Regulation of gelatinases in human airway smooth muscle cells: mechanism of progelatinase A activation

Hussein D. Foda1,2, Suni George1, Ellen Rollo1, Michelle Drews1, Cathleen Conner1, Jian Cao2, Reynold A. Panettieri Jr.3, and Stanley Zucker1,2

1 Department of Medicine and Research, Veterans Affairs Medical Center, Northport 11768; 2 Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8172; and 3 Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-4283


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Matrix metalloproteinases (MMPs) play an important role in tumor metastasis and invasion, inflammatory tissue destruction and remodeling, wound healing, and angiogenesis. The 72-kDa gelatinase A is the most widely distributed of all the MMPs, and along with the 92-kDa gelatinase B, both play an important role in the turnover of basement membrane. The role of MMPs in chronic airway inflammation and remodeling has received scant attention. In this study, we sought to examine the release and activation of gelatinases from human airway smooth muscle (ASM) cells and the effect of tumor necrosis factor-alpha and phorbol 12-myristate 13-acetate (PMA) on this release and activation. The role of membrane type 1 MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP)-2 in activating progelatinase A has been explored. We have demonstrated, using human airway smooth muscle cells in culture, that 1) ASM releases gelatinase A constitutively and when stimulated with PMA and tumor necrosis factor-alpha releases gelatinase B, and the release of gelatinase B is protein kinase C dependent because it is blocked by H-7 and staurosporin; 2) treatment of ASM cells with PMA or concanavalin A failed to activate progelatinase A despite these agents increasing cell expression of MT1-MMP; and 3) the inability of ASM cell membranes to activate progelatinase A is most likely secondary to the high levels of TIMP-2 on the cell membrane. In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.

human; matrix metalloproteinase; membrane-type matrix metalloproteinase; tissue inhibitor of matrix metalloproteinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MATRIX METALLOPROTEINASES (MMPs) play an important role in tumor metastasis and invasion, inflammatory tissue remodeling and destruction, wound healing, and angiogenesis. The 72-kDa gelatinase A (MMP-2) is the most widely distributed of all the MMPs (2). It is expressed constitutively by most connective tissue cells including endothelial cells, fibroblasts, myoblasts, osteoblasts, and chondrocytes as well as invasive tumor cells. Along with the 92-kDa gelatinase B (MMP-9), gelatinase A plays an important role in the turnover of basement membrane type IV collagen (28) and in controlling cell proliferation (24).

MMPs are tightly regulated in their transcription and release (15). They are further regulated at the level of conversion of latent enzymes to their active forms. Gelatinase A is secreted as a latent enzyme, and when it is converted to a 62-kDa active form, it can degrade collagen types IV and V, laminin, and elastin. The mechanism by which progelatinase A is activated physiologically to a 62-kDa enzyme involves a recently described, novel membrane-type matrix metalloproteinase (MT-MMP) (23). Specific tissue inhibitors of metalloproteinase (TIMPs) inhibit the activation of gelatinases. The expression of TIMPs is also tightly regulated (18).

Chronic airway inflammation, reversible airway obstruction, and bronchial hyperresponsiveness characterize bronchial asthma. This airway inflammation involves microvascular leakage and release of several inflammatory mediators including cytokines (3, 10). Pathologically, there is an eosinophilic and mast cell infiltration of the submucosa, hyperplasia of mucous glands, thickening of the subepithelial basement membrane, and hypertrophy and hyperplasia of bronchial smooth muscle. This increase in airway smooth muscle (ASM) mass may be as important as smooth muscle shortening in determining airway responsiveness in patients with asthma (10, 11, 26).

Recent observations have suggested that cytokines acting as mediators of inflammation can stimulate vascular smooth muscle cells to increase their production of gelatinase B (6). Gelatinases are necessary for vascular smooth muscle proliferation (24) by virtue of their degradation of vascular smooth muscle basement membrane, thereby facilitating cell migration and subsequent cell proliferation. Other investigators (29) have found that MMP inhibition decreased vascular smooth muscle migration, proliferation, and intimal thickening in rat arteries after injury. More recently, Ohno et al. (20) found gelatinase B expression to be increased in eosinophils seen on bronchial biopsies from asthmatic subjects.

