Conceived and designed the experiments: MEZ JPW EFR. Performed the experiments: MEZ PS Y-PJ. Analyzed the data: MEZ PS. Contributed reagents/materials/analysis tools: MEZ PS Y-PJ JPW EFR. Wrote the paper: MEZ EFR.
The authors have declared that no competing interests exist.
Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection.
The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin.
Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types as a method for uncovering novel regulators of cytoskeleton changes.
The composition of the eukaryotic cytoskeleton consists mainly of three discrete fibrous structures—actin microfilaments, microtubules and intermediate filaments. These structures provide the framework to maintain cell shape and polarity and also contribute to other fundamental cellular functions such as motility, organelle transport and division
In ECs, the cytoskeleton plays an essential role in providing a sturdy intracellular scaffold, which serves to organize vital membrane proteins within the cell. The cytoskeleton also has the ability to respond to extracellular stimuli and undergo reorganization
When ECs are quiescent, actin creates a cortical ring providing a link between cell-cell and cell-matrix adhesion complexes and intracellular organelles. The cortical actin ring is necessary to sustain the EC barrier function
Understanding how ECs respond to stimuli that mediate cytoskeleton changes is an important topic in organ allograft rejection. Antibodies directed toward HLA class I molecules on the donor endothelium play a major role in antibody-mediated transplant rejection
Here we present the results of a proteomic-based analysis characterizing changes in the cytoskeleton of human aortic ECs following stimulation with thrombin, basic fibroblast growth factor and HLA class I antibodies. The contents of these isolations were characterized by mass spectrometry, annotated and a candidate list of HLA class I-induced cytoskeleton associated proteins was identified. Three of the candidates, TPM4, eIF4A1 and DDX3X, were further characterized. TPM4 was uniquely found in the cytoskeletal fraction of class I stimulated ECs, but absent in the cytoskeleton of ECs stimulated with thrombin and bFGF. The eIF4A1 protein, a regulator of protein synthesis and cell proliferation, was found in association with the actin cytoskeleton by Western blot and showed a higher degree of colocalization with F-actin and paxillin in HLA class I stimulated cells compared to the other treatment groups. This proteomic discovery approach provides candidate targets, which may link HLA class I-induced cytoskeleton changes to downstream cellular functions such as proliferation.
Informed written consent for use of the aortic tissue as an anatomical gift for research was obtained by OneLegacy (a federally-designated organ procurement organization) at the time of organ donation from the next of kin or authorized party. The use of the human aortic tissue for the research described herein was approved by the OneLegacy Biomedical Review Board under the agreement #RS-02-10-2 and UCLA MTA2009-561.
Cytochalasin D was purchased from Sigma-Aldrich. Recombinant human bFGF was purchased from R&D Systems. Human α-thrombin was from Enzyme Research Laboratories. The murine monoclonal antibody W6/32 (IgG2a), reactive with a monomorphic epitope on HLA class I antigens, was obtained from the American Type Culture Collection. The mouse IgG used as an isotype control was supplied by Sigma-Aldrich. Recombinant human FGF basic was from R&D Systems. Antibodies used for Western blot were β-actin, eIF4A1, 4E-BP1 obtained from Cell Signaling and vinculin, β-tubulin, and myosin light chain (all three from Santa Cruz) and β1-integrin and paxillin (BD Biosciences) and TPM4 and DDX3X (both from Acris Antibodies). The eIF4A1 antibody used for confocal microscopy was obtained from Santa Cruz. A second eIF4A1 antibody obtained from Abcam was used to confirm the specificity of the immunofluorescent staining pattern. The fluoresent conjugated secondary antibodies, Alexafluor 488 goat anti-rabbit, Rhodamine Red™-X goat anti-mouse and Texas Red-phalloidin were from Invitrogen.
