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The Journal of Immunology, 2001, 166: 5550-5556.
Copyright © 2001 by The American Association of Immunologists

The Vasodilator-Stimulated Phosphoprotein Is Regulated by Cyclic GMP-Dependent Protein Kinase During Neutrophil Spreading1

Donald W. Lawrence2 and Katherine B. Pryzwansky3

Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The vasodilator-stimulated phosphoprotein (VASP)4 is a 46-kDa membrane-associated protein that is a member of the Enabled (Ena) family of proteins. These proteins are localized to the actin cytoskeleton and are in vitro and in vivo ligands for the focal adhesion-associated proteins zyxin and vinculin and the actin-binding protein profilin (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). VASP is believed to play an important role in controlling the cytoskeletal organization because it binds filamentous actin (F-actin) and profilin, a protein that forms complexes with G-actin and regulates actin dynamics (2, 11, 12). In platelets VASP, in association with profilin, modulates actin polymerization by nucleating actin filaments (6). The phosphorylation of VASP is thought to regulate its affinity for F-actin (11). VASP is phosphorylated by cAMP-dependent protein kinase (cAK) or cGMP-dependent protein kinase (cGK). The phosphorylation of VASP by cGK-1 promotes detachment of VASP and zyxin from focal adhesions in endothelial cells, suggesting that the phosphorylation state of VASP may alter the composition and functional integrity of these structures (10). In addition, the prolonged effects of 8-pCPT-cGMP, a direct activator of cGK, causes a reduction of the actin microfilament system and vinculin at focal adhesions in endothelial cells (10). Recent studies suggest that the placement of zyxin within a cell can affect the distribution of VASP to F-actin-rich sites (8). Knockout mice for VASP have a defect in cyclic nucleotide-mediated platelet disaggregation and integrin {alpha}IIb{beta}3 activation (13, 14). Evidence suggests that type I cGK (cGK-1) phosphorylates VASP and regulates adhesion in platelets, as platelets of knockout mice for cGK-1 lack VASP phosphorylation and have a defect in cGMP-mediated aggregation (15). Others have shown in endothelial cells that VASP is regulated at focal adhesion sites by cGK-1, and that cGK-1 is required for focal adhesion disassembly in endothelial cells and smooth muscle cells (10, 16, 17).

VASP is found in a wide variety of cell types, including platelets, vascular endothelial cells, HL-60 cells, vascular smooth muscle cells, fibroblasts, and cardiomyocytes (7, 10, 17, 18). The expression and function of VASP in neutrophils is unknown. VASP is predominately localized in cells at areas of membrane activity, focal adhesions, stress fibers, cell-matrix, and cell-cell adherens junctions (1, 17). Recent studies suggest that the subcellular distribution of VASP may be important for reorganizing actin during cell adhesion and spreading (8, 10). Neutrophils are highly motile and depend on an actin-based motility to carry out their functions. In neutrophils, cyclic nucleotides regulate actin, adherence, and shape changes that may modulate VASP function (19, 20, 21, 22, 23, 24, 25, 26). In this study, VASP was investigated as a downstream target for cyclic nucleotide-dependent regulation of neutrophil adhesion. We demonstrate that adhesion and spreading induce significant elevations of cGMP in neutrophils and the rapid phosphorylation of VASP by cGK at the onset of cell spreading. Our findings indicate that cGMP/cGK signaling is required for neutrophil spreading, and that VASP is a target of cGK regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Neutrophil isolation

Human neutrophils were isolated from citrated whole blood of healthy human individuals as described previously (27). The cells were suspended at 3 x 106/ml in Gey’s balanced salts buffered with 10 mM HEPES (pH 7.2) and supplemented with 1.5 mM CaCl2, 1 mM MgCl2, and 0.3 mM MgSO4 (GBSS). The cell preparations were >98% viable by trypan blue exclusion and consisted of >95% neutrophils.