In contrast to the active interest in vascular smooth muscle cells, the effect of inflammatory mediators on ASM cell production and activation of MMPs has received little attention. In this study, we sought to systematically examine the release and activation of gelatinases from ASM cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Tumor necrosis factor (TNF)-alpha was purchased from Boehringer Mannheim (Indianapolis, IN). Phorbol 12-myristate 13-acetate (PMA), aminophenylmercuric acetate, EDTA, H-7, staurosporin, concanavalin A (Con A), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO). Precast zymogram gels were purchased from NOVEX (San Diego, CA).

Recombinant human progelatinase A and recombinant (r) TIMP-1 and rTIMP-2 (19) were a generous gift from Celltech (Slough, UK).

Cell Culture

Human ASM Cells. Human tracheae were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania (Philadelphia, PA) Committee on Studies Involving Humans. ASM cells were cultured as previously described (22). Briefly, a segment of trachea just proximal to the carina was removed under sterile conditions, and the trachealis muscle was isolated. With this technique, ~0.5 g of wet tissue was obtained, minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 U/ml of collagenase, 1 mg/ml of soybean trypsin inhibitor, and 10 U/ml of elastase. Enzymatic dissociation was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105-mm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F-12 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 2.5 µg/ml of amphotericin B. Aliquots of the cell suspension were plated at a density of 1.0 × 104 cells/cm2. The cells were refed with supplemented Ham's F-12 medium every 72 h. Cell counts were obtained with 0.5% trypsin in 1 mM EDTA. Human ASM cells in subculture during the second through the fifth cell passages were used because during these cell passages, the cells retain native contractile protein expression as demonstrated by immunocytochemical staining for smooth muscle actin and myosin (22).

Human umbilical vein endothelial cells. Human umbilical vein endothelial cells (HUVECs) were harvested with the method of Jaffe (9) and cultured in gelatin-coated plates as previously described (5). The cells were grown in human endothelial-basal growth medium (GIBCO BRL, Life Technologies, Grand Island, NY) supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin (GIBCO BRL), and 10 ng/ml of endothelial growth factor. All cells were grown in a humidified atmosphere of 95% air-5% CO2 at 37°C and passaged every 7-10 days. Cells from the third to the seventh passages were used.

Experimental Procedure

When ASM cells reached confluence in the wells, the cells were washed three times with serum-free medium. Test substances were added, and the cells were then incubated for 18 h in serum-free medium. Cell morphology (number of adhering cells, granularity, and spread) was examined after each incubation to substantiate that cell viability was not compromised by any of the test substances. The conditioned medium (CM) was then collected, centrifuged at 1,500 g for 10 min to remove any particulate matter and nonadherent cells, and then stored for 1-3 days at -70°C until assayed. All experiments were repeated four to six times, and samples were run in duplicate unless otherwise stated.

Gelatinase Zymography and Reverse Zymography

Substrate zymography was performed in 10% polyacrylamide gels that had been cast in the presence of 0.1% gelatin. SDS-PAGE was performed with Tris-glycine SDS in sample and running buffers as described by the manufacturer (NOVEX). Samples were not boiled before electrophoresis. After electrophoresis, the gels were washed in 2.5% Triton X-100 to remove SDS, thus renaturing the gelatinases. The gels were then incubated in Tris buffer containing NaCl, CaCl2, and Brij 35 for 48 h at 37°C. Zones of enzymatic activity were characterized by the absence of Coomassie blue staining. Protein standards (Bio-Rad, Richmond, CA) were run concurrently, and approximate molecular masses of sample proteins were determined by plotting the relative mobilities of known proteins. Gelatinolytic bands were quantified by gel scanning and densitometry (30) with an Alpha Imager (Alpha Inotech, San Leandra, CA).

For reverse zymography, we used gels impregnated with both 0.05% gelatin and activated gelatinase A (gelatinase A was aminophenylmercuric acetate activated). TIMP-2 bands were identified by Coomassie blue staining (8). Semiquantitative analysis of the amount of TIMP-2 was performed with gel scanning and densitometry.