Primary human aortic ECs were isolated from the aortic rings of explanted donor hearts, as previously described
ECs grown to 80–90% were starved for 2 h in basal medium containing 0.2% FBS followed by treatment with mAb W6/32 (1 µg/ml) (referred to as HLA class I stimulated), isotype control mouse IgG (1 µg/ml) (referred to as unstimulated), thrombin (1 U/ml) or bFGF (25 ng/ml) for 10 min. Treated ECs were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton-X-100. The presence of F-actin was visualized by direct staining with Texas Red–phalloidin. Cells were analyzed with Zeiss Axioplan 2 microscope with the Zeiss FluoArc 100 watt mercury light source. Digital images of fluorescence were obtained with a cooled charge-coupled device camera (SPOT 2, Diagnostic Instruments) and associated software (SPOT 4.5, Diagnostic Instruments).
The method was adapted from a previously published method
Beads containing the cytoskeleton isolation preparations were heated for 5 min at 95°C in SDS sample buffer, electrophoresed on SDS polyacrylamide gels, and transferred to a polyvinylidene difluoride membrane. The membranes were blocked using 5% nonfat dry milk in TBS (pH 7.4) containing 0.1% Tween 20 (TBST) for 20 min at room temperature, and incubated with the appropriate primary antibody overnight at 4°C. The blots were washed with TBST followed by incubation in HRP-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The blots were subsequently washed with TBST and developed with ECL.
Beads containing the cytoskeleton proteins were resuspended in digestion buffer (50 mM ammonium bicarbonate). The sample was first reduced using 100 mM DTT (Sigma-Aldrich). Next, the preparation was alkylated using 100 mM iodoacetamide (Sigma Aldrich). The proteins were then digested overnight using proteomics grade trypsin (Sigma Aldrich) at a concentration of 0.1 µg/µl. Following trypsin digest, the beads were centrifuged at 14,000× g for 1 min and the supernatant was collected.
Magnetic Dynabeads RPC 18 (Invitrogen) were used to bind the cytoskeleton peptides for purification. RPC 18 beads were first washed with binding buffer (0.1% TFA/0.5%ACN). The cytoskeleton peptides were mixed with binding buffer then added to the beads. The beads were washed 4 times with binding buffer and eluted with 60% ACN.
nLC-MS/MS with Collision Induced Dissociation (CID) is performed on a 7 tesla LTQ FT Ultra (Thermo Scientific, Waltham, MA) integrated with an Eksigent nano-LC. A prepacked reverse-phase column (Microtech Scientific C18 with a dimension of 100 µm×3.5 cm) containing reverse-phase resin (Biobasic C18, 5-µm particle size, 300-Å pore size, Microtech Scientific, Fontana, CA) is used for peptide chromatography and subsequent CID analyses. ESI conditions using the nano-spray source (Thermo Fisher) for the LTQ-FT are set as follows: capillary temperature of 220°C, tube lens 110 V and a spray voltage of 2.5 kV. The flow rate for reverse-phase chromatography is 5 µl/min for loading and 300 nl/min for the analytical separation (buffer A: 0.1% formic acid, 1% ACN; buffer B: 0.1% formic acid, 100% ACN). Peptides are resolved by the following gradient: 2–60% buffer B over 40 min, then increased to 80% buffer B over 10 min and then returned to 0% buffer B for equilibration of 10 min. The LTQ FT is operated in data-dependent mode with a full precursor scan at high-resolution (100000 at m/z 400) and six MSMS experiments at low resolution on the linear trap while the full scan is completed. For CID the intensity threshold was set to 5000, where mass range was 350–2000. Spectra are searched using Mascot software (Matrix Science, UK) in which results with p<0.05 (95% confidence interval) were considered significant and indicating identity. The data was also analyzed through Sequest database search algorithm implemented in Discoverer software (Thermo Fisher, Waltham, MA).
