School of Kinesiology and Health Sciences, York University, Toronto, Ontario, Canada
Submitted 28 April 2004 ; accepted in final form 26 October 2004
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ABSTRACT |
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membrane type 1; matrix metalloproteinases; angiogenesis; transcriptional control; extracellular matrix; mitogen-activated protein kinase
Signaling events that initiate production of MMP-2 and MT1-MMP in endothelial cells are not well characterized. Endothelial cell-extracellular matrix interactions play critical roles in the regulation of many cellular processes, including proliferation, migration, cell shape, differentiation, and apoptosis (7, 40), and likely contribute significantly to the initiation of angiogenesis in vivo (37). Stretch of skeletal or cardiac muscle stimulates angiogenesis, and it has been hypothesized that tensional forces within the muscle during stretch are sensed by endothelial cells through perturbations of endothelial cell-extracellular matrix attachments (14, 15). Three-dimensional (3D) culture of microvascular endothelial cells within a type I collagen matrix induces the cells to reorganize themselves and the surrounding matrix, ultimately forming multi-cellular networks (27, 39). In 3D culture, cells contract the matrix, resulting in local increases in extracellular matrix tension detectable by all surrounding cells (16). Although not a complete mimicry of the angiogenic process, this model recapitulates some of the key biochemical changes that are known to occur during initiation of angiogenesis. Critically, both stretch-induced angiogenesis in vivo and network formation within a 3D collagen matrix in vitro involve upregulated production of MMP-2 and MT1-MMP (11, 34). Furthermore, broad-spectrum MMP inhibition interferes severely with capillary sprouting in vivo, and network formation in vitro (11, 41). Thus the 3D collagen matrix model provides a simplified experimental system to analyze the endothelial cell specific signaling events that regulate the production of MMP-2 and MT1-MMP in response to an altered extracellular matrix environment.
The mitogen-activated protein kinase (MAPK) family is a common participant in mediating growth factor receptor as well as adhesive and tensional signals from the cell surface to the nucleus (26). The three most well-characterized MAPK pathways are the extracellular-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and the p38 pathways. MAPKs are activated in response to physiological angiogenic stimuli, such as elevated shear stress and mechanical stretch, and they are responsible for such diverse downstream actions as initiating endothelial cell proliferation, cytoskeletal remodeling, modifying cell adhesion properties, and cell migration (5). Thus we hypothesized that endothelial cell expression of MT1-MMP and MMP-2 is regulated by the MAPK family of enzymes. In this study, we utilized a 3D type I collagen culture system to investigate the changes in activation of MAPKs in response to prolonged extracellular matrix-transduced mechanical stimulation and the effects of these changes on MT1-MMP and MMP-2 expression. We demonstrated that ERK1/2 and JNK activity is increased persistently in response to 3D collagen culture, whereas p38 activity is inhibited. Furthermore, these changes in MAPK signaling contribute significantly to the organization of endothelial cells into networks in 3D type I collagen lattices. Moreover, our findings implicate ERK1/2 as an essential mediator of 3D collagen-induced upregulation of MT1-MMP, its transactivator Egr-1, and MMP-2, and thus as a key regulator of extracellular matrix proteolysis.