VASP immunoprecipitation

Neutrophils in suspension were solubilized on ice for 10 min in a lysis buffer containing 50 mM Tris (pH 7.5), 1% Nonidet P-40, 0.1% deoxycholate, 150 mM NaCl, 50 mM NaF, 1.2 mM p-aminobenzamidine, 2 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A. The lysate was cleared with 35 µl of a 50% protein A-Sepharose slurry (Pharmacia, Piscataway, NJ) for 1 h at 4°C, and the total protein concentration was measured by the bicinchonic acid method (Pierce, Rockland, IL). An aliquot (500 µg) of cell lysate was incubated for 1 h at 4°C with 1.25 µg of mouse anti-VASP or 1.25 µg of mouse anti-focal adhesion kinase (anti-FAK, isotype control; Transduction Laboratories, Lexington, KY). The primary Ab was captured by using a complex of rabbit anti-mouse IgG conjugated to protein A-Sepharose. The immune complexes were washed, heated at 100°C for 5 min in SDS sample buffer, and analyzed by Western blot analysis for VASP.

Western blot analysis for VASP

Neutrophil proteins were solubilized in 2x sample buffer containing 200 mM Tris-base (pH 6.7), 6% SDS, 15% glycerol, 0.03% bromophenol blue, and 10% 2-ME. After separation on a 8% polyacrylamide gel, proteins were transferred to polyvinylidene fluoride membranes. Membranes were blocked with 4% skim milk in PBS containing 0.05% Tween 20 (PBS-T), and then incubated with 1:1500 mouse anti-VASP (Transduction Laboratories) for 1 h at room temperature. The membranes then were washed in PBS-T and incubated with 1:10,000 goat anti-mouse IgG-HRP (Dako, Carpenteria, CA) for 1 h at room temperature. The wash was repeated and VASP was detected by using the SuperSignal chemiluminescence detection system (Pierce).

VASP phosphorylation of adherent neutrophils

Neutrophils were adhered for 1 to 10 min on 100-mm petri dishes coated with human type AB serum. Coating was achieved by incubating plates at 37°C with 5 ml of GBSS supplemented with 50% human type AB serum for 1 h. The plates were rinsed once in GBSS and used immediately. The monolayers were lysed on ice for 10 min by the addition of 70 µl of immunoprecipitation lysis buffer (see above). The plates were scraped and protein concentrations were measured. Aliquots of the lysates were subjected to Western blot analysis for VASP. In some instances, neutrophil suspensions were treated with 1 µM KT 5720 or 10 µM Rp-8-pCPT-cGMPS for 30 min at 37°C to inactivate cAK or cGK, respectively. Phosphorylation shifts the apparent mobility of VASP in SDS-PAGE from a 46-kDa to a 50-kDa species. The autoradiographs were scanned with an Epson Expression 800 Scanner (Epson, Long Beach, CA), and densitometric measurements were performed with NIH Image, version 1.59 (National Institutes of Health, Bethesda, MD). VASP phosphorylation was expressed as the ratio of the density measurements of the 50-kDa band to the 46-kDa band (50 kDa/46 kDa).

Measurements of cGMP in neutrophils

Neutrophils were adhered to serum-coated 60-mm plastic petri dishes from 1 to 10 min. At the desired time point, cGMP levels were measured by RIA as described previously (27). Neutrophil cGMP levels are subject to donor variability. Experiments were performed in triplicate at least three times with different donors. The data represent a common trend present in each experiment.

Neutrophil spreading assay

Neutrophils were adhered to 60-mm petri dishes in the presence of 10% human type AB serum for 10 min at 37°C. The plates were placed at 4°C for 10 min to induce cell retraction. After the majority of the cells were round, as evident by phase microscopy, the plates were incubated at 37°C from 1 to 10 min to induce cell spreading. The monolayers then were fixed in 1% glutaraldehyde in GBSS for 10 min at room temperature. Phase micrographs were captured with an Optronics Engineering DEI-470 color video camera (Optronics International, Chelmsford, MA). In addition, intracellular cGMP levels were measured at each time point, monolayers were fixed for immunofluorescence microscopy, and lysates were obtained for Western blot analysis of VASP.