ECs were grown on 35 mm glass bottom dishes to 80–90% confluence and starved for 2 h in basal medium containing 0.2% FBS followed by treatment with mAb W6/32 (1 µg/ml), isotype control mouse IgG (1 µg/ml), thrombin (1 U/ml) or bFGF (25 ng/ml) for 10 min. Treated ECs were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton-X-100. Cells were incubated with primary antibody (mouse anti-paxillin or rabbit anti- eIF4A1) in 5% BSA in PBS overnight at 4°C washed and incubated with secondary antibody (Alexafluor 488 goat anti-rabbit for eIF4A1 and Rhodamine Red™-X goat anti-mouse IgG for paxillin) for 30 min at room temperature. The presence of F-actin was visualized by direct staining with Texas Red–phalloidin. Cell images were captured using a Zeiss LSM 510 confocal microscope at 63× magnification using the Zeiss LSM 5 PASCAL software (Carl Zeiss MicroImaging GmbH, Germany)
A comparison of F-actin reorganization by immunofluorescence revealed that stimulation with HLA class I antibodies (
Human aortic ECs were treated with either mIgG 1 µg/ml (A), HLA class I antibody (Ab) 1 µg/ml (B), thrombin 1 U/ml (C) or bFGF 25 ng/ml (D) for 10 min. Cells were stained with Texas-Red–phalloidin and analyzed by fluorescence microscopy. (E) The fluorescence intensity and the total area of each cell in each field were measured using ImageJ software (
Previous studies showed that the cytoskeleton and its associated proteins could be selectively enriched using Dynal beads
(A) Human aortic ECs were treated for 10 min with either mIgG 1 µg/ml (lane 1), HLA class I Ab 1 µg/ml (lane 2), thrombin 1 U/ml (lane 3), bFGF 25 ng/ml (lane 4) or treated with cytochalasin D 5 µM (lane 5) for 30 min. The cytoskeleton was isolated from the lysates using tosylactived magnet Dynal beads and the fraction was analyzed by Western blot for components of the cytoskeleton. (B) Human aortic ECs were treated for 10 min with either mIgG 1 µg/ml (lanes 1 and 6), HLA class I Ab 1 µg/ml (lanes 2 and 7), thrombin 1 U/ml (lanes 3 and 8), bFGF 25 ng/ml (lanes 4 and 9) or treated with cytochalasin D 5 µM (lanes 5 and 10) for 30 min. The cytoskeleton was isolated using tosylactived magnetic Dynal beads. The cytoskeleton fraction (lanes 6–10) and the depleted fraction after cytoskeleton isolation (lanes 1–5) were immunoblotted to detect the presence of ß1-integrin and ß-actin as a loading control. The data presented in panels A and B are representative of 5 independent experiments.
Using in-solution trypsin digestion the isolated cytoskeleton fraction from each treatment group was digested into peptides. To identify the proteins in the cytoskeleton isolation preparations, nLC-MS/MS was performed on the peptides and Mascot searches were carried out. The list of proteins identified in each treatment group is summarized in
The identified proteins were classified using the Gene Ontology (GO) enrichment analysis tool Gene Onology Tree Machine (GOTM)
The Gene Ontology (GO) enrichment analysis tool called Gene Onology Tree Machine (GOTM) was used to create the DAG. The gene list was created using the uniprot accession numbers and thus the gene ID type selected was “hsapiens_uniprot_swissprot_accession.” Next the reference for GOTM was selected as “hsapiens_genome.” The statistical method was set to the default “hypergeometric,” the multiple test adjustment was set to be done by Benjamini & Hochberg, the significance level was set to the top 10 and the minimum number of genes for a category was 2. The DAG is a re-creation of the graph generated by GOTM. The shaded boxes indicate categories that were not significantly enriched. The significantly enriched categories found in the HLA class I treated group are specified by the white boxes and include the adjusted p-values and the number of proteins found in each category. The categories found to be uniquely enriched only by HLA class I stimulation are indicated in bold.