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METHODS |
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Collagen cell culture. RECs were plated in monolayer (1 million cells/35 mm dish) on type I collagen-coated cell culture plates (2D) or within 3D type I collagen (2 million cells/ml of collagen). Type I collagen from calf skin (ICN) was dissolved in 10 mM acetic acid to make a final concentration of 2.5 mg/ml collagen. In preparation for 2D cultures, collagen coating of dishes was done by the addition of the acid solubilized collagen to carbonate buffer (pH 9.6) (5 µg collagen/cm2) and incubating the plates with this solution for 1 h at room temperature. The coating solution was aspirated before the cells were plated. For 3D cultures, collagen was neutralized immediately before use by the addition of 1/10th volume of x100 Earle's salt solution (Invitrogen) and drops of 2 M NaOH until the pH indicator was pink. The appropriate number of cells was centrifuged to pellet, excess media were removed, and cells were resuspended in 50 µl of DMEM before being mixed with the neutralized collagen solution. Drops of collagen (100 µl) were placed on noncoated culture dishes and allowed to polymerize at 37°C for 1015 min, followed by the addition of media. Both 2D and 3D cells were subsequently cultured for 24 h at 37°C with 7% CO2 in complete DMEM (DMEM supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, 0.3 mg/ml L-glutamine, and 0.11 mg/ml sodium pyruvate). In experiments involving MAPK inhibitors, cells were pretreated in monolayer for 3 h before 2D/3D culture with either DMSO or drug (50 µM PD-98059, 40 µM SB-203580, or 50 µM SP-600125). A fresh drug was added at the onset of 2D and 3D cultures. For all experiments, total cellular protein or RNA was isolated after 24 h.
Transient transfection.
RECs were plated in monolayer on type I collagen-coated (5 µg/cm2) 6-well cell culture plates (100,000 cells/well). The following day, they were transiently transfected with constructs expressing vector alone, or constitutively active JNK3 [MKK7-JNK3; kindly provided by Dr. L. Heasley (46) and MEK; MEK(N3/ED)-pMM9] or p38 (p38-pMT3-HA, both kindly provided by Dr. J. McDermott) using LipofectAMINE (GIBCO) in the presence of serum-reduced media (Optimem, GIBCO), according to the manufacturer's instructions. Three hours after transfection, the Optimem transfection mixture was replaced with complete DMEM. Cells were then cultured for 72 h at 37°C after which time cells were lysed in 120 mM Tris buffer (pH 8.7) containing 0.1% Triton X-100 and 5% glycerol. Total cellular protein was quantified using the bicinhoninic acid assay (Pierce) according to manufacturer's instructions. Equal quantities of cellular protein (10 µg per lane) were analyzed by gelatin zymography. Proteins were size fractionated under nonreducing conditions through an SDS-polyacrylamide (8%) gel impregnated with 0.04% gelatin (11, 21). Gels were incubated for 20 h in buffer composed of 5 mM CaCl2, 50 mM Tris·HCl, pH 8.0, before being stained with Coomassie brilliant blue. Gels were destained until desired resolution was obtained. It has been demonstrated that the amount of enzymatic activity in the zymography gel, detected as a band of clearing on a blue background, is directly proportional to the amount of MMP-2 protein (21). Gels were digitally imaged with the use of the Fluorchem system (AlphaInnotech). Band intensities were calculated using AlphaEase software, were normalized to "vector" samples, and then were expressed as fold change in MMP-2 protein.
Northern blot analysis.
Total cellular RNA was extracted using TRIzol Reagent (GIBCO) according to manufacturer's instructions and dissolved in formamide. RNA concentration was determined by measuring the optical density at 260 nm. Equal amounts of total cellular RNA extracts were electrophoresed through a formaldehyde-containing, 1% agarose gel, stained with Sybr Green (Sigma) to identify ribosomal RNA, transferred by capillary action to a nylon membrane using 10x standard saline citrate and cross-linked using ultraviolet light (Spectroliner). Membranes were prehybridized in a solution containing 44% Formamide (Fisher), 0.1% BSA, 0.1% Ficoll (Type 400), 0.1% polyvinylpyrrolidone, 0.035 M SDS, 10% Dextran sulfate, 0.075 M NaCl, 0.0075 M NaCitrate, and 0.1 mg/ml salmon sperm DNA (Sigma), and hybridized for 612 h with a cDNA probe specific for MMP-2, MT1-MMP, Egr-1 (11), or GAPDH in prehybridization solution. Probes were labeled using [-32P]dCTP (Amersham Pharmacia) and NEBlot Kit (NEB) according to manufacturer's instructions and purified using ProbeQuant G-50 Micro Columns (Amersham Pharmacia). Blots were exposed to film (MP Hyperfilm, Amersham Pharmacia) and the signal intensity measured by densitometry using AlphaEase (Alpha Innotech) software. Blots were normalized for lane loading with the use of either the 28S ribosomal RNA or GAPDH.