Morphometric analysis

Neutrophils were suspended in GBSS containing 10% human serum and were preincubated with either 1 µM 8-Br-cGMP (BioLog, La Jolla, CA) for 5 min, 1 µM KT5720 (Calbiochem, La Jolla, CA) for 30 min, or 10 µM Rp-8-pCPT-cGMPS (BioLog) for 30 min at 37°C. Aliquots were removed and fixed in 1% glutaraldehyde in GBSS to examine neutrophil morphology before adhesion. The remaining neutrophil suspensions were adhered from 1 to 10 min at 37°C. At the desired time point, the monolayers were fixed in 1% glutaraldehyde in GBSS for 10 min at room temperature. The changes in morphology during adherence were recorded by photography with a Zeiss Axiovert 10 inverted microscope (Zeiss, Oberkochen, Germany) on Kodak Technical Pan film, ASA 100 (Kodak, Rochester, NY). The area and perimeter of a population of cells (n = 250 for each condition) were measured using NIH Image version 1.59 (National Institutes of Health). The formula, circularity = 4 x {pi} x area/(perimeter)2, was used to analyze cell shape (28).

Immunofluorescence microscopy

Monolayers were fixed for immunofluorescence microscopy as described previously (27) and stained for 1 h with 1:25 mouse anti-VASP and for 30 min with rhodamine-labeled goat anti-mouse IgG (Rockland, Gilbertsville, PA). For localization of F-actin and VASP, neutrophil monolayers were stained with mouse anti-VASP and fluorescein-labeled goat anti-mouse IgG (Rockland) as described above and then stained for F-actin with rhodamine phalloidin as described previously (27). Immunofluorescence micrographs were acquired using Adobe Photoshop 3.0 (Adobe Systems, Mountain View, CA) and an Optronics Engineering DEI-470 color video camera (Optronics International).

Statistical analysis

All data were presented as mean ± SD. Statistical differences between means of two populations were determined by Student’s t test, with p < 0.05 regarded as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of VASP in neutrophils

VASP was shown to be expressed in lysates of neutrophil suspensions by immunoprecipitation with an Ab specific for both the dephosphorylated and phosphorylated forms of the protein and by Western blot analysis. The phosphorylation of VASP by cGK or cAK alters the properties of the protein in SDS-PAGE, causing a shift in its apparent molecular mass from 46 kDa to 50 kDa (18). Two proteins at 46 kDa and 50 kDa were detected by Western blot analysis (Fig. 1Go). These molecular mass agree with those published for the dephosphorylated (46 kDa) and phosphorylated (50 kDa) forms of human VASP (18, 29). Western blot analysis of the postimmunoprecipitation lysate for VASP or FAK (isotype-specific Ab control) indicated that VASP immunoprecipitation was complete and specific.



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  		FIGURE 1. VASP immunoprecipitation. Neutrophil lysates were immunoprecipitated with mouse anti-FAK (lane 1) or mouse anti-VASP (lane 2). The lysate before immunoprecipitation is shown in lane 3. Aliquots of the postimmunoprecipitation lysate for both VASP (lane 4) and control Ab (lane 5) also were subjected to Western blot analysis for VASP. The immunoprecipitation reaction was complete, as VASP was not detected in the lysate after immunoprecipitation (lane 4). These data are from a representative experiment repeated twice.

 
Neutrophil adhesion and spreading induce VASP phosphorylation

The phosphorylation state of VASP was investigated in neutrophils during adhesion and spreading onto a serum-coated surface. In neutrophil suspensions at 37°C, VASP was present as a predominantly dephosphorylated protein (Fig. 2Go). When neutrophils were allowed to adhere from 1 to 10 min, VASP became transiently phosphorylated within 1 min of adhesion and remained phosphorylated for at least 5 min. After 10 min, VASP became dephosphorylated, and the phosphorylation profile resembled that of cells in suspension.



