is indicated. (B) After serum starvation for 18 to 20 h, IMCD-3 cells were pretreated for 1 h with LY294002, as in panel A. Cells were then treated for 15 min with BMP7, and cytoplasmic lysates were analyzed for induced levels of GSK3β Ser9 phosphorylation. Numbers below the panel indicate ratios of phospho-GSK3β to total GSK3β by densitimetry. (C) IMCD-3 cells were treated as in the experiment shown in panel B, and cytoplasmic lysates were then subjected to ILK immune complex kinase assays, using MBP as exogenous substrate. All results are representative of three independent experiments.

FIG. 7.

ILK mediates BMP7-dependent activation of p38^MAPK and ATF-2. (A) IMCD-3 cells were infected with the indicated adenoviruses (MOI = 5). At 24 h postinfection, cells were treated for 60 min with 0.25 nM BMP7. Cells were harvested and cytoplasmic lysates were analyzed in Western blots for phosphorylated and total protein levels of p38^MAPK (Thr180/Tyr182) and GSK3β (Ser 9). The panel below indicates lack of p38^MAPK phosphorylation by infection with empty adenovirus and with untreated and Ad-ILK virus infection controls. (B) IMCD-3 cells were infected as indicated in the legend to panel A. Following 15 min of treatment with 0.25 nM BMP7, cells were harvested and cytoplasmic lysates were analyzed for levels of phospho-ATF-2 (Thr71) and total ATF-2. (C) Cells were pretreated for 1 h with or without 10 μM SB203580 p38^MAPK inhibitor and then treated with or without 0.25 nM BMP7 for 15 min. Cytoplasmic lysates were analyzed by Western blotting for levels of total and phospho-GSK3β (pSer9), as a measure of ILK activity. (D) IMCD-3 cells were pretreated for one hour with SB203580 or LY294002 as described above and then treated with BMP7 for 15 min. ILK immune complex kinase assays were performed on cytoplasmic lysates, as above. Numbers below panels B and C are ratios of phospho/total protein, normalized to the strongest signal. Numbers below panel D are relative densitometric units. All results shown in each panel (A to D) are representative of three to five independent experiments.