Immunoblotting.
Total cellular protein was extracted in the presence of 0.1% protease inhibitor cocktail composed of 4-(2-aminoethyl)benzensulfonyl fluoride, aprotinin, bestatin, E-64, leupeptin, pepstatin A (Sigma), and 1 µM sodium orthovanadate, in a 120 mM Tris buffer (pH 8.7) containing 0.1% Triton X-100 and 5% glycerol. Total cellular protein was quantified using the bichoninic acid assay (Pierce) according to manufacturer's instructions. Equal concentrations of protein extracts were electrophoresed through a 12% denaturing SDS-polyacrylamide gel and transferred to a polyvinylidine difluoride membrane (Immobilon-P, Sigma). The membrane was blocked in 0.5% Tween-Tris-buffered saline (TTBS) solution containing 5% nonfat milk and then incubated overnight with antibodies directed against phosphoERK (NEB), phosphoJNK (NEB), or phosphop38 (Santa Cruz), diluted 1:500 in TTBS containing 5% nonfat milk or 5% BSA. The following day, the membrane was incubated in horseradish peroxidase-conjugated antibodies directed against mouse, rabbit, or goat IgG (Pierce) and protein expression detected using enhanced chemiluminescence (SuperSignal West Pico, Pierce). Subsequently membranes were stripped in 62.5 mM Tris containing 2% SDS and 0.7% -mercaptoethanol and reprobed using antibodies directed against total ERK1/2 (NEB), JNK1/2 (NEB), or p38 (Santa Cruz), diluted 1:500 in TTBS containing 5% nonfat milk. Protein expression was visualized using Fluorchem Imager (Alpha Innotech) and quantified by densitometry using Alpha Ease (Alpha Innotech) software. Phospho-MAP kinase band intensities were normalized to nonphospho band intensities then expressed as a ratio to 2D conditions.
Microscopy and photography. Cells were cultured on glass coverslips on 2D or 3D type I collagen matrixes for 24 h in the presence of vehicle or inhibitors. Digital images of the cells were taken using a Canon G5 digital camera mounted to an inverted Zeiss Axiovert 25 microscope, using a x10 objective [numerical aperture (NA) = 0.25] and phase contrast optics. Cells were fixed using 3.75% formaldehyde, then blocked and permeabilized in PBS + 5% normal goat serum + 0.05% Triton X-100. Some conditions were stained with primary antibody corresponding to phosphoERK1/2 (diluted 1:300 in blocking solution), then with secondary goat anti-mouse-Alexa 568 (Molecular Probes; diluted 1:500 in blocking solution). All conditions then were incubated with FITC-conjugated phalloidin (Sigma) diluted 1:500 in blocking solution and counterstained with 4',6'-diamidino-2-phenylindole (DAPI; Molecular Probes) before visualization on an inverted light microscope (Axiovert 200-M, Zeiss) using a x10 (NA = 0.25) or a x63 oil (NA = 1.40) objective. Images were captured using a Quantix digital cooled charge-coupled device camera (Photometrics) and Metamorph Software (Universal Imaging). Network quantification was performed on phalloidin-FITC and DAPI-counterstained images acquired with the x10 objective by quantifying network length using distance calipers and single cells using the object counting tool in Metamorph software. A minimum of three fields of view from three separate experiments were quantified for network lengths and number of single cells. Networks were defined to be composed of more than one cell (based on nuclear stain) connected to each other by end-to-end (rather than lateral) cellular extensions. Statistical analysis was performed on the ratio of the total network length per field of view divided by the number of single cells within that same field of view.
