Am J Physiol Cell Physiol Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 291: C579-C588, 2006. First published May 3, 2006; doi:10.1152/ajpcell.00300.2005
0363-6143/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/4/C579    most recent
00300.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ispanovic, E.
Right arrow Articles by Haas, T. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ispanovic, E.
Right arrow Articles by Haas, T. L.

PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

JNK and PI3K differentially regulate MMP-2 and MT1-MMP mRNA and protein in response to actin cytoskeleton reorganization in endothelial cells

Eric Ispanovic and Tara L. Haas

School of Kinesiology and Health Sciences, York University, Toronto, Ontario, Canada

Submitted 17 June 2005 ; accepted in final form 28 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK-dependent signaling pathway in endothelial cells. The pharmacological actin depolymerization agent cytochalasin D increased expression of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c-Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of phosphoinositide 3-kinase (PI3K), but not JNK, significantly decreased the amount of active MMP-2 following cytochalasin D stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF-induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF-induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signaling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression, whereas PI3K regulates protein levels of both MMP-2 and MT1-MMP.

angiogenesis; mechanotransduction; vascular endothelial growth factor; c-Jun; phosphoinositide 3-kinase; membrane type 1-matrix metalloproteinase

ANGIOGENESIS IS THE GROWTH of new capillaries from preexisting mature ones and occurs through a cascade of events in which disruption of the endothelial adherens junctions by way of actin cytoskeleton reorganization and proteolysis of the basement membrane and interstitial matrix are critical steps. Remodeling of both the basement membrane and interstitial matrix is facilitated by the expression and activation of matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent enzymes (7, 54). Each MMP has substrate specificity for a limited set of extracellular matrix proteins (for review, see Ref. 53). MMPs are key participants in several steps of the angiogenic response, including regulation of endothelial cell permeability, migration, invasion, and tubule formation (16, 23). MMP-2 and membrane type 1 (MT1)-MMP are produced by endothelial cells and can degrade type I and IV collagen (2, 41). MMP-2-deficient mice have reduced tumor, corneal, and retinal angiogenesis (27, 29, 43), whereas MT1-MMP-deficient mice fail to gain weight, have deficient connective tissue metabolism, and die 3–4 wk after birth (26, 61). Combined MMP-2 and MT1-MMP deficiency causes embryonic lethality, highlighting the potential of these MMPs as key targets in controlling angiogenesis (42).

The actin cytoskeleton creates a scaffold that provides structural stability and the organization of signaling molecules. In endothelial cells, shear stress and mechanical stretch cause actin stress fiber reorganization in the direction of flow and perpendicular to the axis of stretch, respectively (8, 11, 17). This reorganization of the actin cytoskeleton increases MMP-2 and MT1-MMP expression (20, 48). Growth factors such as VEGF induce changes in endothelial cell permeability by remodeling the actin cytoskeleton (47). These changes in the actin cytoskeleton may contribute significantly to the initial signaling events during angiogenesis. However, the signaling events that connect actin reorganization to MMP-2 production have yet to be determined.

Numerous signaling cascades may be activated with reorganization of the actin cytoskeleton. These include the mitogen-activated protein kinases (MAPK), specifically extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), focal adhesion kinase (FAK), Rho-family GTPases (Rho, Rac, and Cdc42), and their downstream effector, p21-activated kinase (PAK), as well as phosphoinositide 3-kinase (PI3K) (1, 5, 8, 30, 33, 56). The MAPKs are implicated in controlling a number of angiogenic processes (migration, proliferation) with ERK and JNK specifically implicated in the production and activation of MMP-2 (9, 13, 38, 40). The MAPKs can activate numerous transcription factors, including the AP-1 family, which then initiate gene expression.

