PINCH-1 Promotes Tubular Epithelial-to-Mesenchymal Transition by Interacting with Integrin-Linked Kinase
Yingjian Li,
Chunsun Dai,
Chuanyue Wu and
Youhua Liu
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Correspondence: Dr. Youhua Liu, Department of Pathology, University of Pittsburgh, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: 412-648-8253; Fax: 412-648-1916; E-mail: liuy{at}upmc.edu
Received for publication March 15, 2007.
Accepted for publication May 22, 2007.
PINCH-1 is an adaptor protein that binds to the integrin-linkedkinase (ILK), an intracellular serine/threonine protein kinasethat plays a critical role in mediating tubular epithelial-to-mesenchymaltransition (EMT). To determine whether PINCH-1 is also involvedin the EMT process, we investigated its regulation and functionduring TGF-1–stimulated EMT. TGF-1 induced PINCH-1 mRNAand protein expression in human proximal tubular epithelialcells in a time-dependent fashion, an effect that was largelydependent on intracellular Smad signaling. Overexpression ofPINCH-1 suppressed epithelial markers E-cadherin and ZO-1 andincreased fibronectin expression and extracellular assembly,whereas knockdown of PINCH–1 via small interfering RNAreduced TGF–1–mediated fibronectin expression andpartially restored E–cadherin. PINCH–1 formed aternary complex with ILK at the focal adhesion sites of tubularepithelial cells. Treatment with an ILK inhibitor or disruptionof the ILK/PINCH-1 interaction by overexpressing a dominant-negativeN-terminal ankyrin domain of ILK resulted in reduced fibronectindeposition, indicating that the ability of PINCH-1 to stimulateEMT is ILK-dependent. In a mouse model of obstructive nephropathy,PINCH-1 expression increased in a time-dependent manner, suggestingthat it may play a role in EMT and renal fibrosis in vivo. Weconclude that PINCH-1, through its interaction with ILK, playsan important role in regulating TGF-1–mediated EMT andcould be a potential future therapeutic target to prevent progressionof renal disease.
Tubular epithelial-to-mesenchymal transition (EMT) is increasinglyrecognized as one of the major pathways leading to generationof the matrix-producing effector cells in the fibrotic kidneyafter a diverse array of injuries.1–4 Extensive studiesindicate that TGF-1, a potent fibrogenic cytokine whose expressionis markedly induced in virtually every type of chronic kidneydiseases,5–8 is the driving force behind tubular EMT underpathologic conditions. In vitro, TGF-1, as a single factor,is able to orchestrate the completion of the entire EMT coursethat consists of four key steps.9,10 Blockade of TGF-1 signalingthrough either upregulating Smad transcriptional co-repressorSnoN by hepatocyte growth factor (HGF) or genetic ablation ofSmad3 inhibits tubular EMT and attenuates renal interstitialfibrosis.3,11–13
Although the vital role of TGF-1 in promoting tubular EMT iswidely accepted, the mechanism by which TGF-1 regulates theEMT process remains incompletely understood. We previously identifiedthe integrin-linked kinase (ILK) as a key mediator in TGF-1–inducedEMT.14 ILK is an intracellular serine/threonine protein kinasethat interacts with the cytoplasmic domains of integrins andmediates the integrin signaling in diverse types of cells.15,16Apart from its catalytic kinase activity, ILK is known as anadaptor protein that interacts with numerous intracellular proteins,including PINCH-1 (particularly interesting new cysteine-histidinerich protein-1),17 the founding member of small family of proteinsconsisting of a tandem array of five LIN11, Isl1, and MEC-3(LIM) domains that are involved in mediating protein–proteininteractions.18,19 Previous studies revealed that PINCH-1 andILK form a complex in mammalian cells through an interactionbetween the LIM1 domain of PINCH-1 and the ankyrin (ANK) repeatdomain of ILK.20,21 Disruption of the PINCH-1/ILK complex formationhas been shown to influence cell survival, proliferation, andmatrix deposition in cultured cells.20,22–25 Therefore,it seems that ILK works in concert with its interacting partnerPINCH-1 to elicit its cellular function.
PINCH-1 is expressed in early embryonic development and adulttissues in a ubiquitous manner.24,26 It primarily localizesat the focal adhesion sites of the culture cells, a patternthat overlaps with other focal adhesion proteins, includingILK. Knockout of PINCH-1 gene in mice and other model organismssuch as Caenorhabditis elegans and Drosophila results in developmentaldefects in cell polarity, cell–matrix adhesion, cell proliferation,and apoptosis, leading to early embryonic death,27–29phenotypes that resemble the deletion of ILK or 1-integrin.30–33Despite that PINCH-1 has been shown to express in embryonicand adult kidney, relatively little is known about its regulationand function in the kidney in both physiologic and pathologicsettings.
In this study, we demonstrate that PINCH-1 expression is inducedin tubular epithelial cells by TGF-1 and in the fibrotic kidneyafter ureteral obstruction. We further show that PINCH-1 isfunctionally involved in promoting tubular EMT after chronicinjury, and the action of PINCH-1 is likely operated throughits interaction with ILK.
TGF-1 Induces PINCH-1 Expression in Tubular Epithelial Cells
Northern blot analysis demonstrated that TGF-1 significantlyinduced PINCH-1 mRNA expression in human proximal tubular epithelial(HKC-8) cells. As shown in Figure 1A, the steady-state levelof PINCH-1 mRNA increased at 6 h, sustained at least to 48 hafter TGF-1 treatment. PINCH-1 protein was also induced afterTGF-1 treatment in tubular epithelial cells, as demonstratedby Western blot analyses. As presented in Figure 1B, PINCH-1protein began to increase at 12 h after TGF-1, a time pointthat lagged significantly behind the mRNA induction (Figure 1A).The PINCH-1 protein induction was sustained to at least 72 h.Figure 1C shows the dose-response of PINCH-1 protein inductionto TGF-1 stimulation. Maximal induction was observed when HKC-8cells were treated with 2 ng/ml TGF-1. Further increase in TGF-1concentration did not result in additional induction of PINCH-1in HKC-8 cells.