Statistics. Data are presented as means ± SE. Statistical significance for each data set was tested using the paired, two-tailed Student's t-test or one-way ANOVA, followed by post hoc Tukey testing, with statistical significance set at P < 0.05.
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RESULTS |
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DISCUSSION |
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Microvascular endothelial cells respond to a 3D type I collagen matrix by elongating and forming filopodia-like projections that extend to form contact points with adjacent cells, thus establishing cell-cell networks. This model provides a means to identify the biochemical events that initiate the morphological changes of elongation, sprouting, and network formation. MAPK cascades are known transducers of extracellular mechanical signals (26), and thus it is not surprising that we detected prolonged changes in MAPK activity in endothelial cells cultured in 3D type I collagen lattices. Manipulation of MAPK activity using specific MAPK inhibitors profoundly altered the cellular network formation that normally occurs in microvascular endothelial cells in response to 3D type I collagen culture. Persistent changes in the activation states of the MAPK pathways likely contribute to numerous events that are involved in cellular network formation within a 3D type I collagen matrix.
Inhibition of ERK1/2 using PD-98059 markedly inhibited network formation in endothelial cells cultured in 3D. Thus ERK1/2 activity appears to be critical for the morphogenetic "switch" from a monolayer (2D) to a branching network (3D) in response to a collagen matrix. This is consistent with the current theory that sustained ERK activation induces changes in cytoskeletal organization, thereby influencing cell shape, cellular motility and invasiveness (3, 8, 46). The striking localization of phosphoERK1/2 with actin at adhesion contacts and along stress fibers supports a role for ERK1/2 in the transduction of signals in response to adhesion and cytoskeletal perturbations. This localization of phosphoERK1/2 has been noted in other cell types (20, 25), and several studies indicate that cytoskeletal arrangement critically determines not only the subcellular distribution of ERK1/2 but also the ability of phosphoERK1/2 to translocate to the nucleus (1, 9, 19).
In addition, we demonstrated that ERK1/2 activity is required for the induction of Egr-1, MT1-MMP, and MMP-2 expression in endothelial cells cultured within 3D type I collagen. The ERK1/2 signaling cascade has been reported to regulate MMP-13 expression in response to changes in mechanical stress in fibroblasts (23). Our findings directly support previous studies that have reported that mechanical forces, such as fluid shear stress and cyclic mechanical strain, transiently induce Egr-1 expression in endothelial and epithelial cells through activation of the ERK1/2 MAPK signaling pathway (44). The prolonged elevation of Egr-1 in our study correlates with the sustained phosphorylation of ERK1/2 and JNK1/2 at 24 h of 3D culture. Because inhibition of either ERK1/2 or JNK1/2 significantly reduced Egr-1 levels, it is reasonable to conclude that the continued activation of both these pathways contributes directly to the elevated level of Egr-1 mRNA.
Inhibition of JNK using SP-600125 significantly impaired endothelial cell network formation within 3D type I collagen matrixes, leaving many cells rounded and incapable of elongation. Although JNK activation is most often associated with an apoptotic response, it is also known that JNK regulates cellular migration in a variety cell types, including endothelial cells (35), fibroblasts (18), fish keratocytes, and rat bladder tumor epithelial cells (13). These findings are consistent with our observations that endothelial cells treated with SP-600125 attach but fail to elongate or migrate through the collagen matrixes. In addition, JNK has been linked to cell survival as a positive regulator of vascular endothelial growth factor (VEGF) expression in osteoblast-like MC3T3-E1 cells (38, 42). VEGF is a potent endothelial cell mitogen and in endothelial cells in 3D matrixes VEGF sensitivity is augmented through upregulation of VEGF receptors (33), suggesting that VEGF is important for cell survival in 3D culture. Thus persistent activation of JNK may contribute not only to the migratory abilities of the endothelial cells but also to endothelial cell survival in 3D type I collagen matrixes. Inhibition of JNK activity did not affect MMP-2 or MT1-MMP mRNA levels, although a significant suppression of Egr-1 was detected. This was surprising considering the severe morphological effects of JNK inhibition on 3D cultured endothelial cells but was confirmed by prolonged (48 h) JNK inhibition and higher doses (100 µM) of the JNK inhibitor. Considering that, to date, Egr-1 is the only defined regulator of MT1-MMP transcription in endothelial cells, the failure of JNK inhibition to attenuate MT1-MMP expression despite the downregulation of Egr-1 expression suggests that additional transcription factors are involved in the regulation of MT1-MMP in response to 3D type I collagen. Thus we conclude that JNK contributes to endothelial cell network formation but is not required for the 3D collagen-induced transcriptional activation of MT1-MMP or MMP-2 in these cells.