Immunoblotting

To identify the secreted MMPs detected on the zymograms, immunoblotting of CM for gelatinase A was performed with affinity-purified specific rabbit antibodies to human gelatinase A as previously described (17).

Immunoblotting of cell lysate for membrane type 1 MMP (MT1-MMP) was performed with affinity-purified rabbit antibodies directed against a unique peptide of MT1-MMP (CDGNFDTVAMLRGEM) (5). The cells were washed, then lysed by incubation in 1% SDS for 1 h. The samples were concentrated by precipitation in 6% TCA and resuspended in buffer. Prestained molecular-mass markers were run on each gel to identify molecular mass.

Immunoblotting of cell membranes for TIMP-2 was performed with affinity-purified rabbit antibodies directed against TIMP-2 supplied by Dr. Stetler-Stevenson (National Cancer Institute, Bethesda, MD) (30).

Quantification of Gelatinase A, Gelatinase B, TIMP-1, and TIMP-2

The concentrations of gelatinase A, gelatinase B (both latent and activated enzymes), TIMP-1, and TIMP-2 were determined in ASM CM with enzyme-linked immunosorbent assays (ELISAs) as previously described (12, 32). These experiments were repeated three times with samples run in duplicate.

Preparation of Cell Membranes

Approximately 2 × 107 ASM or endothelial cells were rinsed three times in serum-free medium, mechanically scraped from dishes, pelleted by centrifugation, resuspended in cavitation buffer (25 mM sucrose and 5 mM MgCl2 in 5 mM Tris base, pH 7.4), and subjected to 1,000 psi N2 for 30 min at 4°C as previously described (33). Whole cells and nuclei were removed by centrifugation at 770 g for 10 min; the postnuclear supernatant was collected, and heavy organelles (mitochondria, lysosomes, and endoplasmic reticulum) were recovered by centrifugation at 6,000 g for 15 min. The supernatant was then centrifuged at 100,000 g for 1 h at 4°C to recover the lighter cell organelles in the pellet (i.e., plasma membranes, endoplasmic reticulum, and Golgi); the 100,000-g supernatant was collected and designated cytosol.

Cell Surface Binding of TIMP-2

To examine TIMP-2 binding to ASM and compare it with that of TIMP-2 binding to HUVECs, we used methods previously described (31). Briefly, rTIMP-2 was iodinated to a specific activity of 5.5 ×1010 dpm/mg by adding 0.25 mCi of Na125I to a tube containing 10 µg of rTIMP-2 and 100 µg of chloramine T. After a 5-min incubation on ice, 200 µg of sodium metabisulfite were added, and 125I-labeled TIMP-2 in PBS containing 0.1% BSA was separated from free 125I by chromatography over a G-25 Sephadex column (Pharmacia Biotech, Washington, DC). Binding of 125I-labeled TIMP-2 to cells propagated in 24-well dishes (Corning Costar, Wilmington, MA) to >90% confluence was performed in duplicate (-10% variation between duplicates). The cells were washed thoroughly and treated with 50 mM glycine and 0.1 M NaCl in PBS (pH 3.0) for 3 min to dissociate preformed receptor-ligand complexes. The cells were then washed with cold PBS. For equilibrium binding experiments, dilutions of 125I-labeled TIMP-2 (0.8-16 nM) in PBS-BSA buffer were added to the cells in 200 µl of incubation buffer (total volume) in the presence and absence of excess unlabeled rTIMP-2. After a 3-h incubation, the supernatant fluid was collected, and the dishes were washed three times with PBS; the washes were collected and added to the unbound 125I-TIMP-2 fraction. The cell monolayers were then lysed in 0.1% SDS in 0.5 M NaOH and collected as the bound fraction. Bound and unbound 125I were measured by gamma counting. The residual radioactivity associated with the cells in the nonspecific binding experiment (50-fold excess TIMP-2) was subtracted from the total bound fraction (no unlabeled TIMP-2) to give specific binding. Scatchard plot analysis of binding data employed best-fit curves with the Sigma Plot program (Jandal Scientific, San Rafael, CA). These experiments were repeated three times.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Release and Activation of Progelatinases A and B

Gelatin zymography of CM from untreated ASM showed an incremental increase over time in the release of 72-kDa gelatinolytic bands, consistent with progelatinase A (data not shown). To confirm the identification of the gelatinolytic bands released by ASM, progelatinase A (72 kDa) was identified on immunoblots with specific rabbit anti-human antibodies to gelatinase A (data not shown).

