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1–Mediated Inhibition of HK-2 Cell Migration
Institute of Nephrology, University of Wales College of Medicine, Cardiff, Wales.
Correspondence to Dr. A. O. Phillips, Institute of Nephrology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN. Phone: 44-2920-748411; Fax: 44-2920-748470;
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Abstract |
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1 synthesis. This study examines the effect of TGF-1 on PTC migration. A model of PTC injury was used consisting of mechanically wounding a monolayer of HK2 cells followed by repopulation of the denuded area by time lapse photomicroscopy. Repopulation was the result of cell migration but not proliferation. Addition of TGF-1 led to a marked inhibition of cell migration increased expression of paxillin and vincullin and their incorporation into dense focal adhesion plaques. This was associated with increased association of focal adhesion components with the f-actin cytoskeleton. There was also increased 3 integrin expression and increased synthesis of the matrix component fibronectin. The effect on migration and focal adhesion reorganisation was abrogated by inhibitors of the RhoA downstream target ROCK, suggesting that signaling events resulting from altered 3 integrin expression initiate the TGF-1 response. These results suggest that, by inhibition of cell migration, increased expression of TGF-1 after ischemia delays recovery of proximal tubule structure and function. We speculate that this may contribute to permanent alteration in renal tubular function after severe ischemic injury. E-mail: PhillipsAO@cf.ac.uk |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transforming growth factor– is a 25-kD dimeric polypeptide growth factor with a wide range of biologic effects. There is overwhelming evidence implicating TGF- in the pathogenesis of progressive renal fibrosis associated with numerous diseases. In contrast, very little is known regarding its physiologic role in the kidney and how this may relate to the repair and remodeling of the kidney after acute and reversible renal injury. In vitro studies demonstrated stimulation of proximal tubular cell TGF-1 production when cells were cultured under hypoxic conditions (7). Recent studies performed in a rodent model of ischemic injury also demonstrated elevation of TGF-1 expression, seen predominantly in the proximal tubules (8). This occurred as early as 12 h post-ischemia, during the phase of cellular proliferation, and persisted for as long as 14 d post-ischemia, well into the phase of remodeling. In this model, antibody-mediated inhibition of TGF-1 activity was associated with decreased extracellular matrix synthesis during the recovery phase. These studies therefore suggest that hypoxia-induced PTC TGF-1 may be involved in the post-mitotic remodeling phase of recovery.
Our previous in vitro experiments have demonstrated that TGF-1 induced marked alteration in cell morphology and function. Stimulation of confluent monolayers of PTC also resulted in the loss of cell-cell contact, reorganization of the cell actin cytoskeleton, and a marked alteration in cell phenotype (9,10). To further our understanding of the role of TGF-1 in post-ischemic PTC remodeling, we have extended our previous observations to examine the effect of TGF-1 on cell migration and to define the mechanism by which TGF-1 mediates these effects. The data demonstrate that TGF-1 inhibits cell migration. Furthermore TGF-1 inhibition of cell migration was related to a marked increase in focal adhesion formation coupled with cytoskeletal reorganization and increased integrin expression together with increased matrix deposition. The data therefore suggest that prolonged expression of TGF-1 in the context of renal ischemia, through its anti-migratory effects, does not participate in the recovery/remodeling phase, but rather is likely to delay recovery of normal tubular structure and function. Furthermore, we speculate that increased expression of TGF-1 may contribute to the persistence of abnormal renal structure and function described after severe renal injury.
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Materials and Methods |
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3 integrin (dilution 1:50) from Upstate Inc (Lake Placid, NY). The tyrosine phosphatase inhibitor sodium orthovanadate and the inhibitors of the RhoA target-protein Rho-associated coiled-coil kinase (ROCK) R-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and 5(-isoquinolinesulfonyl)homopiperazine (HA-1077) were purchased from Calbiochem (San Diego, CA). Recombinant TGF-1 was purchased from R&D Systems (Oxford UK).