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  		FIGURE 2. Adhesion-dependent VASP phosphorylation. Neutrophil suspensions were adhered to serum-coated dishes for various times at 37°C. Equal amounts of protein at each time point were analyzed for the VASP Ag by Western blot analysis. Densitometric measurements of the 50-kDa and 46-kDa bands were performed, and phosphorylation of VASP was expressed as the 50 kDa/46 kDa ratio for each condition. Lane 1 is VASP from neutrophil suspensions at 37°C and lanes 2–5 represent cells adhered for 1, 3, 5, and 10 min, respectively. VASP was phosphorylated in actively adhering and spreading neutrophils from 1 to 5 min. By 10 min, the phosphorylation profile resembled that of cells held in suspension. These data are from a representative experiment repeated at least three times.

 
Adhesion and spreading are dynamic processes. Therefore, each of these functions could be responsible for inducing phosphorylation of VASP. To differentiate between adhesion and spreading without pharmacological intervention, neutrophil monolayers were incubated at 4°C for 10 min to promote cell retraction or until the majority of the cells maintained a round morphology, as evidenced by phase microscopy (Fig. 3Go). The retracted cells were then incubated at 37°C for various times to induce cell spreading. After 1 min of re-warming at 37°C, neutrophils demonstrated membrane ruffling and some membrane extension. Within 3 min, neutrophils were well spread and resembled control cells maintained at 37°C. There were no changes in morphology observed after rewarming cells for 5 or 10 min.



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  		FIGURE 3. Phase microscopy of neutrophils induced to spread. Neutrophils were adhered in the presence of 10% human serum for 10 min (A). The monolayers then were placed at 4°C for 10 min to induce cell rounding (B). The cells then were placed at 37°C for 1 min (C) or 3 min (D) to induce cell spreading. There were no changes in cell morphology after 3 min.

 
VASP was phosphorylated at the onset of changes in cell shape from initially round to spread cells. Western blots of neutrophils induced to spread demonstrated that VASP was present as a dephosphorylated 46-kDa protein in monolayers held at 4°C or 37°C (Fig. 4Go). The initiation of neutrophil spreading induced the phosphorylation of VASP within 1 min, and VASP remained phosphorylated throughout the time course. The observed phosphorylation was not attributable to changes in temperature from 4°C to 37°C, as neutrophil monolayers express VASP as a dephosphorylated protein at both temperatures, and VASP is expressed predominantly as a dephosphorylated protein in neutrophil suspensions at 37°C (Fig. 2Go). Thus, VASP is in its dephosphorylated state in round retracted cells and is phosphorylated at the time of neutrophil membrane extension during spreading.



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  		FIGURE 4. VASP phosphorylation in neutrophils induced to spread. Neutrophil monolayers held at 37°C (lane 1) were incubated at 4°C to induce retraction (lane 2). Retracted monolayers then were warmed to 37°C for 1 min (lane 3), 3 min (lane 4), 5 min (lane 5), and 10 min (lane 6). Equal amounts of protein at each time point were analyzed for the VASP Ag by Western blot analysis. Densitometric measurements of the 50-kDa and 46-kDa bands were performed and phosphorylation of VASP was expressed as the 50 kDa/46 kDa ratio for each condition. VASP was phosphorylated in response to the initiation of cell spreading (lane 3). These data are from a representative experiment repeated at least three times.

 
Localization of VASP

VASP was localized by immunofluorescence microscopy in control neutrophil monolayers, retracted cells, and in cells induced to spread (Fig. 5Go), as shown in Fig. 3Go. In control neutrophil monolayers, VASP was localized in punctate structures at a plane below the nucleus, apparently at sites of contact with the substratum. Retracted neutrophils were round, and VASP was localized predominantly at the cell cortex. The punctate staining pattern observed in well-spread neutrophils was not evident in retracted cells. When neutrophils were induced to spread at 37°C for 1 min, there was a redistribution of VASP from the cell cortex to foci at the ventral surface. After 3 min, neutrophils were well spread, and VASP remained localized within focal adhesions and showed no further changes in localization.



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  		FIGURE 5. Localization of VASP in spreading neutrophils by immunofluorescence microscopy. Adherent monolayers (A) were retracted at 4°C (B) and then warmed at 37°C for 1 min (C) or 3 min (D) to induce cell spreading as demonstrated in Fig. 3Go. VASP was localized at the membrane in retracted cells (B). At the onset of cell spreading, VASP redistributed rapidly to focal adhesion complexes at a plane below the nucleus. Original magnification, x125.

