Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-alpha

Ruihua Chen, Baochong Gao, Cancan Huang, Byron Olsen, Robert F. Rotundo, Frank Blumenstock, and Thomas M. Saba

Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-alpha causes a rearrangement of the fibrillar fibronectin (FN) extracellular matrix and an increase in protein permeability. Using calf pulmonary artery endothelial cell layers, we determined whether these changes were mediated by FN multimerization due to enhanced transglutaminase activity after TNF-alpha (200 U/ml) for 18 h. Western blot analysis indicated that TNF-alpha decreased the amount of monomeric FN detected under reducing conditions. Analysis of 125I-FN incorporation into the extracellular matrix confirmed a twofold increase in high molecular mass (HMW) FN multimers stable under reducing conditions (P < 0.05). Enhanced formation of such HMW FN multimers was associated with increased cell surface transglutaminase activity (P < 0.05). Calf pulmonary artery endothelial cells pretreated with TNF-alpha also formed nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF-alpha -induced formation of nonreducible HMW multimers of FN but did not prevent either disruption of the FN matrix or the increase in monolayer permeability. Thus increased cell surface transglutaminase after TNF-alpha exposure initiates the enhanced formation of nonreducible HMW FN multimers but did not cause either the disruption of the FN matrix or the increase in endothelial monolayer permeability.

tumor necrosis factor-alpha ; endothelial cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR (TNF)-alpha is a major inflammatory cytokine released from macrophages and monocytes activated during inflammation and/or gram-negative infection, especially after trauma, burns, and major surgery (10, 49). The endothelium is one of the primary targets for such inflammatory cytokines (31), and high levels of TNF-alpha are believed to contribute to altered integrity of the lung endothelial barrier, resulting in excessive transendothelial leakage of plasma proteins into the interstitium and interstitial pulmonary edema (16, 17, 46, 47). Purified TNF-alpha added to the culture medium of confluent calf pulmonary artery endothelial (CPAE) monolayers increases their protein permeability (6, 50, 51); and the intravenous infusion of TNF-alpha into postsurgical sheep can also increase lung endothelial protein permeability (39), very similar to that observed in postsurgical sheep after Pseudomonas or endotoxin challenge (7, 8, 11, 14). Such observations have suggested that high concentrations of TNF-alpha in the lung microcirculation during pulmonary inflammation and/or bacterial sepsis after surgery or trauma may contribute to the loss of lung endothelial integrity (42).

Studies (16, 50) on the TNF-alpha -induced increase in transendothelial protein permeability with immunofluorescence microscopy have confirmed a dramatic disruption and/or reorganization of the fine fibrillar fibronectin (FN) network in the subendothelial extracellular matrix (ECM) of lung endothelial cell monolayers exposed to TNF-alpha . This includes an apparent aggregation of FN in the matrix temporally associated with both an increase in protein permeability and the formation of gaps between cells in previously confluent monolayers (16, 17, 51). A very similar disruption of the FN matrix is seen after the addition of either monoclonal antibodies to the alpha 5beta 1-integrin complex or soluble Arg-Gly-Asp (RGD)-containing peptides to the culture medium (16, 17).

Addition of endotoxin-free and highly purified human plasma FN to the culture medium can both attenuate and reverse the TNF-alpha -induced increase in endothelial monolayer protein permeability, a protective response that appears to be dependent on the incorporation of the added soluble FN into the ECM (16, 17, 51). Similarly, the intravenous infusion of such commercially purified human FN into bacteremic sheep after surgical trauma to significantly elevate the circulating plasma FN concentration (by 20-40%) can also attenuate the increase in lung protein permeability once the infused human FN becomes incorporated into the lung interstitial ECM (11, 14, 40). Although disruption of the FN subendothelial matrix after TNF-alpha exposure is now well documented, the mechanisms mediating this change in the fibrillar FN matrix are not known. However, an alteration of the FN matrix has the potential to either reduce endothelial cell adhesion or spreading, interrupt integrin-mediated ECM signaling to the cell cytoskeleton, or perhaps modify the integrity of the vascular barrier, including the exclusion properties of the matrix.

The organization of FN in the ECM of lung endothelial cell layers is influenced by several processes that include its assembly into fine FN fibrils within the ECM, its turnover potentially mediated by proteolysis, and the biochemical structuring of the FN lattice within the ECM. Structuring of FN in the normal ECM is influenced by disulfide exchange in the amino-terminal domain of FN and perhaps by transglutaminase-mediated cross-linking of FN based on experiments primarily with fibroblasts (3, 4). We determined whether TNF-alpha could induce changes in the properties of FN within the ECM to explain its influence on both the fine fibrillar structure of the FN matrix and the protein permeability of previously confluent endothelial monolayers. In this regard, we tested the concept that TNF-alpha may have enhanced extracellular transglutaminase activity, resulting in FN multimerization leading to disruption of the FN matrix and increased protein permeability of endothelial monolayers. Our observations indicate that the total amount of FN in the matrix as well as the FN mRNA levels were unchanged after TNF-alpha exposure. Moreover, we were unable to detect the release of FN fragments from the cell layer after TNF-alpha exposure. In contrast, there was a marked increase in the amount of nonreducible high molecular mass (HMW) multimers of FN detected in the ECM of the lung endothelial cell monolayers after TNF-alpha exposure that was associated with enhanced extracellular endothelial cell surface transglutaminase activity. Blocking such enhanced transglutaminase activity attenuated the TNF-alpha -enhanced multimerization of the FN matrix in endothelial monolayers but did not prevent either the rearrangement of the fibrillar FN matrix as analyzed by immunofluorescence microscopy or the increase in protein permeability as quantified by transendothelial 125I-albumin clearance.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fibroblast and endothelial cell cultures. CPAE cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS; HyClone, Logan, UT), 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Before TNF-alpha treatment, the culture medium was replaced with DMEM supplemented with 5% FBS plus the antibiotics. For analysis of the effect on TNF-alpha on the release of FN from the endothelial cell layers, the CPAE monolayers were cultured to confluence in 3-4 days and then washed four times with phosphate-buffered saline (PBS) before the addition of DMEM containing FN-deficient FBS prepared by gelatin-Sepharose affinity chromatography (20). A1-F human foreskin fibroblasts (originally obtained from Dr. Lynn Allen-Hoffman, University of Wisconsin, Madison) were cultured in DMEM supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Both fibroblast and endothelial cell layers were exposed to recombinant human TNF-alpha at a concentration of 200 U/ml medium for 18 h before analysis.

Phase-contrast and immunofluorescent microscopy. Endothelial cells cultured on coverslips were treated with and without 200 U/ml of TNF-alpha for 18 h. The cells were examined and photographed with an inverted microscope (Olympus IX50). Immunofluorescent staining for FN in the cell layers was performed as described by Curtis and colleagues (16, 17) with an Olympus BX60 microscope. CPAE cells cultured on coverslips were fixed, permeabilized, and stained with a rabbit polyclonal antibody to bovine FN (Chemicon International, Temecula, CA) and a fluorescein goat anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR).

Enzyme-linked immunosorbent assay and immunoblot analysis. Standard enzyme-linked immunosorbent assay (ELISA), dot blot, and Western blot analysis of FN were done with a rabbit polyclonal antibody to bovine FN (Calbiochem, San Diego, CA) and a secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (Calbiochem). For ELISA, FN was detected with the 3,3',5,5'-tetramethylbenzidine microwell peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Dot blot and Western blot analyses were performed with a SuperSignal ULTRA chemiluminescent substrate (Pierce, Rockford, IL) with subsequent exposure to Fuji X-ray films (Fuji Medical Systems USA, Stamford, CT).