PMA and the tetravalent lectin Con A are known to induce several cell types to activate progelatinase A (5, 21, 27). When ASM cells were treated with PMA, the zymogram of the CM showed an increase in gelatinolytic activity at 92 kDa (Fig. 1A) and a variable increase at 85 kDa (best visualized in Fig. 1B), consistent with progelatinase B and activated gelatinase B, respectively; however, there was no change in the gelatinolytic activity at 72 kDa (progelatinase A) and no activation. PMA also induced gelatinolytic activity at ~55 kDa, presumably representing collagenase or stromelysin-1 (Fig. 1A). Treatment of ASM cells with Con A likewise did not result in activation of progelatinase A (see Fig. 5).


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Fig. 1.   A: phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor (TNF)-alpha in combination increase airway smooth muscle (ASM) secretion and activation of gelatinase B. ASM conditioned medium was collected after 18 h of incubation with PMA and/or TNF-alpha and run on gelatin zymograms. Lane 1, buffer; lane 2, PMA (20 nM); lane 3, TNF-alpha (104 U); lane 4, TNF-alpha (105 U); lane 5, PMA (20 nM) and TNF-alpha (104 U); lane 6, PMA (20 nM) and TNF-alpha (105 U). B: PMA and TNF-alpha in combination increase ASM secretion and activation of gelatinase B; H-7 inhibits this effect. Lane 1, conditioned medium (18 h) from untreated ASM; lane 2, conditioned medium (18 h) from cells treated with PMA (20 nM); lane 3, conditioned medium from cells treated with TNF-alpha (104 U); lane 4, conditioned medium from cells treated with PMA (20 nM) and TNF-alpha (104 U); lane 5, conditioned medium from cells treated with PMA (20 nM) and TNF-alpha (104 U) in presence of H-7 (40 mM). Nos. on left, molecular mass in kDa.

Based on the known release of numerous cytokines during inflammation, we examined the effect of TNF-alpha on ASM progelatinase A activation. ASM cells were treated with TNF-alpha (103 to 105 U) for 24 and 48 h. TNF-alpha induced a small increase in the 92- and 85-kDa gelatinolytic bands (progelatinase B and activated gelatinase B, respectively; best visualized in Fig. 1B) but had little effect on progelatinase A in the CM. When TNF-alpha was added in combination with PMA to ASM cells, the CM exhibited a large increase in the prominence of the latent and activated gelatinase B bands at 95 and 85 kDa, respectively. This increase in progelatinase B with TNF-alpha and PMA was dose dependent (Fig. 1A) and mediated through protein kinase (PK) C because it was inhibited by the PKC inhibitor H-7 (Fig. 1B) and staurosporin (data not shown). These results were confirmed by an ELISA that demonstrated a large increase in immunoreactive gelatinase B in the CM of cells treated with the combination of PMA and TNF-alpha (data not shown) but no change in measured gelatinase A.

We then compared CM from ASM cells and HUVECs using an ELISA to measure gelatinase A. The levels of gelatinase A in the CM from ASM cells and HUVECs were similar (3.32 ± 1.02 and 3.98 ± 0.44 nM, respectively; Table 1).

                              
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Table 1.   Quantification by ELISA of gelatinase A, TIMP-1, and TIMP-2 released by ASM cells and HUVEC in conditioned medium collected after 18-h incubation

TIMP-1 and TIMP-2 Release From ASM Cells and HUVECs

CM from ASM cells and HUVECs was analyzed with ELISAs to measure TIMP-1 and TIMP-2 levels. We found that ASM cells secreted considerably larger amounts of TIMP-1 compared with HUVECs (323.49 ± 29.3 and 1.31 ± 0.8 nM, respectively). However, TIMP-2 in the CM of ASM cells was lower than that in HUVECs (0.35 ± 0.04 and 0.96 ± 0.09 nM, respectively; Table 1).

