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,
,,1
* Department of Anesthesiology and Intensive Care Medicine, Tübingen University Hospital, Tubingen, Germany;
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA;
Department of Anesthesia and Perioperative Medicine, Childrens Hospital and Harvard Medical School, Boston, Massachusetts, USA; and
Mucosal Inflammation Program, University of Colorado Health Sciences Center, Denver, Colorado, USA
1Correspondence: University of Colorado Health Sciences Center, Biochemistry Research Bldg., Rm. 702, 4200 E. 9th Ave, Denver, CO 80220, USA. E-mail: colgan{at}zeus.bwh.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(hif1a). Somewhat surprisingly, these studies revealed that mutant hif1a mice were protected from hypoxia-induced increases in intestinal permeability in vivo. Guided by microarray analysis of tissues derived from these mutant hif1a mice, we identified HIF-1-dependent repression of vasodilator-stimulated phosphoprotein (VASP), a molecule known to be important in the control of cytoskeletal dynamics, including barrier function. Studies at the mRNA and protein level confirmed hypoxia-elicited repression of VASP in murine tissue, cultured epithelia and endothelia, as well as human saphenous vein ex vivo. Targeted repression of VASP by siRNA recapitulated our findings with hypoxia and directed overexpression of VASP abolished hypoxia-induced barrier dysfunction. Studies in the cloned human VASP promoter revealed hypoxia-dependent transcriptional repression, and functional studies by chromatin immunoprecipitation (ChIP) and site-directed mutagenesis revealed hypoxia-dependent binding of HIF-1 to the human VASP promoter. These studies identify HIF-1-dependent repression of VASP as a control point for hypoxia-regulated barrier dysfunction.—Rosenberger, P., Khoury, J., Kong, T., Weissmüller, T., Robinson, A. M., Colgan, S. P. Identification of vasodilator-stimulated phosphoprotein (VASP) as an HIF-regulated tissue permeability factor during hypoxia.
Key Words: barrier function • actin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. It is thought that hypoxia influences barrier properties though formation of stress fibers and active reorganization of the cytoskeleton in hypoxia (2
3
4)
. For example, in alveolar epithelial cells, exposure to hypoxia results in decreased actin organization associated with decreased zonula occludens-1 (ZO-1) protein levels (5)
. Our own work has demonstrated that as a major actin binding protein, vasodilator-stimulated phosphoprotein (VASP) colocalizes with ZO-1 at tight junctions and plays a key role in establishing and maintaining barrier function (1
, 6)
.
VASP is composed of three major domains: an EVH1 domain, an EVH2 domain, and a central proline-rich region (7)
. As VASP contains both actin binding and actin monomer-nucleating capabilities, this molecule is of particular interest for reorganization of the cytoskeleton. The actin binding and cross-linking capabilities localize to the COOH-terminal EVH2 domain (8)
. Loss of VASP function results in loose associations between epithelia and failure to reseal barriers after insult (9)
. In endothelial cells, VASP functions in membrane ruffling, aggregation, and tethering of actin filaments during the formation of endothelial cell-substrate and cell-cell contacts. Moreover, VASP expression is increased in endothelial cells during angiogenesis and at most phases involving cell shape change (10)
. These earlier investigations objectively demonstrate the importance of VASP in development, organization, and mobility of the cytoskeleton.
In the present study we examined basic elements of tissue permeability in hypoxia. As directed by microarray results from conditional hif1a–– mice, we identified VASP as a crucial molecule in regulating tissue permeability during hypoxia. Molecular analysis revealed a novel HIF-1-dependent repression mechanism. Our data show for the first time a regulatory pathway for VASP expression involving HIF-1-regulated tissue permeability during hypoxia.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Genotyping of mice was carried out at 8–12 wk of age as described (12
, 13)
. After genotyping, littermate controls and conditional hif1a mutant mice were subjected to environments of normoxia (21% O2) or normobaric hypoxia (8% O2, 92% N2) for 8 h. At the beginning of the experiment mice were gavaged with 0.6 mg/g body weight of FITC-dextran (4 kDa, at a concentration of 80 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) to examine intestinal permeability as described previously (14)
. Mice were sacrificed and cardiac puncture was performed for serum analysis of FITC-dextran concentration. Organs were harvested for protein and mRNA analysis. All procedures involving animals were performed according to National Institutes of Health (NIH) guidelines for use of live animals and were approved by the Institutional Animal Care and Use Committee at Brigham and Women’s Hospital and the University of Colorado Health Sciences Center.