Figure 1. TGF-1 induces PINCH-1 mRNA and protein expression in tubular epithelial cells. (A) Northern blot shows that TGF-1 stimulated PINCH-1 mRNA expression in a time-dependent manner. HKC-8 cells were incubated with 2 ng/ml TGF-1 for various periods as indicated. Total RNA was isolated and blotted with cDNA probes for PINCH-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. (B and C) Western blot analyses demonstrate that TGF-1 induced PINCH-1 protein expression in a time- and dosage-dependent manner. HKC-8 cells were treated with 2 ng/ml TGF-1 for various periods as indicated (B) or with various concentrations of TGF-1 for 48 h (C). Total cell lysates were immunoblotted with specific antibodies against PINCH-1 and actin, respectively. Representative Western blots of three experiments.
We next investigated the signal pathway that is responsiblefor the TGF-1–mediated PINCH-1 induction. Previous studiesshowed that TGF-1 activates multiple signal pathways, includingSmad, Akt, and p38 mitogen-activated protein kinase in tubularepithelial cells.14 However, blockade of Akt or p38 mitogen-activatedprotein kinase activation by specific chemical inhibitors didnot affect TGF-1–mediated PINCH-1 induction (Figure 2A).Of note, these inhibitors at the concentrations used were ableto suppress the activation of their respective signaling inHKC-8 cells.14,34 To examine the role of Smad signaling in PINCH-1induction, we investigated the effect of the overexpressionof a Smad transcriptional co-repressor, SnoN. To this end, weestablished several cell lines that overexpress SnoN by stablytransfecting HKC-8 cells with SnoN expression vector (Figure 2B).We found that overexpression of SnoN inhibited the TGF-1–mediatedPINCH-1 induction in tubular epithelial cells (Figure 2C). Comparedwith mock-transfection controls, PINCH-1 expression after TGF-1treatment was reduced substantially in the SnoN-overexpressingcells (Figure 2, C and D), suggesting that PINCH-1 inductionby TGF-1 is largely dependent on intracellular Smad signaling.
Figure 2. PINCH-1 induction by TGF-1 is dependent on intracellular Smad signaling. (A) Pharmacologic inhibition of different signal transduction pathways did not affect PINCH-1 induction by TGF-1. HKC-8 cells were pretreated with either various chemical inhibitors or vehicle (DMSO) for 30 min, followed by incubating in the absence or presence of 2 ng/ml TGF-1 for 48 h. Specific inhibitors for phosphoinositide 3-kinase (10 nM wortmannin), Mek1 (10 µM PD98059), p38 mitogen-activated protein kinase (20 µM SC68376), and protein kinase A inhibitor (0.3 µM protein kinase A inhibitor) were used, respectively. (B) Establishment of stable SnoN-expressing cell lines. HKC-8 cells were stably transfected with SnoN expression plasmid (pHA-SnoN) or empty vector. Overexpression of SnoN in different cell clones was confirmed by Western blot. (C) Western blot showed that overexpression of SnoN suppressed PINCH-1 induction by TGF-1. (D) Graphic presentation of the relative PINCH-1 abundance normalized to actin after TGF-1 treatment. Data are means ± SEM of three experiments. *P < 0.05 versus vector controls.
Ectopic Expression of PINCH-1 Induces Tubular EMT
To investigate the functionality of PINCH-1 induction, we examinedthe effect of overexpression of exogenous PINCH-1 on tubularepithelial cell phenotypes. To this end, we established severalstable cell lines that overexpress PINCH-1 by transfecting witheither the expression vector of the Flag-tagged PINCH-1 or emptyvector. Overexpression of the Flag-tagged PINCH-1 was confirmedby Western blotting with anti-Flag antibody (Figure 3A). Asshown in Figure 3B, ectopic expression of PINCH-1 suppressedepithelial cell marker E-cadherin expression in tubular epithelialcells. In addition, expression of exogenous PINCH-1 inducedfibronectin expression and deposition (Figure 3C). The magnitudeof E-cadherin suppression and fibronectin induction elicitedby overexpressing PINCH-1 was comparable to that induced byTGF-1.
Figure 3. Ectopic expression of PINCH-1 results in loss of the epithelial markers E-cadherin and zonula occludens-1 (ZO-1) and induction of fibronectin. (A) Establishment of stable cell lines overexpressing PINCH-1. HKC-8 cells were stably transfected with either the expression vector of the Flag-tagged PINCH-1 or empty vector. The overexpression of the Flag-tagged PINCH-1 was confirmed in several stable cell lines by Western blotting with anti-Flag antibody (A). (B) Forced expression of PINCH-1 suppressed E-cadherin expression in tubular epithelial cells. TGF-1 treatment (2 ng/ml) of HKC-8 cells was used as positive control. (C) Forced expression of PINCH-1 induced fibronectin expression and assembly. Assembled extracellular fibronectin was extracted and detected by Western blot. Representative Western blots of three experiments. (D) Immunofluorescence staining showed that overexpression of PINCH-1 suppressed epithelial markers E-cadherin and ZO-1 and induced fibronectin expression in tubular epithelial cells. Bar = 5 µm.
Figure 3D shows the immunofluorescence staining of E-cadherinand fibronectin in tubular epithelial cells. Compared with theempty vector controls, overexpression of PINCH-1 resulted indisappearance of E-cadherin and zonula occludens-1 (ZO-1) stainingin plasma membrane (Figure 3D), whereas extracellular matrixcomponent fibronectin expression was markedly induced in PINCH-1–overexpressingcells. Hence, it seems that PINCH-1 triggers E-cadherin andZO-1 suppression and fibronectin induction, the reciprocal changesassociated with the phenotypic transition from epithelial tomesenchyme.