 
VASP is believed to play an important role in controlling the cytoskeletal organization because it binds F-actin and profilin (2, 11). To determine whether VASP was associated with F-actin, well-spread cells were stained simultaneously for F-actin and VASP. An overlay of the immunofluorescence images revealed that VASP was colocalized with F-actin in filopodia and in focal adhesions in the cell body at a plane below the nucleus (Fig. 6Go). No staining was observed in cells stained with secondary Ab alone (not shown). These results suggest that similar to platelets, VASP is associated in neutrophils with actin filaments and focal adhesion complexes (1). The phosphorylation of VASP at the time when VASP was redistributed from the cell cortex to focal adhesions and filopodia suggests that VASP is involved in regulating areas of the cell involved in focal adhesion complex organization and actin dynamics during cell spreading.



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  		FIGURE 6. Localization of VASP and F-actin in adherent neutrophils by immunofluorescence microscopy. Neutrophil monolayers were stained simultaneously for VASP (A) and F-actin (B). An overlay of both images (C) indicated that VASP was colocalized (designated by yellow) with F-actin at the tips of filopodia and within some focal adhesion complexes. Original magnification, x125.

 
VASP is phosphorylated by cGK during neutrophil adherence and spreading

VASP is phosphorylated by both cGK and cAK in cells (1, 30). To identify the kinase responsible for phosphorylation of VASP during neutrophil adhesion and spreading, cell suspensions were preincubated with Rp-8-pCPT-cGMPS (cGK inhibitor) or KT5720 (cAK inhibitor). In untreated neutrophil suspensions VASP was predominantly expressed as a dephosphorylated protein, although some basal levels of VASP phosphorylation were evident (Fig. 7Go). VASP became phosphorylated at elevated levels after neutrophils were allowed to adhere and spread for 3 min, compared with control cells held in suspension. Treatment with Rp-8-pCPT-cGMPS reduced basal levels of VASP phosphorylation and inhibited adhesion-induced VASP phosphorylation 5-fold. KT5720, the cAK inhibitor, did not inhibit the increase in VASP phosphorylation. These results suggest that cGK phosphorylates VASP during neutrophil adhesion and spreading.



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  		FIGURE 7. Effect of cGK and cAK inhibitors on VASP phosphorylation. Neutrophil suspensions were preincubated in the absence (lanes 1 and 2) or presence of Rp-8-pCPT-cGMPS (lanes 3 and 4) or KT 5720 (lanes 5 and 6). An aliquot of each cell suspension was adhered to serum-coated dishes for 3 min at 37°C (lanes 2, 4, and 6). The remainder of the neutrophil suspension was quickly pelleted at room temperature and lysed in SDS lysis buffer. Equal amounts of protein at each time point were analyzed for the VASP Ag by Western blot analysis. Densitometric measurements of the 50-kDa and 46-kDa bands were performed and phosphorylation of VASP was expressed as the 50 kDa/46 kDa ratio for each condition. Rp-8-pCPT-cGMPS inhibited adhesion-induced VASP phosphorylation (lane 4). These data are from a representative experiment repeated at least twice.

 
cGK signaling is required for neutrophil spreading

To determine whether phosphorylation of VASP by cGK is required for neutrophil spreading, morphometric measurements were performed on neutrophils during attachment and spreading in the presence of Rp-8-pCPT-cGMPS. For these experiments, the degree of cell spreading in cell populations (n = 250) was determined based on the degree of circularity of the cell, with circularity values of 1 indicating a perfect circle. Untreated control cells had a circularity value of 0.87 ± 0.08 (Fig. 8Go). At 1 min, adherent control neutrophils were round and had a circularity value similar to cells in suspension. After 3 min, there was a decrease in circularity to 0.36 ± 0.04 as neutrophils spread and extended lamellipodia. Neutrophils maintained this degree of circularity for the remaining time points tested. In contrast to control cells, the changes in neutrophil morphology during spreading from 3 to 10 min were inhibited by Rp-8-pCPT-cGMPs and not by the cAK inhibitor KT5720 (Fig. 8Go). Thus, cGK is required for neutrophil spreading.