Cell Culture and Quantification of Cell Migration
HK2 cells (human renal proximal tubular epithelial cells immortalized by transduction with human papilloma virus (HPV) 16 E6/E7 genes (11)) were cultured in DMEM/Ham’s F12 (Life Technologies, Paisley, UK) supplemented with 10% FCS (Biological Industries Ltd, Cumbernauld, UK), glutamine (Life Technologies Ltd, Paisley, UK), Hepes buffer (Life Technologies BRL, Paisley, UK), insulin, transferring, and sodium selenite (Sigma, Poole, UK). Cells were grown at 37°C in 5% CO2 and 95% air. Fresh growth medium was added to cells every 3 to 4 d until confluent. Cells were grown to confluence and serum-deprived for 48 h before experimental manipulation. All experiments were performed under serum-free conditions. Under these conditions, the cell remained viable in a nonproliferating state. In all aspects of cell biology that we have studied previously, HK-2 cells respond in an identical fashion to primary cultures of human proximal tubular cells (12–15). They are therefore a good model from which general conclusions can be drawn in terms of proximal tubular cell biology.
Cell migration was examined utilizing a monolayer wounding system as described previously (16). Briefly, quiescent cell monolayers were injured by scraping with a sterile 200-µl pipette tip to generate an intersecting area of denuded cells. The monolayer was washed twice with PBS and then incubated with serum-free medium or serum free medium to which either TGF-1 or sodium orthovanadate was added.
Closure of the denuded area was monitored using an Axiovert 100M inverted microscope fitted with a digital camera (Hamamatsu), and images of the wounded area were captured as a digitalized sequence. The rate of motility of cells was calculated as the number of cells entering the central denuded area. Cell number was expressed as cells per mm2 of original denuded area.
Identification of Proliferation
Cell proliferation was evaluated as described previously by immunocytochemistry (16). Cells were wounded as described above, cultured in bromodeoxyuridine (BrdU) at a final concentration of 10-5M and at different time intervals were fixed in acetone:methanol (1:1) for 90 s. The incorporation of BrdU into the DNA of cells was then assessed as follows. After fixation, cells were washed with Tris-buffered saline, pH 7.6 (TBS) for 5 min, incubated with 95% (vol/vol) formamide in 0.15 M trisodium citrate for 45 min at 70°C to denature double-stranded DNA, and washed with TBS. After a blocking step (1% bovine serum albumin), anti-BrdU (diluted 1:1000 in TBS) was added, incubated for 30 min at room temperature, and after an additional washing step, incubated with anti-mouse immunoglobulins for an additional 30 min at room temperature. Cells were washed and incubated with alkaline phosphates-anti-alkaline phosphatase complex (Sigma) for 30 min, and incorporation of BrdU into the nuclei was monitored by staining with FAST RED (Sigma) for 20 min according to the manufacturer’s instructions. Finally, the cells were rinsed with water and mounted in aqueous mounting medium (Glycergel; DAKO ltd) and viewed by light microscopy.
Immunohistochemistry
Immunohistochemistry was performed for examination of the focal adhesion components, integrin expression, and cytoskeleton organization on cells grown in 8-well glass chamber slides (Nunc, Life Technologies/BRL Life Technologies Ltd, Paisley, UK). Cells were grown to confluence and stimulated under serum-free conditions with TGF-1 (10ng/ml) for up to 4 d. Culture medium was subsequently removed, and the cell monolayer was washed with sterile PBS. Cells were fixed in 3.5% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Tween in PBS for 5 min at room temperature. After fixation, slides were blocked with 1% bovine serum albumin (BSA) for 1 h before an additional washing step with PBS. Subsequently slides were incubated with the primary antibody, diluted in 1%BSA/PBS for 1 h at room temperature. Finally slides were incubated with the appropriate FITC-conjugated secondary antibody. Cells were then mounted and analyzed by fluorescence microscopy.
Double immunofluorescence was performed to examine stress fiber organization together with expression of paxillin following TGF-1 stimulation. Fixation and a blocking step as described above cells were incubated with anti-paxillin antibody for 1 h before addition of TRITC-conjugated phalloidin and mouse anti-FITC–conjugated monoclonal antibody to visualize both paxillin and stress fibers.