Knockdown of PINCH-1 Expression Blocks EMT
To characterize further the potential function of PINCH-1 intubular epithelial cells, we investigated the impact of theknockdown of endogenous PINCH-1 in tubular epithelial cells.For this purpose, tubular epithelial HKC-8 cells were transfectedwith PINCH-1–specific small interfering RNA (siRNA) orcontrol siRNA. As shown in Figure 4, A and B, transfection ofHKC-8 cells with specific siRNA resulted in significant reductionof endogenous PINCH-1 mRNA and protein expression. Approximately85% of HKC-8 cells were transfected with siRNA, as illustratedby a transfection efficiency assessment (Figure 4C). We foundthat knockdown of PINCH-1 only partially restored E-cadherinexpression (Figure 4, D and E). This may reflect that anotherdownstream effector of TGF-1 signaling, Id1, also plays a rolein conferring E-cadherin suppression by an ILK-independent mechanism.35However, downregulation of PINCH-1 reduced fibronectin expressionand deposition in basal conditions (Figure 4F, lane 3 versus1) and completely prevented fibronectin overproduction in responseto TGF-1 stimulation (Figure 4F, lane 6 versus 4).
Figure 4. Knockdown of endogenous PINCH-1 expression partially restores E-cadherin expression and inhibits fibronectin induction. (A through C) Knockdown endogenous PINCH-1 expression by small interfering RNA (siRNA) strategy. Reverse transcriptase–PCR (A) and Western blot (B) demonstrated a reduced PINCH-1 mRNA and protein expression after transfection of PINCH-1–specific siRNA. (C) Micrographs showed a high efficiency of siRNA transfection in HKC-8 cells, as illustrated by siGLO transfection indicator (red). (D and E) Knockdown of endogenous PINCH-1 partially restored E-cadherin mRNA (D) and protein (E) expression repressed by TGF-1. HKC-8 cells were transfected with either control or PINCH-1 siRNA, followed by incubation with 2 ng/ml TGF-1 for 48 h. (F) Western blot analysis showed that knockdown of PINCH-1 expression suppressed fibronectin expression in response to TGF-1 stimulation. Representative blots of two (D and F) or three (E) experiments.
PINCH-1 Forms a Complex with ILK at Focal Adhesions
PINCH-1 and ILK have been shown to interact with each other,and together with -parvin, they form a ternary complex thatis important for ILK-mediated cellular processes in other celltype.16,19 To investigate whether PINCH-1 forms a complex withILK in tubular epithelial cells, we first examined the potentialILK–PINCH-1 complex formation by immunoprecipitation.As shown in Figure 5A, when HKC-8 cell lysates were immunoprecipitatedwith anti–-parvin antibody, both ILK and PINCH-1 weredetected in the precipitated complexes. Neither ILK nor PINCH-1was found in the precipitates by normal control IgG. Therefore,ILK can form a ternary complex with PINCH-1 and -parvin in tubularepithelial cells.
Figure 5. PINCH-1 forms a complex with integrin-linked kinase (ILK) at focal adhesion sites. (A) Co-immunoprecipitation demonstrated a ternary complex formation among PINCH-1, ILK, and -parvin. HKC-8 cell lysates were immunoprecipitated with specific antibody against -parvin, followed by immunoblotting with antibodies against PINCH-1, ILK, and -parvin, respectively. mIgG, control mouse IgG. (B) Immunofluorescence staining exhibited the co-localization of PINCH-1 and ILK at the focal adhesion sites. HKC-8 cells were transiently transfected with green fluorescence protein (GFP)-tagged PINCH-1 plasmid, followed by staining with anti-ILK and anti-paxillin antibodies. Arrows indicate the co-localization sites at focal adhesions. Bar = 5 µm.
Immunofluorescence staining revealed a co-localization of ILKand PINCH-1 along the focal adhesion sites in tubular epithelialcells. Transfection of the HKC-8 cells with green fluorescenceprotein–PINCH-1 expression vector illustrated that thegreen fluorescence protein–tagged PINCH-1 was primarilylocalized in focal adhesions, the contact sites of plasma membraneto substrate (Figure 5B). Merging of PINCH-1 and ILK stainingrevealed a clear co-localization. The localization of PINCH-1at the focal adhesion sites was confirmed by its co-stainingwith paxillin, a focal adhesion protein (Figure 5B).
Disruption of the ILK–-PINCH-1 Interaction Inhibits Fibronectin Deposition
Because PINCH-1 forms a ternary complex with ILK in tubularepithelial cells (Figure 5), we sought to investigate whetherPINCH-1 action depends on its interaction with ILK. To thisend, we used a dominant negative approach by overexpressingthe truncated form of ILK that merely contains the ankyrin domain,the binding site of ILK to PINCH-1.22 Overexpression of Flag-ANKfragment in HKC-8 cells was confirmed after infection with recombinantadenovirus harboring Flag-tagged ankyrin domain sequence (Figure 6A).Immunostaining revealed that approximately 95% of the cellswere infected and expressed exogenous protein (data not shown).Because Flag-ANK competes with endogenous ILK to bind PINCH-1,22overexpression of Flag-ANK presumably leads to the disruptionof ILK–PINCH-1 interaction in a dominant negative manner.As shown in Figure 6B, disruption of ILK–PINCH-1 interactionsignificantly reduced fibronectin expression and depositionat both basal and TGF-1–stimulated conditions. Similarresults were obtained when the cells were immunostained withantifibronectin antibody (Figure 6C). These results suggestthat PINCH-1 action dependent on its interaction with ILK.
