Citation
Alderliesten, M. C. (2009, February 11). Focal adhesion signaling in acute renal failure. LACDR, Division of Toxicology, Faculty of Science, Leiden University.
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5
Chapter 5
Focal Adhesion Kinase Signaling is Implicated Ischemia/Reperfusion Induced Acute Renal Failure
Maaike Alderliesten, Yu Qin and Bob van de Water
Abstract
One of the prominent features of acute renal failure is ATP depletion, which leads to dis- ruption of the F-actin cytoskeleton, dephosphorylation of proteins and mislocalization of cell-extracellular matrix adhesion molecules like integrins. Most renal injury is sublethal, that results in recovery of the renal cells and restoration of the renal cell function. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase, activated upon integrin clustering and involved in signaling pathways leading to survival, proliferation and cell adhesion. Here we studied the role of FAK in primary renal cells during recovery from ATP depletion. FAK was conditionally deleted from primary mouse renal cells, using the Cre- LoxP system. Upon tamoxifen treatment FAK was deleted. This did not result in changes in proliferation or cell survival. The phosphorylation and expression levels of several focal adhesion proteins and the expression of total tyrosine phosporylation were unchanged and spreading was impaired. However, focal adhesions of FAK knock out cells were larger in size than those wildtype cells and spreading of the cells was impaired. ATP depletion did not lead to cell death rather it caused dephosphorylation of focal adhesion proteins and a decrease in total tyrosine phosphorylation. Furthermore the F-actin cytoskeleton was disrupted. During recovery, focal adhesions of FAK knockout cells restored slower than those of wildtype cells. The same accounts for the F-actin cytoskeleton reorganiza- tion. These data suggest that FAK is important for the reassembly and recovery of focal adhesions after renal cell injury induced by ATP depletion.
Introduction
Acute renal failure, often caused by ischemia/reperfusion (I/R), is associated with high mortality of up to 50%, partly because of a lack of therapeutic strategies1. Ischemic injury to the kidney results in profound alterations in morphology and function of the renal cells that can lead to improper kidney function. These alterations include reversal of epithelial polarity, changes in actin cytoskeleton dynamics and disruption of cell-cell and cell-extra- cellular matrix (ECM) adhesions subsequently resulting in renal cell detachment with renal failure as the final outcome2-4.
Morphology and correct function of proximal tubular epithelial cells are determined by a structured cytoskeleton as well as organized cell adhesions. ATP depletion for example induced by chemical anoxia causes reorganization of the F-actin cytoskeleton in various cultured kidney epithelial cells3;5. Inactivation of RhoA has been implicated in the disrup- tion of stress fibers caused by ATP depletion in proximal tubular cells6. This maybe related to differential expression and localization of small Rho GTPase family members in the proximal tubule. F-actin disruption compromises adhesion which is related to the exfolia- tion of renal cells during I/R. When injury induced by I/R is sublethal the F-actin cytoskel- eton is rebuilt during recovery, which leads to stabilization of cell adhesion structures as observed in vivo and in vitro7;8.
Cell-ECM adhesions in renal epithelial cells are mediated by focal adhesions (FAs), which are formed by a complex of cytoskeletal, adapter and signaling proteins. These include structural adapter proteins like paxillin and vinculin and kinases like focal adhesion kinase (FAK), Src and the mitogen activated protein kinase (MAPK) family members ERK and JNK9-11. These proteins form a link between the integrins, cell adhesion receptors, and the F-actin cytoskeletal network. FAK is a ubiquitously expressed non-receptor protein tyrosine kinase that interacts with integrin-associated proteins such as paxillin and talin and elicits downstream signaling that mediates, amongst others, the turnover of FAs12;13. FAK is activated via autophosphorylation on tyrosine residue 397 upon integrin clustering.