Western Blot Analyses
Association of focal adhesion components with the actin cytoskeleton was indirectly assessed by examining their triton solubility as described previously (17). Briefly, after seeding of equal number of cells, confluent monolayers were washed once with cold PBS, scraped, and rinsed into 5 ml of cold PBS. After centrifugation at 2500 rpm for 10 min, cells pellets were extracted in buffer (150 mM NaCl, 50 mM Tris-Cl, 0.01% NaN3, 2 mM EDTA, 1 mM sodium orthovandadate, 10 µg/ml leupeptin, 25 µg/m aprotinin) containing 1% Triton X-100 (TX buffer) for 30 min on ice. Samples were centrifuged at 12,500 rpm for 30 min, and the supernatant (Triton-soluble component including membrane and cytosolic fraction) was transferred to a separate tube and kept at -70°C until use.
Fibronectin concentration in cell culture supernatant as well as expression of focal adhesion components was determined by Western analysis performed by standard methodologies. Briefly, equal amounts of proteins were prepared in sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min at 95°C before loading onto 5 to 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gradient gels, and electrophoresis was carried out under reducing conditions according to the procedure of Laemmli (18). After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane (Amersham, Little Chalfont, UK). The membrane was blocked with Tris-buffered saline containing 5% nonfat powdered milk for 1 h and then incubated with the primary antibody in Tris-buffered saline containing 1% bovine serum albumin and 0.1% Tween 20 (Tris-buffered saline–Tween) for 1 h at room temperature. The blots were subsequently washed in Tris-buffered saline-Tween and then incubated with an appropriate HRP-conjugated secondary antibody in Tris-buffered saline–Tween. Proteins were visualized using enhanced chemiluminescence (Amersham, UK) according to the manufacturer’s instructions.
Statistical Analyses
Statistical analyses were performed using the unpaired t test, with a value of P < 0.05 considered to represent a significant difference. The data are presented as means ± SD of n experiments. For each individual experiment, the mean of duplicate determinations was calculated.
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Results |
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1 and Migration1 led to a marked inhibition of cell migration as compared with those seen in control experiments (Figure 1, lower panels). Decrease in migratory cell number was seen at all doses of TGF-1 between 1 and 10 ng/ml (Figure 2). This was exemplified by a significant inhibition of cell migration at all time points beyond 24 h after mechanical injury of the monolayer and addition of 1 ng/ml of TGF-1 (Figure 2) (24 h: control 66.2 ± 9 versus TGF-1 29.3 ± 7.7, P = 0.0007; 72 h: control 222.5 ± 35.2 versus TGF-1 95.6 ± 5.1, P = 0.0003, mean cell number mm2 ± SD, n = 3]. Following addition of TGF-1,a confluent cell monolayer did not reform over the time course studied.
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1 were not dependent on any cytotoxic effects related to culture under these conditions, the effect of TGF-1 after monolayer wounding was also assessed in the presence of 0.1% FCS. The data confirmed previously published work demonstrating enhanced epithelial cell migration in the presence of serum, and it also confirmed the anti-migratory effect of TGF-1 in the presence of serum (24 h: 0.1% FCS control 98.6 ± 26 versus 0.1% FCS+TGF-1 79.09 ± 5.2; 72 h: 0.1% FCS control 310.7 ± 29.9 versus 0.1% FCS+TGF-1 156.5 ± 11.1, P < 0.05, mean cell number mm2 ± SD, n = 3].
The contribution of cell proliferation in reformation of the cell monolayer after mechanical wounding was examined by incubation of cells with BrdU and use of immunocytochemical staining to detect its incorporation into cellular DNA. BrdU is only introduced into the S phase of the cell cycle; therefore, the method offers an accurate estimation of cell proliferation. Figure 3 demonstrates little uptake of BrdU at the margins of the wound for up to 6 d after mechanical wounding. Moreover, there were no differences in BrdU uptake after wounding under serum-free conditions (Figure 3, A and B) or after addition of TGF-1 under serum-free conditions (Figure 3, D and E). This is in contrast to assessment of cell proliferation away from the denuded area in which prominent cell proliferation was seen (Figure 3, C and G).