To determine which proteins are important for inducing HLA class I cytoskeleton changes, the proteins found under the unique categories of the HLA class I DAG were examined (
Accession | Protein Name | Gene Name | GO Categories |
Q8WZ74 | Cortactin-binding protein 2 | CTTNBP2 | protein polymerization |
cellular protein complex assembly | |||
macromolecular complex assembly | |||
protein complex biogenesis | |||
O15145 | Actin-related protein 2/3 complex subunit 3 | ARPC3 | protein polymerization |
cellular protein complex assembly | |||
macromolecular complex assembly | |||
protein complex biogenesis | |||
actin cytoskeleton | |||
Q99880 | Histone H2B type 1-L | HIST1H2BL | macromolecular complex assembly |
P83731 | 60S ribosomal protein L24 | RPL24 | macromolecular complex assembly |
P04114 | Apolipoprotein B-100 | APOB | macromolecular complex assembly |
P11142 | Heat shock cognate 71 kDa protein | HSPA8 | hydrolase activity, acting on acid anhydrides |
O00571 | ATP-dependent RNA helicase DDX3X | DDX3X | hydrolase activity, acting on acid anhydrides |
P60842 | Eukaryotic initiation factor 4A-I | EIF4A1 | hydrolase activity, acting on acid anhydrides |
P35249 | Replication factor C subunit 4 | RFC4 | hydrolase activity, acting on acid anhydrides |
P49790 | Nuclear pore complex protein Nup153 | NUP153 | hydrolase activity, acting on acid anhydrides |
P06576 | ATP synthase subunit beta, mitochondrial | ATP5B | hydrolase activity, acting on acid anhydrides |
P67936 | Tropomyosin alpha-4 chain | TPM4 | actin cytoskeleton |
Among the 12 candidates identified, 11 of the proteins were phosphoproteins with the exception of RCF4 (
Kinase Family | Kinases | Candidate Phosphoproteins |
ACTR2_ACTR2B_TGFbR2 | TGF-beta type II receptor | HSPA8, NUP153 |
Activin receptor type II | DDX3X | |
AMPK | AMPK alpha-1 chain | RPL24 |
AMPK alpha-2 chain | RPL24 | |
ATM_ATR | Serine-protein kinase ATR | HSPA8, DDX3X |
Serine-protein kinase ATM | HSPA8, DDX3X | |
AuroraA | Serine/threonine-protein kinase 6 | HIST1H2BL |
AuroraC_AuroraB | Serine/threonine-protein kinase 12 | DDX3X |
CaMKIIalpha_CaMKIIdelta | CaM-kinase II alpha chain | RPL24 |
CaMKIIbeta_CaMKIIgamma | CaM-kinase II gamma chain | NUP153 |
CDK2_CDK3 | Cell division protein kinase 2 | APOB, EIF4A1, NUP153 |
CDK4_CDK6 | Cell division protein kinase 4 | HSPA8 |
Cell division protein kinase 6 | HSPA8 | |
CK2 | Casein kinase II, alpha' chain | CTTNBP2, HSPA8, ATP5B, APOB, RPL24, DDX3X, NUP153 |
Casein kinase II, alpha chain | CTTNBP2, HSPA8, ATP5B, APOB, RPL24, DDX3X, NUP153 | |
CLK | Dual specificity protein kinase CLK1 | DDX3X, HIST1H2BL |
Dual specificity protein kinase CLK2 | DDX3X, HIST1H2BL | |
DMPK | Myotonin-protein kinase | RPL24 |
EGFR | Receptor tyrosine-protein kinase erbB-1, erbB-2, erbB-3, erbB-4 | HSPA8 |
EphA7_EphA6_EphA4_EphA3_EphA5 | Ephrin type-A receptor 4, 7, 3 | DDX3X, TPM4 |
FLT3_CSF1R_Kit | Macrophage colony stimulating factor I receptor | ARPC3, DDX3X |
Mast/stem cell growth factor receptor | ARPC3, DDX3X | |
GSK3 | Glycogen synthase kinase-3 beta | HSPA8, DDX3X, NUP153 |