PY397 is a docking site for the SH2 domain of Src kinase. Src in its turn can further phos- phorylate FAK resulting in increased FAK activity, providing binding places for (cytoskele- ton-associated) adaptor proteins14-17. However, little is known about the alterations in FAs in the proximal tubules during renal I/R. Previously, we and others showed that induction of ischemia in rats induced rapid dephosphorylation of FA proteins like FAK followed by rephosphorylation during the reperfusion period. In vivo, this reversible dephosphoryla- tion of FAK was associated with loss of FAs during the early phases of reperfusion and the reformation of these structures at later time points in the reperfusion phase14-18. So far, the role of FAK in restructuring of the F-actin cytoskeleton and FAs after ATP depletion in renal epithelial cells is not known.
In this study, we used conditional knockout of FAK in primary mouse renal cells to study the role of FAK in ATP depletion-induced toxicity and recovery. We report that deletion of FAK under normal culturing conditions leads to increased FA size and shorter and thicker stress-fibers. Furthermore, deletion of FAK leads to impaired spreading but has no influ- ence on migration as shown by a wound healing assay. ATP depletion leads to a decrease in tyrosine phosphorylation and disruption of the F-actin cytoskeleton. During recovery the FAs are increased in size and the F-actin stress fibers are thicker compared to the control.
In addition, FAK deletion leads to a delay in FA recovery and F-actin stress fiber formation to normal phenotype. These data suggest that FAK is necessary for proper spreading of renal cells possibly because of its role on FA turnover and cytoskeleton organization.
Material and methods Materials
Dulbecco’s modified Eagles medium/Ham’s F12 1:1, PBS, cholera toxin and penicilin/
streptomycin/amphotericin B (PSA) were from Invitrogen. Fetal bovine serum (FBS) was from Life technologies (Grand Island, NY). Collagen (type I, rat tail) and epidermal growth factor (EGF) were from Upstate Biotechnology (Lake Placeid, NY). Antimycin A and 2-de- oxy-glucose were purchased from Sigma (St. Louis, MO).
Mice and genotyping
Mice were maintained and bred at the animal facility of the Leiden University Gorlaeus Laboratories in accordance with institutional guidance and national health standards. The mice were regularly monitored and had free access to water and standard mice chow. All experiments using mice were approved by the local animal experimental committee of the Leiden University. Mice were genotyped as described in chapter 4. Briefly, FAKloxP and FAKwt bands (400bp and 290bp respectively) are detected using 5’-GAGAATCCA- GCTTTGGCTGTTG-3’ and 5’-GAATGCTACAGGAACCAAATAAC-3’ primers. Cre-ERT2 bands are detected using 5’-GTT CAG GGA TCG CCA GGC G-3’ and 5’-GCT GGC TGG TGG CAG ATG G -3’. PCR products were separated at 2% agarose gels.
Isolation and culture of primary mouse renal cells
Mouse renal cells were isolated from the kidneys of male mice (25gram) with Rosa-Cre- ERT2//FAKLoxP/LoxP genotype. The kidneys were minced and digested in a collagenase solu- tion in Hank’s Hepes balanced salt solution (137mM NaCl, 5mM KCl, 0.8 mM MgSO4·7H2O, 0.4mM Na2HPO4·2H2O, 0.4mM KH2PO4, 1.3mM CaCl2, 4mM NaHCO3, 25mM HEPES, 5mM D-glucose, pH 7.4) for half hour at 37ºC. The cell suspension was washed three times in Hank’s Hepes. After the washing steps cells were resuspended in Dulbecco´s Eagles medium-Ham F12 1:1, 1% FBS (v/v), 0.5 mg/ml bovine serum albumin, 10 ng/ml EGF, 10 ng/ml cholera toxin, 50nM hydrocortisone, 15mM HEPES, 2mM glutamine, 1x Insulin-transferrin sodium selenite supplement and 1% (v/v) penicillin, streptomycin and amphotericin B (PSA) and plated on collagen coated dishes. The cells were maintained at 37ºC in a humidified atmosphere of 95% air/5% CO2 and fed every other day. Cells were exposed to 1μM 4-hydroxy-tamoxifen (4-OHT) to induce FAK recombination every other day of culturing together with refreshing the medium. Cells were used for experiments when they reached a confluent monolayer after 9 days of culturing.