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  		FIGURE 8. Effect of cGK and cAK inhibitors on neutrophil morphology. Neutrophil suspensions were preincubated with Rp-8-pCPT-cGMPS ({triangleup}), KT5720 ({blacksquare}), or tissue culture medium alone (•) for 30 min at 37°C. Cells then were adhered from 1 to 10 min at 37°C, and the circularity was measured by using digitized photomicrographs. Rp-8-pCPT-cGMPS inhibited shape changes during spreading (*, p <= 0.001). No changes in neutrophil shape were observed with KT5720. Data are circularity ± SD for one of two representative experiments.

 
It is well established that cGK is inactive until activated by cGMP (31). To determine whether elevating intracellular levels of cGMP affected neutrophil morphology, neutrophils were preincubated for 5 min with the membrane-permeable analog 8-Br-cGMP, a direct activator of cGK. Membrane extension and ruffling were induced in neutrophil suspensions preincubated with 8-Br-cGMP. A change in cell shape was evident by a decrease in circularity from 0.87 ± 0.08 (control cells) to 0.67 ± 0.10 (8-Br-cGMP-treated cells; Fig. 9Go). Furthermore, 8-Br-cGMP caused neutrophils to change from a round to a polarized morphology at an earlier time during adhesion (1 min vs 3 min). The circularity value of cells treated with 8-Br-cGMP remained equivalent to control cells for the remainder of the time course.



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  		FIGURE 9. The effect of 8-Br-cGMP on neutrophil morphology during adherence. Neutrophils were preincubated for 5 min in the absence ({blacksquare}) or presence ({triangleup}) of 1 µM 8-Br-cGMP, and adhered from 1 to 10 min. Circularity was measured by using digitized negative images. Preincubation of neutrophils in suspension with 8-Br-cGMP induced cell polarization and significantly accelerated changes in cell shape during adhesion (p <= 0.001 at 1 min). Data are circularity ± SD for one of two representative experiments.

 
Our observations that cGK inhibitors suppress cell spreading and that cGMP analogs enhance cell spreading strongly suggest that cGMP/cGK signaling is required for neutrophil spreading.

Adhesion and spreading elevate cGMP levels in neutrophils

An increase in cGMP levels in neutrophils during adhesion and spreading would provide further evidence that this process is regulated by a cGMP/cGK signaling pathway. Therefore, intracellular levels of cGMP were measured in neutrophils adhered for various times as described above. A dramatic increase (180%) in cGMP levels was observed within 1 min of adherence (Fig. 10Go). This increase was temporal, as cGMP levels decreased within 3 min and remained at those levels for the remainder of the time course. However, even from 3 to 10 min, cGMP levels were significantly elevated when compared with cells held in suspension (p <= 0.001).



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  		FIGURE 10. Levels of cGMP in neutrophils during adherence. Cyclic GMP levels were measured in neutrophils adhered to serum-coated plates for various times. Adhesion stimulated a transient 180% increase in cGMP levels within 1 min. Levels of cGMP remained significantly elevated over that of cells held in suspension for the remainder of the time course (p <= 0.001). Data are fM cGMP/mg protein ± SD of triplicate determinations for one of three representative experiments.

 
Spreading also induced elevations of cGMP in neutrophils. A significant increase in cGMP levels (72% increase) was observed when retracted cells were allowed to spread at 37°C for 1 min (Fig. 11Go). This elevation of cGMP began during the time that cells extended their lamellipodia (Fig. 3Go). The levels of cGMP in adherent neutrophils induced to spread were not as high as when cells adhered and spread simultaneously.



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  		FIGURE 11. Levels of cGMP in neutrophils induced to spread. Cyclic GMP levels were measured in retracted neutrophils and in neutrophils induced to spread as described in Figs. 3Go and 4Go. Retracted adherent cells are designated as time 0. The induction of cell spreading significantly increased cGMP levels within 1 min when compared with retracted cells (*, p <= 0.009). Data are fM cGMP/mg protein ± SD of triplicate determinations for one of three representative experiments.

