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Research Article
Kindlin-2 regulates podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and integrins
Hong Qu, Yizeng Tu, Xiaohua Shi, Hannu Larjava, Moin A. Saleem, Sanford J. Shattil, Koichi Fukuda, Jun Qin, Matthias Kretzler, Chuanyue Wu
Journal of Cell Science 2011 124: 879-891; doi: 10.1242/jcs.076976
Hong Qu
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Yizeng Tu
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Xiaohua Shi
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Hannu Larjava
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Moin A. Saleem
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Sanford J. Shattil
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Koichi Fukuda
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Jun Qin
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Matthias Kretzler
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Chuanyue Wu
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  • For correspondence: carywu@pitt.edu
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Summary

Kindlin-2 is a FERM and PH domain-containing integrin-binding protein that is emerging as an important regulator of integrin activation. How kindlin-2 functions in integrin activation, however, is not known. We report here that kindlin-2 interacts with multiple phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate. Although integrin-binding is essential for focal adhesion localization of kindlin-2, phosphoinositide-binding is not required for this process. Using biologically and clinically relevant glomerular podocytes as a model system, we show that integrin activation and dependent processes are tightly regulated by kindlin-2: depletion of kindlin-2 reduced integrin activation, matrix adhesion and fibronectin matrix deposition, whereas overexpression of kindlin-2 promoted these processes. Furthermore, we provide evidence showing that kindlin-2 is involved in phosphoinositide-3-kinase-mediated regulation of podocyte-matrix adhesion and fibronectin matrix deposition. Mechanistically, kindlin-2 promotes integrin activation and integrin-dependent processes through interacting with both integrins and phosphoinositides. TGF-β1, a mediator of progressive glomerular failure, markedly increased the level of kindlin-2 and fibronectin matrix deposition, and the latter process was reversed by depletion of kindlin-2. Our results reveal important functions of kindlin-2 in the regulation of podocyte-matrix adhesion and matrix deposition and shed new light on the mechanism whereby kindlin-2 functions in these processes.

Introduction

Cell–extracellular matrix (ECM) adhesion and fibronectin matrix deposition are fundamental cellular processes that are mediated primarily by integrins (Hynes, 2002). The ligand binding activity of integrins and subsequent cell adhesion to ECM and fibronectin matrix deposition can be regulated by intracellular signaling events (Calderwood, 2004; Ginsberg et al., 2005; Ma et al., 2007; Wegener and Campbell, 2008). We recently showed that kindlin-2 (also known as Mig-2), a member of the kindlin protein family (for reviews, see Lai-Cheong et al., 2010; Larjava et al., 2008; Moser et al., 2009; Plow et al., 2009), binds β1- and β3-integrin cytoplasmic tails (Shi et al., 2007). Kindlin-2 is highly concentrated at integrin-rich cell-ECM adhesions (Tu et al., 2003). Using a mutational strategy, we have shown that integrin binding is essential for kindlin-2 localization to cell–ECM adhesions (Shi et al., 2007).

Kindlin-2 is emerging as an important regulator of integrins (Dowling et al., 2008a; Dowling et al., 2008b; Lai-Cheong et al., 2010; Larjava et al., 2008; Montanez et al., 2008; Moser et al., 2009; Plow et al., 2009; Tu et al., 2003). In C. elegans, loss of UNC-112, the C. elegans ortholog of kindlin-2, results in an embryonic lethal phenotype caused by defects in muscle attachments (Rogalski et al., 2000). In mice, loss of kindlin-2 causes peri-implantation lethality resulting from severe detachment of the endoderm and epiblast from the basement membrane (Dowling et al., 2008a; Montanez et al., 2008). The functions of kindlin-2 in differentiated cells, however, are complex and appear to be cell-type- and integrin-type-dependent (Harburger et al., 2009; Ma et al., 2008; Montanez et al., 2008; Shi et al., 2007).

Recent studies suggest that kindlin-2 cooperates with talin in integrin activation (Ma et al., 2008; Montanez et al., 2008) but the mechanism is poorly understood. Kindlin-2 contains no catalytic domains but instead multiple molecular interaction motifs including a FERM (four-point-one, ezrin, radixin, moesin) domain, which comprises four (F0, F1, F2 and F3) subdomains, and a PH (pleckstrin homology) domain inserted within F2 (Tu et al., 2003). Thus, kindlin-2 probably cooperates with talin in integrin activation through mediating multiple molecular interactions. However, our current understanding of kindlin-2-mediated interactions is incomplete, which hampers elucidation of the mechanism whereby kindlin-2 functions.

The goal of this study was to identify and better characterize molecular interactions mediated by kindlin-2 using the biologically and clinically relevant glomerular podocytes as a model system. Glomerular podocytes contribute to synthesis and deposition of glomerular ECM and, together with endothelial cells and glomerular basement membrane, form a filtration barrier that is essential for kidney glomerular function (Barisoni and Mundel, 2003; Faul et al., 2007; Pavenstadt et al., 2003). Podocytes are known to be targets of fibrogenic cytokines such as transforming growth factor β1 (TGF-β1), a key mediator of progressive glomerular failure (for a review, see Wolf and Ziyadeh, 2007). Treatment of podocytes with TGF-β1 promotes fibronectin matrix deposition (Li et al., 2008; Sam et al., 2006; Ziyadeh and Wolf, 2008), which probably contributes to podocyte dysfunction in progressive renal diseases. Although it has been well documented that alterations of podocyte adhesion and ECM deposition are crucially involved in glomerular diseases, the molecular mechanisms through which podocytes regulate these processes are not fully understood.

In this study, we have identified a novel interaction between kindlin-2 and phosphoinositides. Furthermore, we have mapped the binding site to the kindlin-2 PH domain and demonstrated a combined requirement for phosphoinositide- and integrin-binding in podocyte integrin activation, ECM adhesion and deposition. In addition, we provide evidence showing that kindlin-2 is involved in phosphoinositide 3-kinase (PI3K)-mediated regulation of podocyte-ECM adhesion and fibronectin matrix deposition. Finally, we show that the level of kindlin-2 in podocytes is upregulated by TGF-β1, which contributes to a TGF-β1-induced increase of fibronectin matrix deposition. Our results suggest that kindlin-2 is an important component of the cellular machinery that controls integrin activation, podocyte adhesion and fibronectin matrix deposition, and shed new light on the mechanism whereby kindlin-2 regulates these processes.

Results

Expression and localization of kindlin-2 in human podocytes

The mammalian kindlin protein family consists of three members, namely kindlin-1, -2 and -3. Kindlin-1 and -2, but not kindlin-3, were detected in kidney tissues (Siegel et al., 2003; Ussar et al., 2006). As an initial step in investigating the functions of kindlins in podocytes, we analyzed the expression of kindlin-1 and -2 in these cells. Because mature podocytes are unable to proliferate in culture, conditionally immortalized human podocytes (Saleem et al., 2002) were used. We detected kindlin-2 in undifferentiated, proliferating podocytes cultured under permissive conditions (Fig. 1A, lane 1). Consistent with previous studies (Saleem et al., 2002), after induction of differentiation, these cells expressed a higher level of cyclin kinase inhibitor p27 (Fig. 1A, lane 2) and lost their proliferative capacity. Concomitantly, the level of kindlin-2 was increased (Fig. 1A, lane 2). By contrast, no kindlin-1 was detected in podocytes either under differentiation or undifferentiation conditions (Fig. 1B, lanes 1 and 2). In control experiments, abundant kindlin-1 was detected in HaCaT keratinocytes (Fig. 1B, lane 3). These results suggest that kindlin-2 is the sole member of the kindlin family expressed by podocytes.

