Immunohistochemical analysis of embryonic mouse kidney revealed strong ILK immunoreactivity in the uretric bud and metanephric mesenchyme of E13.5 kidneys (Fig. ). By E17.5, ureteric bud cells have differentiated into collecting duct cells in the cortex and medulla. At this stage as well, ILK is strongly expressed throughout the collecting duct system. Thus, the temporal and spatial pattern of ILK in embryonic kidney is consistent with a functional role for ILK during RBM. To test whether ILK activity plays a role in renal morphogenesis, we infected embryonic mouse kidney explants with adenoviruses carrying wild-type ILK (Ad-ILKWT), or a dominant negative ILK mutant (Ad-ILKE359K). We confirmed that control, Ad-ILKWT, and Ad-ILKE359K viruses infected the kidney explants efficiently, judging by the expression of virally encoded GFP in whole-mount explants (Fig. ). Interestingly, we noted that Ad-ILKWT-infected kidneys appeared slightly larger and those infected with Ad-ILKE359K appeared smaller than control virus (AdCONTROL)-infected explants. This is consistent with a role for ILK in regulating cell proliferation in the intact kidney. After 5 days in culture, formation of collecting ducts was readily apparent in the control virus-infected kidneys. Infection with the Ad-ILKWT virus stimulated an increase in the number of UB branches formed, and UB formation was markedly inhibited in kidneys that had been infected with Ad-ILKE359K (Fig. ). These expression and functional data suggest an important role for ILK signaling in RBM.
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 (more ...)
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 (more ...)
The cellular complexity and dynamic epithelial-mesenchymal interactions in the developing kidney make it difficult to discriminate direct and indirect effects of growth factors. To directly examine a role of ILK in renal cell morphogenesis, we first tested whether BMP7 induced ILK activity in IMCD-3 cell cultures. We used EGF as a positive control for ILK stimulation and induction of tubule progenitors, since it potently induces ILK activity in other epithelial cells (unpublished data). Growth factor-dependent morphogenesis was quantified by culturing the IMCD-3 cells in three-dimensional collagen gels containing 5% serum, 0.25 nM BMP7, or 20 ng of EGF/ml. In this morphogenetic assay system, IMCD-3 cells forms structures at 48 h comprising two or more cells, with cellular processes that form the basis for multicellular branches, which are observed after 5 to 7 days in culture. Again, both these growth factors enhanced IMCD-3 morphogenesis significantly over serum-induced (control) levels. BMP7 induced a 1.4-fold increase, and EGF induced a 2-fold increase, over serum-induced levels (Fig. ). We then assayed for growth factor activation of ILK, using immune complex kinase assays. BMP7 (0.25 nM) and EGF (20 ng/ml) induced ILK activity to from five- to sixfold over unstimulated levels (Fig. ). Thus, ILK activation is an early event during BMP7-dependent IMCD-3 cell morphogenesis, and these results therefore raised the question of whether ILK acts to stimulate morphogenesis. We directly tested whether ILK promotes morphogenesis by infecting IMCD-3 cells with Ad-ILKWT
. Infection with Ad-ILKWT
stimulated tubule progenitor formation 2.8-fold relative to serum-induced levels (Fig. ), indicating that ILK is a positive mediator of IMCD-3 morphogenesis. We also noted an increase in cell numbers in Ad-ILKWT
-infected cultures; thus, like BMP7, ILK controls both proliferation and morphogenesis of renal epithelial cells. To test whether morphogenesis requires ILK activity, we inhibited ILK by infecting IMCD-3 cells with adenovirus expressing a dominant negative ILK mutant, ILKE359K
. We (28
) and others (33
) have shown that the ILKE359K
mutant exerts dominant inhibition of growth factor- or ECM-induced ILK activity in a number of cell lines. Infection of IMCD-3 cells with the Ad-ILKE359K
virus inhibited tubule progenitor formation by approximately twofold over serum control levels (Fig. ). Thus, ILK is sufficient to induce IMCD-3 morphogenesis, and inhibition of ILK blocks this response.
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 (more ...)
FIG. 4. ILK activity induces IMCD-3 tubule progenitors. IMCD-3 cells were infected with Ad-ILKWT, dominant negative Ad-ILKE359K, or “empty” AdCONTROL viruses (multiplicity of infection [MOI] = 5). At 24 h postinfection, cells were cultured (more ...)
We next tested whether ILK mediates BMP7-dependent morphogenesis by quantifying BMP7-induced formation of tubule progenitors in Ad-ILKE359K-infected cultures. BMP7-induced morphogenesis in Ad-ILKE359K-infected cells was decreased by approximately sixfold, relative to that of AdCONTROL virus-infected cells. Similarly, EGF-stimulated morphogenesis was decreased about threefold by Ad-ILKE359K, relative to that of control virus-infected cultures (Fig. ). These results demonstrate that ILK activity mediates morphogenetic signaling by BMP7.
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 (more ...)
As a second, independent method of inhibiting ILK we used KP-392, a small molecule that selectively inhibits ILK activity (18
). IMCD-3 cells were cultured in collagen gels supplemented with 5% serum or 20 ng of EGF/ml, with or without 5 μM KP-392 (50
). After 48 h, morphogenesis was visually quantified (Fig. ). Serum stimulated morphogenesis was inhibited by about 2 fold, and EGF stimulation was inhibited by 4.5 fold in KP-392-pretreated cells. We could not determine an effect of KP-392 on BMP7-dependent morphogenesis, since treatment with a dimethyl sulfoxide vehicle at the appropriate control concentration abrogated BMP7 activity. These results, coupled with those showing stimulation of tubule progenitors by Ad-ILK, indicate that ILK activity plays a role in IMCD-3 morphogenesis.