 
These studies demonstrate that the second messenger, cGMP, is significantly elevated during neutrophil adhesion and spreading. It is important to note that others reported that cAMP levels decrease in neutrophils during adherence, and that low levels of cAMP were required to achieve neutrophil spreading (23). The observations that VASP phosphorylation and neutrophil spreading are impaired by cGK inhibitors and not cAK inhibitors strongly suggest that cGK is activated by cGMP during neutrophil adhesion and spreading.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expression and phosphorylation state of VASP was investigated in neutrophils during cell adherence. Adhesion is an essential process for neutrophil migration from the peripheral blood to sites of inflammation. During the process of adhesion, neutrophils adhere and spread without any clear stopping point between these two processes. Therefore, it was important to determine whether VASP was phosphorylated in response to signals involved in adhesion and/or spreading. In this report, we demonstrate that VASP is a target for cGK regulation of neutrophil spreading. We showed that VASP was in its dephosphorylated form in retracted round neutrophils and was rapidly phosphorylated by cGK at the onset of cell spreading. Both adherence and the onset of cell spreading induced significant elevations of cGMP in neutrophils. When neutrophils were incubated with 8-Br-cGMP, a direct activator of cGK, cells became more polarized in suspension, and spread more rapidly during adhesion. Our observations that adhesion and spreading induced significant cGMP elevations in neutrophils and that cell spreading was impaired by Rp-8-pCPT-cGMPS (cGK inhibitor) support the premise that cGK is a major mediator in regulating neutrophil spreading and that VASP is a target for cGK regulation.

VASP is a substrate in vitro and in vivo for cGK and cAK (32). Evidence that VASP was phosphorylated by cGK in neutrophils during cell spreading was that Rp-8-pCPT-cGMPS and not KT5720 (cAK inhibitor) inhibited VASP phosphorylation and that adhesion and the onset of spreading dramatically induced elevations of cGMP. Furthermore, it is unlikely that cAK was activated, as others report that low levels of cAMP were required to mediate neutrophil spreading and that activation of neutrophils with cAMP elevating agents promoted cell retraction (23). However, phosphorylation of VASP may be only one of the effects of cGK on the neutrophil cytoskeleton. Presently, vimentin is the only reported cytoskeletal protein that is phosphorylated by cGK in neutrophils (33). Therefore, the identification of other targets for cGK regulation in neutrophils during adhesion and spreading necessitates further investigation.

Although this is the first report to demonstrate that adherence elevates cGMP in neutrophils, adhesion-induced modulation of cyclic nucleotide levels has been reported in platelets. Integrin-dependent adhesion of platelets induced an increase in cGMP and a simultaneous decrease in cAMP levels (34, 35). The high levels of cGMP in neutrophils observed in this report and low levels of cAMP observed by others (23) suggest a mechanism similar to platelets for cyclic nucleotide regulation of neutrophil adhesion and spreading. Furthermore, the phosphorylation of VASP by cGK in both neutrophils and platelets (13, 15), and similar localization of VASP to focal adhesions and filopodia during spreading (1) suggest that VASP is a target for cGK regulation of adhesion and spreading in both neutrophils and platelets.

During neutrophil spreading, VASP rapidly redistributed from the cell margin to the tips of filopodia and focal adhesion complexes containing F-actin. Neutrophil spreading and VASP phosphorylation were dependent on cGK activation, as the cGK inhibitor Rp-8-pCPT-cGMPS impaired neutrophil spreading and inhibited VASP phosphorylation. Neutrophil spreading is a dynamic process that involves actin polymerization/depolymerization and focal adhesion assembly and disassembly. These results suggest that cGK plays a role in regulating regions of the cell where actin filaments are highly dynamic and support recent studies suggesting that the subcellular positioning of VASP is important when the actin cytoskeleton is reorganized during cell spreading (8).