We next analyzed subcellular localization of kindlin-2. Immunofluorescent staining showed that kindlin-2 was clustered at focal adhesions (FAs) where actin stress fibers were anchored (Fig. 1C–E). Kindlin-2 can localize to cell-cell contacts in certain cell types including mouse embryonic fibroblasts and keratinocytes (Ussar et al., 2008; Ussar et al., 2006). In podocytes, however, no obvious localization of kindlin-2 to β-catenin-rich cell-cell contacts was detected (Fig. 1F–H). Thus, kindlin-2 is expressed and highly concentrated at FAs in terminally differentiated human podocytes.

Kindlin-2 regulates integrin activation and podocyte–ECM adhesion

To analyze the functions of kindlin-2 in podocytes, we depleted kindlin-2 in podocytes by transfecting them with two different kindlin-2 siRNAs. The level of kindlin-2, but not that of talin, integrin-linked kinase (ILK) or migfilin, was substantially reduced in kindlin-2 siRNA transfectants (Fig. 2A,B, lanes 1 and 2). Despite presence of normal levels of talin, ILK and migfilin, knockdown of kindlin-2 significantly reduced podocyte-ECM adhesion (Fig. 2C), suggesting a crucial role of kindlin-2 in this process.

To gain insight into the mechanism by which kindlin-2 regulates podocyte-ECM adhesion, we analyzed the effects of kindlin-2 on cell surface expression and activation of integrins. Fluorescence-activated cell sorting showed that knockdown of kindlin-2 modestly reduced the cell surface levels of β1- and β3-integrins (Fig. 2D,E). No changes in the levels of total (i.e. cell surface plus intracellular) β1- and β3-integrins, however, were observed (Fig. 2A,B), suggesting that kindlin-2 might play a role in promotion or retention of cell surface localization of β1- and β3-integrins. Analyses of the cells with HUTS-4 and WOW-1, which are known to recognize activated β1- and β3-integrins, respectively, showed that β1- and β3-integrin activation was impaired in response to knockdown of kindlin-2 (Fig. 2F,G), suggesting that a proper level of kindlin-2 is required for optimal integrin activation in podocytes.

Fig. 1.
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Fig. 1.

Expression and localization of kindlin-2 in podocytes. (A) Podocytes were cultured under permissive (lane 1) or nonpermissive conditions (lane 2) for 14 days. Cell lysates were analyzed by western blotting with antibodies (Abs) recognizing kindlin-2 (2 μg proteins/lane) or p27 (10 μg proteins/lane). Equal loading was confirmed by probing the membrane with anti-tubulin Ab. (B) Lysates (45 μg proteins/lane) of podocytes (lanes 1 and 2) and HaCaT cell lysates (lane 3) were analyzed by western blotting with Abs recognizing kindlin-1 or Coomassie Blue staining. (C–H) Differentiated podocytes were dually stained with mouse anti-kindlin-2 mAb (C) and FITC-conjugated phalloidin (D) or mouse anti-kindlin-2 mAb (F) and rabbit anti-β-catenin Ab (G). The images in panels E and H were merged using the NIH ImageJ program. Scale bars: 10 μm.

We next sought to test the effects of increased expression of kindlin-2 in podocytes. To do this, we infected the cells with an adenoviral vector encoding kindlin-2 (Fig. 3A, lane 2). Overexpression of kindlin-2 significantly enhanced podocyte-ECM adhesion (Fig. 3B). Slight increases in cell surface expression of β1- and β3-integrins were observed in kindlin-2 overexpressing cells (Fig. 3C,D), albeit quantification of results from three independent experiments showed that the increases did not reach statistical significance (P-values for the increases in cell surface expression of β1- and β3-integrins were 0.156 and 0.106, respectively). The levels of talin, ILK and migfilin were not altered (Fig. 3A). Despite expression of normal levels of talin, ILK and migfilin, overexpression of kindlin-2 significantly increased activation of β1- and β3-integrins (Fig. 3E,F). These results suggest that increased expression of kindlin-2 is sufficient to promote integrin activation and ECM adhesion in podocytes.

Kindlin-2 interacts with phosphoinositides through its PH domain

To better understand the mechanism whereby kindlin-2 functions, we sought to identify new binding partners of kindlin-2. Molecular modeling analysis using the Rosetta program (Chivian et al., 2005) suggests that kindlin-2 contains a PH-like structure (Fig. 4A). PH domains are protein modules that could potentially mediate interactions with phosphoinositides (Lemmon, 2008). To test this, we generated GST–kindlin-2 fusion proteins that either contained (GST–F2-PH) or lacked (GST–ΔPH) the PH domain (Fig. 4B, lanes 1 and 2). Incubation of purified GST–F2-PH with various phospholipids showed that it bound strongly to phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], moderately to phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] and weakly to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol 3-phosphate [PI(3)P] (Fig. 4C). No interactions with other phospholipids, including lysophosphatidic acid (LPA), lysophosphocholine (LPC), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine-1-phosphate (S1P), phosphatidic acid (PA) and phosphatidylserine (PS), were detected (Fig. 4C). Deletion of the PH domain eliminated the ability of GST–F2 to interact with phosphoinositides (Fig. 4D), confirming that the PH domain mediates the phosphoinositide binding.

Fig. 2.
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Fig. 2.

Knockdown of kindlin-2 inhibits β1- and β3-integrin activation. (A,B) Differentiated podocytes were transfected with control RNA (lane 1), KD1 (A, lane 2) or KD2 (B, lane 2). The lysates (5 μg proteins/lane) were analyzed by western blotting with Abs recognizing talin, β1-integrin, β3-integrin, kindlin-2, ILK, migfilin or tubulin. Relative levels (RLs) of kindlin-2 (means ± s.d. from five independent experiments) in A and B were calculated as described in the Materials and Methods. (C) Cell adhesion to fibronectin or vitronectin was analyzed as described in the Materials and Methods. Bars represent means ± s.d. from three independent experiments. *P<0.05 versus the control. **P<0.01 versus the control. (D–G) Cell surface expression (D,E) and activation (F,G) of β1- (E,G) and β3- (D,F) integrins in kindlin-2 knockdown and control cells were analyzed as described in the Materials and Methods. Bars represent means ± s.d. from three independent experiments. *P<0.05 versus the control.

Fig. 3.
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Fig. 3.

Kindlin-2 promotes integrin activation and ECM adhesion through interactions with phosphoinositides and integrins. (A) Podocytes cultured under non-permissive condition for 12 days were infected with adenoviral vectors encoding β-galactosidase (lane 1), kindlin-2 (lane 2), Q614W615AA (lane 3), ΔPH (lane 4) or K390A (lane 5) mutants. The lysates (5 μg proteins/lane) were analyzed by western blotting with Abs recognizing talin, kindlin-2, ILK, migfilin or tubulin. Relative levels (RLs) of wild-type or mutant forms of kindlin-2 (means ± s.d. from five independent experiments) were quantified as described in the Materials and Methods. (B) Cell adhesion to fibronectin was analyzed as described in the Materials and Methods. Bars represent means ± s.d. from five independent experiments. *P<0.05 versus the control. **P<0.01 versus the control. (C–F) Cell surface expression (C,D) and activation (E,F) of β1- (D,F) and β3- (C,E) integrins were analyzed as described in the Materials and Methods. Bars represent means ± s.d. from four independent experiments. *P<0.05 versus the control. **P<0.01 versus the control.