Mechanisms of ASM Progelatinase A Activation

In contrast with ASM cells, PMA and Con A treatment of HUVECs results in activation of secreted progelatinase A (5). Cell membranes isolated from HUVECs readily activated progelatinase A in HUVEC CM (13), thus implicating a plasma membrane activator, presumably MT1-MMP (23).

To investigate the inability of PMA or Con A to activate ASM progelatinase A while being effective in activating progelatinase A from HUVECs, cell membrane fractions from HUVECs were incubated for 18 h with CM from ASM cells. The addition of the plasma membrane-enriched fraction (100,000-g pellet) from HUVECs to the ASM CM resulted in a dose-dependent increase in gelatinolytic bands at 64 and 62 kDa, which is consistent with the activation of progelatinase A (Fig. 2A). When the experiment was altered so that cell membranes from ASM cells were incubated with CM from ASM, no activation of progelatinase A was noted (Fig. 2B).


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Fig. 2.   Human umbilical vein endothelial cell (HUVEC) plasma membranes, but not ASM plasma membranes, activate progelatinase A secreted by ASM cells. Conditioned medium was collected at 18 h from ASM cells, incubated in a cell-free environment for another 18 h, and run on gelatin zymograms. A: lane 1, untreated conditioned medium; lane 2, ASM conditioned medium incubated with HUVEC membranes (20 µg/ml); lane 3, ASM conditioned medium incubated with HUVEC membranes (12 µg/ml); lane 4, HUVEC membranes only (20 µg/ml); lane 5, HUVEC membranes only (12 µg/ml). B: lane 1, untreated ASM conditioned medium; lane 2, ASM conditioned medium incubated with ASM membranes (20 µg/ml); lane 3, ASM conditioned medium incubated with ASM membranes (12 µg/ml); lane 4, ASM plasma membranes only (20 µg/ml); lane 5, ASM plasma membranes only (12 µg/ml). Nos. on left, molecular mass.

To examine whether HUVEC membrane activation of gelatinase A was specific, we added HUVEC plasma membranes to CM from ASM in the presence and absence of TIMP-2, TIMP-1, PMSF (serine proteinase inhibitor), and E-64 (cysteine proteinase inhibitor). We found that TIMP-2 inhibited the HUVEC membrane activation of progelatinase A, but TIMP-1 did not (Fig. 3). PMSF and E64 were also unable to prevent this activation (data not shown). These results suggest that the plasma membranes of HUVECs, but not of ASM, contain an essential component required for the activation of progelatinase A. 


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Fig. 3.   HUVEC plasma membrane activation of progelatinase A secreted by ASM cells is inhibited by tissue inhibitor of matrix metalloproteinase (TIMP)-2. Conditioned medium was collected at 18 h from ASM cells, incubated in a cell-free environment for another 18 h, and run on gelatin zymograms. Top: lane 1, untreated conditioned medium; lane 2, conditioned medium incubated with HUVEC membranes (12 µg/ml); lane 3, conditioned medium incubated with HUVEC membranes (12 µg/ml) and TIMP-2 (6 µM); lane 4, conditioned medium incubated with HUVEC membranes (12 µg/ml) and TIMP-2 (24 µM); lane 5, conditioned medium incubated with HUVEC membranes (12 µg/ml) and TIMP-1 (24 µM). Nos. on right, molecular mass. Bottom: densitometry results from above zymogram. Progelatinase A activation after addition of HUVEC membranes (lane 2) is considered 100% activation. TIMP-2, but not TIMP-1, inhibited progelatinase A activation.

To determine whether culture conditions affected progelatinase A activation, ASM cells were cultured on a gelatin substrate. This had no effect on gelatinase A release or activation (data not shown).

A recent study (23) has suggested that MT1-MMP, an intrinsic membrane protein, is responsible for cell-mediated activation of progelatinase A. Based on this information, cell membrane preparations from ASM cells and HUVECs were examined by immunoblotting for the presence of MT1-MMP. Our immunoblot results suggest that ASM and HUVEC plasma membranes contain approximately equivalent amounts of MT1-MMP (Fig. 4). Northern blot analysis revealed an upregulation of MT1-MMP mRNA in ASM cells treated with PMA or Con A (Fig. 5). In HUVECs, PMA caused an increase in MT1-MMP mRNA, whereas Con A did not (data not shown).