By contrast, p38 inhibition supported endothelial cell network formation and completely negated the effects of ERK1/2 inhibition. We found that suppression of p38 activity resulted in enhanced ERK1/2 phosphorylation, indicating the existence of cross-talk between these signaling pathways in endothelial cells. Significantly, inhibition of p38 in combination with ERK1/2 inhibition reversed the negative effect of ERK1/2 inhibition on MMP-2 and MT1-MMP mRNA levels. Numerous researchers have reported that p38 suppresses ERK1/2 signaling (36, 47, 48). This suppression may occur through inhibition of upstream activators of ERK1/2 or through direct physical interaction between phospho-p38 and ERK1/2 (35). Consistent with our findings, inhibition of p38 has been reported to enhance vascular sprouting and tube formation, promote migration and reduce endothelial cell permeability, whereas activation of p38 promotes apoptosis (17, 29, 45). Thus we suggest that the functional significance of persistent p38 inhibition in response to 3D-induced mechanical stimulation may be to promote network formation and stability primarily through relieving the inhibition of ERK1/2.
Transient transfection of constitutively active MAPK constructs complimented the results obtained using pharmacological inhibitors. The p38 and JNK constructs did not affect MMP-2 protein production, whereas expression of constitutively active MEK1 induced MMP-2 protein level but not activation. However, we found that MT1-MMP mRNA was elevated, implying that MT1-MMP transcription was stimulated by the activated MEK but that cell surface MT1-MMP was not capable of activating MMP-2. In support of this observation, Galvez and colleagues (10) demonstrated that endothelial cells plated to confluency on collagen type I exhibited minimal activation of MMP-2, despite the presence of MT1-MMP on the cell surface, and further provided evidence that association of MT1-MMP with 1 integrin inhibited MT1-MMP-dependent activation of MMP-2. Considering that the organization of cellular adhesions and cytoskeletal components differs markedly between cells cultured in 2D compared with those cultured in 3D (6), it is possible that MT1-MMP association with
1-integrin is disrupted in 3D culture, leading to increased activation of MMP-2. Further studies are necessary to examine this relationship. Our results contrast with a recent report that ERK1/2 activity is critical and sufficient to induce MMP-2 expression in fibroblasts, but has no effect on MT1-MMP expression (22), but are in agreement with another study by Montesano et al. (30), which reported that expression of constitutively active MEK1 results in significant increases in both MT1-MMP and MMP-2 expression in canine epithelial cells.
In summary, our study demonstrates the prolonged activation of ERK1/2, JNK, and inhibition of p38 MAPK in response to a persistent 3D type I collagen stimulation. These changes in signaling contribute significantly to the process of microvascular endothelial cell network formation in 3D type I collagen matrixes. Functionally, we have demonstrated that ERK1/2 activation is essential for upregulation of both MT1-MMP (via Egr-1) and MMP-2 expression in response to the 3D collagen matrix. These findings provide a link toward increasing our understanding of the coordinated role that these signaling cascades play in the initiation of angiogenesis. Our results provide evidence that, through regulation of MT1-MMP and MMP-2 expression, the ERK1/2 signaling cascade is a potential target for therapeutic intervention in the regulation of matrix proteolysis, cell invasion, and capillary formation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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