To further test this, we incubated GST–F2-PH with membrane immobilized with different amounts of phosphoinositides. Again, GST–F2-PH interacted strongly with PI(3,4,5)P3 [binding was detected with 6.2 pmoles of PI(3,4,5)P3], moderately with PI(3,5)P2 [25 pmoles of PI(3,5)P2 was required in order to detect the interaction with GST–F2-PH], and weakly with PI(4,5)P2 and PI(3)P [100 pmoles of PI(4,5)P2 or PI(3)P was required in order to detect the interaction with GST–F2-PH] (Fig. 4E). Next, we performed NMR experiments using purified kindlin-2 PH domain and phosphoinositides. Structural modeling predicts that the head groups of phosphoinositides interact with kindlin-2 PH (Fig. 4A). D-myo-inositol 1,3,4,5-tetraphosphate and D-myo-inositol 1,4,5-triphosphate, which represent the head groups of PI(3,4,5)P3 and PI(4,5)P2 (two phosphoinositides that exhibited the highest and lowest kindlin-2 PH-binding activities, respectively, in our biochemical assay) were used. Titration of kindlin-2 PH with D-myo-inositol 1,3,4,5-tetraphosphate caused large chemical shift changes of kindlin-2 PH residues (Fig. 4F). Titration of kindlin-2 PH with D-myo-inositol 1,4,5-triphosphate also caused chemical shift changes. However, despite addition of more D-myo-inositol 1,4,5-triphosphate, the extent of the chemical shift changes was less than those induced by D-myo-inositol 1,3,4,5-tetraphosphate (Fig. 4F), suggesting that phosphorylation at the 3′ position of the inositol ring enhances the interaction with kindlin-2. Collectively, the biochemical and NMR studies demonstrate that the kindlin-2 PH domain can directly interact with multiple phosphoinositides, preferentially with PI(3,4,5)P3.

Fig. 4.
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Fig. 4.

Kindlin-2 interacts with phosphoinositides through the PH domain. (A) Superposition of the Akt PH domain (yellow) bound to the PI(3,4,5)P3 head group (purple; PDB1UNQ) with a model of the PH domain of kindlin-2 (light cyan) showing that the kindlin-2 PH domain K390 (red) is positioned to potentially interact with the phosphate group of the PI(3,4,5)P3 head group. Model inspection and analysis of the kindlin-2 PH domain were performed using the PYMOL program (Schwede et al., 2003). (B) GST fusion proteins containing the kindlin-2 F2 subdomain with (lane 1) or without (lane 2) the PH insert, or the GST–kindlin-2 F2 subdomain containing the PH insert bearing the K390A mutation (lane 3) were analyzed by SDS-PAGE and Coomassie Blue staining. (C–E,G) PIP strips immobilized with different phospholipids (C,D,G) and a PIP array immobilized with different amounts of phosphoinositides (E) were incubated with 1 μg/ml of purified GST–F2-PH (C,E), GST–ΔPH (D) or GST–K390A (G). GST-fusion proteins bound to immobilized phosphoinositides were detected with an anti-GST Ab. (F) A representative HSQC spectrum of kindlin-2 PH domain showing D-myo-inositol 1,3,4,5-tetraphosphate and D-myo-inositol 1,4,5-triphosphate-induced chemical shift changes of the kindlin-2 PH domain. HSQC spectra were acquired as described in the Materials and Methods. Black, kindlin-2 PH domain alone; red, kindlin-2 PH domain: D-myo-inositol 1,3,4,5-tetraphosphate=1:2 (molar ratio); blue, kindlin-2 PH domain: D-myo-inositol 1,4,5-triphosphate=1:5 (molar ratio). (H) Podocyte lysates were incubated with PI(3,4,5)P3 beads (lane 3) or control beads lacking PI(3,4,5)P3 (lane 2). Kindlin-2 precipitated with PI(3,4,5)P3 beads was detected by western blotting with anti-kindlin-2 mAb. Lane 1 was loaded with 1 μg of podocyte lysates. (I) Lysates of podocytes expressing FLAG–kindlin-2, FLAG–K390A or FLAG–ΔPH were incubated with PI(3,4,5)P3 beads (lanes 5–7) or control beads lacking PI(3,4,5)P3 (lane 4) as indicated. The lysates (3 μg proteins/lane) of podocytes expressing FLAG–kindlin-2 (lane 1), FLAG–K390A (lane 2) or FLAG–ΔPH (lane 3) and proteins pulled down by PI(3,4,5)P3 beads (lanes 5–7) or the control beads (lane 4) were analyzed by western blotting with anti-kindlin-2 mAb.

We next sought to identify kindlin-2 residues that are crucial for phosphoinositide-binding. Previous studies of other PI(3,4,5)P3-binding PH domains suggest that positively charged residues within the β1–β2 loop are essential for recognizing PI(3,4,5)P3 (Ferguson et al., 2000; Isakoff et al., 1998; Lemmon and Ferguson, 2000; Wakamatsu et al., 2006). Based on structural modeling, we predict that K390 within the kindlin-2 PH domain could be involved in phosphoinositide-binding (Fig. 4A). To test this, we substituted K390 with alanine (Fig. 4B, lane 3) and found that it indeed abolished the interactions with PI(3,4,5)P3 and other phosphoinositides (Fig. 4G).

To test whether kindlin-2 expressed in podocytes interacts with PI(3,4,5)P3, we incubated podocyte lysates with PI(3,4,5)P3 coated beads or control beads lacking PI(3,4,5)P3. PI(3,4,5)P3 beads (Fig. 4H, lane 3), but not the control beads (Fig. 4H, lane 2), pulled down kindlin-2, suggesting that kindlin-2 expressed by podocytes, like bacterially expressed kindlin-2, interacts with PI(3,4,5)P3. To determine whether the interaction is mediated by the PH domain, we expressed FLAG-tagged wild-type (Fig. 4I, lane 1) or mutant forms of kindlin-2 bearing K390A (Fig. 4I, lane 2) or PH deletion (Fig. 4I, lane 3) mutations in podocytes. As expected, PI(3,4,5)P3 beads (Fig. 4I, lane 5), but not the control beads (Fig. 4I, lane 4), pulled down FLAG–kindlin-2. By contrast, neither FLAG–K390A (Fig. 4I, lane 6) nor FLAG–ΔPH (Fig. 4I, lane 7) bound PI(3,4,5)P3. These results confirm that kindlin-2 expressed in podocytes interacts with PI(3,4,5)P3 through its PH domain.

Kindlin-2-mediated regulation of integrin activation and ECM adhesion involves both integrin- and phosphoinositide-binding

Next, we overexpressed kindlin-2 mutants lacking either the integrin-binding activity (i.e. Q614W615AA) or the phosphoinositide-binding activity (i.e. ΔPH or K390A) in podocytes (Fig. 3A, lanes 3–5). Elimination of either integrin-binding or phosphoinositide-binding significantly impaired the ability of kindlin-2 to promote podocyte-ECM adhesion (Fig. 3B). Expression of the integrin-binding-defective or the phosphoinositide-binding-defective mutants did not significantly alter cell surface expression of β1- and β3-integrins (Fig. 3C,D). Consistent with the effects on podocyte-ECM adhesion (Fig. 3B), elimination of integrin-binding or phosphoinositide-binding impaired the ability of kindlin-2 to enhance β1- and β3-integrin activation (Fig. 3E,F). Collectively, these results suggest a combined requirement for phosphoinositide- and integrin-binding in podocyte integrin activation and ECM adhesion.