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Fig. 4.   Immunoblotting with rabbit polyclonal antibodies to membrane type 1 matrix metalloproteinase (MT1-MMP) reveals localization of MT1-MMP in plasma membranes of ASM cells. Lane 1, molecular-mass standards (nos. on left); lane 2, plasma membranes of HUVECs; lane 3, plasma membranes of ASM cells.



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Fig. 5.   PMA and concanavalin A (Con A) do not activate ASM progelatinase A despite increasing MT1-MMP mRNA as shown by Northern blot (A) and zymogram (B). A: lane 1, untreated ASM cells; lane 2, ASM treated with Con A (40 µg/ml); lane 3, ASM treated with PMA (20 nM). B: lane 1, conditioned medium from ASM collected at 18 h; lane 2, conditioned medium from ASM treated with Con A (40 µg/ml); a faint band at 92 kDa is visualized; lane 3, conditioned medium from ASM treated with PMA (20 nM).

When membrane preparations from ASM were combined with HUVEC membrane preparations (100,000-g pellet), the ability of HUVEC membranes to activate progelatinase A in ASM CM was diminished; this result suggests the presence of an inhibitor of progelatinase A activation in the membranes of ASM cells. The inhibitory effect of ASM membranes on HUVECs was diminished with further dilution of ASM membranes (Fig. 6).


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Fig. 6.   Progelatinase A from ASM is activated by HUVEC plasma membranes; this activation is inhibited by ASM plasma membranes. Conditioned medium collected at 18 h from ASM cells was incubated in a cell-free environment for another 18 h with ASM and HUVEC plasma membranes and run on gelatin zymograms. Top: lane 1, untreated ASM conditioned medium; lane 2, ASM conditioned medium incubated with HUVEC membranes (12 µg/ml); lane 3, ASM conditioned medium incubated with both HUVEC (12 µg/ml) and ASM (12 µg/ml) plasma membranes; lane 4, ASM conditioned medium incubated with both HUVEC (12 µg/ml) and ASM (6 µg/ml) plasma membranes; lane 5, ASM conditioned medium incubated with HUVEC (12 µg/ml) and ASM (2 µg/ml) plasma membranes. Nos. on right, molecular mass. Bottom: densitometry results from above zymogram. Progelatinase A activation after addition of HUVEC membranes alone (lane 2) is considered 100% activation.

To further investigate the differences between crude plasma membrane preparations from HUVECs and ASM cells, we examined gelatinase A immunoblots. In HUVEC membranes, gelatinase A was identified primarily as a 64-kDa band, with a weaker dimer at ~72 kDa, whereas in ASM membranes, gelatinase A was present primarily as a 72-kDa band (Fig. 7). These data are consistent with activation of progelatinase A after binding to HUVEC plasma membrane. In ASM, progelatinase A binding to plasma membranes was minimal and no enzyme activation was noted.


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Fig. 7.   Activation of gelatinase A on HUVEC plasma membranes but not on ASM plasma membranes. HUVEC and ASM plasma membrane fractions (100,000 g) were run on SDS-PAGE, and immunoblotting was performed with affinity-purified rabbit polyclonal antibodies. Lane 1, recombinant gelatinase A demonstrating 72-, 64-, and 62-kDa forms (nos. on left); lane 2, HUVEC plasma membranes; lane 3, ASM plasma membranes.

TIMP-2 in ASM and HUVEC Membranes

Using immunoblotting and reverse zymography, we identified TIMP-2 bands on ASM and HUVEC crude membranes. Our densitometry results show that seven to nine times more TIMP-2 (21 kDa) was associated with ASM than with HUVEC membranes (Fig. 8). Reverse zymography also identified a 50-kDa band in both ASM and HUVEC membranes that has been previously reported (16). In the TIMP-2 Western blot of ASM membranes, there were several bands between 30 and 42 kDa. These bands have been previously described in other biological samples (17) and are of unknown significance. To determine whether the bands identified on reverse zymography were TIMP-1, we performed Western blots on CM and membranes from ASM and HUVECs. TIMP-1 was readily detected in the CM from ASM cells but not in the CM from HUVECs; TIMP-1 was not detected in the cell membrane of either cell type (Fig. 9).