Endothelial macromolecule paracellular permeability assay
Using a modification of methods described (15)
, HMEC-1 were seeded on polystyrene-permeable inserts (0.4 µm pore, 6.5 mm diameter; Costar Corp., Acton, MA, USA) and studied 5–7 days after seeding (2–3 days after confluency). Inserts were exposed to normobaric hypoxia (pO2=20 torr) for up to 48 h. After this, inserts were placed in Hanks buffered saline (HBSS+) -containing wells (1.0 ml), and HBSS+ was added to inserts apically (100 µl). At the start of the assay (t=0), FITC-labeled dextran 70 kDa (concentration 3.5 µM) was added to fluid within the insert. The size of FITC-dextran (70 kDa) approximates that of human albumin, both of which have been used in similar endothelial paracellular permeability models (16)
. Fluid from an opposing well (reservoir) was sampled (50 µl) over 60 min (t=20, 40, and 60 min) after exposure to hypoxia for 24 h and 48 h. Fluorescence intensity of each sample was measured (excitation, 485 nm; emission, 530 nm; Cambrex FLX 800) and FITC-dextran concentrations were determined from standard curves generated by serial dilution of FITC-dextran. Paracellular flux was calculated by linear regression of sample fluorescence (15)
Transcriptional analysis
Semiquantitative analysis was performed using real-time PCR RT-PCR (iCycler; Bio-Rad Laboratories Inc., Hercules, CA, USA) to examine VASP expression levels in HMEC-1 and T84 cells after confluent cells were exposed to 4 h, 8 h, 24 h, and 48 h hypoxia. Primer sets contained 10 pM each of the sense primer 5'-GAA AAC CCC CAA GGA TGA AT-3' and the antisense primer 5'-GGA AGT GGT CAC CGA AGA AG-3'. The primer set was amplified using increasing numbers of cycles of 94°C for 1 min, 60°C for 2 min, 72°C for 4 min, and a final extension of 72°C for 7 min. Samples were controlled for ß-actin using the following primers: sense 5'-GGT GGC TTT TAG GAT GGC AAG-3', antisense 5'-ACT GGA ACG GTG AAG GTG ACA G-3', 162 bp). After approval by the institutional review board, saphenous vein material was obtained from patients undergoing coronary artery bypass grafting. Equal portions of dissected vein tissue were subjected to normoxia or hypoxia (pO2 20 torr) for 8 h. After hypoxic exposure, RNA was isolated from the tissues and real-time PCR was performed as described above.
Mouse analysis for mRNA levels was performed using real-time PCR using sense primer 5-TGG GCT ACA GGC TTG TCA CT-3 and 5-GAG AAG AAA AGC CAC ACA GGT T-3 antisense primer. Mouse ß-actin expression was evaluated with sense 5'-CTC TCC CTC ACG CCA TCC TG-3', antisense 5'-TCA CGC ACG ATT TCC CTC TCA G-3'.
Human and mouse protein analysis
Cell culture and mouse tissue samples were normalized for protein levels before applying them in nonreducing conditions to SDS containing polyacrylamide gels. Antibodies used for Western blot included mouse polyclonal anti-VASP (BD Transduction Laboratories, Lexington, KY, USA) for human VASP analysis and rabbit polyclonal anti-VASP (Alexis Biochemicals, San Diego, CA, USA) for mouse VASP analysis. Actin was stained using rabbit anti-actin (Sigma-Aldrich). Blots were washed and species-matched, peroxidase-conjugated secondary antibody was added. Labeled bands from washed blots were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Blots were scanned and densitometry analysis was performed using NIH Image J Software.