Deletion of PH compromises kindlin-2 localization to FAs but phosphoinositide-binding is not essential for this process

We previously showed that F3-mediated integrin-binding is essential for kindlin-2 localization to FAs (Shi et al., 2007). To test whether the PH domain is involved in this process, we expressed GFP-tagged wild-type and mutant forms of kindlin-2 in podocytes. GFP–K390A and GFP–ΔPH, like GFP–kindlin-2, interacted with β1- and β3-integrin tails (Fig. 5A–D, lanes 6–8). In control experiments, GFP–kindlin-2 did not interact with GST (Fig. 5A-D, lane 5). Furthermore, as we showed previously (Shi et al., 2007), a Q614W615→AA mutation within F3 abolished the interaction of kindlin-2 with β1 and β3 tails (Fig. 5A–D, lane 9), confirming the specificity of the binding assay. Consistent with our previous studies (Shi et al., 2007), GFP–kindlin-2 readily localized to FAs where abundant vinculin was present (Fig. 5E–G). Interestingly, the ability of GFP–ΔPH to localize to FAs was compromised. In many cells, we failed to detect GFP–ΔPH in FAs where abundant vinculin was detected (a representative cell is shown in Fig. 5H–J). However, we did occasionally observe GFP–ΔPH in FAs (Fig. 5K–M). To further test this, we analyzed the localization of GFP–K390A and found that it readily localized to FAs (Fig. 5N–P). Collectively, these results suggest that phosphoinositide-binding is not required for FA localization of kindlin-2, albeit the PH domain might influence this process through a mechanism that is independent of phosphoinositide-binding.

Fig. 5.
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Fig. 5.

Deletion of the PH domain compromises kindlin-2 clustering at FAs but phosphoinositide-binding is not essential for this process. (A–D) Human podocytes were transfected with vectors encoding GFP–kindin-2 (lanes 1, 5 and 6), GFP–K390A (lanes 2 and 7), GFP–ΔPH (lanes 3 and 8) or GFP–Q614W615AA mutant (lanes 4 and 9). A GST pull-down experiment was performed using cells expressing GFP-tagged wild-type or mutant forms of kindlin-2 as indicated in the figure. The cell lysates (2 μg proteins/lane, lanes 1–4), GST precipitates (lane 5), GST–β1 precipitates (A,C, lanes 6–9) and GST–β3 precipitates (B,D, lanes 6–9) were analyzed by western blotting with anti-kindlin-2 mAb (A,B) or Coomassie Blue staining (C,D). The samples in lane 10 were prepared as those in lanes 6–9 except cell lysates were omitted. (E–P) Podocytes were transfected with vectors encoding GFP–kindlin-2 (E–G), GFP–ΔPH (H–M) or GFP–K390A (N–P). The transfectants were plated on fibronectin-coated cover slips and stained with a mouse anti-vinculin mAb and a Rhodamine-Red-conjugated anti-mouse IgG Ab. The cells were observed under a fluorescence microscope equipped with GFP (E,H,K,N) and Rhodamine (F,I,L,O) filters. The GFP and Rhodamine images were merged in panels G, J, M and P using the NIH ImageJ program. Scale bar: 10 μm.

In addition to localizing to FAs, kindlin-2 was sometimes detected in regions of plasma membrane (for example, see Fig. 5E). The membrane localization was particularly obvious in cells transfected with a vector encoding a constitutively active form of PI3K (p110*) (Klippel et al., 1996) (arrowheads in Fig. 6A). Expression of p110* did not increase membrane localization of the K390A mutant (Fig. 6B,C). To confirm that kindlin-2 localized to PI(3,4,5)P3-rich membrane regions, we co-transfected podocytes with vectors encoding p110* and GFP–Akt-PH, which has been widely used as a sensor for monitoring subcellular distribution of PI(3,4,5)P3 (for a review, see Lemmon and Ferguson, 2000). Staining of the cells with anti-kindlin-2 monoclonal antibody (mAb) showed that kindlin-2 was concentrated in not only FAs but also membrane regions in which GFP–Akt-PH was enriched (arrowheads in Fig. 6G–I). Much lower levels of kindlin-2 were detected in membrane regions in which GFP–Akt-PH was not highly concentrated (arrow in Fig. 6G–I). In cells that lacked high concentrations of membrane PI(3,4,5)P3 (as indicated by the lack of high concentrations of membrane GFP–Akt-PH), we detected abundant kindlin-2 in FAs (Fig. 6D–F,J–L), suggesting that a high level of PI(3,4,5)P3 is not necessary for clustering of kindlin-2 at FAs.

Fig. 6.
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Fig. 6.

Membrane localization of kindlin-2 and kindlin-2 PH domain. (A–C) Human podocytes that were transfected with GFP–kindlin-2 vector and p110* vector (A), GFP–K390A vector and p110* vector (B) or GFP–K390A vector and a control vector lacking the PI3K sequence (C) were plated on fibronectin-coated cover slips and fixed. (D–L) Podocytes that were transfected with GFP–Akt-PH vector and p110* vector (G–L) or GFP–Akt-PH vector and a control vector lacking the PI3K sequence (D–F) were plated on fibronectin-coated cover slips, fixed and stained with anti-kindlin-2 mAb and a Rhodamine-Red-conjugated anti-mouse IgG Ab. (M–U) Podocytes that were transfected with GFP–Akt-PH vector, tdTomato-kindlin-2-PH vector and p110* vector (P–U) or GFP–Akt-PH vector, tdTomato-kindlin-2-PH vector and a control vector lacking the PI3K sequence (M–O) were plated on fibronectin-coated cover slips and fixed. Cells in J–L and S–U were treated with 0.1 μM wortmannin for 1 hour prior to fixation. Cells were observed under a fluorescence microscope equipped with GFP (A–D,G,J,M,P,S) and Rhodamine and tdTomato (E,H,K,N,Q,T) filters. The GFP and Rhodamine and tdTomato images were merged in panels F, I, L, O, R and U using the NIH ImageJ program. Arrows indicate membrane regions lacking a high concentration of GFP–Akt-PH. Arrowheads indicate membrane regions containing high concentrations of GFP–Akt-PH. Scale bar: 10 μm.

To further analyze this, we compared membrane localization of the kindlin-2 PH domain with that of Akt PH. To do this, vectors encoding tdTomato-tagged kindlin-2-PH and GFP–Akt-PH, together with a vector encoding p110* or a control vector lacking the PI3K sequence, were transfected into podocytes. The results showed that tdTomato-kindlin-2-PH colocalized with GFP–Akt-PH in the membrane (Fig. 6M–R). This is particularly obvious in p110* transfectants in which tdTomato-kindlin-2-PH was highly concentrated in membrane regions where GFP–Akt-PH was enriched (arrowheads in Fig. 6P–R) but not in membrane regions lacking GFP–Akt-PH (arrow in Fig. 6P–R). The level of tdTomato-kindlin-2-PH in the membrane, like that of GFP–Akt-PH, was reduced in cells treated with PI3K inhibitor wortmannin (Fig. 6S–U). Thus, consistent with strong PI(3,4,5)P3 binding activity detected in biochemical and NMR analyses, the kindlin-2 PH domain, like the Akt PH domain, localizes to PI(3,4,5)P3-rich membranes in cells.