We hypothesized that expression of endothelial MMP-2 and MT1-MMP in response to depolymerization of the actin cytoskeleton requires MAPKs. We have shown that in primary cultures of rat skeletal muscle endothelial cells (SMEC), treatment with cytochalasin D activates JNK and increases both MMP-2 and MT1-MMP mRNA, which results in increased MMP-2 production and activation. These increases in MMP-2 and MT1-MMP involve JNK- and PI3K-dependent pathways. Downstream of JNK activation, we have identified c-Jun as a transcriptional activator regulating MMP-2. Furthermore, the potent angiogenic factor VEGF increases MMP-2 mRNA and MMP-2 and MT1-MMP protein production, also through JNK- and PI3K-dependent pathways. Together, these data implicate JNK/c-Jun, in conjunction with PI3K, as physiological regulators of MMP-2 and MT1-MMP in endothelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. SMEC were isolated from extensor digitorum longus muscles and cultured as previously described (21), using protocols approved by the York University Animal Care Committee and in compliance with the American Physiological Society's "Guiding Principles in the Care and Use of Animals." Cells were used for experiments between passages 4 and 11 and plated on type I collagen (12.5 µg collagen/ml coating buffer)-coated culture dishes. For inhibitor studies, SMEC were pretreated for 3 h with either 50 µM PD-98059, 10 µM U0126, 50 or 100 µM SP600125, 40 µM SB-203580, or 50 µM LY-294002 and then treated with either 1 or 10 µM cytochalasin D for 24 h, 25 ng/ml VEGF or 1 µg/ml anisomycin (Sigma) for 7 h. All inhibitors were purchased from Calbiochem.

Immunofluorescence staining. Cells were plated on collagen-coated glass coverslips and cultured in the presence or absence of 25 ng/ml VEGF for 24 h at 37°C before staining. Cells were fixed with 3.75% paraformaldehyde and then blocked and permeabilized in PBS plus 5% normal goat serum and 0.05% Triton X-100. Cells were then stained with primary phospho-JNK antibody (1:300 dilution; Upstate) and then with secondary goat anti-rabbit Alexa-568 (1:400 dilution; Molecular Probes). Nuclei were counterstained with 4',6'-diaminodino-2-phenylindole (1:1,500 dilution; Molecular Probes). Cells were visualized using fluorescence microscopy (Zeiss Axiovert 200M). Images were captured using a cooled digital charge-coupled device camera (Quantix 57) and imaging software (Metamorph; Universal Imaging).

Gelatin zymography. Cells were lysed in 120 mM Tris·HCl (pH 8.7), 0.1% Triton X-100, and 5% glycerol supplemented with protease inhibitors (Sigma) and sodium orthovandate (lysis buffer). Protein (10 µg), as determined using bicinchoninic acid assay (BCA; Pierce), was separated on an 8% SDS-polyacrylamide gel containing 0.02% gelatin (20). The gel was incubated at 37°C for 20–24 h in buffer containing 50 mM Tris·HCl buffer (pH 7.6) with 5 mM CaCl2, after which the gels were fixed with 50% methanol and 10% acetic acid and, finally, stained with 0.25% Coomassie blue protein stain. Gels were visualized and imaged using the Fluorchem gel doc system and analyzed using Alphaease software (Alpha Innotech). Total MMP-2 protein expression was calculated as the sum of the latent (72 kDa) and active (62 kDa) bands, whereas the amount of active MMP-2 was calculated as the percentage of active compared with total MMP-2 protein. For PI3K inhibitor treatments, the amount of active MMP-2 (62 kDa) was expressed as the increase relative to cytochalasin D.

Northern blot. Total RNA was isolated by lysing cells in TRIzol reagent (GIBCO) and quantified using spectrophotometry at 260 nm. RNA (10 µg) was separated by formaldehyde-containing 1% agarose gel electrophoresis, and gels were stained with SYBR green (Sigma) to visualize 28S/18S ribosomal RNA bands. Gels were transferred overnight to a nylon membrane (GeneScreen Plus; NEN Life Science Products) and probed with 32P-labeled MMP-2 or MT1-MMP cDNA (26). The membrane was exposed to film for 1 h to 7 days at –80°C. The film was developed and scanned, and densitometry was performed using Alphaease software. Loading was normalized to the 28S band.

Western blot. SMEC were lysed using lysis buffer or 1x loading buffer (37.75 mM Tris·HCl, 65 mM DTT, 3.75% SDS, 5% glycerol, and 0.003% bromphenol blue), and 10 µg of protein or 30 µl were separated by SDS-PAGE, respectively. Proteins were transferred to PVDF membrane (Millipore) using the semidry transfer method. Primary antibodies (phospho-c-Jun, beta-actin, and phospho-ERK/ERK, Cell Signaling; phospho-p38/p38, Santa Cruz Biotechnology; MT1-MMP, Chemicon) were incubated overnight at 4°C with gentle agitation and secondary antibodies (Amersham) for 1 h at room temperature. Bound antibodies were detected using chemiluminescence (SuperSignal West Pico Chemiluminescent; Pierce). The film was developed and scanned, and densitometry was performed using Alphaease software. Phosphorylated values were normalized to total values to account for variability in loading.