Figure 6. Disruption of the ILK–PINCH-1 interaction inhibits fibronectin expression and deposition. (A) HKC-8 cells were infected with either adenovirus harboring the Flag-tagged ILK N-terminal ANK fragment (Ad Flag-ANK) or control adenovirus containing -galactosidase (Ad -gal). Overexpression of Flag-ANK was confirmed by Western blot. (B) Forced expression of dominant negative Flag-ANK reduced fibronectin expression and deposition at both basal and TGF-1–stimulated conditions. Representative Western blots of three experiments. (C) Immunofluorescence staining showed that disruption of the ILK–PINCH-1 interaction by dominant negative Flag-ANK inhibited fibronectin expression and assembly in HKC-8 cells. Bar = 5 µm.
Small Molecule ILK Inhibitor Abrogates PINCH-1–Mediated EMT
To establish further the involvement of ILK in mediating PINCH-1action, we examined the effects of ILK inhibition on tubularEMT induced by PINCH-1 overexpression. As shown in Figure 7A,a highly selective small molecule ILK inhibitor, QLT0267, restoredE-cadherin expression in the PINCH-1–overexpressing tubularepithelial cells. Likewise, QLT0267 inhibited fibronectin expressioninduced by PINCH-1 in tubular epithelial cells in a dosage-dependentmanner (Figure 7B). Immunofluorescence staining also showedthat QLT0267 inhibited fibronectin expression and largely restoredZO-1 expression in the PINCH-1–overexpressing cells (Figure 7C).Therefore, inhibition of ILK abolishes the phenotypic conversionof tubular epithelial cells induced by PINCH-1 overexpression.
Figure 7. Small molecule ILK inhibitor QLT0267 blocks tubular epithelial-to-mesenchymal transition (EMT) induced by PINCH-1. (A) Small molecule ILK inhibitor restored E-cadherin expression in PINCH-1–overexpressing tubular epithelial cells. Stable PINCH-1–overexpressing (PINCH-1) and control (Vector) cells were treated with for 48 h at various concentrations as indicated. (B) QLT0267 inhibited fibronectin expression in the PINCH-1–overexpressing cells in a dosage-dependent manner. Representative Western blots of two experiments. (C) Immunofluorescence staining showed that small molecule ILK inhibitor QLT0267 inhibited fibronectin expression and largely restored ZO-1 expression in the PINCH-1–overexpressing cells. Bar = 5 µm.
PINCH-1 Expression Is Induced in the Fibrotic Kidney
To investigate the relevance of PINCH-1 induction to renal fibrosisin vivo, we examined the expression of PINCH-1 in the evolutionof renal interstitial fibrosis induced by ureteral obstruction.As shown in Figure 8, Western blot analyses revealed that PINCH-1was markedly induced in the fibrotic kidney in a time-dependentmanner. PINCH-1 protein began to increase as early as day 1after unilateral ureteral obstruction (UUO), and significantinduction was observed at 3 d (Figure 8A), a time point precedingthe onset of EMT in this model.36 Quantitative determinationexhibited approximately eight-fold induction of the relativeabundance of PINCH-1 protein in the obstructed kidney at 14d after UUO, when compared with the sham controls (Figure 8B).Hence, PINCH-1 induction correlates with tubular EMT and renalinterstitial fibrogenesis in vivo.
Figure 8. PINCH-1 is induced in the fibrotic kidney after unilateral ureteral obstruction (UUO). (A) Western blot analysis showed a marked induction of PINCH-1 in the fibrotic kidney induced by UUO in a time-dependent manner. Kidney tissue lysates were prepared from the obstructed kidneys at different time points after UUO as indicated. The samples were immunoblotted with specific antibodies against PINCH-1 and GAPDH, respectively. (B) Graphic presentation of the relative abundance of PINCH-1 normalized to GAPDH in the obstructed kidneys at different time points after UUO. Data are means ± SEM (n = 4). P < 0.05 versus sham control.
The results presented in this study demonstrate that PINCH-1,a LIM domain-only adaptor protein that interacts with ILK, playsa critical role in mediating tubular EMT induced by TGF-1. Wehave shown that PINCH-1 expression is induced during TGF-1–mediatedEMT, and such induction is dependent on, at least in part, Smadsignaling, an intracellular signal pathway that is essentialfor EMT.12,37 Moreover, forced expression of exogenous PINCH-1mimics TGF-1 action, resulting in loss of E-cadherin and ZO-1and induction of fibronectin. Conversely, knockdown of PINCH-1completely blocks TGF-1–mediated fibronectin expressionand partially restores E-cadherin. Furthermore, PINCH-1 co-localizeswith ILK at the focal adhesion sites of tubular epithelial cellsand physically interacts with ILK, and disruption of PINCH-1–ILKinteraction causes a complete blockade of the TGF-1–mediatedfibronectin induction. Finally, PINCH-1 is upregulated in theobstructed kidney, and its expression is associated with tubularEMT and renal interstitial fibrosis in vivo. These observations,together with previous studies,14 have established that PINCH-1–ILKcomplex may act as a functional element that plays a crucialrole in mediating TGF-1–triggered tubular EMT, therebyhaving significant implications in the pathogenesis of renalinterstitial fibrosis.
PINCH-1 is expressed early in embryonic development and in adultkidney26; however, little is known from the literature aboutits regulation and function in renal tissues. This study providesclear evidence that PINCH-1 expression at both mRNA and proteinlevels is induced in the process of tubular EMT triggered byTGF-1. The intuitive association among PINCH-1, TGF-1, and EMTis also observed in the obstructed kidney in vivo, in whichboth TGF-1 and PINCH-1 are upregulated and tubular EMT is abundant.9,38PINCH-1 induction by TGF-1 most likely occurs at the gene transcriptionlevel, because its steady-state mRNA abundance is increasedafter TGF-1 stimulation (Figure 1). Consistent with this, PINCH-1mRNA induction significantly precedes an elevation of its proteinabundance (Figure 1). Therefore, it seems clear that PINCH-1is a target gene of TGF-1/Smad signaling. This is also corroboratedby the observation that blocking Smad signaling by overexpressingSnoN reduces PINCH-1 induction by TGF-1 (Figure 2).