Immunofluorescent staining
HMEC-1 were grown to confluence on acid washed in 12 mm glass coverslips. Monolayers were washed once in phosphate-buffered saline (PBS) and fixed for 10 min at room temperature in 1% paraformaldehyde in cacodylate buffer (0.1M sodium cacodylate; pH 7.4, 0.72% sucrose). The monolayers were permeabilized for 10 min in PBS containing 0.2% Triton X-100 and 3% BSA. After washing twice with PBS, the cells were stained for 1 h with a monoclonal anti-VASP (12.5 µg/ml, BD Transduction Labs). After washing, the monolayers were incubated with goat anti-mouse Oregon Green (1 µg/ml, Molecular Probes, Eugene, OR, USA). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, 10 µg/ml; Molecular Probes). Coverslips were mounted in polyvinylalcohol and viewed at 400 x on a fluorescence microscope (Nikon, Melville, NY, USA). Image pixel densities were calculated using Image J (NIH, Bethesda, MD, USA).
VASP pGL3-reporter assay
The VASP 5' flanking region of the human VASP gene was cloned by PCR, ligated into a pGL3 firefly luciferase reporter vector, and sequenced with basic pGL3 vector primer GL2. As a control for hypoxia, cells were transfected with a pGL3-based HRE plasmid containing four tandem HIF-1 enhancer sequences from the 3'-region of the erythropoietin gene (17)
. HMEC-1 and HeLa cells were transfected using standard methods with Lipofectamine 2000 for 5 h. After transfection, media was changed and cells were subjected to hypoxia or normoxia for 24 h. Luciferase activity was assessed (Turner Designs, Sunnyvale, CA, USA) using a luciferase assay kit (Promega, Madison, WI, USA). All firefly luciferase activity was normalized with respect to the constitutively expressed protein. In a subset of experiments, HIF-1 binding site mutations were introduced in VASP pGL3 reporter plasmids to evaluate the functional importance of the HIF-1 binding. Mutations were performed using the Quickchange in vitro site-directed mutagenesis system (Stratagene, San Diego, CA, USA). The original sequence in position 22–25 containing the HIF-1 binding site CGTG was mutated to GCCA and the pGL3 vector was amplified with Top Ten fast-growing bacteria. After extraction from bacteria, the pGL3 vector containing the mutation was sequenced again to confirm mutation at the specified site. Again, HeLa cells were transfected with the original VASP pGL3 reporter, and the mutated reporter and firefly luciferase assay were performed after exposure of cells to 24 h of normobaric hypoxia.
Chromatin immunoprecipitation (ChIP) assay
HMEC and T84 cells were fixed with 1% paraformaldehyde. Chromatin derived from isolated nuclei was sheared by using a F550 micro-tip cell solicitor (Fisher Scientific, Pittsburgh, PA, USA). After centrifugation, supernatants containing sheared chromatin were incubated with an anti-HIF-1 antibody (Novus Biologics, Littleton, CO, USA) or control IgG (OE). Protein A Sepharose was then added, incubated overnight, and immune complexes were eluted. Complexes were next treated with Raze and protein K, extracted with phenol/chloroform, then with chloroform. DNA was precipitated, washed, dried, resuspended in water, and analyzed by PCR (33 cycles). The primers used in this analysis spanned 165 bp around the putative HIF-1 binding site within the VASP promoter (sense, 5'-CCA CTT CCT CCT CAC CTT CC-3', and antisense, 5'-TTC TCC CTG AAA TCC CAT CTT-3').
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These earlier studies revealed a protective role for intestinal epithelial HIF-1 in a murine model of colitis. Since epithelial cells are the primary determinant of mucosal barrier function, we compared intestinal permeability to 4 kDa FITC dextran in wild-type and conditional hif1a mutants in response to hypoxia (8 h at 8% O2; 92% N2), a condition we previously showed to significantly increase intestinal permeability. As shown in Fig. 1
A, while wild-type animals showed a predictable increase in permeability to fluorescent tracer (P<0.025), conditional hif1a mutant animals were unexpectedly protected from increased intestinal permeability (P=not significant), thereby implicating HIF-1 in increased intestinal permeability during hypoxia.