RNA isolation and Northern blot analysis. Poly(A)+ RNA was isolated directly from cell extracts of CPAE cells with the FastTrack kit (Invitrogen, San Diego, CA). RNA samples (1 µg) were electrophoresed on a 1.3% agarose gel containing 0.66 M formaldehyde and transferred to nitrocellulose as described by Davis et al. (18). DNA probes were radiolabeled with [alpha -32P]dCTP with the Random Primer Plus Extension Labeling System (New Life Science Products, Boston, MA), and hybridization was performed as previously described (19). Equal loading of mRNA samples on Northern blots was verified by simultaneous probing with a cDNA specific to mouse gamma -actin.

Detection of HMW multimers of 125I-FN incorporated in the matrix of endothelial cells. FN purified from fresh human plasma by gelatin-Sepharose affinity chromatography (20, 38) was iodinated as described by Rebres et al. (38). The iodinated FN was confirmed to be intact (440 kDa) by SDS-PAGE and autoradiography. At 48 h after CPAE cells were seeded at a density of 10,000 cells/cm2, the medium of the preconfluent cells was changed to FN-deficient medium supplemented with 3 µg/ml of 125I-FN. After an 18-h incubation with and without TNF-alpha , the cells were washed four times with PBS and solubilized in SDS-PAGE sample buffer [0.125 M Tris (pH 6.8), 4% SDS, and 10% glycerol]. Samples with equal radioactivity were analyzed by SDS-PAGE and autoradiography. Polyacrylamide gradient gels (4-15%) with a 3% stacking gel were used for PAGE analysis. When analyzed under reducing conditions, the concentration of beta -mercaptoethanol in the sample buffer was 2.5% (vol/vol). The gels were then dried and exposed to Kodak Bio-Max films with an intensifying screen. When the effect of transglutaminase inhibitors on the formation of nonreducible HMW FN was examined, monodansylcadaverine (MDC; Sigma) at 0.1 mM or cystamine (Cys; Sigma) was added during TNF-alpha treatment. Both MDC and Cys are primary amine substrates that have been routinely used to inhibit transglutaminase activity (24, 41, 44).

Assay of transglutaminase activity in cell lysate. Transglutaminase activity was determined on the basis of [3H]putrescine incorporation into dimethylated casein as previously described by Korner et al. (29). Cells were washed twice with cold PBS, once with Tris-EDTA-dithiothreitol (TED) buffer (50 mM Tris · HCl, pH 7.4, 1 mM EDTA, and 1 mM dithiothreitol) supplemented with 150 mM NaCl (TED-buffered saline), and then scraped into 1 ml TED-buffered saline/dish. The cells were collected by centrifugation; resuspended in 200 µl of TED buffer containing 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml of pepstatin, and 5 µg/ml of leupeptin; and then lysed by three cycles of freezing and thawing. The final reaction mixture (120 µl) consisted of 50 mM Tris · HCl, pH 7.4, 10 mM CaCl2, 15 mM dithiothreitol, 0.5 mM (1 µCi) putrescine, 50 mM NaCl, and 0.8 mg of dimethylated casein, and the cell lysates that contained 80-130 µg of total protein. After incubation at 37°C for 9-15 min, the proteins were precipitated with TCA, and free putrescine was removed by passage through a filter paper as described by Korner et al. Each sample had a negative control in which CaCl2 in the reaction mixture was replaced with 50 mM EDTA. Putrescine incorporation, used as a measure of transglutaminase activity (29), was quantified by scintillation counting.

Assay of cell surface transglutaminase activity. Cell surface transglutaminase activity was specifically determined by measuring the incorporation of biotinylated cadaverine (Pierce) into FN on precoated 96-well plates as described by Verderio et al. (48). Briefly, the plates were first coated with purified human FN at 10 µg/ml. Then endothelial cells that were either not exposed to TNF-alpha (control) or pretreated with TNF-alpha (200 U/ml) were seeded at various densities from 10,000 to 50,000 cells/well and incubated at 37°C for 3 h in the presence of 0.5 mM biotinylated cadaverine. The reaction was first stopped by rinsing the wells with 2 mM EDTA in PBS, and then the cell layers were removed by incubation for 20 min with 0.1% sodium deoxycholate in PBS at room temperature. Cadaverine incorporation was quantified with horseradish peroxidase-conjugated streptavidin (Amersham, Piscataway, NJ) and the 3,3',5,5'-tetramethylbenzidine color-developing kit (Kirkegaard & Perry Laboratories).

Preparation of preformed endothelial cell matrices and assay of cell adhesion and spreading. Confluent endothelial monolayers cultured on glass coverslips in 12-well plates were treated for 18 h with either TNF-alpha (200 U/ml) alone or TNF-alpha in conjunction with the transglutaminase inhibitor Cys (0.05 mM) to obtain two preformed matrices: one in which the fine fibrillar FN matrix was disrupted or reorganized with the parallel formation of the HMW FN multimers inhibited (TNF-alpha and cystamine treated), and the other in which the FN matrix was disrupted in parallel with the TNF-alpha -induced formation of nonreducible HMW FN multimers (TNF-alpha treated).

To determine cell adhesion and spreading on the preformed matrices, the cell layers were first removed by a 10-min rinse in PBS containing 0.5% sodium deoxycholate at 4°C. The coverslips were examined with an inverted microscope to ensure complete removal of the cell layers. Then, normal CPAE cells were suspended by trypsinization and seeded onto two different types of preformed matrices at 105 cells/well. The plates were incubated at 37°C for 30 min, and nonadherent cells were removed by gentle rinsing of the wells three times with PBS. The coverslips were then processed for immunofluorescence microscopy. The matrices and adhered cells were stained with both an antibody against FN and an antibody against the alpha 5beta 1-integrin (Chemicon International) as well as the required secondary antibodies (Molecular Probes). Both adherent and spread CPAE cells were quantified by analysis of 12 random areas with an immunofluorescence microscope.

Radiolabeling of albumin and assay of transendothelial protein permeability. Transendothelial protein permeability was assessed by measuring the diffusive protein (125I-albumin) permeability across the endothelial monolayer with a dual-chamber monolayer system as previously described (15-17, 50, 51). This technique allows for the measurement of transendothelial albumin flux in the absence of a hydrostatic or oncotic pressure gradient (15). This in vitro model system consists of a luminal chamber containing the "tissue culture ready" filter that is covered with a confluent monolayer of CPAE cells. A styrofoam collar around the luminal chamber allows it to float in a larger abluminal chamber. This allows the fluid height in both chambers to be maintained at an identical level to eliminate the convective flux of albumin across the monolayers. During each experiment, the fluid in the abluminal chamber was stirred constantly, and both chambers were kept at 37°C with a thermostatically controlled water bath.

Bovine albumin (Sigma) was iodinated with Na125I with the chloramine T method (50, 51). Five millicuries of 125I were combined with 100 mg of albumin. 125I-albumin was maintained in dialysis against PBS at pH 7.4 until used. The ratio of free to protein-bound 125I was periodically analyzed to ensure that the preparations of albumin used had <0.5% free 125I.