Kindlin-2 functions downstream of TGF-β1 to regulate fibronectin matrix deposition

Integrin activation controls not only cell–ECM adhesion but also deposition of fibronectin matrix (Wu et al., 1995). The finding that kindlin-2 stimulates integrin activation prompted us to test whether kindlin-2 plays a role in this process. To do this, we isolated ECM fractions from podocytes. Western blotting analyses showed that fibronectin was deposited into ECM fractions (Fig. 7A,B, lane 2). Double immunofluorescent staining with rabbit anti-fibronectin antibodies (Abs) and mouse mAb SNAKA51, which is known to preferentially recognize active α5β1-integrins in fibrillar adhesions (Clark et al., 2005), confirmed that fibronectin and active α5β1-integrins were clustered at fibrillar adhesions (Fig. 7C,D). Next, we depleted kindlin-2 from podocytes and found that the amount of fibronectin matrix was reduced in response to downregulation of kindlin-2 (Fig. 7A,B, lane 1; Fig. 7G,H,K,L), suggesting that kindlin-2 is involved in fibronectin matrix deposition. Consistent with this, the level of total cell-associated (i.e. intracellular plus assembled) fibronectin was also reduced in kindlin-2 knockdown cells (Fig. 7A,B, lane 1).

Fig. 7.
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Fig. 7.

Kindlin-2 functions in TGF-β1-induced fibronectin matrix deposition in podocytes. Differentiated human podocytes were transfected with kindlin-2 siRNA KD1, kindlin-2 siRNA KD2 or control RNA and cultured in the presence or absence of TGF-β1 (5 ng/ml) as indicated in the figure. (A,B) Cell lysates and ECM fractions were analyzed by western blotting with Abs specific for kindlin-2, tubulin (as a loading control) or fibronectin as indicated in the figure. The densities of fibronectin, kindlin-2 and tubulin bands were quantified using the NIH ImageJ program. Relative levels (RLs) of kindlin-2 and fibronectin in total cell lysates or ECM fractions (means ± s.d. from three independent experiments in A and two independent experiments in B) were quantified as described in the Materials and Methods. (C–N) Cells (as indicated in the figure) were dually stained with mouse anti-α5-integrin mAb SNAKA51 and rabbit anti-fibronectin Abs. The mouse and rabbit Abs were detected with secondary Rhodamine-Red-conjugated anti-mouse IgG Ab and FITC-conjugated anti-rabbit IgG Ab. The cells were observed under a fluorescence microscope equipped with Rhodamine (C,E,G,I,K,M) and FITC (D,F,H,J,L,N) filters. Scale bar: 10 μm.

TGF-β1 promotes fibronectin matrix deposition in podocytes (Li et al., 2008). To test whether kindlin-2 is involved in this process, we compared the level of kindlin-2 in podocytes that were treated with or without TGF-β1. Treatment of podocytes with TGF-β1 increased the level of kindlin-2 (Fig. 7A,B, lane 3) and fibronectin matrix deposition (Fig. 7A,B, lane 3; Fig. 7E,F). The increase of fibronectin matrix deposition induced by TGF-β1 was reversed by knockdown of kindlin-2 (Fig. 7A,B, lane 4; Fig. 7I,J,M,N), suggesting that kindlin-2 contributes to TGF-β1-induced fibronectin matrix deposition.

To further test the role of kindlin-2 in fibronectin matrix deposition, we overexpressed kindlin-2 (Fig. 8A, lane 2), Q614W615AA (Fig. 8A, lane 3), ΔPH (Fig. 8A, lane 4) or K390A (Fig. 8A, lane 5) in podocytes. Consistent with a positive role for kindlin-2 in integrin activation, overexpression of kindlin-2 increased fibronectin matrix deposition (Fig. 8A, lane 2). Elimination of integrin-binding (Fig. 8A, lane 3) or phosphoinositide-binding (Fig. 8A, lanes 4 and 5) substantially reduced the ability of kindlin-2 to promote fibronectin matrix deposition, suggesting that the interactions of kindlin-2 with integrins and phosphoinositides are crucially involved in this process.

Depletion of kindlin-2 reduces PI3K-induced upregulation of cell-ECM adhesion and fibronectin matrix deposition

The finding that kindlin-2 preferentially binds PI(3,4,5)P3 prompted us to test whether kindlin-2 is involved in cellular processes that are regulated by PI3K. To do this, we transfected podocytes with a vector encoding Myc–p110* (Klippel et al., 1996). Expression of Myc–p110* was confirmed by western blotting (Fig. 8B, lane 4). Expression of constitutively active PI3K stimulated the activation (indicated by the increase of Thr308- and Ser473-phosphorylation), but not the protein level, of Akt (Fig. 8B, compare lane 4 with lane 3). PI3K-induced activation of Akt was not inhibited by knockdown of kindlin-2 (Fig. 8B, compare lanes 1 and 2 with lane 4). Of note, expression of constitutively active PI3K increased the amount of fibronectin matrix (Fig. 8B, compare lane 4 with lane 3). Knockdown of kindlin-2 substantially reversed the PI3K-induced increase in fibronectin matrix deposition (Fig. 8B, compare lanes 1 and 2 with lane 4). Consistent with this, the level of total cell-associated (i.e. intracellular plus assembled) fibronectin was reduced in kindlin-2 knockdown cells (Fig. 8B, compare lanes 1 and 2 with lane 4). Additionally, expression of constitutively active PI3K enhanced cell–ECM adhesion, which was effectively reversed by knockdown of kindlin-2 (Fig. 8C).

Fig. 8.
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Fig. 8.

The kindlin-2-phosphoinositide interaction promotes cell–ECM adhesion and fibronectin matrix deposition. (A) Human podocytes cultured under nonpermissive condition for 12 days were infected with adenoviral vectors encoding β-galactosidase (lane 1), kindlin-2 (lane 2), Q614W615AA (lane 3), ΔPH (lane 4) or K390A (lane 5). Kindlin-2, GAPDH (as a loading control), total cell-associated fibronectin and fibronectin in the ECM fractions were analyzed by western blotting with Abs as indicated. Relative levels (RLs) of kindlin-2 and fibronectin (means ± s.d. from two independent experiments) were calculated as described in the Materials and Methods. (B) Human podocytes were transfected with kindlin-2 siRNA KD2 (lane 1), KD1 (lane 2), control RNA (lanes 3 and 4) and a DNA vector encoding Myc-p110* (lanes 1, 2 and 4) or a control DNA vector lacking the PI3K sequence (lane 3). The cell lysates or ECM fractions were analyzed by western blotting with Abs specific for Myc, kindlin-2, Akt, phospho-Akt (Ser473), phospho-Akt (Thr308), fibronectin or GAPDH (as a loading control). Relative levels of fibronectin in total cell lysates and ECM fractions (means ± s.d. from two independent experiments) were calculated as described in the Materials and Methods. (C) Adhesion of podocytes that were transfected with control DNA vector and control RNA (control), p110* vector and control RNA (p110*), p110* vector and KD1 (p110* + KD1), and p110* vector and KD2 (p110* + KD2) to fibronectin was analyzed as described in the Materials and Methods. Error bars represent means ± s.d. from three independent experiments. *P<0.05 versus the control.

Discussion

Kindlin-2 is a widely expressed and evolutionarily conserved FA protein that is emerging as an important regulator of integrin-mediated cell-ECM adhesion (for reviews, see Lai-Cheong et al., 2010; Larjava et al., 2008; Moser et al., 2009; Plow et al., 2009). Despite strong evidence for a role for kindlin-2 in integrin-mediated cell-ECM adhesion, the specific functions of kindlin-2 in this process appear to be integrin- and cell-type-dependent. For example, overexpression of kindlin-2 in CHO cells exogenously expressing αIIbβ3 integrin enhances its activation (Ma et al., 2008; Shi et al., 2007). However, overexpression of kindlin-2 in the same type of cells inhibits endogenous α5β1-integrin activation (Harburger et al., 2009). In mouse embryonic stem cells, kindlin-2 is required for β1-integrin activation and ECM adhesion (Montanez et al., 2008). However, in human colon cancer cells, kindlin-2 does not appear to influence integrin activation but instead promotes maturation of cell-ECM adhesions (Shi et al., 2007). Because defects in podocyte-ECM adhesion are intimately associated with glomerular diseases, we set out to determine the functions of kindlin-2 in podocytes.