The magnitude of PINCH-1 protein induction after TGF-1 treatment(four-fold) seems significantly greater than its mRNA increase(2.3-fold) in tubular epithelial cells. This is consistent withrecent studies demonstrating that the assembly of PINCH-1–ILKcomplex mutually stabilizes each other by preventing the proteolyticdegradation of PINCH-1 and ILK.23 It should be stressed thatILK is also induced concurrently by TGF-1 in tubular epithelialcells during EMT.14 Increased ILK will form complex with PINCH-1,which occurs before their localization to the cell–matrixadhesion sites,39 thereby enhancing PINCH-1 protein stabilityand increasing its abundance. Accordingly, depletion of ILKvia siRNA inhibition has been shown to reduce PINCH-1 protein,but not mRNA, levels in other cell types.23 It therefore isconceivable that PINCH-1 and ILK depend on each other for maintenanceof their protein levels in mammalian cells. Needless to say,the simultaneous induction of both PINCH-1 and ILK expressionafter TGF-1 treatment will result in a substantial increasein PINCH-1–ILK complex formation during the process oftubular EMT.
One of the important observations in this study is that thefunctionality of PINCH-1 in tubular EMT is dependent on itsinteraction with ILK. This notion is supported by several observations.First, PINCH-1 physically interacts with ILK and forms a complexwith it at focal adhesion sites (Figure 5). Second, disruptionof PINCH-1–ILK interaction by overexpression of the ILK-ANKfragment, which results in competitive binding to endogenousPINCH-1 and reduces native PINCH-1–ILK interaction ina dominant negative manner,22 abolishes fibronectin induction,suggesting that PINCH-1 action necessitates its interactionwith ILK (Figure 6). Third, inhibition of ILK activity by selectivesmall molecule inhibitor40 abrogates PINCH-1–mediatedE-cadherin suppression and fibronectin induction, providinga direct evidence for the involvement of ILK in PINCH-1 functionin tubular epithelial cells. The dependence of PINCH-1 on ILKto promote tubular EMT is also highly consistent with severalprevious studies demonstrating the importance of PINCH-1–ILKcomplex formation in eliciting their biologic function.22,23,39Structurally, PINCH-1 protein merely consists of a tandem arrayof five LIM domains and a short C-terminal tail.16 Such simplestructural arrangement suggests that PINCH-1 may function primarilythrough its ability to interact with other proteins. It is plausibleto postulate that PINCH-1 promotes tubular EMT mainly throughbinding to ILK, which leads to the recruitment of ILK to theclose proximity of plasma membrane and presentation of it tothe integrins. However, at this stage, we cannot completelyrule out the possibility that PINCH-1 may promote tubular EMTthrough other mechanisms as well.
This study represents the first demonstration that PINCH-1,an adaptor protein that interacts with ILK, is dysregulatedin the fibrotic kidney after obstructive injury. The concomitantinduction of both PINCH-1 (as presented in this study) and ILK(as reported previously14) underscores that PINCH-1–ILKcomplex may play a fundamental role in mediating tubular EMTin the obstructed kidney, in which more than one third of thematrix-producing fibroblasts are known to derive from tubularepithelial cells through EMT.38 In addition, the characteristickinetic of PINCH-1 and ILK induction further strengthens thisnotion. Undoubtedly, further investigations on the regulation,function, and mechanism of PINCH-1 in the setting of renal fibrosisare warranted to gain mechanistic insights into the regulationof tubular EMT and to offer new clues for designing rationalstrategies for the treatment of chronic kidney diseases.
Cell Culture and Cytokine Treatment
HKC-8 was provided by Dr. L. Racusen (Johns Hopkins University,Baltimore, MD). Cells were cultured in DMEM-Ham's F12 mediumsupplemented with 5% FBS. Serum-starved HKC-8 cells were treatedwith recombinant TGF-1 (R & D Systems, Minneapolis, MN)for various periods of time at the concentration of 2 ng/mlexcept as otherwise indicated. The cells were then collectedfor Northern and Western blot analyses and immunofluorescencestaining. In some experiments, cells were pretreated for 30min with various chemical inhibitors at the concentrations specified,followed by incubating in the absence or presence of 2 ng/mlTGF-1. PD98059, wortmannin, myristoylated protein kinase A inhibitor,and SC68376 were purchased from Calbiochem (La Jolla, CA). Smallmolecule ILK inhibitor QLT0267 was provided by QLT (Vancouver,BC, Canada).41 All other chemicals were of analytic grade andwere obtained from Sigma (St. Louis, MO) or Fisher (Pittsburgh,PA) unless otherwise indicated.
Establishment of Stable Cell Lines
HKC-8 cells were transfected with HA-tagged SnoN expressionvector (provided by Dr. R. Weinberg, MIT, Cambridge, MA)42 andFlag-tagged PINCH-1 expression plasmid,23 respectively, usingthe Lipofectamine 2000 according to the instructions specifiedby the manufacturer (Invitrogen, Carlsbad, CA). The empty vectorpcDNA3 (Invitrogen) or p3XFlag-CMV (Sigma) was used as a mock-transfectioncontrol. Twenty-four hours after transfection and every 3 to4 d thereafter, the cells were refed with fresh selective mediumcontaining G418 (Geneticin; Invitrogen) at a final concentrationof 0.8 mg/ml. Neomycin-resistant clones were selected and expandedindividually. Ectopic expression of SnoN and PINCH-1 in thestable cell lines was confirmed by Western blot analysis.