Calculation of 125I -albumin clearance (in µl/min) was used to measure the changes in the diffusive permeability of albumin across the lung endothelial monolayers as described by Cooper et al. (15). To conduct this procedure, 25 ml of low-glucose DMEM supplemented with 0.5% BSA were added to the abluminal chamber. Medium from the luminal chamber was first changed after exposure of the endothelial monolayer to TNF-alpha (200 U/ml), and 200 µl of the same BSA-containing DMEM solution supplemented with the 125I-albumin tracer were added to the luminal chamber. For measurement of albumin clearance (16, 17, 50, 51), we collected 400-µl aliquots of the labeled medium from the abluminal chamber every 5 min over a 60-min interval and assayed these serial aliquots for 125I radioactivity with a TM Analytic 1193 gamma counter. The clearance, expressed in microliters per minute, was determined by weighted least squares nonlinear regression for all 5-min experimental time intervals over the 5- to 60-min test period.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TNF-alpha causes a decrease in monomeric FN detectable under reducing conditions. Previous immunofluorescent studies (16, 50) revealed a dramatic reorganization of the FN matrix under the endothelial cell monolayers after TNF-alpha exposure. The matrix FN changed from a fibrillar meshwork of fine FN fibers into a matrix with predominantly thick bundles of FN after TNF-alpha exposure. To determine whether this reorganization of the FN matrix was related to a change in the quality and/or quantity of FN in the matrix after TNF-alpha exposure, we analyzed the FN in cell lysates by Western blotting under reducing conditions. Unexpectedly, with limited film exposure (Fig. 1A), we observed that the lysate of TNF-alpha -treated endothelial cells contained almost no monomeric FN detectable at 220 kDa. More extensive exposure (Fig. 1B) revealed the presence of monomeric FN that was still much less after TNF-alpha exposure than in control cells. We initially speculated that the reduction in FN monomers could be due to a drastic detachment of cells, a pronounced increase in FN degradation, and/or a decrease in FN synthesis. We therefore examined the endothelial cell monolayers by both phase-contrast and immunofluorescent microscopy using an antibody to FN. Figure 2 shows that after an 18-h exposure to TNF-alpha (200 U/ml), the endothelial cell layer was still intact, although intercellular gaps in the previously confluent monolayer became very noticeable (compare Fig. 2, A and B), and the fine FN subendothelial matrix was disrupted, with thick bundles of FN apparent (Fig. 2, C and D). Thus the negligible detection of monomeric FN under reducing conditions was not due to the loss of CPAE cells from the monolayer after TNF-alpha exposure.


View larger version (102K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of tumor necrosis factor (TNF)-alpha on the amount of monomeric fibronectin (FN) detected under reducing conditions by Western blot analysis. Confluent calf pulmonary artery endothelial (CPAE) cells were incubated with and without 200 U/ml of TNF-alpha for 18 h (n = 4 wells/group). Cells were washed and scraped into SDS-PAGE sample buffer. Aliquots of cell lysates containing equal amounts of total protein were analyzed for FN by Western blotting under reducing conditions with a polyclonal antibody to bovine FN. Purified plasma FN standard (Std) was used to indicate the position of the well-characterized 220-kDa monomer of FN. A: limited exposure of the film for development. B: exposure for a more extensive interval.



View larger version (172K):
[in this window]
[in a new window]
 
Fig. 2.   Endothelial cell monolayers were still visually intact and matrix FN was mostly in the form of thick bundles after TNF-alpha exposure. Confluent endothelial cells were treated with (B and D) and without (A and C) 200 U/ml of TNF-alpha for 18 h. Cells were either photographed under an inverted microscope (A and B) or fixed, permeabilized, and stained for FN (C and D). TNF-alpha caused the formation of gaps between endothelial cells (B) and the loss of the fine fibrillar FN matrix (D) but did not elicit significant cell loss from the monolayer.

We then determined whether TNF-alpha had caused an increase in FN proteolytic activity by studying the release of either intact dimeric FN (440 kDa) or fragments of FN into the medium with an ELISA method with cells cultured in a FN-deficient medium. As shown in Fig. 3A, after incubation with TNF-alpha for 3, 6, or 18 h, the FN content in the medium of the lung endothelial monolayer had increased in a pattern essentially identical to that seen in control cells not exposed to TNF-alpha . The slightly higher level seen in the TNF-alpha -treated group at 18 h was not significant (P > 0.05). The five- to sixfold increase in medium concentration of FN over 18 h is consistent with a previous study (42) on the release of FN by endothelial cells in culture. In addition, we detected no significant fragmentation of the FN released into the medium after TNF-alpha exposure as studied by Western blot analysis under reducing conditions with a polyclonal antibody to bovine FN (Fig. 3B), a finding in agreement with our recent documentation of the lack of proteolytic fragmentation of FN in either the medium or endothelial cell layer after TNF-alpha exposure (16).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   TNF-alpha did not change either the FN content or FN mRNA levels in CPAE cell layers. Confluent CPAE cells were incubated in FN-deficient medium with and without (control) 200 U/ml of TNF-alpha for 18 h. A: at 3-, 6-, or 18-h time points, 15 µl of the culture medium were collected and assayed for FN content by ELISA with a polyclonal antibody against bovine FN. n, No. of samples. B: reduced samples of the culture medium harvested from both control (CON) and TNF-alpha -treated cells (done in duplicate) were analyzed by Western blot with a polyclonal antibody against bovine FN. C: confluent CPAE monolayers (n = 4) that had been incubated with and without TNF-alpha were washed and scraped into SDS-PAGE sample buffer, and cell lysates (containing an equal amount of total protein) were assayed for FN content by dot blot analysis under reducing conditions. D: CPAE monolayers were assayed at 18 h for mRNA content by Northern blot with a rat FN cDNA probe, with actin mRNA used as a loading control.

Because the total FN in the cell layers could not be carefully quantified by Western blot (Fig. 1), we then used dot blot analysis to assess the amount of FN in the cell lysates. As shown (Fig. 3C), the FN content in the endothelial cell lysate (which includes FN in the CPAE cells and the matrix) did not change in any consistent manner after exposure to TNF-alpha . To determine whether TNF-alpha may have caused a decrease in FN synthesis, we quantified the FN mRNA in both control and TNF-alpha -treated endothelial cells and observed that the FN mRNA level was also not significantly changed after 18 h of TNF-alpha treatment (Fig. 3D). Collectively, all these observations suggested that our finding of minimal amounts of monomeric FN under reduced conditions in CPAE cell layers treated with TNF-alpha (Fig. 1) was not caused by loss of CPAE cells, increased FN proteolysis, or decreased FN synthesis.

TNF-alpha caused an increase in nonreducible HMW FN multimers in endothelial cell layers. Because the total FN content in the cell lysate was also unchanged after TNF-alpha (Fig. 3C), we hypothesized that the reduced monomeric FN in TNF-alpha -treated cells could mean that much of the FN may have existed as nonreducible HMW complexes, which were perhaps not transferred onto the nitrocellulose membrane and therefore not detectable by subsequent antibody probing.

To test this possibility, we again analyzed the cell lysate by Western blot coupled with densitometric scanning, but in this experiment, we used Pronase to aid in the transfer of HMW multimeric FN onto the nitrocellulose membrane. With the use of Pronase, we observed a fivefold increase in the percentage of multimeric FN transferred onto the nitrocellulose membrane from the cell lysates of TNF-alpha -treated cells compared with those from control cells (data not shown). It is difficult to accurately quantify the percentage of HMW FN with the Pronase-aided Western blotting because the results could vary with the level of proteolysis; i.e., higher levels of proteolysis will cause the loss of protein during the transfer, especially of monomeric FN, whereas lower levels of proteolysis will result in incomplete transfer, especially of HMW FN. Thus to further refine our analysis of the percentage of FN multimerized during TNF-alpha treatment, we then analyzed the multimerization of soluble FN newly incorporated into cell layers exposed to TNF-alpha . To perform this experiment, purified 125I-FN (3 µg/ml) was added to the FN-deficient culture medium of the endothelial cell layers that were being exposed to TNF-alpha or were sham treated, and the formation of nonreducible HMW FN complexes was assessed by autoradiographic analysis.

Figure 4 shows the autoradiographic analysis of control and TNF-alpha -treated endothelial cell layers under both nonreducing (A) and reducing (B) conditions. The gels revealed that under nonreducing conditions, the FN migrated mainly as HMW complexes or the well-known 440-kDa dimer (Fig. 4A), whereas under reducing conditions, more FN migrated as the 220-kDa monomer in control cells than in TNF-alpha -treated cells (Fig. 4B). The autoradiographs were also quantified by densitometric scanning (Fig. 4C). Our results showed that 73% of the total 125I-FN in control cells and 84% of the total 125I-FN in TNF-alpha -treated cells were present in a HMW form under nonreducing conditions. In contrast, when tested under reducing conditions, 57% of the total FN was still detected as HMW complexes in the TNF-alpha -treated cells compared with only 28% in control cell layers (Fig. 4C). In essence, TNF-alpha amplified the formation of HMW complexes of FN in the endothelial cell layers (P < 0.01), which were resistant to reduction to the monomeric 220-kDa form of FN. In preliminary experiments, we have also demonstrated that TNF-alpha -treated nonconfluent endothelial cell layers increase the ability of the cells to both bind soluble 125I-FN and incorporate soluble 125I-FN into their ECM (unpublished data).