Using complementary approaches of genetic knockdown and overexpression, we have found that kindlin-2 plays a crucial role in promotion of β1- and β3-integrin activation and ECM adhesion in podocytes. Depletion of kindlin-2 reduces integrin activation and podocyte-ECM adhesion, whereas overexpression of kindlin-2 promotes these processes. Exposure of podocytes to TGF-β1, a mediator of progressive kidney glomerular failure, increased the kindlin-2 level. TGF-β1 is known to promote fibronectin matrix deposition (Li et al., 2008). Because fibronectin matrix deposition is positively regulated by integrin activation (Wu et al., 1995), and increased expression of kindlin-2 in podocytes promoted integrin activation (Fig. 3), our results suggest that kindlin-2 is probably involved in TGF-β1-induced upregulation of fibronectin matrix deposition. Consistent with this, siRNA-mediated knockdown of kindlin-2 substantially reversed the TGF-β1-induced increase of fibronectin matrix deposition.

In addition to identifying TGF-β1 as an upstream regulator of kindlin-2 and demonstrating an important role for kindlin-2 in the regulation of integrin activation and podocyte-ECM adhesion, the present study sheds new light on the mechanism by which kindlin-2 functions. Because kindlin-2 contains multiple molecular interaction domains but no catalytic domain, we reasoned that kindlin-2 probably promotes integrin activation through mediating multiple interactions. Previous studies by us and others have identified three kindlin-2-mediated interactions, namely with integrins, ILK and migfilin (for reviews, see Lai-Cheong et al., 2010; Larjava et al., 2008; Moser et al., 2009; Plow et al., 2009). Among these interactions, only the site that mediates the interaction with integrins has been determined (Shi et al., 2007). Integrin-binding-defective kindlin-2 mutants were unable to promote activation of αIIbβ3 that is exogenously expressed in CHO cells (Ma et al., 2008). Whether or not interactions with ILK and migfilin are involved in integrin activation remains to be determined. In the current study, we have shown that integrin-binding is essential for kindlin-2-induced activation of endogenous β1- and β3-integrin in podocytes. Furthermore, we have identified a novel interaction between kindlin-2 and phosphoinositides. We have mapped the phosphoinositide-binding site to the PH domain and shown that K390, which is probably located at the β1–β2 loop region, is essential for phosphoinositide-binding. Deletion of PH or substitution of K390 with alanine, which eliminates phosphoinositide-binding, impaired the ability of kindlin-2 to promote β1- and β3-integrin activation. Thus, kindlin-2-induced integrin activation requires interactions with not only integrins but also phosphoinositides. These findings are consistent with recent studies in CHO cells showing that deletion of the PH domain reduces kindlin-2-induced αIIbβ3-integrin activation (Ma et al., 2008). We have found that deletion of the PH domain compromised FA localization of kindlin-2. However, because the phosphoinositide-binding-defective K390A mutant, like wild-type kindlin-2, localizes to cell–ECM adhesions (Fig. 5N), phosphoinositide-binding per se is not essential for this process. Although the mechanism by which the PH domain influences this process remains to be determined, PH domains could potentially influence FA localization of kindlin-2 through mediating protein-protein interactions.

How does the interaction of kindlin-2 with phosphoinositides contribute to integrin activation? Recent studies indicate that optimal integrin activation requires not only separation of α- and β-integrin tails and transmembrane domains but also tethering of β tails to the membrane (Ma et al., 2007; Wegener et al., 2007; Wegener and Campbell, 2008; Goksoy et al., 2008; Yang et al., 2009). Thus, it is conceivable that kindlin-2, through PH-mediated phosphoinositide-binding (the current paper) and F3-mediated integrin-binding (Shi et al., 2007; Ma et al., 2008; Montanez et al., 2008), might help to tether integrin β tails to the plasma membrane and consequently enhance talin-mediated integrin activation.

PI3K is known to play important roles in the regulation of integrins and cell–ECM adhesion (Byzova et al., 2000; Greenwood et al., 1998; Greenwood et al., 2000; Jackson et al., 2004; Kovacsovics et al., 1995; Orr et al., 2006; Shimizu et al., 1995; Zhang et al., 1996) but the underlying mechanisms are incompletely understood. By analyzing interactions of the kindlin-2 PH domain with different phospholipids using biochemical and NMR methods, we have found that multiple phosphoinositides including PI(3,4,5)P3, PI(3,5)P2 and PI(4,5)P2 can bind the kindlin-2 PH domain, among which PI(3,4,5)P3 displays the strongest binding activity. Thus, although PI(3,4,5)P3 is not the only phosphoinositide that can mediate membrane localization of kindlin-2, an increase of PI(3,4,5)P3 synthesis could boost this process. Using the Akt PH domain as a sensor for PI(3,4,5)P3, we have found that kindlin-2 and the kindlin-2 PH domain, like the Akt PH domain, are indeed enriched in PI(3,4,5)P3-rich membrane regions in response to increased PI(3,4,5)P3 synthesis (Fig. 6). In addition, we have found that activation of PI3K promotes podocyte-ECM adhesion and fibronectin matrix deposition in a kindlin-2-dependent manner. These findings, together with our biochemical and NMR studies showing that PI(3,4,5)P3 is preferentially recognized by kindlin-2 and our mutational studies showing that phosphoinositide-binding is crucial for kindlin-2 regulation of ECM adhesion and fibronectin matrix deposition, suggest that kindlin-2, through its interactions with PI(3,4,5)P3 and integrins, can sense activation of PI3K and link it to changes in cell behavior (i.e. increase of ECM adhesion and fibronectin matrix deposition).

Materials and Methods

Antibodies and other reagents

Mouse anti-kindlin-2 mAb 3A3, anti-kindlin-1 mAb 4A5.14 and anti-migfilin mAb 43 have been previously described (Papachristou et al., 2007; Tu et al., 2003). Anti-talin mAb was from Chemicon. Antibodies recognizing p27, ILK, β1-integrin and β3-integrin were from BD Transduction Laboratories. Anti-αvβ3 integrin mAb LM609 was from Millipore. Monovalent ligand-mimetic anti-αvβ3 Ab WOW-1 has been previously described (Pampori et al., 1999). Anti-β1-integrin mAb P5D2 was from Santa Cruz Biotech. Anti-β1 integrin mAb HUTS-4 was from Chemicon. Rabbit anti-fibronectin Ab has been previously described (Wu et al., 1993). Anti-α5 integrin mAb SNAKA51 was a gift from Martin J. Humphries (University of Manchester, Manchester, UK). Rabbit Abs against Akt, phospho-Akt (Ser473) and phospho-Akt (Thr308) were from Cell Signaling Technology. FITC-phalloidin, anti-β-catenin Ab and anti-vinculin mAb were from Sigma. R-Phycoerythrin-conjugated F(ab1)2 goat anti-mouse IgG Ab, Allophycocyanin-conjugated F(ab1)2 goat anti-mouse IgG Ab, Rhodamine Red-conjugated goat anti-mouse IgG Ab, FITC-conjugated anti-rabbit IgG Ab and horseradish-peroxidase-conjugated secondary Abs were from Jackson ImmunoResearch Laboratories. Human TGF-β1 was from Chemicon. Wortmannin was from MP Biomedicals. Cell culture media were from Mediatech/Cellgro (Herndon, VA).

Podocyte culture

Conditionally immortalized human glomerular podocytes were propagated under permissive condition at 33°C as previously described (Saleem et al., 2002). To induce differentiation, cells were switched to 37°C (nonpermissive condition) for 10–14 days.