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To search for hif1a-dependent genes involved in epithelial barrier regulation, we profiled conditional hif1a mutant animals (n=5) and littermate controls (n=5) subjected to hypoxia (4 h at 8% O2; 92% N2) by microarray analysis. This analysis revealed that although the vast majority of genes (>96%) were unchanged, one barrier-related gene was significantly different among these animal populations. Specifically, these studies revealed that VASP was significantly repressed by hypoxia in wild-type animals but not in conditional hif1a mutant animals (Fig. 1B
). We and others had previously identified VASP as an actin binding protein important in the dynamic regulation of barrier function (18
19
20)
, and therefore we hypothesized that VASP repression in hypoxia represents an HIF-regulated pathway important in barrier regulation. Initially, we verified our microarray findings in conditional hif1a mutant animals. As shown in Fig. 1C
, VASP mRNA levels were significantly reduced in colonic epithelia of hif1a wild-type (P<0.025) but not in hif1a mutant animals after subjection to hypoxia (P=not significant). This difference was reflected only in the Fabpl4x at–132/Cre conditionally deleted tissue (colon; see Fig. 1C
); other organs demonstrated a wild-type phenotype with regard to VASP repression in hypoxia, including the lung (Fig. 1D
), liver, and kidney (data not shown).
We next compared VASP protein expression between conditional hif1a mutant animals and littermate controls. As shown in Fig. 1E
, Western blot analysis was used to examine VASP expression and revealed that protein expression paralleled changes in mRNA, wherein wild-type animals expressed significantly less VASP than hif1a mutant littermates, thereby implicating a hif1a-dependent repression of VASP expression. Densitometry verified these results (Fig. 1F
), reflecting the hypoxia-dependent down-regulation of VASP (P<0.05) and the unchanged VASP expression profile in conditional hif1a mutant tissues (P=not significant).
Hypoxia-dependent repression of VASP in human tissue and human cell lines
We next extended our findings in the mouse to examine whether hypoxia similarly regulates human VASP expression. First, we addressed whether hypoxia influenced VASP expression in human saphenous vein ex vivo. As shown in Fig. 2
A, explants of human saphenous vein exposed to hypoxia (pO2 20 torr for 8 h) resulted in a 56 ± 11% decrease in VASP mRNA expression relative to actin controls (P<0.025). Second, we used two individual barrier cell types—namely, human microvascular endothelia (HMEC-1) and human colonic epithelia (T84 cells)—to examine VASP expression in hypoxia. As shown in Fig. 2B
, exposure of T84 and HMEC-1 to a time course of hypoxia (0–48 h) revealed a sustained and significant decrease in VASP mRNA for both cell lines examined (P<0.025 by ANOVA).
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Localization of VASP by immunofluorescence in HMEC-1 revealed a prominent loss of VASP (63±11% decrease in pixel density, n=14, P<0.025) in hypoxia relative to normoxia (Fig. 2C, D
). A confirmatory decrease in VASP protein expression by Western blot was evident in both HMEC-1 and T84 cells (Fig. 2C
), suggesting that similar to our findings in murine models in vivo (Fig. 1)
, hypoxia represses VASP protein and mRNA expression in human cells and tissues.
Impact of VASP expression on barrier function
We next sought to determine whether a functional cause-and-effect relationship exists between hypoxia and VASP repression. First, we determined whether conditions that repress VASP protein (i.e., hypoxia; see Fig. 2D, E
) influence permeability in human cells in vitro. For these purposes, we exposed HMEC-1 to hypoxia (0–48 h) and examined paracellular permeability to FITC-labeled dextran. As shown in Fig. 2F
, increasing time points of hypoxia increased paracellular permeability in a time-dependent manner (P<0.01 by ANOVA).
We next induced repression of VASP through use of siRNA and examined changes in permeability (i.e., independent of hypoxia). As shown in Fig. 3
A, loading of HMEC-1 with VASP siRNA, but not a nonspecific control siRNA, decreased VASP mRNA and protein by nearly 80%. Examination of permeability in these cells (Fig. 3B
) revealed a 68 ± 8% increase in paracellular permeability (P<0.025 compared with control monolayers).