Recombination PCR
The presence of FAKΔloxP/ΔloxP (band at 327 bp) in cell lysates was detected as described in chapter 4 using 5’-GACCTTCCAACTTCTCATTTCTCC-3’ and 5’-GAATGCTACAGGAAC- CAAATAAC-3’ primers PCR products were separated on a 2% agarose gel.
ATP depletion and repletion of primary mouse renal cells
Cells were washed once with PBS and then exposed to 10 μM antimycin A (AA) and 5 mM 2-Deoxyglucose (DOG) in glucose free Dulbecco’s modified Eagles medium (DMEM) for periods up to 2 hours at 37ºC in a humidified atmosphere of 95% air/5% CO2. For repletion of ATP medium was refreshed with normal primary mouse renal cell culturing medium as described above for indicated periods at 37ºC in a humidified atmosphere of 95% air/5% CO2.
Immunohistochemistry of Focal Adhesion Proteins
Cells were cultured on collagen coated glass coverslips in 24-wells plates. Cells were fixed in 4% formaldehyde for 10 minutes followed by 3 washes with PBS. The cells were permeabilized and blocked in TBP (PBS, 0.2% (w/v) Triton X-100, 0.5% (w/v) bovine se- rum albumin, pH 7.4) for 1 hour and incubated overnight at 4ºC with primary antibody; anti- FAK (Upstate), anti-vinculin (Transduction Laboratories, anti-paxillin and anti-PY118-paxillin (BioSource International, Camarillo,CA). Thereafter, coverslips were washed 3 times in TBP and incubated for 1 h with secondary antibody; Alexa488-labeled goat anti-rabbit (In- vitrogen, Carlsbad, CA). Rhodamin-phalloidin (Invitrogen) was used for F-actin cytoskele- ton staining. After removing the secondary antibody, slides were mounted on glass slides using Poly Aquamount (Polysciences, Inq., Warington, PA). Images were made using a Bio-Rad Radiance 2100 confocal system (Bio-Rad, Hercules,CA) with a 60x Plan Apo (NA 1.4:Nikon, Tokyo) objective lens. All images were processed with Image-Pro® Plus (Ver- sion 5.1 Media Cybernetics, Bethesda, MD).
Western blot analysis
For Western blot analyis cells were washed twice with PBS and once with TSE (10mM Tris-HCl, 250 mM sucrose, I mM EGTA, pH 7.4). Then cells were scraped in 100 μl TSE with inhibitors (1 mM dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM so- dium vanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride) and boiled in sample preparation buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol (w/v), 4% SDS (w/v), bromophenol blue ). Protein concentrations were determined using a Bradford assay with IgG as a standard. Equal amounts of protein were separated by SDS-PAGE and trans- ferred to PVDF membrane (immobilon-P, Millipore). Blots were blocked in 5% (w/v) BSA
in TBS-T (0.5 M NaCl, 20 mM Tris-HCl and 0.05 % Tween-20(v/v) pH 7.4) for 1h. Primary antibody incubation was performed overnight at 4 ºC in anti-PY99 (0.04 μg/ml, mono- clonal, Santa Cruz, Santa Cruz), anti-FAK (monoclonal, 1 μg/ml, Transduction Laborato- ries), anti-PY118-paxillin (0.75 μg/ml, polyclonal, Biosource), anti-paxillin (monoclonal, 0.5 μg/ml, Transduction Laboratories), anti-vinculin, anti-PY416-Src ( polyclonal 0.5μg/ml, Bio- source), anti-ERK1/2, anti-p-ERK1/2 (Cell Signaling) and anti-tubulin (Sigma, St. Louis, MO). Thereafter blots were incubated with horseradish peroxidase conjugated secondary antibody (GE Healthcare, Little Chalfont Buckinghamshire, UK) or fluorescent CY5 con- jugated secondary antibody (Jackson laboratories) in TBS-T for 1h at room temperature.