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Fig. 8.   A: TIMPs identified in ASM and HUVEC membranes (15 µg protein/lane) by reverse zymography. Lane 1, HUVEC membrane fraction (100,000 g); lane 2, ASM cell membrane fraction (100,000 g). B: TIMP-2 identified in ASM and HUVEC membranes (15 µg protein/lane) by immunoblotting of equal protein amounts of plasma membranes with rabbit polyclonal antibodies to TIMP-2. Lane 1, HUVEC membrane fraction (100,000 g); lane 2, ASM cell membrane fraction (100,000 g). Nos. on left, molecular mass.



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Fig. 9.   TIMP-1 identified in ASM conditioned medium by immunoblotting with rabbit polyclonal antibodies to TIMP-1 (15 µg protein/lane). Lane 1, conditioned medium from ASM; lane 2, plasma membranes from ASM; lane 3, conditioned medium from HUVECs; lane 4, plasma membranes from HUVECs; lane 5, recombinant (R) TIMP-1; lane 6, molecular-mass standards (nos. on right).

125I-Labeled TIMP-2 Binding to ASM Cells and HUVECs

125I-labeled TIMP-2 binding to PMA-treated ASM cells demonstrated a specific and saturable pattern of binding, with a dissociation constant (Kd) of 5.37 nM and a maximum binding capacity (Bmax) of 0.048 nM (Fig. 10, A and B). The number of receptors per ASM cell was triple-bond 480,000 (0.43 fmol/µg membrane protein). This binding of 125I-labeled TIMP-2 to ASM cells was considerably less than that of 125I-labeled TIMP-2 to Con A-treated HUVECs, which exhibited a Kd of 0.77 nM and Bmax of 0.075 nM (Fig. 10, A and B). The number of receptors per endothelial cell was triple-bond 180,000 (2.67 fmol/µg membrane protein). The greater number of receptors per microgram of membrane protein for HUVECs is consistent with the smaller size of HUVECs compared with ASM cells. Maximum binding of 125I-labeled TIMP-2 to cells occurred if ASM cells were pretreated with PMA and if HUVECs were pretreated with Con A. Without PMA or Con A treatment, binding was considerably less (data not shown). These data indicate that 125I-labeled TIMP-2 binds approximately seven times more efficiently to HUVECs than to ASM cells.



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Fig. 10.   Linear plots of 125I-TIMP-2 binding showing total (T), specific (S), and nonspecific (NS) binding to ASM (A) and HUVECs (B). Maximum binding capacity (Bmax) for ASM is 0.048 nM and for HUVECs is 0.075 nM. C: 125I-labeled TIMP-2 binds to HUVECs more efficiently than to ASM cells as shown by Scatchard plot analysis of TIMP-2 binding data for ASM cells and HUVECs. Kd, dissociation constant; bound/free, ratio of bound to free TIMP-2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MMPs are important in inflammatory tissue destruction and remodeling. In this study, we systematically investigated the release and activation of gelatinases A and B from ASM cells and further used these cells to study the mechanism of progelatinase A activation. Our results demonstrate that ASM cells secrete progelatinase A constitutively and when stimulated with PMA and/or TNF-alpha , these ASM cells were induced to release gelatinase B. The release of gelatinase B through a PKC-dependent mechanism is similar to gelatinase B regulation in aortic smooth muscle (4). CM from ASM cells had large amounts of TIMP-1 and a relatively small amount of TIMP-2 compared with those in HUVECs.