JNK kinase assay. JNK was immunoprecipitated by incubating 150–200 µg of protein from total cell lysates with 2 µg of JNK antibody (Upstate) for 1 h at 4°C with gentle rocking. Protein A-agarose beads (Pierce) were then added to the lysates and allowed to bind to the JNK antibody for 1 h at 4°C with gentle rocking. The antibody-protein A-agarose complex was collected by centrifugation, and the pellet was washed. The lysates were then incubated in the presence of 1x assay buffer (20 mM MOPS, 25 mM beta-glycerolphosphate, pH 7.2, 1 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol), magnesium/ATP cocktail (10 µM nonradioactive ATP and 75 mM MgCl2 in assay buffer), 1 µCi/µl {gamma}-[32P]ATP (Amersham) and 2 µg/µl glutathione S-transferase-c-Jun (substrate; Cell Signaling) at 37°C for 30 min with gentle shaking. The reaction was terminated by the addition of denaturing loading buffer, and the proteins were separated by 10% SDS-PAGE as previously described. The gels were fixed with 50% methanol, 10% acetic acid, and 3% glycerol, transferred to Wattman paper, and vacuum dried overnight. Dry gels were then exposed to X-ray film for 1–7 days at –80°C and then developed, scanned, and analyzed using Alphaease software.

Surface biotinylation. SMEC were cultured on type I collagen for 24 h and then stimulated with 25 ng/ml VEGF for 60 min. Cells were washed with ice-cold PBS and incubated with 1 mg/ml Sulfo-NHS-biotin in PBS for 30 min on ice. The reaction was terminated by washing the cells with 100 mM glycine for 20 min. Cells were then lysed as previously described, and 75 µg of protein from total cell lysates were incubated with streptavidin-agarose beads (Pierce) overnight at 4°C with gentle rocking. The streptavidin-agarose complex was collected by centrifugation, and the pellet was washed with PBS containing 0.1% Nonidet P-40. Next, 50 µl of 1x loading buffer was added, the samples were boiled, and the proteins were separated by 10% SDS-PAGE as previously described.

Reverse transcription and quantitative real-time PCR. cDNA from SMEC was produced without RNA isolation using the Cells-to-cDNA kit (Ambion, TX). Briefly, SMEC cells were washed three times in sterilized PBS at 4°C and then heated (75°C for 15 min) in cell lysis buffer II. Next, the cell lysate was treated with DNase 1 (0.004 U/µl) to degrade genomic DNA (37°C for 15 min), followed by inactivation of DNase (75°C for 5 min). The cell lysate was stored at –20°C until it was used for the reverse transcription reaction. Cell lysate (10 µl) was reverse transcribed in a 20-µl reaction by using reagents from the Cells-to-cDNA kit according to the manufacturer's protocols. The cDNA was diluted fourfold with RNase-free water. Quantitative real-time PCR (Q-PCR) was performed using the ABI PRISM 7700 sequence detection system. VIC-labeled control rRNA and a FAM-labeled MT1-MMP probe and primers set were purchased from Applied Biosystems (catalog nos. P/N4308329 and Mm00485954-m1, respectively). Primers and TaqMan FAM-labeled probes for MMP-2 were designed using PrimerExpress 1.0 software (PerkinElmer Life Sciences): MMP-2 probe, 6FAM-caa tgc tga tgg aca gcc ctg ca-MGBNFQ; forward primer, CCA TGA AGC CTT GTT TAC CA; reverse primer, CTG GAA GCG GAA CGG AAA (for siRNA experiments, FAM-labeled MMP-2 probe and primers set was purchased from Applied Biosystems, catalog no. Rn01538174_m1). A 25-µl reaction mixture contained 12.5 µl of TaqMan universal PCR master mix (PCR Mix; Applied Biosystems), 4 µl of cDNA template, and the appropriate concentrations of gene-specific primers and probe sets. PCR was performed with thermal conditions as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Cycle threshold (Ct) values were used to determine the amount of MMP-2 and MT1-MMP mRNAs and 18S rRNA for all groups. The mean Ct values of triplicate samples from each group were determined, and then {Delta} Ct(sample) was calculated according to the equation {Delta}Ct(sample) = average Ct(rRNA) – average Ct(sample). Changes in MMP-2 and MT1-MMP mRNA expression following VEGF or anisomycin treatment were calculated using the {Delta}{Delta}Ct method as described in the Applied Biosystems manual (as described and validated in Ref. 39). The mRNA expression levels of target genes were expressed relative to the appropriate untreated control, which was set to 1.0.