Protein signals were detected with ECL plus method (GE Healthcare) or using fluores- cence of the secondary antibody followed by scanning of the blots with the Typhoon 9400 (GE Healthcare).
Cell spreading and wound closure assay
Cells were trypsinized and resuspended in DMEM/HAM F12 1:1 without FBS. Next, cells were plated on collagen coated glass coverslips in 24 well plates and incubated at 37ºC for 150 minutes. Unattached cells were removed and the cells were fixed and stained for PY-99 as described in the section immunocytochemistry. Attached cells were scored for spreading; not spread, partially spread and fully spread. Scores are presented as percent- age of all attached cells.
Confluent monolayer of cells was serum starved for 1 hour and subsequently wounded with a pipet tip. Phase contrast pictures were taken at indicated periods followed by mea- suring the width of the wound.
Caspase-3 activity assay
Cells were scraped in medium and collected by centrifugation. The cell pellet was taken up in lysisbufer (10 mM HEPES, 40 mM ß-glycerophosphate, 50 mM NaCl, 2 mM MgCl2 and 5 mM EGTA) followed by 3 cycles of freezing in liquid nitrogen and thawing. The sus- pension was centrifuged at 13,000 rpm in a microfuge for 30 min. The supernatant was collected and used for protein concentration determination by Bradford analysis using IgG as a standard. Equal amounts of cell protein (10 μg) were used for measuring caspase-3 activity using Ac-DEVD-AMC as a substrate (25 μM). AMC fluorescence was followed in time using a fluorescence plate reader (HTS 7000 Bio assay reader; Perkin Elmer). Cas- pase activity was calculated as pmol/mg cell protein/minute using AMC as a standard.
Cell cycle analysis
Apotosis was determined by cell cycle analysis. Medium containing floating cells was col- lected. Adherent cells were washed twice with PBS 1 mM EDTA and trypsinized. Floating and trypsinized adherent cells were pooled, centrifuged for 10 minutes at 2000rpm, resus- pended in 100 μl PBS followed by fixation in ice-cold absolute ethanol. Fixed cells were centrifuged and washed with PBS-EDTA and resuspened in PBS-EDTA containing 7.5 μM PI and 10 μg/ml RNAse A. After 30 minutes at room temperature cells were analysed by flow cytometry (FACS-Calibur, Beckton Dickenson). The amount of cells in subG0/G1 was calculated using cellquest software (Beckton Dickenson).
LDH leakage assay
Medium of cells was collected in a 96 wells plate to determine free LDH during ATP deple- tion and repletion. For 100 values the cells were lysed in 0.7% Triton X-100 in medium.
After addition of the LDH assay solution (200mM Tris-HCl pH 7.4, 1mM pyruvate, 0.4mM NADH) LDH activity was measured at fluorescence plate reader (HTS 7000 Bio assay reader; Perkin Elmer). Values are presented as percentage of total LDH activity.
Statistics
All values are provided as mean ±SEM. Statistical analysis was performed by student T- Test. Significant difference was set when P-value was <0.05.
Results
FAK is effectively knocked out in primary mouse renal cells after 4-OHT treatment To delete fak in primary mouse renal cells we used a conditional FAK knockout mouse model. Mice with LoxP sites flanking the second exon of the fak gene (FAKloxP/loxP) (Beggs etal. 2003) were crossed with Rosa-Cre-ERT2 mice that express Cre recombinase in every cell type to generate FAKloxP/loxP//Rosa-Cre-ERT2 mice. Renal cells were isolated from male FAKloxP/loxP//Rosa-Cre-ERT2 mice and 1 μM 4-hydroxy-tamoxifen (4-OHT) was added to the culturing medium to induce Cre recombinase mediated recombination of the fak alleles.