Glycogen synthase kinase-3 alpha | HSPA8, DDX3X, NUP153 | |
InsR | Insulin-like growth factor 1 receptor | HSPA8, DDX3X, NUP153, TPM4 |
Insulin receptor | HSPA8, DDX3X, NUP153, TPM4 | |
JNK | Mitogen-activated protein kinase 8 | NUP153 |
Mitogen-activated protein kinase 10 | NUP153 | |
Mitogen-activated protein kinase 9 | NUP153 | |
MAP2K6_MAP2K3_MAP2K4_MAP2K7 | Dual specificity mitogen-activated protein kinase kinase 4 | HSPA8, DDX3X |
Dual specificity mitogen-activated protein kinase kinase 3 | HSPA8, DDX3X | |
Dual specificity mitogen-activated protein kinase kinase 6 | HSPA8, DDX3X | |
MAPK3_MAPK1_MAPK7_NLK | Mitogen-activated protein kinase 3 | NUP153 |
Mitogen-activated protein kinase 1 | NUP153 | |
Mitogen-activated protein kinase 7 | NUP153 | |
MAPKAPK | MAP kinase-activated protein kinase 5 | RPL24 |
MAP kinase-activated protein kinase 2 | RPL24 | |
MAP kinase-activated protein kinase 3 | RPL24 | |
Met | Hepatocyte growth factor receptor | HSPA8 |
NEK1_NEK5_NEK3_NEK4_NEK11_NEK2 | Serine/threonine-protein kinase Nek2 | HSPA8, DDX3X, NUP153 |
p38 | Mitogen-activated protein kinase 14 | NUP153 |
Mitogen-activated protein kinase 11 | NUP153 | |
Mitogen-activated protein kinase 13 | NUP153 | |
p70S6K | Ribosomal protein S6 kinase 1 | DDX3X, NUP153 |
PAKA | Serine/threonine-protein kinase PAK 1 | HSPA8, DDX3X, NUP153 |
Serine/threonine-protein kinase PAK 2 | HSPA8, DDX3X, NUP153 | |
Serine/threonine-protein kinase PAK 3 | HSPA8, DDX3X, NUP153 | |
PAKB | Serine/threonine-protein kinase PAK 4 | DDX3X |
Serine/threonine-protein kinase PAK 7 | DDX3X | |
PDGFR | Beta platelet-derived growth factor receptor | HSPA8 |
Alpha platelet-derived growth factor receptor | HSPA8 | |
Pim2 | Serine/threonine-protein kinase Pim-2 | RPL24, DDX3X |
Pim3_Pim1 | Proto-oncogene serine/threonine-protein kinase Pim-1 | DDX3X |
PKA | cAMP-dependent protein kinase, beta-catalytic subunit | NUP153 |
cAMP-dependent protein kinase, alpha-catalytic subunit | NUP153 | |
PKC | Protein kinase C, delta type | HIST1H2BL, NUP153 |
Protein kinase C, zeta type | HIST1H2BL, NUP153 | |
Protein kinase C, iota type | HIST1H2BL, NUP153 | |
Protein kinase C, theta type | HIST1H2BL, NUP153 | |
Protein kinase C, alpha type | HIST1H2BL, NUP153 | |
Protein kinase C, gamma type | HIST1H2BL, NUP153 | |
PKD | Protein kinase, D1 type | HSPA8 |
Tec | Tyrosine-protein kinase ITK/TSK | ARPC3 |
Tyrosine-protein kinase BTK | ARPC3 | |
Tyrosine-protein kinase Tec | ARPC3 | |
TLK | Serine/threonine-protein kinase tousled-like 1 | DDX3X, HIST1H2BL |
To determine the role these kinases may play in HLA class I cytoskeleton activation the 70 protein kinases were annotated using GOanna
The 70 predicted kinases thought to have the potential to phosphorylate the 11 candidate phosphoproteins were annotated using GOanna and the annotations were categorizied using CateGOrizer which mapped the kinases based on EGAD2GO. The GO terms mapped to 47 EGAD2GO ancestor terms and this graph represents the top 17 definitions and the rest are grouped into the “other” category.