siRNA. c-Jun siRNA (30 nM; Ambion) or equal amounts of negative control were incubated in 50 µl of Opti-MEM containing siPORT-neoFX (3 µl/ml; Ambion) for 15 min at room temperature per the manufacturer's instructions. The complexes were then added to 45,000 SMEC plated in 24-well plates or 90,000 SMEC plated in 12-well plates and incubated at 37°C for 48 or 72 h for mRNA or protein analysis, respectively.

Statistics. Data were normalized to control values and are presented as means ± SE relative to controls. Student's t-test or one-way ANOVA, followed by Tukey's post hoc tests, was applied to determine statistical significance (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Depolymerization of the actin cytoskeleton induces MMP-2 and MT1-MMP expression. Treatment of endothelial cells for 24 h with 10 µM cytochalasin D resulted in cell rounding, consistent with complete depolymerization of the actin cytoskeleton (data not shown). Previous studies showed that cytochalasin D treatment increases production and activation of MMP-2 in fibroblasts, and we verified this finding in microvascular endothelial cells (18). Treatment of cells with cytochalasin D increased total MMP-2 protein (1.96 ± 0.29-fold vs. control, P ≤ 0.05) and the percentage of active MMP-2 protein (42.87 ± 5.6 vs. 5. 28 ± 4.42%, P ≤ 0.05) as detected by gelatin zymography. Cytochalasin D treatment also increased MMP-2 and MT1-MMP mRNA expression (1.59 ± 0.06- and 1.90 ± 0.19-fold vs. control, respectively, P ≤ 0.05).

JNK, but not ERK1/2 or p38, regulates MMP-2 mRNA in response to actin cytoskeleton reorganization. The MAPKs are known to be key regulators of MMP-2 and MT1-MMP in response to various stimuli (1, 4, 45). To determine the involvement of the MAPKs in controlling MMP-2 and MT1-MMP production induced by actin cytoskeleton rearrangement, we pretreated endothelial cells with specific inhibitors to JNK, ERK1/2, or p38 before cytochalasin D treatment. Cytochalasin D induced JNK activity in SMEC, with a trend for increased JNK activity seen as early as 30 min posttreatment and a significant increase at 4 h (Fig. 1A). Treatment of SMEC with SP600125 resulted in a nonsignificant attenuation of cytochalasin D-induced MMP-2 protein expression (P = 0.37) and had no effect on active MMP-2 (cytochalasin D: 45.54 ± 1.67% vs. cytochalasin D + SP600125: 45.80 ± 1.69%) (Fig. 1B). Inhibition of JNK significantly attenuated MMP-2 mRNA and MT1-MMP mRNA (Fig. 1, C and D). Inhibition of ERK1/2 with either PD-98059 or U0126 did not affect MMP-2 production or activation in response to cytochalasin D treatment (data not shown). Inhibition of p38 with SB203580 increased MMP-2 protein expression and induced MMP-2 activation without altering endothelial cell morphology (Fig. 2). Addition of cytochalasin D to endothelial cells pretreated with SB further augmented MMP-2 protein production, suggesting that inhibition of p38 stimulates MMP-2 protein production by a pathway distinct from that initiated by cytochalasin D (Fig. 2).


Figure 1
View larger version (37K):
[in this window]
[in a new window]
  		Fig. 1. Cytochalasin D increases JNK activity, and JNK regulates matrix metalloproteinase-2 (MMP-2) and membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA expression. Rat skeletal muscle endothelial cells (SMEC) treated with cytochalasin D showed a significant increase in JNK activity as assessed by kinase assay (A). SMEC pretreated with 100 µM SP600125 were treated with 10 µM cytochalasin D for 24 h. MMP-2 production and activation was measured using gelatin zymography (B). MMP-2 (C) and MT1-MMP mRNA (D) were quantified using Northern blot analysis. C, control; CD, cytochalasin D; SP, SP600125. Values are means ± SE, n = 3. *P < 0.05 vs. control. #P < 0.05 vs. cytochalasin D.