DNA vectors and transfection

DNA vectors encoding kindlin-2 and the Q614W615→AA mutant were previously described (Shi et al., 2007; Tu et al., 2003). To generate vectors encoding GFP– or FLAG–kindlin-2 ΔPH mutant, a kindlin-2 cDNA fragment in which the PH (residues 381–477) coding sequence was deleted, was prepared by PCR and inserted into pEGFP-C2 (Clontech) or pFLAG-CMV-2 vector (Sigma). A K390A point mutation was generated using a QuickChange Site-Directed Mutagenesis System (Stratagene). All mutations were confirmed by DNA sequencing. A DNA vector (pEF-BOS/myc-p110*) encoding a constitutively active form of PI3K (p110*) (Klippel et al., 1996) was kindly provided by Lawrence Kane (University of Pittsburgh). To generate vectors encoding GFP–Akt PH and tdTomato-kindlin-2 PH, Akt PH (residues 21–153) or kindlin-2 PH (residues 370–481) was inserted into pEGFP-C2 or pCMV-tdTomato vector. Podocytes were transfected with DNA vectors using Lipofectamine 2000 (Invitrogen). Two days after transfection, the cells were analyzed as specified in each experiment.

Kindlin-2 siRNA and transfection

The sequence of kindlin-2 siRNA KD1 has been previously described (Tu et al., 2003). KD2 was a mixture of three kindlin-2 siRNAs (Oligo ID HSS116968/116969/116970) from Invitrogen. Podocytes were transfected with KD1, KD2 or an irrelevant small RNA (as a control) twice using Lipofectamine 2000. In some experiments, the cells were transfected with a DNA vector (pEF-BOS/myc-p110*) 8 hours after the second siRNA transfection. Two days after the second siRNA transfection, the cells were analyzed as specified in each experiment. To quantify the efficiencies of kindlin-2 knockdown, the densities of kindlin-2 and tubulin (as a loading control) bands were quantified using the NIH ImageJ program. The relative levels of kindlin-2 (defined as densities of the kindlin-2 band/densities of the tubulin band) in kindlin-2 knockdown cells were compared with those in control cells (normalized to 1).

Adenoviral vectors and infection

Adenoviral vectors encoding wild-type or the Q614W615→AA mutant of kindlin-2 were previously described (Shi et al., 2007) and those encoding kindlin-2 ΔPH or K390A were generated using the AdEasy system following a previously described protocol (Guo and Wu, 2002). Podocytes that were cultured under nonpermissive conditions for 12 days were infected with the adenoviruses. The infection efficiency was monitored by the expression of GFP encoded by the adenoviral vectors, which typically reached 80–90% within 2 days. Overexpression of wild-type or mutant forms of kindlin-2 was confirmed by western blotting. To compare relative expression levels of mutant forms of kindlin-2 with that of wild-type kindlin-2, the densities of wild-type or mutant forms of kindlin-2 and tubulin (as a loading control) bands were quantified using the NIH ImageJ program. The relative levels (RLs) of mutant forms of kindlin-2 (defined as densities of kindlin-2 mutant bands/densities of the tubulin band) were compared with that of wild-type kindlin-2 (normalized to 1).

Computer modeling of kindlin-2 PH domain

The kindlin-2 sequence was submitted to the web server http://robetta.bakerlab.org for structure prediction. The result shows a PH-like structure in all lowest energy structures inserted within the kindlin-2 FERM F2 domain.

GST–kindlin-2 and GST–integrin fusion proteins

To prepare GST–kindlin-2 fusion proteins, DNA fragments encoding wild-type or mutant forms of kindlin-2 were inserted into pGEX-5x-1 (Pharmacia). The recombinant vectors were used to transform E. coli cells. The expression of GST–kindlin-2 fusion proteins was induced with IPTG and the proteins were purified with glutathione–Sepharose 4B as described (Tu et al., 2003). GST fusion proteins containing an integrin β1 tail (residues 775–786) or β3 tail (residues 716–762) were prepared as described (Shi et al., 2007).

Biochemical phosphoinositide-binding assays

Membranes immobilized with different phospholipids (PIP strips, Echelon Biosciences) or different amounts of phosphoinositides (PIP array, Echelon Biosciences) were blocked in PBST (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.4) supplemented with 1% milk for 2 hours at room temperature. The membranes were then incubated with 1 μg/ml GST–F2-PH (containing kindlin-2 residues 281–572), GST–ΔPH (containing kindlin-2 residues 281–380 and 478–572) or GST–K390A (containing kindlin-2 F2-PH bearing a K390A mutation) for 2 hours at room temperature. After washing, GST-fusion proteins bound to phosphoinositides were detected with anti-GST mAb and an HRP-conjugated goat anti-mouse IgG Ab.

To test whether endogenous kindlin-2 interacts with PI(3,4,5)P3, podocytes were lysed on ice with binding buffer [10 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% (v/v) Nonidet P-40] and centrifuged at 20,000 g for 20 minutes at 4°C. The supernatants were incubated with PI(3,4,5)P3 beads (Echelon Biosciences) or an equal amount of beads lacking PI(3,4,5)P3 as a control overnight at 4°C. After washing, kindlin-2 bound to PI(3,4,5)P3 beads was detected by western blotting.

To confirm that kindlin-2 PH mediates phosphoinositide-binding, podocytes were transfected with vectors encoding FLAG-tagged kindlin-2, ΔPH or K390A. The transfectants were lysed on ice with the binding buffer and centrifuged at 20,000 g for 20 minutes at 4°C. The supernatants were mixed with PI(3,4,5)P3 beads or an equal amount of control beads for 3 hours at 4°C. After washing, FLAG-kindlin-2 protein bound to PI(3,4,5)P3 beads was detected by western blotting.

NMR spectroscopy of the kindlin-2 PH domain

A cDNA encoding the kindlin-2 PH domain (residues 367–500) was subcloned into pET15b (Novagen). 15N-labeled hexahistidine-tagged kindlin-2 PH was expressed in E. coli and purified using a Ni-affinity column followed by Resource-S and HiLoad 16/60 Superdex 200 prep grade chromatography columns (GE Healthcare). The hexahistidine tag was removed by thrombin cleavage. Purified PH domain was dialyzed in a buffer consisting of 10 mM HEPES, pH 7, 100 mM NaCl and 2 mM DTT. The NMR samples of the kindlin-2 PH domain in the presence of D-myo-inositol 1,3,4,5-tetraphosphate or D-myo-inositol 1,4,5-triphosphate (Echelon), which represent the head groups of PI(3,4,5)P3 and PI(4,5)P2, respectively, were prepared by mixing at 1:2 or 1:5 molar ratio using the dialysis buffer and a 10% (v/v) D2O supplement. The 1H–15N heteronuclear single quantum correlation (HSQC) spectra were acquired at 25°C on a Bruker Avance Ice 600 MHz spectrometer equipped with a cryoprobe. Data were processed and analyzed by NMRPipe (Delaglio et al., 1995).

Integrin binding

Integrin binding was analyzed as described (Shi et al., 2007). Briefly, podocytes expressing GFP-tagged wild-type or mutant forms of kindlin-2 were lysed with 1% Triton X-100 in 20 mM Tris (pH 7.1). The lysates were incubated with glutathione-Sepharose beads containing GST, GST–β1-integrin tail or GST–β3-integrin tail overnight at 4°C. The glutathione–Sepharose beads were precipitated by centrifugation. After washing, the samples were analyzed by western blotting and Coomassie Blue staining.