Northern Blot Analysis and Reverse Transcriptase–PCR
Total RNA was extracted from HKC-8 cells by using TRIzol reagent,according to the instructions specified by the manufacturer(Invitrogen). Northern blot analysis for gene expression wascarried out by the procedures described previously.3632P-labeledDNA probes were prepared by the random primer labeling kit (Stratagene,La Jolla, CA). After autoradiography, membranes were strippedand rehybridized with glyceraldehyde-3-phosphate dehydrogenaseprobe to ensure equal loading of each lane.
For reverse transcriptase–PCR, the first strand of cDNAwas synthesized using 2 µg of RNA in 20 µl of reactionbuffer by reverse transcription using AMV-RT (Promega, Madison,WI) and random primers at 42°C for 30 min. PCR was carriedout using a standard PCR kit on 1-µl aliquots of cDNAand HotStarTaq polymerase (Qiagen, Valencia, CA) with specificprimer pairs designed for PINCH-1, E-cadherin, and -actin, asdescribed previously.36 The sequences of primer pairs were asfollows. PINCH-1 forward 5'-AAGAACGACCCCTACCATCC and reverse5'-TGGCTTCATGTCAAACTCCA; E-cadherin forward 5'-TGGCTGAAGGTGACAGAGCand reverse 5'-CGTTAGCCTCGTTCTCAGG; and -actin forward 5'-TCAAGATCATTGCTCCTCCTGAGCand reverse 5'-TGCTGTCACCTTCACCGTTCCAGT.
Western Blot Analysis
Western blot analysis for specific protein expression was performedessentially according to an established procedure.11 The extracellularassembly of fibronectin in the deoxycholate-insoluble fractionwas quantitatively determined by an established method as describedpreviously.14 The primary antibodies used were as follows: Rabbitpolyclonal anti-PINCH-1 as described previously,20 anti-ILK(05-575; Upstate Biotechnology, Charlottesville, VA), anti-SnoN(sc-9595), and anti-actin (sc-1616; Santa Cruz Biotechnology,Santa Cruz, CA); anti–-smooth muscle actin (-SMA; clone1A4; Sigma); anti–E-cadherin (clone 36) and anti-fibronectin(clone 10; BD Transduction Laboratories, Lexington, KY); anti-Flag(M2) and anti–-tubulin (T9026; Sigma); and anti–glyceraldehyde-3-phosphatedehydrogenase (Ambion, Austin, TX) and anti–-parvin.39Quantification was performed by measurement of the intensityof the bands with the use of National Institutes of Health Imageanalysis software.
Immunofluorescence Staining
Indirect immunofluorescence staining was performed using anestablished procedure.11 Briefly, cells cultured on coverslipswere washed with cold PBS twice and fixed with cold methanol:acetone(1:1) for 10 min at –20°C. After extensive washingwith PBS containing 0.5% BSA for three times, the cells wereblocked with 20% normal donkey serum in PBS buffer for 30 minat room temperature and then incubated with the specific primaryantibodies against E-cadherin, fibronectin, and ILK as describedpreviously, as well as anti–ZO-1 (61–7300; Invitrogen)and anti-paxillin (610052; BD Transduction). For visualizationof the primary antibodies, cells were stained with cyanine Cy2-or Cy3-conjugated secondary antibodies (Jackson ImmunoResearchLaboratories, West Grove, PA). As a negative control, the primaryantibody was replaced with nonimmune IgG, and no staining occurred.Cells were double stained with DAPI (4',6-diamidino-2-phenylindole,HCl) to visualize the nuclei. Stained cells were mounted withVectashield mounting medium (Vector Laboratories, Burlingame,CA) and viewed under Nikon Eclipse E600 Epi-fluorescence microscopeequipped with a digital camera (Melville, NY).
Immunoprecipitation
Immunoprecipitation experiments were performed using similarmethods as described previously.43 Briefly, HKC-8 cell lysateswere centrifuged at 12,000 x g for 10 min at 4°C. The resultingsupernatants were collected for immunoprecipitation. After preclearingwith normal host IgG, the lysates were immunoprecipitated overnightat 4°C with 2 µg of anti–-parvin or normal mouseIgG, followed by precipitation with 30 µl of protein A/GPlus-Agarose (Santa Cruz Biotechnology) for 3 h at 4°C.The precipitated complexes were boiled for 5 min in SDS samplebuffer, followed by immunoblotting with various antibodies asindicated.
siRNA Inhibition
The PINCH-1–specific siRNA duplex was purchased from Ambionor generated in our laboratory by using Silencer siRNA constructionkit (Ambion), as described previously.44 The oligonucleotidetemplate for silencing PINCH-1 gene contains 21-nt target sequence5'-AAGGTGATGTGGTCTCTGCTC-3' and 8-nt leader sequence 5'-CCTGTCTC-3'that is complementary to the T7 promoter primer. An unrelatedsiRNA template (with no sequence homology) was also used togenerate negative control siRNA. PINCH-1–specific andcontrol siRNA duplexes were transiently transfected into HKC-8cells by using the siPORT NeoFX transfection reagents (Ambion).siGLO Red Transfection Indicator (Dharmacon, Chicago, IL) wasused for transfection efficiency assessment. Whole-cell lysateswere prepared and subjected to various analyses.
Animal Model
Male CD-1 mice weighing approximately 20 to 22 g were obtainedfrom Harlan Sprague-Dawley (Indianapolis, IN). UUO was performedusing an established procedure, as described previously.45 Micewere killed at various time points as indicated after surgery,and kidneys were removed. PINCH-1 expression was examined byWestern blot analysis of whole kidney lysates.
Strutz F, Zeisberg M: Renal fibroblasts and myofibroblasts in chronic kidney disease.