The cultured mouse renal cells were checked for recombination using a non-quantitative
Figure 1. Tamoxifen successfully recombines loxed fak alleles and deletes FAK in primary mouse renal cells. To show recombination renal cells of FAKloxP/loxP//Rosa-Cre-ERT2 mice were isolated and treated with 4-OHT and genomic DNA was extracted and checked for recombination by PCR (FAKΔloxP/
ΔloxP). Recombination band appears at 327 bp (A). Protein levels of FAK during culturing were detected by Western blot (B). The protein levels of FAK, PY397-FAK and vinculin after 9 days of culturing and 4-OHT treatment were detected by Western blot analysis (C). To determine the structure of the FAs of FAKΔloxP/ΔloxP renal cells FAKΔloxP/ΔloxP and FAKloxP/loxP primary renal cells were stained for FAK and vin- culin (D). All images were made using confocal laser scanning microscope. Images are representative for 3 experiments.
recombination PCR. The FAKloxP/loxP//Rosa-Cre-ERT2 renal cells showed full recombination of fak alleles (FAKΔloxP/ΔloxP) by the appearance of a recombination band at 327 bp only after 4-OHT treatment (Fig. 1A). 4-OHT induced FAKΔloxP/ΔloxP resulted in disappearance of FAK protein on western blot after 9 days of culturing (Fig. 1B). FAK as well as PY397-FAK were absent in the mouse renal cells after addition of 4-OHT (Fig. 1C) resulting in the disap- pearance of FAK from the FAs (Fig. 1D). These data show that the FAKloxP/loxP//Rosa-Cre- ERT2 mouse renal cells are a successful inducible FAK knockout model.
FAK deletion in renal cells does not affect cell survival and migration
FAK is implicated in many signaling pathways including adhesion, proliferation, migration and survival signaling. Therefore, we studied how deletion of FAK affected these cellular processes in our model. After 9 days of culturing both the FAKΔloxP/ΔloxP cells and the FAKloxP/
loxP cells formed a nice monolayer. The morphology of the FAKΔloxP/ΔloxP cells is less cubodial shaped than the morphology of FAKloxP/loxP cells (Fig. 2A). Proliferation assays showed that during these 9 days proliferation is not significantly different and that cell cycle distribution is the same (data not shown). Some studies reported increased apoptosis after deletion of FAK (ref). However in our model FAK deletion did not result in increased cell death as measured by FACS analysis (Fig. 2B). This suggests that under normal culturing condi- tions FAK deletion does not influence cell survival. FAK is also implicated in cell migration, however migration of FAKloxP/loxP and FAKΔloxP/ΔloxP cells did not show a significant difference as evaluated using a wound healing assay (Fig. 2C).
Figure 2. Chemical anoxia and recovery induced injury in FAKΔloxP/ΔloxP and FAKloxP/loxP primary renal cells. FAKloxP/loxP FAKΔloxP/ΔloxP primary renal cells were cultured for 9 days. Phase-contrast pic- tures of the cells were made (A). Apoptosis was determined using flow cytometry analysis (B). A wound healing assay was performed in monolayers of FAKloxP/loxP FAKΔloxP/ΔloxP primary renal cells.
Wound closure was measured after 8 and 24 h (C). Data shown represent the mean ±SEM for three independent experiments (n=3).
FAK deletion in renal cells does not affect FA protein expression or phosphorylation
FAK is known to control FA dynamics therefore we studied the effect of FAK knockout on FA arrangements in primary renal cells. In FAKloxP/loxP mouse renal cells, small FAs are located at the edge of the cell and some thicker and elongated FAs are located more perinuclear. In FAKΔloxP/ΔloxP cells FAK had disappeared from the FAs and the size of the FA was increased. These FAs were located mainly perinuclear and less at the edge
(Fig. 3A). In addition, the F-actin cytoskeleton in FAKloxP/loxP cells has a cortical ring and stress fibers that end in the focal adhesions (Fig. 3B). Deleting FAK results in increased
number of thicker, shorter stress fibers and a denser cortical ring (Fig. 3B).