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1. These studies demonstrated that tyrosine phosphorylation of TGF-1 receptor associated E-cadherin led to disassembly of adherens junctional protein complexes and loss of cell-cell contact (20). Furthermore, our recent studies suggest that sodium othovanadate by inhibition of tyrosine phosphatase activity increased tyrosine phosphorylation of E-cadherin and mimicked the effect of TGF-1 on the disassembly of adherens junction complexes and reorganisation of the F-actin cytoskeleton. In the current study, we therefore sought to examine the role of adherens junction disassembly on cell migration using sodium orthovanadate in our mechanical wounding experimental system. In contrast to the inhibitory effect of TGF-1, addition of sodium orthovanadate led to marked increased rate of cell migration relative to the serum-free control conditions (Figure 4). Significant increase in migratory cell number entering the denuded area was seen at all time point beyond 48 h of monolayer wounding (Figure 5) (48 h: control 100.2 ± 37.1 versus sodium orthovanadate 283.2 ± 110.5, P = 0.04; 72 h: control 222.6 ± 35.1 versus sodium orthovanadate 408.2 ± 86.9, P = 0.02, mean cell number mm2 ± SD, n = 3). As with TGF-1, closure of the denuded area was not associated with cell proliferation as assessed by BrDU staining (data not shown).
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1 and sodium orthovandate cause a similar reorganization of PTC F-actin cytoskeleton. To examine how this relates to cell-matrix interaction and hence cell migration, we next sought to examine regulation of focal adhesions after addition of TGF-1 or sodium orthovanadate to wounded cells, as these form the link between the matrix-attached trans-membrane integrin receptors and the actin cytoskeleton.
Addition of TGF-1 led to a marked increase in the levels of paxillin and vinculin and their incorporation into dense focal adhesion plaques situated around the whole of the periphery of the cells in the monolayer, including those at the wound margin as assessed by immunocytochemistry and confocal microscopy (Figure 6). When cultured cells are extracted with non-ionic detergent, all soluble proteins, lipids, and most organelles are released into solution, whereas the nucleus, cytoskeleton, and cytoskeleton–associated proteins remain insoluble (17). Addition of TGF-1 to confluent monolayers of HK-2 cells resulted in decreased levels of Triton-soluble talin and vinculin, suggesting increased association with the Triton-insoluble cytoskeleton (Figure 7). The association of focal adhesion components with the cytoskeleton was further examined by double immunocytochemistry. The experiments demonstrated that TGF-1–induced increased paxillin levels co-localized with F-actin (Figure 8).
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In addition to alteration in focal adhesion expression and cytoskeletal rearrangement, addition of TGF-1 also led to incorporation of 3 integrin into focal adhesions (Figure 9). This was not associated with alteration in 3 integrin levels detectable by Western analysis, suggesting rather redistribution of 3 integrin to cell surface sites (data not shown). In addition, there was an increase in the level of the matrix component fibronectin as assessed by immunohistochemistry of extracellular matrix generated by the cells and also by Western analysis of cell culture supernatant after addition of TGF-1 (Figure 10). In contrast, addition of sodium orthovanadate did not increase the expression of either 3 integrin or fibronectin.
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1 (10 ng/ml) in the presence of either 1.4 µM Y-27632 or 20 µM HA-1077 (Figure 11). TGF-1 stimulation of cells in monolayer culture led to a marked increase in the expression of paxillin and its incorporation into dense focal adhesion plaques as seen in the wounding system (Figure 11c). In addition, recombinant TGF-1 also resulted in a reorganization of the actin filament architecture with coalescence of actin fibers into stress fibers with direct extension of these fiber formations from cell to cell as we have previously described (Figure 11d) (10). TGF-1–mediated formation of dense focal adhesion plaques and reorganization of the f-actin cytoskeleton were inhibited by both inhibitors, supporting the notion that RhoA and its downstream target ROCK are important components of the signaling events that alter cell-matrix association. In addition, the inhibitory effect of TGF-1 on cell migration after monolayer wounding was also abrogated when cells were stimulated by TGF-1 in the presence of either of the ROCK inhibitors, Y-27632 or HA-1077 (Figure 12). Addition of either inhibitor alone did not influence wound closure in the absence of TGF-1 (data not shown).