The mechanism of cell surface-mediated activation of progelatinase A is of considerable current interest. Unlike what Foda et al. (5) and others (7) have reported with HUVECs, ASM progelatinase A is not activated by cells treated with PMA or Con A. We took advantage of this difference to elucidate the activation of progelatinase A. PMA- and Con A-induced HUVEC progelatinase A activation occurs through a plasma membrane-dependent mechanism (5). Progelatinase A in ASM CM was readily activated when incubated with HUVEC membrane preparations but was not activated when incubated with ASM membranes, suggesting a lack of an important "inducing factor" or the presence of an inhibitor in the membranes of ASM. The activation of progelatinase A by HUVEC membranes was specific because it was inhibited by the addition of excess TIMP-2 but not by TIMP-1. Furthermore, it is unlikely that the initial progelatinase A activation step (72-64 kDa) induced by HUVEC membranes was due to autoactivation because it was not inhibited by excess TIMP-1, which inhibits autoactivation of soluble progelatinase A (30). The data in Fig. 6, indicating that small amounts of ASM membrane have an inhibitory effect on HUVEC membrane-induced progelatinase A activation, is consistent with the presence of an inhibitory factor in ASM membranes.

Strongin et al. (25) and Zucker et al. (31) have proposed that the plasma membrane-dependent activation of progelatinase A on HT 1080 fibrosarcoma cells and endothelial cells required the formation of a trimolecular complex of progelatinase A, TIMP-2, and MT1-MMP on the cell surface (23). Free MT1-MMP (not complexed with TIMP-2) is required to convert 72-kDa progelatinase to a 64-kDa intermediate (31), which is followed by autoactivation of 64- to 62-kDa-activated gelatinase A (1, 25). We demonstrated 64-kDa gelatinase A on crude plasma membranes from HUVECs but not from ASM, which suggests both lack of binding and activation of progelatinase A on ASM. Our results indicate that although there is increased mRNA expression of MT1-MMP in ASM cells, activation of progelatinase A was not induced. In contrast, the addition of Con A to HUVECs leads to the activation of progelatinase A (5) but no increase in MT1-MMP mRNA.

Cell membranes of ASM contain a considerable amount of TIMP-2 that we suspect totally saturates the MT1-MMP binding capacity for TIMP-2. Therefore, no free MT1-MMP is available to activate gelatinase A bound to the triplex of MT-MMP1, TIMP-2, and gelatinase A. We therefore conclude that the excess TIMP-2 in the membrane contributes to the inhibition of gelatinase A activation in ASM. The relatively low levels of TIMP-2 in ASM CM is presumably secondary to high amounts of TIMP-2 bound to ASM membranes. In the experimental conditions employed for evaluating 125I-TIMP-2 binding, the cell surface is stripped of bound TIMP-2 by lowering the pH, thus permitting maximum binding of radiolabeled ligand. This effect was exaggerated with ASM compared with that with HUVECs (data not shown) and is consistent with excess endogenous TIMP-2 bound to ASM membranes. Additional studies are needed to explain the mechanism for intense binding of endogenous TIMP-2 to ASM membranes even though TIMP-2 levels are higher in HUVEC CM than in ASM CM. An abnormality of MT1-MMP function on insertion in the plasma membrane is suspected; a higher Kd for 125I-TIMP-2 binding to ASM cells compared with that to HUVECs supports this assumption.

Lohi et al. (14) have recently reported findings similar to ours in embryonic lung fibroblasts where PMA did not activate progelatinase A despite an increase in MT1-MMP mRNA. The authors suggested that this was due to the lack of a "cooperating factor"; however, they did not consider the possibility of an inhibitory phenomenon.

In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.


    ACKNOWLEDGEMENTS

We thank Dr. William G. Stetler-Stevenson (National Cancer Institute, Bethesda, MD) and Dr. Andrew J. P. Docherty (Celltech Ltd., Slough, UK) for their generous gifts of material.


    FOOTNOTES

This work was supported by funds from the American Lung Association of New York and the American Heart Association (to H. D. Foda) and by Veterans Affairs Merit Review Grant Funds (to S. Zucker).

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.

Address for reprint requests and other correspondence: H. D. Foda, Pulmonary and Critical Care Medicine, SUNY at Stony Brook, Health Science Center, Stony Brook, NY 11794-8172 (E-mail: hfoda{at}mail.som.sunysb.edu).

Received 31 August 1998; accepted in final form 10 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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