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 4.   TNF-alpha increases the formation of nonreducible high molecular mass (HMW) FN complexes in lung endothelial monolayers. Preconfluent endothelial cells were incubated in FN-deficient medium containing 3 µg/ml of 125I-FN with and without 200 U/ml of TNF-alpha for 18 h. Cell layers were washed 4 times and scraped into SDS-PAGE sample buffer. Aliquots of cell lysates that contained equal 125I radioactivity were analyzed by SDS-PAGE and autoradiography under both nonreducing (A) and reducing (B) conditions. The autoradiographs from a representative experiment were scanned, and the percentage of total FN that existed as HMW FN was quantified (C). Percentage of total FN retained as HMW FN multimers under reducing conditions was 28.3 ± 3.4% in control monolayers (n = 3) and 57.0 ± 0.5% in TNF-alpha treated monolayers (n = 3). The amount of HMW FN multimers detected under reducing conditions (C) in the TNF-alpha -treated group was significantly greater than that in control group (*P < 0.05).

The increase in nonreducible HMW FN multimers is associated with an increase in transglutaminase activity in TNF-alpha -treated endothelial cells. FN can be processed into HMW complexes by a transglutaminase-mediated cross-linking mechanism (29, 32). Thus one possible explanation for the observed increase in nonreducible HMW FN complexes detected after exposure of the endothelial cell layers to TNF-alpha is an increase in transglutaminase activity, although this has not been previously documented. Accordingly, we measured transglutaminase activity in whole endothelial cell lysates by quantifying the incorporation of [3H]putrescine into dimethylated casein (29). Transglutaminase activity increased in the endothelial cell layers in a dose-dependent manner after TNF-alpha exposure (P < 0.05; Fig. 5B) in association with the appearance of nonreducible HMW FN multimers (Fig. 5A), suggesting that the larger amounts of nonreducible HMW FN complexes detected in TNF-alpha -treated endothelial cells may be due to an unexpected elevation in transglutaminase activity.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5.   Ability of TNF-alpha treatment of endothelial cells to cause a dose-dependent increase in both HMW FN multimers and transglutaminase activity. Preconfluent endothelial cells incubated in FN-deficient medium supplemented with 125I-FN (3 µg/ml) were exposed to increasing concentrations of TNF-alpha for 18 h. After the 18-h incubation, the cell layer was washed 4 times and scraped into reducing SDS-PAGE sample buffer. Aliquots of the cell lysates that contained equal radioactivity were analyzed by SDS-PAGE and autoradiography under reducing conditions. A: representative autoradiographic film. B: lysed cell layers assayed for transglutaminase activity by measurement of [3H]putrescine incorporation. Values are means ± SE of 3 wells/TNF-alpha dose. Transglutaminase activity was greater after 50 or 800 U/ml of TNF-alpha compared with that in control layers (P < 0.05).

To support this conclusion, we evaluated the effect of two different transglutaminase inhibitors, i.e., MDC and Cys, on the TNF-alpha -induced increase in HMW FN complexes. Both MDC and Cys at concentrations of 0.10 and 0.05 mM, respectively, were able to attenuate the TNF-alpha -induced increase in the formation of nonreducible HMW FN complexes (Fig. 6). The inhibitory response was especially apparent after the addition of Cys, consistent with previous findings on this inhibitor (9, 37). Indeed, Cys actually decreased the basal amount of nonreducible HMW FN complexes detected in control endothelial cell layers, which is consistent with the previous suggestions that transglutaminase-mediated cross-linking of matrix proteins may be a constitutive process in normal endothelial cells (29, 32).


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 6.   Inhibitors of transglutaminase can attenuate TNF-alpha -induced formation of HMW FN in endothelial cell layers. Preconfluent CPAE cell layers were incubated in FN-deficient medium containing 3 µg/ml of 125I-FN in the absence and presence of 2 different transglutaminase inhibitors, 0.10 mM monodansylcadaverine (MDC) and 0.05 mM cystamine (Cys). After 18 h of incubation with TNF-alpha (200 U/ml) at 37°C, the cells were washed 4 times and scraped into SDS-PAGE sample buffer, and cell lysates were analyzed by SDS-PAGE and autoradiography under reducing conditions (A). For quantification, the autoradiographic films in A were scanned, and the percentage of the total FN that existed as nonreducible HMW FN multimers was calculated (B). Values are means ± SE of 3 repeat experiments, each of which had 2 wells containing no inhibitor and 2 wells with each inhibitor (total n = 6/each).

The amount of nonreducible FN multimers corresponds to the level of transglutaminase activity in both endothelial cells and fibroblasts. If a TNF-alpha -induced increase in transglutaminase activity was actually the basis for the significant increase in nonreducible HMW complexes of FN detected in the endothelial matrix, we predicted that there should be very little formation of such HMW FN complexes with fibroblast cell layers because fibroblasts have limited transglutaminase activity. In a side-by-side comparative analysis, we observed that nearly all of the FN in the fibroblast ECM existed as HMW species under nonreducing condition (data not shown). More importantly, the basal level of transglutaminase activity in control fibroblast cell layers was only ~20% of that detected in endothelial cell layers (Fig. 7). Furthermore, the transglutaminase activity in the fibroblast cell layers remained low even after treatment with TNF-alpha for 18 h, whereas with endothelial cell layers, transglutaminase activity increased in a dose-dependent manner in response to TNF-alpha (P < 0.05; Fig. 7). This finding was consistent with two additional observations; first, most of the HMW FN multimers in the fibroblast cultures were readily reducible to FN monomers (Fig. 8A), and second, TNF-alpha was unable to further increase the formation of nonreducible HMW FN multimers in the ECM of the fibroblasts, although it could readily do so with endothelial cells (Fig. 8B). These results suggest a correlation between the formation of HMW nonreducible complexes of FN in the ECM and the level of transglutaminase activity. These findings also support the conclusion that a TNF-alpha -induced increase in transglutaminase activity in the CPAE cell layers may have contributed to the increased formation of such nonreducible HMW FN complexes in the matrix.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7.   Comparative analysis of transglutaminase activity in fibroblast and endothelial cell layers as influenced by TNF-alpha . Confluent cell layers of endothelial cells and fibroblasts were incubated with TNF-alpha for 18 h and then assayed for transglutaminase activity by measurement of [3H]putrescine incorporation. Values are means ± SE; nos. in parentheses, total no. of determinations. For endothelial cells, there were 3 experiments, each with triplicate wells. For fibroblasts, there were 3 experiments, each with duplicate wells. Transglutaminase activity in normal fibroblasts was ~20% of that in endothelial cells. TNF-alpha caused a dose-dependent increase in transglutaminase activity in endothelial monolayers (P < 0.05) but had no significant effect on transglutaminase activity in fibroblasts (P > 0.05).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 8.   TNF-alpha did not stimulate the formation of increased amounts of nonreducible HMW FN multimers in fibroblast cell layers. Confluent fibroblasts were incubated in FN-deficient medium containing 3 µg/ml of 125I-FN in the presence and absence of 200 U/ml of TNF-alpha for 18 h. After incubation at 37°C, the cell layers were washed 4 times and scraped into SDS-PAGE sample buffer. Aliquots of the fibroblast cell lysate (containing equal amounts of radioactivity) were analyzed by SDS-PAGE and autoradiography under reducing conditions (A). The autoradiograph was scanned, and the percentage of total FN that existed as HMW FN in the fibroblast cell layers was quantified and compared with that in endothelial cells (B). Values are means ± SE from 2 experiments, each of which had 3 control and 3 TNF-alpha wells. TNF-alpha increased the formation of nonreducible HMW FN in CPAE cells (P < 0.01) but not in fibroblasts.