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Discussion |
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1 expression during the recovery phase of renal ischemia in rodent models suggests that hypoxia-induced PTC TGF-1 may be involved in the post-mitotic remodeling phase of recovery (8,24,25). Although the kidney exhibits a remarkable ability to recover from acute ischemic injury, it is also clear that kidney function may not completely return to normal after severe ischemic injury. Furthermore, studies in rats have shown that incomplete recovery from acute injury may be followed by progressive renal injury (24,26,27). Reports that impairment of graft function immediately after transplantation is associated with increased risk of late graft loss suggest that a similar predisposition to progressive renal disease may also occur in humans after severe ischemic injury (28,29). It has been proposed that endogenous renal TGF-1 serves to promote early tissue regeneration after acute injury via both autocrine and paracrine mechanisms (8). Neutralization of TGF-1 in vivo during the early phase of recovery also attenuates remodeling of the extracellular matrix (25). More recent studies have also demonstrated that increased expression of TGF-1 at a later stage after severe ischemic injury may be associated with permanent alterations in renal structure (24). Our aim was therefore to further examine the role of TGF-1 in proximal tubule repair after injury.
Our previous in vitro experiments have demonstrated that TGF-1 stimulation of confluent monolayers of PTC resulted in the loss of cell-cell contact, reorganization of the cell actin cytoskeleton, and a marked alteration in cell phenotype (20). Work by others has also demonstrated that TGF-1 regulates PTC apoptosis (30). Coordinated regulation of production and turnover of extracellular matrix components is essential for normal tissue homeostasis, and is known to influence events such as proximal tubular cell growth, proliferation, and differentiation (31). We have demonstrated that addition of TGF-1 to PTC led to the induction of type IV collagen mRNA, stimulation of collagen synthesis, and its incorporation into the extracellular matrix (10). It is clear therefore that TGF-1 receptor activation regulates numerous different intracellular signaling events implicated in the different phases of the repair and remodeling that occurs during PTC recovery from hypoxic injury. In this study, we have addressed a specific aspect of the role of TGF-1 related to recovery from ischemic injury, namely cell migration.
In the present study, we have described a simple and reproducible model with which to investigate PTC response to mechanical injury. Repair in this model occurred in the absence of proliferation at the leading edge, because the immunocytochemical studies with BrdU did not reveal an increase in cell number synthesizing DNA at the wound edge, thus the model is ideally suited to examine factors regulating PTC migration. The data clearly demonstrate that TGF-1 inhibits PTC cell migration and repair of the monolayer after mechanical injury. Much of the work on the role of TGF-1 in epithelial cell migration has been done in the context of keratinocyte migration and wound healing. The role of TGF-1 in re-epithelialization however remains unclear. In vitro experiments with keratinocytes have shown that TGF-1 stimulates the expression of integrins, which facilitates migration over a provisional basement membrane (32,33). More recent studies however have shown accelerated wound healing in Smad3-null mice (34) and also accelerated skin wound re-epithelialization in transgenic mice expressing a dominant negative type II TGF-1 receptor (35). Our data derived from PTC is therefore consistent with these later observations, suggesting that TGF-1 impairs cutaneous re-epithelialization. Our studies however are in contrast to a recent published report of increased tubular epithelial cell migration in response to TGF-1 (36). The discrepancy between our observations and this study may be explained both by the experimental system and also the stimuli used, in that chemotactic migration was studied in an in vitro Boyden chamber system, rather that migration after "wounding;" furthermore the pro-migratory effects were most pronounced after stimulation with a combination of TGF-1 and EGF.