J Am Soc Nephrol 17
: 2992
–2998, 2006[Free Full Text]
Liu Y: Epithelial to mesenchymal transition in renal fibrogenesis: Pathologic significance, molecular mechanism, and therapeutic intervention.
J Am Soc Nephrol 15
: 1
–12, 2004[Abstract/Free Full Text]
Kalluri R, Neilson EG: Epithelial-mesenchymal transition and its implications for fibrosis.
J Clin Invest 112
: 1776
–1784, 2003[CrossRef][Medline]
Neilson EG: Mechanisms of disease: Fibroblasts—A new look at an old problem.
Nat Clin Pract Nephrol 2
: 101
–108, 2006[CrossRef][Medline]
Bottinger EP, Bitzer M: TGF-beta signaling in renal disease.
J Am Soc Nephrol 13
: 2600
–2610, 2002[Abstract/Free Full Text]
Schnaper HW, Hayashida T, Hubchak SC, Poncelet AC: TGF-beta signal transduction and mesangial cell fibrogenesis.
Am J Physiol Renal Physiol 284
: F243
–F252, 2003[Abstract/Free Full Text]
Liu Y: Renal fibrosis: New insights into the pathogenesis and therapeutics.
Kidney Int 69
: 213
–217, 2006[CrossRef][Medline]
Yang J, Liu Y: Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.
Am J Pathol 159
: 1465
–1475, 2001[Abstract/Free Full Text]
Fan JM, Ng YY, Hill PA, Nikolic-Paterson DJ, Mu W, Atkins RC, Lan HY: Transforming growth factor-beta regulates tubular epithelial-myofibroblast transdifferentiation in vitro.
Kidney Int 56
: 1455
–1467, 1999[CrossRef][Medline]
Yang J, Liu Y: Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.
J Am Soc Nephrol 13
: 96
–107, 2002[Abstract/Free Full Text]
Yang J, Dai C, Liu Y: A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition.
J Am Soc Nephrol 16
: 68
–78, 2005[Abstract/Free Full Text]
Sato M, Muragaki Y, Saika S, Roberts AB, Ooshima A: Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction.
J Clin Invest 112
: 1486
–1494, 2003[CrossRef][Medline]
Li Y, Yang J, Dai C, Wu C, Liu Y: Role for integrin-linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis.
J Clin Invest 112
: 503
–516, 2003[CrossRef][Medline]
Hannigan G, Troussard AA, Dedhar S: Integrin-linked kinase: A cancer therapeutic target unique among its ILK.
Nat Rev Cancer 5
: 51
–63, 2005[CrossRef][Medline]
Wu C: PINCH, N(i)ck and the ILK: Network wiring at cell-matrix adhesions.
Trends Cell Biol 15
: 460
–466, 2005[CrossRef][Medline]
Campana WM, Myers RR, Rearden A: Identification of PINCH in Schwann cells and DRG neurons: Shuttling and signaling after nerve injury.
Glia 41
: 213
–223, 2003[CrossRef][Medline]
Wu C, Dedhar S: Integrin-linked kinase (ILK) and its interactors: A new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes.
J Cell Biol 155
: 505
–510, 2001[Abstract/Free Full Text]
Wu C: The PINCH-ILK-parvin complexes: Assembly, functions and regulation.
Biochim Biophys Acta 1692
: 55
–62, 2004[Medline]
Tu Y, Li F, Goicoechea S, Wu C: The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells.
Mol Cell Biol 19
: 2425
–2434, 1999[Abstract/Free Full Text]
Jung KY, Chen K, Kretzler M, Wu C: TGF-beta1 regulates the PINCH-1-integrin-linked kinase-alpha-parvin complex in glomerular cells.
J Am Soc Nephrol 18
: 66
–73, 2007[Abstract/Free Full Text]
Guo L, Wu C: Regulation of fibronectin matrix deposition and cell proliferation by the PINCH-ILK-CH-ILKBP complex.
FASEB J 16
: 1298
–1300, 2002[Abstract/Free Full Text]
Fukuda T, Chen K, Shi X, Wu C: PINCH-1 is an obligate partner of integrin-linked kinase (ILK) functioning in cell shape modulation, motility, and survival.
J Biol Chem 278
: 51324
–51333, 2003[Abstract/Free Full Text]
Yang Y, Guo L, Blattner SM, Mundel P, Kretzler M, Wu C: Formation and phosphorylation of the PINCH-1-integrin linked kinase-alpha-parvin complex are important for regulation of renal glomerular podocyte adhesion, architecture, and survival.
J Am Soc Nephrol 16
: 1966
–1976, 2005[Abstract/Free Full Text]
Legate KR, Montanez E, Kudlacek O, Fassler R: ILK, PINCH and parvin: The tIPP of integrin signalling.
Nat Rev Mol Cell Biol 7
: 20
–31, 2006[Medline]
Braun A, Bordoy R, Stanchi F, Moser M, Kostka GG, Ehler E, Brandau O, Fassler R: PINCH2 is a new five LIM domain protein, homologous to PINCH and localized to focal adhesions.
Exp Cell Res 284
: 239
–250, 2003[Medline]
Liang X, Zhou Q, Li X, Sun Y, Lu M, Dalton N, Ross J Jr, Chen J: PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes.
Mol Cell Biol 25
: 3056
–3062, 2005[Abstract/Free Full Text]
Clark KA, McGrail M, Beckerle MC: Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes.
Development 130
: 2611
–2621, 2003[Abstract/Free Full Text]
Hobert O, Moerman DG, Clark KA, Beckerle MC, Ruvkun G: A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans.
J Cell Biol 144
: 45
–57, 1999[Abstract/Free Full Text]
Sakai T, Li S, Docheva D, Grashoff C, Sakai K, Kostka G, Braun A, Pfeifer A, Yurchenco PD, Fassler R: Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation.