 

Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Inhibition of p38 increases MMP-2 production and activation. SMEC pretreated with SB-203580 were treated with 1 µM cytochalasin D for 24 h. MMP-2 production and activation were measured using gelatin zymography. SB, SB-203580. Values are means ± SE, n = 3. *P < 0.05 vs. control. #P < 0.05 vs. cytochalasin D.

 
Activation of JNK using anisomycin increased both MMP-2 and MT1-MMP mRNA expression as evidenced by RT-PCR (Fig. 3, A and B). On the basis of data supporting a role for JNK in the regulation of MMP-2 and MT1-MMP, we examined the involvement of c-Jun as a transcriptional regulator of MMP-2 and MT1-MMP. Transfection of c-Jun siRNA into SMEC decreased basal levels of c-Jun protein by 40% (data not shown). c-Jun siRNA inhibited the anisomycin-induced MMP-2 mRNA expression (Fig. 3C).


Figure 3
View larger version (5K):
[in this window]
[in a new window]
  		Fig. 3. Activation of JNK is sufficient to induce MMP-2 and MT1-MMP mRNA expression and requires c-Jun. SMEC were treated with 250 ng/ml or 1 µg/ml anisomycin to induce JNK activation. MMP-2 (A) and MT1-MMP mRNA (B) were measured using TaqMan real-time RT-PCR. C: SMEC were treated with 30 nM c-Jun or negative control small interference RNA (siRNA) and then stimulated with 1 µg/ml anisomycin. aniso, Anisomycin; si, siRNA. Values are means ± SE, n = 3. *P < 0.05 vs. control. #P < 0.05 vs. anisomycin.

 
PI3K is required for MMP-2 protein production and activation. We considered that PI3K could be involved in the regulation of MMP-2 and MT1-MMP in response to cytoskeletal remodeling, either by directly activating JNK or by activating a parallel pathway by way of Akt (49). Inhibition of PI3K with LY-294002 did not affect cytochalasin D-induced MMP-2 mRNA expression (Fig. 4A) or cytochalasin D-induced MT1-MMP mRNA expression (Fig. 4B). On the other hand, total MMP-2 protein was decreased with PI3K inhibition (Fig. 4C), as was the amount of active MMP-2 protein (Fig. 4D).


Figure 4
View larger version (35K):
[in this window]
[in a new window]
  		Fig. 4. MMP-2 protein production is dependent on phosphoinositide 3-kinase (PI3K). SMEC pretreated with the PI3K inhibitor LY-294002 were treated with 1 µM cytochalasin D for 24 h. MMP-2 (A) and MT1-MMP mRNA (B) were quantified using Northern blot analysis. Total (C) and active MMP-2 (D) was measured using gelatin zymography. LY, LY-294002. Values are means ± SE, n = 3. *P < 0.05 vs. control. #P < 0.05 vs. cytochalasin D.

 
Simultaneous inhibition of PI3K and JNK (100 µM SP600125 and 50 µM LY-294002) abolished the cytochalasin D-induced increase in total MMP-2 protein expression (Fig. 5A) with a significant decrease in the amount of active MMP-2 protein (Fig. 5B). The decrease in active MMP-2 protein (shown in Figs. 4D and 5B) suggested that there was a concurrent decrease in MT1-MMP protein, and this was verified with Western blotting (Fig. 5C). Similar to the effect of JNK inhibition alone (Fig. 1), dual inhibition of JNK and PI3K prevented a cytochalasin D-mediated increase in MMP-2 (cytochalasin D: 1.81 ± 0.12 above control vs. cytochalasin D + LY-294002/SP600125: 0.94 ± 0.23 relative to control) or MT1-MMP mRNA expression (cytochalasin D: 1.46 ± 0.13 above control vs. cytochalasin D + LY-294002/SP600125: 1.1 ± 0.23 above control).


Figure 5
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5. Increased MMP-2 and MT1-MMP protein production in response to cytochalasin D is mediated by PI3K/JNK. SMEC pretreated with 50 µM SP600125 and 50 µM LY-294002 were treated with 1 µM cytochalasin D for 24 h. MMP-2 production (A) and activation (B) were measured using gelatin zymography. MT1-MMP protein level (C) was measured by Western blotting. Values are means ± SE, n = 3. *P < 0.05 vs. control. #P < 0.05 vs. cytochalasin D.