TNF-alpha -induced, transglutaminase-mediated FN cross-linking activity is located on the surface of the endothelial cell. If the enhanced formation of nonreducible HMW complexes of FN in the ECM was indeed caused by TNF-alpha -induced transglutaminase activity, we would expect to see an increase in extracellular FN cross-linking activity on the surface of the endothelial cells and/or in the culture medium. Thus we examined the ability of both the endothelial cells and their conditioned medium to cross-link 125I-FN that was precoated on a cultured surface. Lung endothelial cells were first preincubated with and without TNF-alpha (200 U/ml) for 18 h, gently detached by mild trypsinization, washed in FN-deficient medium, and then plated onto the 125I-FN coated wells and allowed to adhere. After 3 h, the cells and their 125I-labeled matrices were dissolved and analyzed for the presence of HMW 125I-FN complexes.

As shown in Fig. 9, when analyzed under nonreducing conditions, the majority of 125I-FN within the cell layer was processed into a HMW form in both control and TNF-alpha -treated endothelial cell layers. However, under reducing conditions, only 35% of the HMW 125I-FN complexes formed by control endothelial cells were nonreducible, whereas ~65% of the HMW 125I-FN complexes formed in the TNF-alpha -treated cell were nonreducible. In contrast, the conditioned medium from both control and TNF-alpha -treated CPAE cell layers contained no significant FN cross-linking activity, and, as expected, no HMW FN complexes were detected under either reducing or nonreducing conditions in the absence of the endothelial cells (data not shown). Collectively, these results suggest that a significant amount of the additional transglutaminase activity detected in the CPAE cell layers after TNF-alpha exposure was localized to the cell surface. Indeed, it would appear that the cross-linking of FN mediated by such additional transglutaminase activity could likely occur either during the process of FN matrix assembly or shortly after the soluble FN became incorporated into the matrix.


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 9.   TNF-alpha -treated endothelial cells can enhance the cross-linking and multimerization of FN precoated on a culture surface in the absence of conditioned medium. CPAE cells with and without TNF-alpha (200 U/ml) pretreatment for 18 h were trypsinized, washed once with FN-deficient medium, and seeded onto 12-well culture plates precoated with 125I-FN. After 3 h of incubation, cells were washed 4 times, scraped into SDS-PAGE sample buffer, and then analyzed by SDS-PAGE and autoradiography under nonreducing (A) or reducing (B) conditions. The autoradiographs were scanned, and the percentage of total cell layer FN that existed as nonreducible HMW FN was calculated (C). Values were derived from 2 repeat experiments.

To specifically determine whether TNF-alpha had caused an increase in cell surface transglutaminase activity, we then measured the incorporation of biotin-labeled cadaverine into FN precoated on the culture surface. As shown in Fig. 10, the cell surface transglutaminase activity on the CPAE cells was increased two- to threefold after these cells were treated with TNF-alpha (P < 0.05). Thus a TNF-alpha -induced increase in cell surface transglutaminase activity appears to have been the basis for the increased formation of nonreducible HMW multimers of FN that we detected in the ECM of these endothelial cell layers.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 10.   TNF-alpha caused an increase in cell surface transglutaminase activity on endothelial cells as determined by analysis of the incorporation of biotinylated cadaverine into FN precoated on culture surfaces of 96-well plates with human FN at 10 µg/ml. Endothelial cells pretreated with and without (control) TNF-alpha were seeded at the indicated no. of cells/well and incubated at 37°C in the presence of 0.5 mM biotinylated cadaverine for 3 h. The incorporated cadaverine was quantified by measuring optical density (OD) at 450 nm after treatment with horseradish peroxidase-streptavidin and ELISA color development. Values are means ± SD from 6 experiments. Activity in TNF-alpha -treated groups was greater than in control groups at all 3 levels of cell seeding (P < 0.05).

Effect of the inhibitors of transglutaminase on TNF-alpha -induced reorganization of the FN matrix, cell adhesion and spreading on the matrix, and endothelial protein permeability. Tissue transglutaminase activity has been found in many cell types including endothelial cells (13, 26). Regulated expression of transglutaminase may also influence FN incorporation and play an important role in the regulation of cell attachment and apoptosis (48). Because exposure of the endothelial monolayers to TNF-alpha can induce both a disruption of the fine FN fibers in the matrix and an increase in endothelial protein permeability, we determined whether these events were dependent on the increase in transglutaminase activity and the enhanced formation of nonreducible HMW FN complexes in the ECM. Accordingly, we treated the lung endothelial cell layers with TNF-alpha (200 U/ml) in the absence and presence of the transglutaminase inhibitor Cys to attenuate the formation of nonreducible HMW FN multimers in the ECM of TNF-alpha -treated cells. Then we used these two different matrices to study cell adhesion and cell spreading. First, as shown in Fig. 11, Cys at concentrations that blocked the formation of nonreducible HMW FN complexes was unable to prevent the TNF-alpha -induced reorganization of the FN matrix as studied by immunofluorescent microscopy (Fig. 11, A and B). We next studied cell adhesion and cell spreading on these preformed matrices. To do this, the original cell layers were gently removed with 0.5% sodium deoxycholate, and normal CPAE endothelial cells were then seeded onto these preformed matrices. This allowed us to determine whether CPAE cells adhere or spread differently on the cross-linked FN matrix containing the nonreducible HMW multimers compared with the non-cross-linked FN matrix. As shown in Fig. 11, C-F, endothelial cell adhesion and cell spreading were essentially identical on the subendothelial matrices preformed by TNF-alpha -treated CPAE cells in the presence and absence of the transglutaminase inhibitor (Fig. 11, C and D). Cell adhesion to the FN network is shown by overlays of staining for alpha 5beta 1-integrins (green) and the matrix FN (red; Fig. 11, E and F). Direct microscopic counting of 12 areas/slide confirmed that cell adhesion and cell spreading were basically similar on these two different matrices (Fig. 12).


View larger version (117K):
[in this window]
[in a new window]
 
Fig. 11.   Matrices preformed by CPAE cell layers exposed to TNF-alpha in the presence and absence of a transglutaminase inhibitor supported similar adhesion and spreading of normal CPAE cells. Cell matrices were prepared over 3 days in culture by confluent endothelial cell monolayers treated with TNF-alpha (A) or TNF-alpha plus 0.05 mM Cys (B). Cell layers were removed with 0.5% sodium deoxycholate. Control cells were then seeded on both matrices and incubated at 37°C for 30 min in the adhesion spreading assay. After removal of nonadhered cells, the coverslips were fixed and stained with both an anti-FN (red; A and B) and an anti-alpha 5beta 1-integrin (green; C and D) antibody. A and C: same viewing field of cells adhering to TNF-alpha -treated FN matrix. E: computer overlay of A and C to show cell spreading on FN. B and D: same viewing field of cells adhering to TNF-alpha plus Cys-treated FN matrix. F: computer overlay of B and D to show cell spreading on FN.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 12.   Comparative analysis of CPAE cell adhesion and cell spreading on subendothelial matrices preformed by TNF-alpha -treated endothelial monolayers in the presence and absence of Cys. Both matrices manifest a reorganization of their fine fibrillar FN network but with reduced HMW FN multimerization in those supplemented with Cys. Cell adhesion and cell spreading were assayed by microscopy with a fluorescent microscope. Cells were stained with the anti-alpha 5beta 1-integrin antibody. Values are means ± SD of 12 areas counted.