To further characterize FAKΔloxP/ΔloxP renal cells we analyzed other known FA proteins, like Src and paxillin. Src kinase binds to tyrosine residue Y397 of FAK and is subsequently involved in further activation and phosphorylation of FAK. The FAK-Src complex is also known to phosphorylate paxillin, a binding partner of FAK. Src kinase was not differentially expressed or phosphorylated in FAKΔloxP/ΔloxP cells (Fig. 3C). Paxillin was also not differen- tially expressed or phosphorylated in the absence of FAK (Fig. 3C). Also no differences were observed for vinculin, as well as overall tyrosine phosphorylation expression (Fig.
3C). This suggests that at least the expression and phosphorylation of FA proteins in renal cells is not dependent on the presence of FAK under normal culturing conditions.
Figure 3. Focal adhesion size and F-actin organization changed in primary renal FAKΔloxP/ΔloxP
cells compared to FAKloxP/loxPcells. FAKΔloxP/ΔloxP and FAKloxP/loxP cells were stained for paxillin (green) and and PY118-paxillin (red) (A) and pTyr (green) and F-actin (red) (B), colocalization of the proteins is shown in yellow. To determine the expression of several known FA proteins, cell lysates were made and Western blots were stained for FAK, paxillin, PY118-paxillin, PY1416-Src, vinculin and pTyr. Tubulin was used as a loading control. Data shown represent three independent experiments (n=3).
FAK deletion affects localization and size of FAs and impairs spreading in pri- mary mouse renal cells
FAK is involved in cell spreading. To study this we trypsinized cells and let them adhere and spread on collagen IV-coated glass coverslips in serum free medium to exclude the contribution of growth factor signaling. Significantly less FAKΔloxP/ΔloxP cells were completely spread after 2 h compared to wildtype cells (Fig.: 4A). Staining the adhered and spread cells, treated with and without 4-OHT, for PY118-paxillin, paxillin, total tyrosine phosphoryla- tion and the F-actin cytoskeleton showed that the fully spread FAKΔloxP/ΔloxP cells showed different FA and F-actin cytoskeleton organization. FAKloxP/loxP cells contained small FAs, pointing outward, located at the edge of the cell. The FAs of the FAKΔloxP/ΔloxP cells were thicker and larger in size. The F-actin cytoskeleton formed a cortical ring and had small thin stress fibers that end in tyrosine phosphorylated FAs. In FAKΔloxP/ΔloxP cells the cy- toskeleton was more irregular with more stress fiber like structures and a very thick corti- cal ring compared to the control cells (Fig.: 4B).
Primary mouse renal cells recover from chemically induced ATP depletion
Next, we studied how FAK signaling is involved in the ischemic injury and recovery pro- cess of mouse renal cells. Chemical anoxia was used as a model for ischemic renal cell injury and includes ATP depletion in the cells by using an inhibitor of the complex III of the respiratory chain, antimycin A (AA) and a glycolysis inhibitor, 2-deoxyglucose (DOG). The use of AA (10μM) and DOG (5mM) resulted in complete ATP depletion within 2 h in proxi- mal tubule epithelial cells (data not shown). During ATP depletion cells lost typical phase- Figure 4. FAKΔloxP/ΔloxP primary renal cells spread slower compared to FAKloxP/loxP cells. FAKΔloxP/ΔloxP and FAKloxP/loxP cells were let to adhere to collagen IV- coated glass coverslips and scored for the level of adherence (A). Fully adhered cells are stained for paxillin, PY118-paxillin, F-actin and pTyr (B).
Images were made using confocal laser scan microscopy. Data shown represent three independent experiments (n=3).
contrast features flatten (Fig.: 5A) while some parts of the monolayer were detached most- likely due to loss of cell-ECM contacts. Recovery from the ATP depletion for 4 h (80% ATP recovery) already regained normal phase-contrast prospectus in both FAKΔloxP/ΔloxP and FAKloxP/loxP and the monolayer was almost completely restored. Mainly in FAKΔloxP/ΔloxP renal cells we still observed gaps in the monolayer. After 24 h of recovery, monolayers of both cell types were completely restored and were comparable to the control. Again FAKΔloxP/
ΔloxP cells showed a less cubodial phenotype compared to FAKloxP/loxP (Fig.: 5A).