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Third, we determined whether forced overexpression of VASP might protect against hypoxia-elicited increases in permeability. To do this, we transfected HMEC-1 with VASP cDNA on a heterologous viral promoter (CMV). Such transient transfections resulted in a nearly 6-fold increase in VASP protein expression over mock-transfected controls (Fig. 3C
), with no significant changes associated with hypoxia. Using these cells, we examined the influence of hypoxia on paracellular permeability to 70 kDa FITC-dextran. As shown in Fig. 3D
, forced overexpression of VASP in HMEC-1 resulted in a 22 ± 5% decrease in baseline permeability compared with mock-transfected controls (P<0.05). Moreover, when exposed to hypoxia, cells overexpressing VASP were protected from hypoxia-induced changes in permeability (Fig. 3D
). Indeed, while hypoxia increased the permeability of control-transfected cells by 40 ± 6% (P<0.025, Fig. 3D
), cells transfected with VASP and exposed to hypoxia showed a 34 ± 6% decrease in permeability (P<0.05). Taken together, these findings suggest that VASP is a limiting factor for endothelial permeability during episodes of hypoxia.
Cloning of the VASP promoter and role of HIF in repression during hypoxia
We next examined mechanisms of VASP repression by hypoxia. In view of the likelihood of a transcription-mediated repression of VASP during hypoxia, attention was directed at the 5'-region of the VASP gene for potential hypoxia-regulated transcription factor sequences. Available public databases (21)
and analysis of full-length cDNA [Genbank NM_003370] identified the transcription start site of VASP at position –260 relative to the first codon. Based on these reports, we cloned a genomic fragment extending from positions –975 to +238 (Fig. 4
A) into pGL3 luciferase reporter vector and examined hypoxia-dependent activity (24 h hypoxia) after transient transfection in HMEC-1 and in HeLa cells. Consistent with mRNA and protein analysis (Fig. 3)
, VASP promoter analysis revealed transcriptional repression in response to hypoxia. As shown in Fig. 4B
, transient transfection in HMEC-1 and HeLa cell resulted in a 62 ± 5% and 64 ± 10% loss of activity in hypoxia (P<0.025 for both), respectively, thus implicating transcriptional repression of the VASP promoter by hypoxia.
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Analysis of our cloned region of VASP revealed the existence of a potential binding site for HIF-1 (core sequence 5'-CGTG-3') at position –522 to –519 relative to the transcription start site and an HIF-1 ancillary sequence (22)
located 12 bp in the 3' direction. Based on these findings and recent studies indicating that HIF can function as a transcriptional repressor (23
24
25)
, we determined whether HIF-1 would bind the VASP promoter. For these purposes, we used ChIP analysis to examine binding of HIF-1 to the VASP promoter spanning the putative HIF-1 binding site in intact cells. As shown in Fig. 4C
, this analysis in two separate cell lines (HMEC-1 and T84) revealed a prominent band of 165 bp in nuclei derived from hypoxic, but not normoxic, cells. No bands were evident in the beads-only or H20 controls, and preimmunoprecipitation samples revealed equivalent DNA input, thereby indicating specific binding of HIF-1 to the proximal VASP promoter.
To determine the functional implications of the HIF-1 binding site in the VASP promoter, site-directed mutagenesis was used. We targeted the central HIF binding site in cloned promoter (see Fig. 4A
); as shown in Fig. 4D
, a three-nucleotide mutation (
HIF consensus motif 5'-CACGTGG-3' mutated to 5'-CAATCGG-3' within HIF-1 site) resulted in a complete loss of repression. Indeed, hypoxia-dependent promoter activity was repressed by 53 ± 6% in the wild-type construct (P<0.01 compared with normoxia) and induced by 26 ± 7% in the HIF construct (P=not significant compared with normoxia). As an HIF reference control, cells were transfected with a plasmid expressing the HRE from the 3' region of the EPO (termed HRE) and revealed a 46 ± 6-fold increase in activity with exposure to hypoxia (P<0.01 compared with normoxia). Taken together, these DNA binding and mutagenesis studies strongly suggest that the central HIF site is critical for hypoxia-dependent repression of VASP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Guided by results from microarray analysis, we identified HIF-regulated expression of VASP in vivo as a regulatory mechanism of tissue permeability during hypoxia. VASP is a key actin binding protein with demonstrated importance in junctional reorganization. Extensions of these studies in vitro and with epithelial conditional hif1a–– mice revealed that HIF-dependent repression of VASP contributes to the increased tissue permeability observed in various hypoxic models.