ILK activation, by growth factors or integrin-mediated cell adhesion, is dependent on the activity of PI 3-K. Thus, genetic or pharmacologic inhibition of PI 3-K signaling blocks activation of ILK by a variety of stimuli in epithelial cells, platelets, and myoblasts (9
). To test for the involvement of PI 3-K activity in the BMP7 stimulation of ILK, we used the selective PI 3-K inhibitor LY294002. We pretreated IMCD-3, cultured in collagen gels, with LY294002 and then quantified induction of tubule progenitors by BMP7 and EGF. LY294002 pretreatment inhibited BMP7 induction of IMCD-3 tubule progenitors by 2.3 fold and inhibited EGF induction by 3.3 fold (Fig. ), suggesting that PI 3-K activity is required in BMP7- and ILK-dependent morphogenesis. We therefore tested whether LY294002 inhibited BMP7-dependent ILK activation by two independent assays of ILK activity. LY294003 effectively suppressed BMP7-activated GSK3β Ser9 phosphorylation, a known cellular target of ILK (Fig. ). Similarly, pretreatment of IMCD-3 cells with LY294002 inhibited ILK activity as measured by ILK immune complex kinase assays (Fig. ). Our results indicate that BMP7 stimulation of both morphogenesis and ILK activity requires PI 3-K activity.
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. , with and without 10 μM LY294002. Tubule progenitors were quantified (more ...)
We recently reported that stimulatory doses of BMP7 activate p38MAPK
in IMCD-3 cells and that the selective p38MAPK
inhibitor, SB203580, blocks BMP7-dependent morphogenesis (22
). In the light of our present results showing ILK-dependent IMCD-3 morphogenesis, we examined whether ILK stimulates p38MAPK
activity and whether blocking ILK signaling with a dominant negative ILK mutant blocks stimulation of p38MAPK
by BMP7. Ligand-induced phosphorylation on Thr180 and Tyr182 activates p38MAPK
. To test for ILK-dependent stimulation of p38MAPK
/ATF-2, we assayed levels of phospho-p38MAPK
in IMCD-3 cultures infected with AdCONTROL
, or the dominant negative Ad-ILKE359K
mutant virus (Fig. ). Forty-eight hours after infections, cells were harvested and analyzed by Western blotting with phosphospecific antibodies to p38MAPK
(Thr180/Tyr182). Cells were treated for 1 h with BMP7 to induce p38MAPK
). As a positive control, we assessed ILK-dependent GSK3β Ser9 phosphorylation in the same lysates. Phospho-p38MAPK
levels were increased in Ad-ILKWT
- but not Ad-ILKE359K
- or AdCONTROL
-infected cultures. Infection with Ad-ILK also stimulated phosphorylation of GSK3β on Ser9, whereas cells infected with AdCONTROL
showed no increase in pSer9 levels (not shown). BMP7 also stimulated p38MAPK
phosphorylation in control, but not in Ad-ILKE359K
infected cells. As a positive control, phosphorylation of the known ILK target, PKB Ser473, was stimulated by Ad-ILKWT
and blocked by Ad-ILKE359k
(Fig. ). These data indicate that ILK can activate p38MAPK
independently of ligand and that induction of p38MAPK
by BMP7 is ILK dependent.
FIG. 7. ILK mediates BMP7-dependent activation of p38MAPK 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 (more ...)
Activating phosphorylation of the transcription factor, ATF-2, on Thr71 is mediated by p38MAPK
. Our previous work showed that BMP7 stimulates ATF-2 phosphorylation in IMCD-3 cells and that pretreatment of cells with the p38MAPK
inhibitor SB203580 blocks ATF-2 phosphorylation (22
). We treated IMCD-3 cells with BMP7 for 15 and 60 min and found that although ATF-2 phosphorylation was evident at 60 min, it was not appreciably induced at 15 min (data not shown). We reasoned that BMP7 signaling at this relatively early time point would be more robust in ILK-expressing cells and therefore tested whether BMP7-dependent ATF-2 phosphorylation was affected by increased ILK expression. IMCD-3 cells infected with either Ad-ILKWT
viruses were treated for 15 min with BMP7 and assayed for ATF-2 phosphorylation. Ad-ILKWT
but not Ad-ILKE359K
stimulated ATF-2 Thr71 phosphorylation (Fig. ), similar to p38MAPK
(Fig. ). BMP7 treatment for 15 min potentiated ATF-2 phosphorylation in Ad-ILKWT
- but not in Ad-ILKE359K
-infected cells (Fig. ). Thus, increased ILK expression accelerated BMP7-induced ATF-2 phosphorylation, further indicating that ILK acts in the BMP7/p38/ATF-2 signaling axis.
Our data suggested that ILK lies upstream of p38MAPK
in the BMP7 morphogenetic pathway; therefore, we tested the effects of SB203580 p38MAPK
inhibitor on BMP7-induced ILK activity. Cells were pretreated for 60 min with 0 or 10 μM SB203580 and subsequently treated for 15 min with 0.25 nM BMP7. As a measure of ILK activation, we assayed phospho-GSK3β (pSer9) levels. SB203580 pretreatment did not inhibit BMP7-induced Ser9 phosphorylation (Fig. ). These lysates were also subjected to ILK immune complex kinase assays (28
), which showed lack of inhibition of BMP7-induced ILK activity by SB203580, whereas LY294002 inhibited ILK activity (Fig. ). These results place p38MAPK
downstream of ILK activation by BMP7. Together with the results showing inhibition of BMP7-induced p38MAPK
phosphorylation and morphogenesis by ILKE359K
, these data identify a novel BMP7/ILK/p38MAPK
/ATF-2 signaling axis mediating epithelial cell morphogenesis.