Immunofluorescence staining

Podocytes were cultured under nonpermissive conditions for 13 days and replated on fibronectin-coated (10 μg/ml) cover slips. Twenty-four hours after replating, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and 1 mg/ml BSA. The cells were stained with mouse and/or rabbit primary Abs as specified. The primary Abs were detected with secondary Rhodamine-Red-conjugated anti-mouse IgG Ab and FITC-conjugated anti-rabbit IgG Ab. Actin filaments were detected with FITC-conjugated phalloidin. In some experiments, podocytes were transfected with various expression vectors as specified. One day after transfection, the cells were plated on fibronectin-coated (10 μg/ml) cover slips and stained with Abs as described above. In some experiments (as specified), cells were treated with 0.1 μM wortmannin for 1 hour prior to fixation. For immunofluorescent staining with anti-fibronectin Ab, cells were plated on Matrigel-coated (30 μg/ml) cover slips (to avoid fibronectin background staining), fixed and stained with Abs as described above.

Cell–ECM adhesion assay

Cell–ECM adhesion was performed as we described (Shi et al., 2007). Briefly, 96-well plates (Greiner Bio-One) were coated with fibronectin (10 μg/ml) or vitronectin (5 μg/ml) and incubated with 40 mg/ml heat-denatured BSA for 30 minutes. Kindlin-2 knockdown and control cells were labeled with Calcein-AM for 30 minutes and seeded (2.5×104 cells/well) in triplicates in the wells. To overexpress wild-type or mutant forms of kindlin-2 in podocytes, the cells were infected with corresponding adenoviral vectors. Two days after infection, the cells were seeded (2.5×104 cells/well) in triplicates. The fluorescent signals from the total seeded cells were measured using a GENios Pro Fluorescence Microplate Reader (Tecan) (excitation wavelength=485 nm; emission wavelength=535 nm). The plates were centrifuged with a Sorvall RT7 Plus centrifuge at 60.4 g for 10 minutes at 4°C to facilitate cell settlement. The plates were then centrifuged upside-down at 60.4 g for 1 minute. After removing detached cells, the fluorescent signals from attached cells were measured again. Cell adhesion was calculated as the fluorescence reading of attached cells divided by the fluorescence reading of the total seeded cells. The adhesion of kindlin-2 knockdown or overexpressing podocytes was compared with that of the control cells (normalized to 1).

Cell surface expression and activation of integrins

Cell surface expression and activation of β1-integrin were analyzed as described (Luque et al., 1996). Briefly, cells were suspended on ice in RPMI 1640 medium for 30 minutes and then incubated with P5D2 at 4°C or HUTS-4 in HEPES/NaCl buffer (20 mM HEPES, 150 mM NaCl, 2 mg/ml D-glucose, pH 7.4) at 37°C for 30 minutes. After washing, the cells were incubated on ice for 30 minutes with R-Phycoerythrin- or Allophycocyanin-conjugated F(ab1)2 anti-mouse IgG Ab. The cells were analyzed using a BD LSR II flow cytometer. The cell surface expression of β1-integrin in kindlin-2 knockdown or overexpressing podocytes was assessed by P5D2 staining and compared with that of the control cells. The integrin activation index is defined as the mean fluorescence intensities of HUTS-4 staining (active β1-integrin) divided by the mean fluorescence intensities of P5D2 staining (total β1-integrin). The effect of kindlin-2 on β1-integrin activation was assessed by comparing the activation index in kindlin-2 knockdown or overexpressing podocytes with that of the control cells (normalized to 100%).

Cell surface expression and activation of β3-integrin was analyzed as described (Byzova et al., 2000; Pampori et al., 1999). Briefly, cells were suspended on ice in RPMI 1640 medium for 30 minutes and then incubated on ice with anti-αvβ3 mAb LM609 in 137 mM NaCl, 2.7 mM KCl, 3.3 mM NaH2PO4, 3.8 mM HEPES, 1 mM MgCl2, 5.5 mM glucose and 1 mg/ml bovine serum albumin, pH 7.4 (the incubation buffer) for 30 minutes. After washing, the cells were incubated for 30 minutes on ice with R-Phycoerythrin- or Allophycocyanin-conjugated F(ab1)2 anti-mouse IgG Ab, washed and analyzed using a BD LSR II flow cytometer. To analyze β3-integrin activation, cells were incubated with WOW-1 Fab in the incubation buffer for 30 minutes. After washing, the cells were incubated for 30 minutes with R-Phycoerythrin- or Allophycocyanin-conjugated F(ab1)2 anti-mouse IgG Ab. After washing, the cells were analyzed using a BD LSR II flow cytometer. The activation index is defined as the mean fluorescence intensities of WOW-1 staining divided by the mean fluorescence intensities of LM609 staining. The effect of kindlin-2 on β3-integrin activation was assessed by comparing the activation index in kindlin-2 knockdown or overexpressing podocytes with that of the control cells (normalized to 100%).

Fibronectin matrix deposition

Fibronectin matrix deposition was analyzed as described (Guo and Wu, 2002). Briefly, cells were harvested and extracted sequentially with: (1) 3% Triton X-100 in PBS containing 1 mM AEBSF; (2) 100 μg/ml DNase I in 50 mM Tris-HCl, pH 7.4, 10 mM MnCl2, 1 M NaCl and 1 mM AEBSF; and (3) 2% deoxycholate in Tris, pH 8.8 and 1 mM AEBSF. Fibronectin in deoxycholate-insoluble ECM fractions was analyzed by western blotting. In parallel experiments, cells were lysed with 1% SDS in PBS. Fibronectin in SDS lysates (total fibronectin) was analyzed by western blotting. For TGF-β1 treatment, cells were serum-starved for 24 hours and then switched to serum-free RPMI 1640 medium containing 5 ng/ml TGF-β1 or RPMI 1640 medium lacking TGF-β1 (as a control) for 48 hours. To quantify the changes of fibronectin in total cell lysates and ECM fractions, the densities of fibronectin and tubulin (or GAPDH) bands were quantified using the NIH ImageJ program. The relative levels of fibronectin (defined as densities of the fibronectin bands/densities of the tubulin or GAPDH bands) in TGF-β1-treated, p110*-expressing, kindlin-2 mutant overexpressing and/or kindlin-2 knockdown cells were compared with that in control cells (normalized to 1).

Statistical analysis

Student's t-test was used for statistical analyses of the results. P-values <0.05 were considered statistically significant.

Acknowledgments

This work was supported by NIH grants GM65188 and DK54639 (to C.W.), HL56595 and HL78784 (to S.J.S.) and DE016099 (to H.L.). We thank Martin J. Humphries (University of Manchester), Linton Traub (University of Pittsburgh) and Lawrence Kane (University of Pittsburgh) for providing antibodies and DNA constructs. Deposited in PMC for release after 12 months.

  • Accepted November 12, 2010.
  • © 2011.

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Research Article
Kindlin-2 regulates podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and integrins
Hong Qu, Yizeng Tu, Xiaohua Shi, Hannu Larjava, Moin A. Saleem, Sanford J. Shattil, Koichi Fukuda, Jun Qin, Matthias Kretzler, Chuanyue Wu
Journal of Cell Science 2011 124: 879-891; doi: 10.1242/jcs.076976
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Research Article
Kindlin-2 regulates podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and integrins
Hong Qu, Yizeng Tu, Xiaohua Shi, Hannu Larjava, Moin A. Saleem, Sanford J. Shattil, Koichi Fukuda, Jun Qin, Matthias Kretzler, Chuanyue Wu
Journal of Cell Science 2011 124: 879-891; doi: 10.1242/jcs.076976

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