At the tissue and cellular level, HIF mediates the expression of an array of genes pivotal to survival in low oxygen states (27
, 28)
. Consensus HIF binding sequences termed hypoxia-responsive enhancers/elements (HRE) have been identified in a growing number of hypoxia-responsive genes (28)
, some of which directly affect epithelial barrier function. Initial studies from our laboratory revealed that barrier function in cell lines derived from different tissues may be influenced by hypoxia to different degrees, with cell lines derived from colonic tissue being the most resistant (14)
. These studies indicated the differential expression of barrier protective genes in intestinal tissue, and candidate genes for this effect were consecutively identified in ITF (14)
, CD73 (29)
, adenosine receptors (30)
, and MDR-1 (31)
. These studies and others have implicated HIF-1 as an adaptive barrier mechanism for both epithelial and endothelial cells. Although increased permeability is widely observed in tissues subjected to hypoxic stress, less is known about direct mechanisms for such changes in permeability. Several lines of evidence implicate HIF-dependent VASP repression as a component of increased tissue permeability during hypoxia. First, epithelial conditional hif1a–– mice are generally resistant to increased permeability induced by hypoxia. Second, hypoxia-induced VASP repression was demonstrable in murine tissue, human tissue, and various human cell lines. Third, molecular manipulation of VASP expression (e.g., siRNA and heterologous plasmid expression) independent of hypoxia resulted in a predictable change in barrier function. Fourth, cloning of the VASP promoter identified HIF-1 binding and function at the transcriptional level. Thus, it is likely that HIF-dependent VASP regulation contributes at least in part to changes in tissue permeability associated with hypoxia.
VASP plays a central role in mediating actin dynamics within endothelial and epithelial cells and is involved in stress fiber formation (10)
. Endothelial and epithelial formation of stress fibers or destruction of cytoskeletal structures is associated with an increase in permeability (5
, 32
, 33)
. In addition, passive cell retraction as a result of cytoskeleton rearrangement and attenuation of cell-cell and cell-substrate junctions also plays a key role in mediating cellular contractile response and changes in paracellular permeability (34
, 35)
. This rearrangement transposes its force on cell-cell junctions through indirect attachment of actin fibers with tight and adherens junctions. Previous investigations have focused on demonstrating the influence of hypoxia on cytoskeletal arrangement and development, but focused primarily on its influence in the central nervous system (36
37
38)
. Chemically induced hypoxia through ATP depletion results in disruption of the actin cytoskeleton in renal epithelial cells. Ankyrin and spectrin are dissociated from the actin cytoskeleton in low ATP states, causing intracellular redistribution (39
40
41)
. A redistribution of actin in cells exposed to chemical hypoxia persists for >3 h and is associated with increased paracellular flux (42)
. In addition, ischemia is a cause for cells to lose their polarity, open tight junctions, and thus increase paracellular permeability (43)
. ZO-1 plays a critical role in tight junction complex assembly. Modification of ZO-1 and other tight junctional protein expression leads to increased paracellular permeability (5
, 44
, 45)
. This shows that the regulation of cytoskeletal proteins during hypoxia can have a direct influence on barrier properties of endothelial and epithelial cells, and so is consistent with decreased VASP expression (5)
.
Taken together, our results demonstrate a fundamental control process for VASP as a central mechanism of barrier regulation. Efforts to better understand a possible beneficial role of VASP in barrier function could result in therapeutic modalities for a variety of disease processes that are associated with impaired barrier function.
Received for publication December 28, 2006. Accepted for publication March 1, 2007.
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REFERENCES |
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