Blocking transglutaminase activity with Cys was also unable to prevent the TNF-alpha -induced increase in endothelial monolayer protein permeability as quantified by measurement of protein clearance using the transendothelial flux of 125I-albumin (Fig. 13). We also detected no direct effect of exogenous transglutaminase on endothelial protein permeability when added directly to confluent monolayers (data not shown). This observation indicated that the TNF-alpha -induced increase in transglutaminase activity leading to the enhanced formation of nonreducible HMW FN multimers is not the primary basis for the increase in protein permeability across the lung endothelial cell monolayers.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 13.   Effect of inhibitors of transglutaminase on the TNF-alpha -induced increase in transendothelial protein permeability. Confluent CPAE cell monolayers on tissue culture-ready filter wells were treated with and without TNF-alpha (200 U/ml) for 18 h in the presence and absence of 2 transglutaminase inhibitors, 0.10 mM MDC and 0.05 mM Cys. Transendothelial protein permeability was determined by measuring the clearance of 125I-labeled albumin across endothelial monolayers. Values are means ± SE of 4 separate wells that were typically completed in each separate experiment. The experiment was repeated 5 separate times. Protein clearance was significantly increased (P < 0.05) after TNF-alpha and not blocked (P > 0.05) by either inhibitor.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study indicates that TNF-alpha causes an increase in the formation of nonreducible HMW FN complexes in endothelial cell layers, potentially due to an enhanced transglutaminase activity on the surface of endothelial cells. This conclusion is supported by at least three observations. First, both the increased formation of nonreducible HMW matrix FN complexes and the increase in cellular transglutaminase activity in the endothelial cell layers was TNF-alpha dose dependent. Indeed, fibroblast cell layers with low basal levels of transglutaminase activity contained a matrix with little nonreducible HMW FN, and we observed no increase in transglutaminase activity in fibroblast cell layers after TNF-alpha exposure. Second, inhibition of transglutaminase activity abolished the TNF-alpha -induced increase in nonreducible HMW matrix FN while also reducing the constitutive amounts of such HMW FN complexes detected in control endothelial cells not exposed to TNF-alpha . Third, TNF-alpha -treated endothelial cells displayed high levels of cell surface transglutaminase activity and were capable of cross-linking substrate FN precoated on culture plates at a much greater level compared with that of untreated control endothelial cell layers.

TNF-alpha is released from activated mobile monocytes and macrophages, from sessile macrophages such as hepatic Kupffer cells (12), and from cells within the gut during bacteremia and/or endotoxemia (30). TNF-alpha is believed to make a positive contribution to the inflammatory and generalized host response to sepsis (12, 30) by increasing endothelial permeability, thus allowing certain plasma proteins important for both wound repair and immune function to enter the extravascular space. However, high levels of TNF-alpha can also elicit pathophysiological effects in that they can disrupt the integrity of the lung endothelial barrier (16, 50, 51) and elicit apoptosis of endothelial as well as of tumor cells (36). Intracellular transglutaminase activity is elevated when cells undergo apoptosis, and transglutaminase-mediated cross-linking of proteins has been suggested to contribute to the regulation of programmed cell death (21, 27).

The present study documents an increase in extracellular transglutaminase activity in the bovine lung endothelial cell layer after exposure to TNF-alpha . It is not likely that this increase in transglutaminase activity was due to the nonspecific release of the enzyme from apoptotic endothelial cells because it was only found associated with the harvested CPAE cells and not in their conditioned medium. Indeed, our findings indicate that the increased transglutaminase activity that was likely mediating the enhanced FN multimerization after TNF-alpha exposure was on the cell surface and not in the culture medium. Extracellular tissue transglutaminase has been clearly documented in many cell types including endothelial cells (1, 2, 5, 21, 32, 43). A recent study by Gaudry et al. (22) has shown for the first time the secretion and expression of transglutaminase on the surface of human umbilical vein endothelial cells. Our studies not only confirm these novel findings but also establish their pathological relevance, demonstrating an increased surface expression of transglutaminase activity on endothelial cells after exposure to the inflammatory cytokine TNF-alpha .

For most adherent cell types, FN in the ECM is organized into HMW complexes primarily by interchain disulfide bonds. However, the cross-linking of FN by extracellular transglutaminase appears to provide an additional mechanism especially available to endothelial cells, although it can be seen to a limited degree in fibroblast cell layers. Transglutaminase-mediated cross-linking of FN (26, 32, 48), fibrinogen (33), and proteoglycans (28) into HMW complexes can take place constitutively in endothelial cells. TNF-alpha has also been shown to upregulate transglutaminase expression in other cell types such as brain astrocytes (34). Our present findings, which suggest increased cell surface transglutaminase in TNF-alpha -treated lung endothelial cell layers, provide a mechanism for the enhanced FN multimerization observed in the monolayers after exposure to this cytokine.

Our results show that the majority of matrix FN (~80%) was in the form of HMW complexes in the endothelial cell layers, with ~30% of these HMW FN multimers being stable under reducing conditions. This is in contrast to cultured fibroblasts where, although a major portion of the FN in the ECM also exists in HMW form, most of the HMW FN complexes in the ECM of fibroblasts will readily become monomeric (220 kDa) under reducing conditions. Consistent with this observation is the fact that it appears that fibroblast cell layers have very low levels of transglutaminase activity to mediate such FN multimerization compared with CPAE cells. However, Barry and Mosher (3, 4) have shown that activated factor XIII, also known as plasma transglutaminase, can stimulate the cross-linking of FN in the ECM of fibroblasts. They speculated that both FN already assembled in the ECM and FN in the process of being incorporated can be acted on by activated factor XIII. Based on studies with fibroblast cell layers, it has been proposed that the cross-linking of FN by transglutaminase may work in conjunction with disulfide-bonded FN multimerization to stabilize the assembly of the FN matrix (3).

An unexpected observation was that the increased transglutaminase activity induced by TNF-alpha did not cause additional HMW FN complexes to be seen under nonreducing conditions. This finding suggests that transglutaminase-mediated cross-linking of FN in CPAE cell layers is primarily influencing those FN molecules already incorporated within the matrix due to disulfide linkage. This potential extracellular function of transglutaminase, which has been released and localized to the cell surface of endothelial cells after exposure to TNF-alpha , may enable the endothelium to stabilize its subendothelial matrix especially in response to local vascular inflammation, causing the release of TNF-alpha , a conclusion consistent with the report by Gentile et al. (23) that indicates that cell-matrix interactions can be stabilized by transglutaminase activity. The speculation that constitutively expressed extracellular transglutaminase can stabilize cell-matrix interactions is further supported by the findings that cells deficient in transglutaminase are less able to adhere to an ECM compared with normal cells. However, it is also possible that the enhanced transglutaminase cross-linking of matrix FN may have pathological effects, including alteration of the signaling properties of the ECM or perhaps a reduction in FN turnover in the ECM.

TNF-alpha has been shown to alter the subendothelial FN matrix and reduce the barrier function of the endothelial cell monolayers (16, 17, 50, 51). After exposure of CPAE cells to TNF-alpha , the matrix FN is characterized by the formation of nonreducible HMW FN multimers and the appearance of thick FN bundles deep within the matrix (16, 17). There is also reduced colocalization of endothelial cell surface FN alpha 5beta 1-integrins with the fine FN fibers in the matrix (16, 17). Our current findings suggest an important role for enhanced endothelial cell surface transglutaminase activity in the formation of nonreducible HMW complexes of FN in the matrix after TNF-alpha exposure. However, our findings also suggest that such cross-linking of matrix FN is not the basis for either the increase in endothelial protein permeability or the reorganization of the fine fibrillar FN matrix because transglutaminase inhibitors did not prevent either of these TNF-alpha -induced changes. Moreover, endothelial cell adhesion and cell spreading on the FN-rich matrix was also not altered by Cys treatment to block transglutaminase.