Genes Dev 17
: 926
–940, 2003[Abstract/Free Full Text]
Yasunaga T, Kusakabe M, Yamanaka H, Hanafusa H, Masuyama N, Nishida E: Xenopus ILK (integrin-linked kinase) is required for morphogenetic movements during gastrulation.
Genes Cells 10
: 369
–379, 2005[Abstract/Free Full Text]
Stephens LE, Sutherland AE, Klimanskaya IV, Andrieux A, Meneses J, Pedersen RA, Damsky CH: Deletion of beta1 integrins in mice results in inner cell mass failure and peri-implantation lethality.
Genes Dev 9
: 1883
–1895, 1995[Abstract/Free Full Text]
Fassler R, Meyer M: Consequences of lack of beta1 integrin gene expression in mice.
Genes Dev 9
: 1896
–1908, 1995[Abstract/Free Full Text]
Dai C, Yang J, Liu Y: Transforming growth factor-beta1 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling.
J Biol Chem 278
: 12537
–12545, 2003[Abstract/Free Full Text]
Li Y, Yang J, Luo JH, Dedhar S, Liu Y: Tubular epithelial cell dedifferentiation is driven by the helix-loop-helix transcriptional inhibitor Id1.
J Am Soc Nephrol 18
: 449
–460, 2007[Abstract/Free Full Text]
Yang J, Shultz RW, Mars WM, Wegner RE, Li Y, Dai C, Nejak K, Liu Y: Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy.
J Clin Invest 110
: 1525
–1538, 2002[CrossRef][Medline]
Li JH, Zhu HJ, Huang XR, Lai KN, Johnson RJ, Lan HY: Smad7 inhibits fibrotic effect of TGF-beta on renal tubular epithelial cells by blocking Smad2 activation.
J Am Soc Nephrol 13
: 1464
–1472, 2002[Abstract/Free Full Text]
Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG: Evidence that fibroblasts derive from epithelium during tissue fibrosis.
J Clin Invest 110
: 341
–350, 2002[CrossRef][Medline]
Zhang Y, Chen K, Tu Y, Velyvis A, Yang Y, Qin J, Wu C: Assembly of the PINCH-ILK-CH-ILKBP complex precedes and is essential for localization of each component to cell-matrix adhesion sites.
J Cell Sci 115
: 4777
–4786, 2002[CrossRef][Medline]
Yoganathan N, Yee A, Zhang Z, Leung D, Yan J, Fazli L, Kojic DL, Costello PC, Jabali M, Dedhar S, Sanghera J: Integrin-linked kinase, a promising cancer therapeutic target: Biochemical and biological properties.
Pharmacol Ther 93
: 233
–242, 2002[CrossRef][Medline]
Koul D, Shen R, Bergh S, Lu Y, de Groot JF, Liu TJ, Mills GB, Yung WK: Targeting integrin-linked kinase inhibits Akt signaling pathways and decreases tumor progression of human glioblastoma.
Mol Cancer Ther 4
: 1681
–1688, 2005[Abstract/Free Full Text]
Sun Y, Liu X, Ng-Eaton E, Lodish HF, Weinberg RA: SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor beta signaling.
Proc Natl Acad Sci U S A 96
: 12442
–12447, 1999[Abstract/Free Full Text]
Zhang X, Li Y, Dai C, Yang J, Mundel P, Liu Y: Sp1 and Sp3 transcription factors synergistically regulate HGF receptor gene expression in kidney.
Am J Physiol Renal Physiol 284
: F82
–F94, 2003[Abstract/Free Full Text]
Yang J, Zhang X, Li Y, Liu Y: Downregulation of Smad transcriptional corepressors SnoN and Ski in the fibrotic kidney: An amplification mechanism for TGF-beta1 signaling.
J Am Soc Nephrol 14
: 3167
–3177, 2003[Abstract/Free Full Text]
Yang J, Dai C, Liu Y: Hepatocyte growth factor gene therapy and angiotensin II blockade synergistically attenuate renal interstitial fibrosis in mice.
J Am Soc Nephrol 13
: 2464
–2477, 2002[Abstract/Free Full Text]
This article has been cited by other articles:
Y. Li, X. Tan, C. Dai, D. B. Stolz, D. Wang, and Y. Liu Inhibition of Integrin-Linked Kinase Attenuates Renal Interstitial Fibrosis
J. Am. Soc. Nephrol.,
September 1, 2009;
20(9):
1907 - 1918.
[Abstract][Full Text][PDF]
J. A. Ardura, R. Berruguete, D. Ramila, M. V. Alvarez-Arroyo, and P. Esbrit Parathyroid hormone-related protein interacts with vascular endothelial growth factor to promote fibrogenesis in the obstructed mouse kidney
Am J Physiol Renal Physiol,
August 1, 2008;
295(2):
F415 - F425.
[Abstract][Full Text][PDF]
R. S. Al-Lamki, J. Wang, A. M. Tolkovsky, J. A. Bradley, J. L. Griffin, S. Thiru, E. C.Y. Wang, E. Bolton, W. Min, P. Moore, et al. TL1A Both Promotes and Protects from Renal Inflammation and Injury
J. Am. Soc. Nephrol.,
May 1, 2008;
19(5):
953 - 960.
[Abstract][Full Text][PDF]
Top
Introduction
RESULTS
1–stimulated EMT. TGF-
-parvin, they form a ternary complex that is important for ILK-mediated cellular processes in other cell type.16,19 To investigate whether PINCH-1 forms a complex with ILK in tubular epithelial cells, we first examined the potential ILK–PINCH-1 complex formation by immunoprecipitation. As shown in Figure 5A, when HKC-8 cell lysates were immunoprecipitated with anti–