Importantly, 2 h of chemical anoxia did not induce an increase in apoptosis or necrosis
Figure 5. FAKΔloxP/ΔloxP and FAKloxP/loxP primary renal cells do not die during chemical anoxia and recovery. Primary renal cells were subjected to chemical anoxia using 10μM AA and 5mM DOG for 2 followed by recovery in complete medium. Phase-contrast pictures were made using light microscopy (A). Apoptosis was determined using flow cytometry analysis (B). Necrosis was determined with and LDH release assay (C). Data shown represent the mean ±SEM for n=3.
as determined by cell cycle analysis and LDH release, respectively (Fig. 5B-C). This indi- cates that the changes induced by 2 h chemical anoxia were sub-lethal and allowed us to study the process of F-actin and FA loss and recovery in more detail.
Impaired recovery of stress fibers and FAs after ATP depletion in FAK
ΔloxP/ΔloxPprimary mouse renal cells
During normal culturing conditions cells display dense but long stress fibers, though the FAKΔloxP/ΔloxP cells display a bit shorter stress fibers. After 2 h of ATP depletion by chemical anoxia the F-actin cytoskeleton is disrupted and aggregates of actin are seen as dense spots in the cytoplasm in both cell types. Recovery in normal medium for 1 h still displays a disrupted cytoskeleton and aggregates in the cytoplasm. However 4 h after chemical an- oxia showed recovery of the stress fibers. These were shorter and denser in the FAKΔloxP/
ΔloxP cells compared to the FAKloxP/loxP cells. After 24 h the stress fibers of the FAKloxP/loxP cells had retured to normal, as seen in unexposed cells weheras the stress fibers of the FAKΔloxP/ΔloxP cells were still very dense and short compared to control cells (Fig. 7A). Next, we studied the changes in FA structure and localization during ATP depletion and reple- tion. Cells were stained for PY118-paxillin. FAs of both FAKΔloxP/ΔloxP and FAKloxP/loxP cells were occupied with phosphorylated paxillin (Fig. 7B). Directly after ATP depletion phosphoryla- tion of paxillin was lost from the FAs. During recovery phosphorylation of paxillin on the FAs returned in both cell types. However, in the FAKloxP/loxP cells pY118-Paxillin returned on the FAs after 1 h of recovery, while in FAKΔloxP/ΔloxP cells phosphorylated FA did not appear.
Clear, large phosphorylated FAs appeared after 4 h, the FAs of FAKΔloxP/ΔloxP cells were much larger with plaque like structures at the edges of the cells while the FAK of the wild- type cells were smaller and more FA like structures (Fig. 7B). After 24 h of recovery the FAs were still increased in number in both cell types. The size of the wildtype cells was comparable to the control situation, but the FA of the knockout cells were stil enlarged (Fig. 6B).
Our previous studies showed dephosphorylation and rephosphorylation of proteins after ATP depletion. Directly after ATP depletion FA proteins and total tyrosine phosphoryla- tion as well as ERK phosphorylation decreased as seen by western blot analysis (Fig. 6).
Total tyrosine phosphorylation including phosphorylation of Src, paxillin and ERK returned during recovery (Fig. 6). Furthermore total tyrosine phosphorylation was increased during recovery compared to control and shows a differential expression pattern after 24 h (Fig.
6). However, no clear difference in pattern of phosphorylation between FAKloxP/loxP and FAKΔloxP/ΔloxP was observed.
Figure 6. FAKΔloxP/ΔloxP primary renal cells show delayed recovery of FAs and F-actin cytoskel- eton after chemical anoxia. FAKΔloxP/ΔloxP and FAKloxP/loxP primary renal cells were subjected to chemi- cal anoxia using 10μM AA and 5mM DOG for 2 followed by recovery in complete medium. Cells were stained for F-actin (A) and paxillin and PY118-paxillin (B). Images were made using confocal laser scan microscopy. To determine protein expression and phosphorylation after chemical anoxia and during recovery cell lysates were made and Western blots were stained for PY416-Src, P-ERK1/2, ERK1/2, PY118-paxillin, and pTyr. Tubulin was used for loading control. Data shown represent three independent experiments (n=3).