We have demonstrated that TGF-1–mediated alteration in cell-cell contacts and disassembly of adherens junctional complexes directly alter cell phenotype and that this was unrelated to nuclear events as a result of either Smad or -catenin–dependent signaling pathways. Similarly, addition of sodium orthovanadate also resulted in alteration of cell morphology, which resembled addition of TGF-1. Cells lost cell-cell contact and became elongated and spindle shaped. Associated with loss of cell-cell contact, sodium orthovanadate resulted in re-localization of E-cadherin from the cell periphery, which was associated with increase in its tyrosine phosphorylation (10). These findings are identical to our recently published observations on the effects of addition of recombinant TGF- on adherens junction complex disassembly (20). Addition of sodium orthovanadate also resulted in a reorganization of the actin filament architecture with coalescence of actin fibers into stress fibers with direct extension of these fiber formations from cell to cell similar to that seen after addition of TGF-1. Despite these similarities between the effect of TGF-1 and sodium orthovanadate, they have opposite effects on cell migration, suggesting that loss of cell-cell contact is not the primary regulators of PTC motility.
Previous studies have demonstrated that regulation of cell migration requires cooperative effects of formation of focal adhesion and cytoskeleton reorganization. Addition of TGF-1 to PTC results in re-organization of the actin cytoskeleton (10) and also increase in both focal adhesion number and size. Migration rates are likely to require adhesion of intermediate strength with the underlying matrix (37). Strong adhesion is likely to prevent release of cells from the substratum during migration, whereas weak adhesions inhibit the development of traction. TGF-1–stimulated cells, which form larger than normal focal adhesions, may therefore form matrix attachments that are sufficiently strong to suppress migration. In contrast, sodium orthovanadate results in decreased expression of focal adhesion proteins and their association with the cytoskeleton. This is likely to result in the formation of focal adhesions of intermediate strength, which therefore encourage contact support and traction, thus facilitating migration. This is similar to that observed in studies of primary fibroblast, in which stimuli that decreased the percentage of cells containing focal adhesion led to enhanced locomotion (38). Interestingly, in this system TGF-1 was also associated with a marked increase in the percentage of cells possessing focal adhesions.
Integrins are trans-membrane receptors that mediate cell adhesion by forming links between the extracellular matrix and the cytoskeleton. In the current study, we have shown that decreased cell motility after addition of TGF-1 was associated with increased cell surface expression of 3 integrins and also its matrix ligand fibronectin. This is consistent with previous studies of myofibroblast differentiation, in which initial cell adhesion to fibronectin is followed by cell spreading. It has been suggested that different 
and integrin subunits may regulate different intracellular signaling events. After binding with its ligand, the cytoplasmic domain of integrin subunits interact with focal adhesion proteins, causing the activation of focal adhesion kinase and Src via Rho family GTPases (39). Furthermore, Rho GTPases are known to be differentially regulated by 1 and 3 integrins (39). More specifically, overexpression of a mutant 1-3-1 integrin, in which the extracellular I domain–like sequence is replaced with the corresponding sequence of 3 integrin enhances Rho activity and the formation of stress fibers in Chinese hamster ovary cells, whereas 1 integrin overexpression leads to Rac activity. It is interesting to speculate, therefore, that TGF-1–mediated stimulation of fibronectin and 3 integrin expression may be the initiating stimulus for inhibition of proximal tubular epithelial cell migration. This hypothesis is supported by our observations that TGF-1–mediated focal adhesion and actin cytoskeleton reorganization, as well as alteration in cell motility, were abrogated by inhibitors of RhoA target proteins.
In conclusion, we have shown that HK2 cells in serum-free medium culture conditions can be used as a model to follow the response of proximal tubular cells to injury. The data also demonstrate clearly that TGF-1 through increasing the strength of the interaction of cells with the underlying extracellular matrix. This alteration in the relationship of the cells to the matrix is likely to be the result of the combination of altered cytoskeletal reorganization, increased expression of focal adhesion, and integrins coupled to increased matrix deposition. Given the anti-migratory role of TGF-1, it seems likely therefore that sustained increase in TGF-1 expression seen after severe ischemic injury contributes to permanent structural and functional abnormalities. Furthermore TGF-1 may contribute to a tendency toward the development of progressive renal fibrosis.
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Acknowledgments |
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References |
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