Another possible factor contributing to the altered ECM is the degradation of matrix FN by proteases. Partridge et al. (35) showed by zymography that medium from TNF-alpha -treated microvessel endothelial cells contains metalloproteinase that can cleave FN and other matrix proteins. TNF-alpha has also been shown to induce the release of metalloproteinases such as gelatinase and collagenase (35), which have the potential to cause matrix degradation and/or matrix FN reorganization. But TNF-alpha also causes endothelial cells to release protease inhibitors such as plasminogen activator inhibitor-1 and -2 and tissue inhibitor of metalloproteinases (25, 45), which can also block matrix proteolysis. In this regard, the recent findings by Curtis et al. (17) clearly document that local proteolysis of matrix FN is not the biochemical mechanism causing disruption and/or reorganization of FN in the ECM of CPAE monolayers after their 18-h exposure to TNF-alpha , a protocol also used in the present study.

In the present study, we did not detect by Western blot analysis or tracer 125I-FN experiments any significant increase in the amount of FN fragments in CPAE cell layers or their culture medium after TNF-alpha exposure. These findings confirm and extend the recent study from our laboratory (17) documenting that although conditioned medium from TNF-alpha -treated CPAE cells contains proteolytic activity that can degrade both gelatin (denatured collagen) as well as FN, no FN fragments were found in either the cell lysate or the conditioned medium. Obviously, the proteolytic balance within the medium and/or matrix will determine the presence or absence of FN fragments after TNF-alpha exposure.

The possibility that the increase in the cross-linking of FN matrix could be a protective response of endothelial cells to the presence of TNF-alpha warrants consideration. For example, cross-linking of matrix proteins by extracellular transglutaminase can promote cell-matrix interaction (17) and potentially stabilize matrix integrity. Accordingly, increased extracellular transglutaminase activity leading to the enhanced cross-linking of FN assembled in the matrix via disulfide-bonded multimer formation may provide endothelial cells an additional mechanism to resist the cytotoxic effects of TNF-alpha , especially in regard to the integrity of the ECM, which is vital to cell adhesion and endothelial barrier function. The rapid normalization of barrier function that can be seen after the addition of excess soluble FN to the culture medium followed by its ECM incorporation (16, 17, 50, 51) may reflect the fact that a large portion of the endogenous FN already assembled in the ECM became cross-linked after TNF-alpha exposure.


    ACKNOWLEDGEMENTS

The secretarial assistance of Debbie Moran and Wendy Ward is extremely appreciated.


    FOOTNOTES

These studies were supported primarily by National Institute of General Medical Sciences Grant GM-21447 (to T. M. Saba) and in part by American Lung Association Research Grant RG-133N (to B. Gao).

R. Chen was a postdoctoral fellow supported by National Heart, Lung, and Blood Institute Grant HL-07529 and is currently a Research Instructor in the Department of Medicine, Medical University of South Carolina (Charleston, SC). C. Huang and R. F. Rotundo were postdoctoral fellows supported by National Heart, Lung, and Blood Institute Grant HL-07529. C. Huang is currently a Research Associate in the Department of Microbiology and Immunology, Medical University of South Carolina. R. F. Rotundo is currently a Research Associate in the Department of Physiology and Cell Biology, Albany Medical College (Albany, NY).

Address for reprint requests and other correspondence: T. M. Saba, Dept. of Physiology and Cell Biology (MC-134), Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: sabat{at}mail.amc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 7 June 1999; accepted in final form 3 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aeschlimann, D, and Paulsson M. Cross-linking of lamin-nidogen complexes by tissue transglutaminase. A novel mechanism for basement membrane stabilization. J Biol Chem 266: 15308-15317, 1991[Abstract/Free Full Text].

2.   Aeschlimann, D, Wetterwald A, Fleisch H, and Paulsson M. Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation of chondrocytes. J Cell Biol 120: 1461-1470, 1993[Abstract].

3.   Barry, ELR, and Mosher DF. Factor XIII cross-linking of fibronectin at cellular matrix assembly sites. J Biol Chem 263: 10464-10469, 1988[Abstract/Free Full Text].

4.   Barry, ELR, and Mosher DF. Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers. Cross-linking of cellular and plasma fibronectin and of amino-terminal fibronectin fragments. J Biol Chem 264: 4179-4185, 1989[Abstract/Free Full Text].

5.   Barsigian, C, Stern AM, and Martinez J. Tissue (type II) transglutaminase covalently incorporates itself, fibrinogen, or fibronectin into high molecular weight complexes on the extracellular surface of isolated hepatocytes. Use of 2-[(2-oxopropyl)thio] imidazolium derivatives as cellular transglutaminase inactivators. J Biol Chem 266: 22501-22509, 1991[Abstract/Free Full Text].

6.   Brett, J, Gerlach H, Nawroth P, Steinberg S, Godman G, and Stern D. Tumor necrosis factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins. J Exp Med 169: 1977-1991, 1989[Abstract].

7.   Brigham, KL, Bowers RE, and Haynes J. Increased sheep lung vascular permeability caused by E. coli endotoxin. Circ Res 45: 292-297, 1979[Abstract].

8.   Brigham, KL, Woolverton WC, Blake LA, and Staub NC. Increased sheep lung vascular permeability caused by Pseudomonas bacteremia. J Clin Invest 54: 792-804, 1974[ISI][Medline].

9.   Bungay, PJ, Potter JM, and Griffin M. The inhibition of glucose-stimulated insulin secretion by primary amines. A role for transglutaminase in the secretory mechanism. Biochem J 219: 819-827, 1984[ISI][Medline].

10.   Camussi, G, Albano E, Tetta C, and Bussolino F. The molecular action of tumor necrosis factor-alpha . Eur J Biochem 202: 3-14, 1991[Abstract].

11.   Charash, WE, Vincent PA, McKeown-Longo PJ, Saba TM, Lewis E, and Lewis MA. Kinetics of plasma fibronectin: increased lung tissue incorporation after postoperative bacteremia. Am J Physiol Regulatory Integrative Comp Physiol 260: R553-R562, 1991[Abstract/Free Full Text].

12.   Chaudry, IH, Zellweger R, and Ayala A. The role of bacterial translocation of Kupffer cell immune function following hemorrhage. Prog Clin Biol Res 392: 209-218, 1995[Medline].

13.   Chowdhury, ZA, Barsigian C, Chalupowicz GD, Bach TL, Garcia-Manero G, and Martinez J. Colocalization of tissue transglutaminase and stress fibers in human vascular smooth muscle cells and human umbilical vein endothelial cells. Exp Cell Res 231: 38-49, 1997[ISI][Medline].

14.   Cohler, LF, Saba TM, Lewis EP, Vincent PA, and Charash WE. Plasma fibronectin therapy and lung protein clearance with bacteremia after surgery. J Appl Physiol 63: 623-633, 1987[Abstract/Free Full Text].

15.   Cooper, JA, Del Vecchio PJ, Minnear FL, Burhop KE, Selig WM, Garcia JGN, and Malik AB. Measurement of albumin permeability across endothelial monolayers in vitro. J Appl Physiol 62: 1076-1083, 1987[Abstract/Free Full Text].

16.   Curtis, TM, McKeown-Longo PJ, Vincent PA, Homan SM, Wheatley EM, and Saba TM. Fibronectin attenuates increased endothelial monolayer permeability after RGD peptide, anti-alpha 5beta 1, or TNF-alpha exposure. Am J Physiol Lung Cell Mol Physiol 269: L248-L260, 1995[Abstract/Free Full Text].

17.   Curtis, TM, Rotundo RF, Vincent PA, McKeown-Longo PJ, and Saba TM. Matrix FN disruption and increased endothelial permeability after TNF-alpha is independent of FN proteolysis. Am J Physiol Lung Cell Mol Physiol 275: L126-L138, 1998[Abstract/Free Full Text].

18.   Davis, LG, Gibner MD, and Battey JF. Basic Methods in Molecular Biology. New York: Elsevier Science, 1986, p. 143-146.

19.   Dumin, J, Wilcox BD, Otterness I, Melendez JA, Huang C, and Jeffrey JJ. Serotonin-mediated production of interstitial collagenase by uterine smooth muscle cells requires interleukin-1alpha , but not interleukin-1beta . J Biol Chem 273: 25488-25494, 1998[Abstract/Free Full Text].