To confirm the presence of the candidate proteins in the cytoskeleton fraction, we performed Western blot experiments with antibodies to 3 of these candidate proteins, DDX3X, TPM4 and eIF4A1 (
(A) The EC cytoskeleton was isolated for the 4 treatment groups, mIgG 1 µg/ml (lane 1), HLA class I 1 µg/ml (lane 2), thrombin 1 U/ml (lane 3), bFGF 25 ng/ml (lane 4) and ECs that were treated with cytochalasin D (5 µM, 30 min) (lane 5). The total cell lysate and the cytoskeleton fraction were subjected to SDS-PAGE separation followed by Western blot to examine the presence of MLC, DDX3X and TPM4 proteins. (B) The EC cytoskeleton isolation was isolated for the 4 treatment groups, mIgG 1 µg/ml (lane 1), HLA class I 1 µg/ml (lane 2), thrombin 1 U/ml (lane 3) and bFGF 25 ng/ml (lane 4). The total cell lysates, the depleted fractions and the cytoskeleton isolated fractions were subjected to SDS-PAGE separation followed by Western blot to examine ß-actin, total MLC and eIF4A1 protein levels in the different cell fractions. (C) The cytoskeleton isolation was performed on ECs treated with mIgG 1 µg/ml for 10 min (lane 1) or with cytochalasin D (lane 2) 5 µM for 30 min. The total cell lysates, depleted fractions and cytoskeleton isolated fractions were subjected to SDS-PAGE separation followed by Western blot to examine eIF4A1, total MLC and ß1-integrin in the cell fractions. The results presented in panel A and B are representative of 5 and 3 independent experiments, respectively.
Among the list of candidates, eIF4A1 was selected for further characterization because it functions as a downstream target of mammalian target of rapamycin (mTOR)
We next examined HLA class I-induced eIF4A1 interactions with the cytoskeleton by performing cellular colocalization studies. For this, confocal microscopy was performed to determine the degree of colocalization of eIF4A1 with F-actin and paxillin, both of which are known regulators of HLA class I cytoskeleton changes
(A) The localization of eIF4A1 and F-actin in the EC was determined by confocal microscopy. Cell were treated and prepared for staining and microscopy. The scale bar is equal to 10 µm. Data is representative of 4 independent experiments. (B) eIF4A1 and F-actin colocalization was determined by the the ImageJ plugin Colocalization Finder (
(A) The localization of eIF4A1 and paxillin in the EC was determined by confocal microscopy. Cell were treated and prepared for staining and microscopy. The scale bar is equal to 10 µm. Data is representative of 4 independent experiments. (B) eIF4A1 and paxillin colocalization was determined by the ImageJ plugin Colocalization Finder. Mander's Overlap coefficients were 0.977 (mIgG), 0.929 (HLA class I), 0.933 (thrombin) and 0.888 (bFGF). The data represents 4 independent experiments. (C) Intensities of the colocalization of 3 images per group were determined for each independent experiment (Avg ± SD): mIgG (14.3±4.3), HLA class I (30.2±4.1), thrombin (19.1±7.8) and bFGF (11.7±3.0). The colocalization intensity of eIF4A1 and paxillin in the HLA class I treated group was significantly increased compared to the unstimulated and bFGF groups; mIgG (p = 0.01), thrombin (p = 0.09) and bFGF (p = 0.003) as determined by student t-test.