Discussion
In this study we set up a model that allowed us to investigate the role of FAK under normal conditions and during injury and repair after simulated ischemia in primary renal cells. We show firstly, that the 4-OHT inducible Cre-LoxP system leads to successful deletion of fak in primary mouse renal cells. Secondly, deletion of fak affects the size and localization of the FAs and the organization of the F-actin stress-fibers but not the phosphorylation and expression of some known FA proteins. Thirdly, FAK deletion leads to impaired cell spreading. Most importantly we show that during recovery from ATP depletion fak deletion results in a delay in recovery of phosphorylated FAs.
Our loss of function study showed that the localization and structure of the FAs was mark- edly changed in FAKΔloxP/ΔloxP cells compared to the FAKloxP/loxP cells. The small FAs seen in FAKloxP/loxP cells were replaced by large FAs in FAKΔloxP/ΔloxP cells. The actin stress fibers were more dense and short while the stress fibers of the FAKloxP/loxP cells converge in a lon- ger and more parallel manner. These data suggest that FAK is important for FA dynamics and distribution and is involved in F-actin cytoskeleton organization. FAK is reported to be required for FA disassembly. FAK knockout fibroblasts show a much slower disassembly of FAs than wildtype cells19. In addition, Schober etal. suggest FAK deletion results in per- turbation impaired suppression of Rho signaling leading to induced stress fibers and more stable FAs20. The atypically organized FAs and F-actin stress fibers can be the basis of the impaired spreading of the FAKΔloxP/ΔloxP cells, because spreading involves a more flexible FA formation than the FAKΔloxP/ΔloxP cells are capable of. The tension in the cell provided by the cytoskeleton is very important for the spreading process21;22.
We show that deletion of FAK in primary mouse renal cells affected their ability to recover from ATP depletion as shown by a delay in the formation of phosphorylated FAs and re- covery to normal FAs and stress fibers after ATP depletion. As mentioned above, FAK is necessary for FA disassembly. Deletion of FAK leads to a decrease in FA dynamics. This subsequently affected the recovery of the cell-ECM adhesions, F-actin cytoskeleton re- organization and thus recovery. This has its implications for repair in vivo after ischemia.
These data on impaired recovery of the FAs after ATP depletion together with the changes we report in FAs, F-actin stress fiber formation and consequently spreading must have its implications for recovery of the renal tubules after ischemia.
We found clear dephosphorylation during ATP depletion and rephosphorylation with thick FA and increased number of stress fibers during recovery. These observations are com- parable to the in vivo situation as we have seen in chapter 2. This suggest that the pro- cesses of FA dynamics and F/actin organization are comparable between the in vivo and in vitro situation making ATP depletion and recovery in primary mouse renal cells a good model for future research on the role of FA proteins like FAK during ischemia induced re-
nal injury and recovery. Furthermore ATP depletion in several other cell types, like PTCs of the rat or MDCKs leads to cell death both by apoptosis and necrosis; however we reported no cell death after ATP depletion and during recovery. In vivo cell death is not one of the main processes that affect kidney function. A large part of the decline in renal function is due to sublethally injured renal cells that are able to recover but because of the injury are impaired in their cellular function. Our chemical anoxia and recovery model in primary renal cells is therefore a good model to study this sublethally injured cells and their recovery after ATP depletion. In conclusion we suggest that FAK signaling has an important role in recovery of renal cell after injury.
Acknowledgements
We thank the members of the Division of Toxicology of the Leiden/Amsterdam Center for Drug Research for valuable discussion and support. This work was supported by grants from the Netherlands Organization for Scientific Research (grant 908-02-107).
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