20.   Engvall, E, and Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer 20: 1-5, 1977[ISI][Medline].

21.   Fesus, L, Madi A, Balajthy Z, Nemes Z, and Szondy Z. Transglutaminase induction by various cell death and apoptosis pathways. Experientia 52: 942-949, 1996[ISI][Medline].

22.   Gaudry, CA, Verderio E, Jones RA, Smith C, and Griffin M. Tissue transglutaminase is an important player at the surface of human endothelial cells: evidence for its externalization and its colocalization with the beta 1 integrin. Exp Cell Res 252: 104-113, 1999[ISI][Medline].

23.   Gentile, V, Thomazy V, Piacentini M, Fesus L, and Davies PJ. Expression of tissue transglutaminase in Balb-C 3T3 fibroblasts: effects on cellular morphology and adhesion. J Cell Biol 119: 463-474, 1992[Abstract].

24.   Igarashi, S, Koide R, Shimohata T, Yamada M, Hayashi Y, Takano H, Date H, Oyake M, Sato T, Sato A, Egawa S, Ikeuchi T, Tanaka H, Nakano R, Tanaka K, Hozumi I, Inuzuka T, Takahashi H, and Tsuji S. Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch. Nat Genet 18: 111-117, 1998[ISI][Medline].

25.   Ito, A, Sato T, Iga T, and Mori Y. Tumor necrosis factor bifunctionally regulates matrix metalloproteinases and tissue inhibitor of metalloproteinases (TIMP) production by human fibroblasts. FEBS Lett 269: 93-95, 1990[ISI][Medline].

26.   Jones, RA, Nicholas B, Mian S, Davies PJA, and Griffin M. Reduced expression of tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion, and reduced polymerization of fibronectin. J Cell Sci 110: 2461-2472, 1997[Abstract/Free Full Text].

27.   Karsan, A, Yee E, and Harlan JM. Endothelial cell death induced by tumor necrosis factor-alpha is inhibited by the Bcl-2 family member, A1. J Biol Chem 271: 27201-27204, 1996[Abstract/Free Full Text].

28.   Kinsella, MG, and Wight TN. Formation of high molecular weight dermatan sulfate proteoglycan in bovine aortic endothelial cell cultures. Evidence for transglutaminase-catalyzed cross-linking to fibronectin. J Biol Chem 265: 17891-17898, 1990[Abstract/Free Full Text].

29.   Korner, G, Schneider DE, Purdon MA, and Bjornsson TD. Bovine aortic endothelial cell transglutaminase. Enzyme characterization and regulation of activity. Biochem J 262: 633-641, 1989[ISI][Medline].

30.   Mainous, MR, Ertel W, Chaudry IH, and Deitch EA. The gut: a cytokine-generating organ in systemic inflammation. Shock 4: 193-199, 1995[ISI][Medline].

31.   Mantovani, A, Bussolin F, and Dejana E. Cytokine regulation of endothelial cell function. FASEB J 6: 2591-2599, 1992[Abstract/Free Full Text].

32.   Martinez, J, Chalupowicz DG, Rough RK, Sheth A, and Barsigian C. Transglutaminase-mediated processing of fibronectin by endothelial cell monolayers. Biochem J 33: 2538-2545, 1994.

33.   Martinez, J, Rich E, and Barsigian C. Transglutaminase-mediated cross-linking of fibrinogen by human umbilical vein endothelial cells. J Biol Chem 264: 20502-20508, 1989[Abstract/Free Full Text].

34.   Monsonego, A, Shani Y, Friedmann I, Paas Y, Eizenberg O, and Schwartz M. Expression of GTP-dependent and GTP-independent tissue-type transglutaminase in cytokine-treated rat brain astrocytes. J Biol Chem 272: 3724-3732, 1997[Abstract/Free Full Text].

35.   Partridge, CA, Jeffrey JJ, and Malik AB. A 96-kDa gelatinase induced by TNF-alpha contributes to increased microvascular endothelial permeability. Am J Physiol Lung Cell Mol Physiol 265: L438-L447, 1993[Abstract/Free Full Text].

36.   Polunosky, VA, Wendt CH, Ingbar DH, Peterson MS, and Bitterman PB. Induction of endothelial cell apoptosis by TNFalpha : modulation by inhibitors of protein synthesis. Exp Cell Res 214: 584-594, 1994[ISI][Medline].

37.   Rao, UR, Mehta K, Subrahmanyam D, and Vickery AC. Brugia malayi and Acanthocheilonema viteae: antifilarial activity of transglutaminase inhibitors in vitro. Antimicrob Agents Chemother 35: 2219-2224, 1991[ISI][Medline].

38.   Rebres, RA, McKeown-Longo PJ, Vincent PA, Cho E, and Saba TM. Extracellular matrix incorporation of normal and NEM-alkylated fibronectin: liver and spleen deposition. Am J Physiol Gastrointest Liver Physiol 269: G902-G912, 1995[Abstract/Free Full Text].

39.   Redl, H, Schlag G, and Lamche H. TNF- and LPS-induced changes of lung vascular permeability: studies in unanesthetised sheep. Circ Shock 31: 183-192, 1990[ISI][Medline].

40.   Resnikoff, M, Brien TP, Vincent PA, Rotundo RF, Lewis E, McKeown-Longo PJ, and Saba TM. Lung matrix incorporation of fibronectin reduces vascular permeability in postsurgical bacteremia. Am J Physiol Lung Cell Mol Physiol 277: L749-L759, 1999[Abstract/Free Full Text].

41.   Robinson, NA, and Eckert RL. Identification of transglutaminase-reactive residues in S100A11. J Biol Chem 273: 2721-2728, 1998[Abstract/Free Full Text].

42.   Saba, TM. Kinetics of plasma fibronectin: relationship to phagocytic function and lung vascular injury. In: Fibronectin, edited by Mosher DF.. San Diego, CA: Academic, 1989, p. 395-439.

43.   Sane, DC, Moser TL, and Greenberg CS. Vitronectin in the substratum of endothelial cells is cross-linked and phosphorylated. Biochem Biophys Res Commun 174: 465-469, 1991[ISI][Medline].

44.   Smethurst, PA, and Griffin M. Measurement of tissue transglutaminase activity in permeabilized cells system: its regulation by Ca2+ and nucleotides. Biochem J 313: 803-808, 1996[ISI][Medline].

45.   Unemori, EN, Bouhana KS, and Werb Z. Vectorial section of extracellular matrix proteins, matrix-degrading proteinases, and tissue inhibitor of metalloproteinases by endothelial cells. J Biol Chem 265: 445-451, 1990[Abstract/Free Full Text].

46.   Van der Poll, T., and Lowry SF. Tumor necrosis factor in sepsis: mediator of multiple organ failure or essential part of host defense? Shock 3: 1-12, 1995[ISI][Medline].

47.   Vassalli, P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol 10: 411-452, 1992[ISI][Medline].

48.   Verderio, E, Nicholas B, Gross S, and Griffin M. Regulated expression of tissue transglutaminase in Swiss 3+3 fibroblasts: effects on the processing of fibronectin, cell attachment, and cell death. Exp Cell Res 239: 119-138, 1998[ISI][Medline].

49.   Vilcek, J, and Lee TH. Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions. J Biol Chem 266: 7313-7316, 1991[Free Full Text].

50.   Wheatley, EM, McKeown-Longo PJ, Vincent PA, and Saba TM. Incorporation of fibronectin into matrix decreases the TNF-induced increase in endothelial monolayer permeability. Am J Physiol Lung Cell Mol Physiol 265: L148-L157, 1993[Abstract/Free Full Text].

51.   Wheatley, EM, Vincent PA, McKeown-Longo PJ, and Saba TM. Effect of fibronectin on permeability of normal and TNF-treated lung endothelial cell monolayers. Am J Physiol Regulatory Integrative Comp Physiol 264: R90-R96, 1993[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 279(1):L161-L174
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society