The actin cytoskeleton is an important regulator of various cellular functions in ECs including proliferation, migration and permeability
Among the list of candidates, we identified proteins or related proteins involved in cytoskeleton dynamics. For example, cortactin is a known actin-binding protein and transduces signaling to the cytoskeleton
In the past, we established that HLA class I signaling cascades involve various phosphorylation events and have explored several key proteins responsible for these events
Another kinase family predicted to be involved in the phosphorylation of the candidate proteins is 70-kDa S6 protein kinase (p70S6k) family, where the specific kinase was ribosomal protein S6 kinase 1 (S6RP). HLA class I ligation leads to the phosphorylation of p70S6k and S6RP, which are downstream of mTOR complex 1
TPM4 was identified as a candidate protein in the HLA class I treated group by mass spectrometry and confirmed by Western blot to be present only in the cytoskeleton fraction of HLA class I stimulated ECs. Tropomyosins are among the most abundant cytoskeletal proteins in ECs
The eIF4A1 protein, functions downstream of mTOR complex 1, which has been shown to phosphorylate 4E-BP1 following class I ligation
Although eIF4A1 and DDX3X were found by mass spectrometry to be exclusively in the HLA class I treated EC, surprisingly these proteins were found at approximately equal levels in all of the treatment groups following cytoskeleton isolation and Western blotting. The most likely explanation for the detection of eIF4A1 and DDX3X by mass spectrometry only in the class I treated group is the difference in the sensitivity of these assays. In the ECs treated with bFGF or thrombin, other more abundant cytoskeletal proteins may have been present which reduced the relative amount of DDX3X and eIF4A1, precluding their detection by mass spectrometry. Indeed, even when highly sensitive mass spectrometers are used to analyze complex biological samples and bodily fluids, high-abundance proteins obscure the detection of lower-abundance proteins
An alternative explanation for these discrepant findings is that different protein:protein interactions in the treatment groups influence the ability to detect a protein by mass spectrometry. For example, the function of eIF4A1 has little to do with its protein level and more to do with whether or not it is being inhibited
We found that treatment with HLA class I antibodies or thrombin stimulated varying degrees of colocalization between eIF4A1 and F-actin and paxillin suggesting that eIF4A1 may interact with specific compartments of the cytoskeleton in a unique manner. Consistent with this concept, recent studies by our laboratory identified two different signaling pathways leading to MLC phosphorylation and stress fiber formation in ECs, depending upon the nature of the stimulus (ME Ziegler, unpublished). Stimulation with thrombin at 1 U/ml induced a robust increase in the intracellular Ca2+ concentration, increased phosphorylation of MLC and promoted stress fiber formation via MLCK and ROK in an ERK-independent manner. In contrast, stimulation of ECs with a low dose of thrombin (1 mU/ml) or HLA class I antibodies did not promote any detectable change in intracellular Ca2+ concentration, but induced MLC phosphorylation and stress fiber assembly via MLCK and ROK in an ERK1/2-dependent manner. HLA class I ligation requires the recruitment of integrin ß4 in order to activate proliferation and migration
An important functional consequence of HLA class I ligation on ECs is stimulation of cell proliferation, which we previously reported to occur in subconfluent ECs
A key question is how signal transduction is orchestrated through these molecular interactions to stimulate actin cytoskeletal remodeling. Our previous publications and current data are consistent with a model whereby molecular aggregation of HLA class I molecules with antibodies leads to recruitment of integrin ß4 and the subsequent activation of intracellular signals that increase Rho-GTP activity, induce phosphorylation of ROK and trigger the assembly and phosphorylation of FAK, Src and paxillin at the focal adhesions to stimulate actin reorganization
In conclusion, these studies provide new information that can be applied to the exploration of known pathways. Given that phosphoprotein signal transduction is essential to HLA class I EC activation, not only are the proteins relevant, but also their corresponding kinases. Thus, validation of these proteins and examination of their activation state will be important in future studies. Overall these studies may reveal more specific targets in understanding the mechanisms of HLA class I induced antibody-mediated rejection. Additionally, these methods can be applied to other cell types and agonists as an effort to understand the role of cytoskeleton changes in many pathways.
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