The specific regulatory role of VASP phosphorylation is as yet uncertain. In Listeria monocytogenes, a model for actin-based motility, the phosphorylation of VASP by cAK on serine 157 increases its affinity for F-actin 40-fold. The phosphorylation state of VASP was proposed to transform actin polymerization into active movement based on the frequent attachment/detachment of VASP to F-actin (11). In mammalian cells, proteins of the Ena/VASP family are localized at focal contacts and in regions where actin filaments are highly dynamic (4). These proteins interact with the focal adhesion proteins zyxin and vinculin, and the actin-binding protein profilin (1, 2, 4, 5, 9, 36, 37). In a recent report, VASP phosphorylation by cAK was shown to have a negative effect on actin dynamics but not on its interaction with profilin, vinculin, and zyxin (38). There is evidence that cGMP regulates actin polymerization in neutrophils. F-actin content is increased by elevating cGMP levels in neutrophils (19), and cGK activation was reported to enhance neutrophil motility (chemotaxis) and granule secretion (27, 39, 40). Interestingly, cAMP/cAK signaling has a countereffect on the neutrophil cytoskeleton and is reported to inhibit actin polymerization, cell morphology, cell spreading, adhesion, migration, and neutrophil functions (20, 21, 22, 23, 24, 25, 41, 42, 43, 44). Neutrophils are highly motile and are dependent on an actin-based motility to carry out their functions. The transient phosphorylation of VASP by cGK in neutrophils may provide a mechanism to spatially regulate actin filament polymerization during adhesion and spreading.

There is evidence that cyclic nucleotide-mediated phosphorylation of VASP regulates cell-matrix (focal adhesions) and cell-cell contacts. Platelets contain significant concentrations of VASP, and the phosphorylation of VASP by cAK and cGK correlates with an inhibition of platelet aggregation (13, 45). Mice deficient in VASP or cGK-1 demonstrate defects in cyclic nucleotide-mediated platelet aggregation (13, 15) and inhibition of fibrinogen receptor activation (46). Studies with VASP-deficient platelets suggest that VASP is a negative modulator of platelet and integrin {alpha}IIb{beta}3 activation (13, 14). In cGK-1-deficient platelets, there is an increase in platelet adhesion and aggregation during ischemia/reperfusion injury, suggesting that cGK-1 is required for platelet disaggregation (15). In cultured endothelial cells, VASP is concentrated with microfilaments at cell-cell contacts and at sites of focal contacts and is phosphorylated by cGK in response to cGMP-elevating agents (17). Murphy-Ullrich et al. (16) reported that cGK is required for focal adhesion disassembly of cultured smooth muscle cells and endothelial cells. Phosphorylation of VASP by cGK-1 in endothelial cells transfected with cGK-1{beta} was shown to promote detachment of VASP and zyxin from focal adhesions and to inhibit haptotactic migration (10). Our studies extend to neutrophils the concept that VASP is a target for cGK regulation of neutrophil focal adhesion as well. Future investigations are required to determine whether, similar to platelets and endothelial cells, cGK promotes focal adhesion disassembly in neutrophils.


   Acknowledgments
 
We thank Dr. Patricia Maness (University of North Carolina, Chapel Hill, NC) for her helpful suggestions and comments on the manuscript, and Elizabeth Merricks and Susan Jones for their excellent technical assistance.


   Footnotes
 
1 This work was supported by National Science Foundation Grant MCB-9421731. It was conducted in partial fulfillment of a doctoral dissertation by D.W.L., who was partially supported by National Institutes of Health Research Education Support Grant GM55336-02 and National Institutes of Health Predoctoral Training Grant ES07017. Back

2 Current address: Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Room 704, Thorn Building, 20 Shattuck Street, Boston, MA 02115. Back

3 Address correspondence and reprint requests to Dr. Katherine Pryzwansky, Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599-7525. Back

4 Abbreviations used in this paper: VASP, vasodilator-stimulated phosphoprotein; F-actin, filamentous actin; cAK, cyclic AMP-dependent protein kinase; cGK, cyclic GMP-dependent protein kinase; FAK, focal adhesion kinase; GBSS, Gey’s balanced salt solution. Back

Received for publication November 29, 2000. Accepted for publication February 16, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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