 
VEGF induces production and activation of MMP-2 via JNK and PI3K. VEGF, which plays a significant role as a growth factor in activity-induced angiogenesis in skeletal muscle, induces modification of the actin cytoskeleton and influences endothelial cell permeability and migration during angiogenesis (15, 24, 58). Because VEGF activates JNK (47), we hypothesized that VEGF mediates increased MMP-2 and MT1-MMP expression in SMEC via JNK. Stimulation of SMEC with VEGF (25 ng/ml) caused nuclear translocation of phospho-JNK and increased total cellular phospho-JNK staining (Fig. 6, A and B). MMP-2 protein secretion, production, and activation were increased in response to VEGF treatment for 24 h (Fig. 6, C–E). Similar to the cytochalasin D-induced MMP-2 response, VEGF-induced protein production and activation of MMP-2 were inhibited by pretreatment with 10 µM LY-294002 (Fig. 6, D and E). RT-PCR analysis showed that VEGF increased MMP-2 mRNA after 24 h, and JNK inhibition with SP600125 blocked the VEGF-induced increase in MMP-2 mRNA expression (Fig. 6F). c-Jun siRNA treatment also significantly attenuated the VEGF-induced increase in MMP-2 mRNA expression (Fig. 6G).


Figure 6
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 6. VEGF-induced MMP-2 mRNA expression is dependent on JNK. SMEC in the absence (A) or presence (B) of 25 ng/ml VEGF were stained for phospho-JNK (red) and 4',6'-diaminodino-2-phenylindole (blue, nuclei). Arrows denote nuclear localization of phospho-JNK. Media were collected from SMEC treated with 25 ng/ml VEGF, and MMP-2 production was analyzed using gelatin zymography (C). SMEC pretreated with 50 µM SP600125 and/or 50 µM LY-294002 were treated with 25 ng/ml VEGF for 24 h. MMP-2 production (D) and activation (E) were measured using gelatin zymography. SMEC pretreated with 50 µM SP600125 (F) or 30 nM c-Jun or negative control siRNA (G) were then stimulated with 25 ng/ml VEGF for 24 h, and MMP-2 mRNA was measured using TaqMan real-time RT-PCR. Values are means ± SE, n = 3 (n = 5 for C). *P < 0.05 vs. control. #P < 0.05 vs. VEGF.

 
VEGF increases MT1-MMP protein, but not mRNA, expression. Because VEGF induced activation of MMP-2, we tested the effects of VEGF on MT1-MMP. Interestingly, VEGF did not induce an upregulation in MT1-MMP mRNA (Fig. 7A). Total MT1-MMP was increased after 24 h of VEGF stimulation and was abrogated by pretreatment with 50 µM LY-294002 (Fig. 7B). VEGF is known to increase the amount of MT1-MMP on the cell surface and to cause its localization to caveolae (34, 35). Consistent with these reports, we found that short exposure (60 min) to VEGF increased the amount of MT1-MMP on the cell surface as measured by surface biotinylation, with no change in the amount of MT1-MMP protein in total cell lysates (Fig. 7C).


Figure 7
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 7. VEGF-induced MT1-MMP protein production is dependent on PI3K. SMEC pretreated with 50 µM SP600125 were then stimulated with 25 ng/ml VEGF for 24 h. MT1-MMP mRNA was measured using TaqMan real-time RT-PCR (A). SMEC pretreated with 50 µM SP600125 and/or 50 µM LY-294002 were treated with 25 ng/ml VEGF for 24 h. MT1-MMP protein level was measured using Western blotting (B). Cell surface MT1-MMP protein in response to short-term VEGF treatment (25 ng/ml for 60 min) was measured using surface biotinylation (C). Values are means ± SE, n = 3. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study provides novel evidence that JNK regulates both MMP-2 and MT1-MMP mRNA expression in response to actin cytoskeleton reorganization in microvascular endothelial cells. Downstream of JNK, c-Jun is a regulator of MMP-2 transcription. VEGF, a potent vascular permeability factor and angiogenic mediator, induces MMP-2 mRNA expression via JNK and increases MMP-2 protein production and activation. PI3K appears to regulate MMP-2 and MT1-MMP protein processing independently of mRNA synthesis.

Reorganization of the endothelial actin cytoskeleton occurs very early in the initiation of angiogenesis, in response to growth factors, cytokines, and inflammatory mediators or as a result of mechanotransduction (22, 31, 47, 55). This reorganization may contribute to the process by which quiescent endothelial cells become activated and make the "angiogenic switch." Initially, we avoided the pleiotropic signals initiated by growth factors by using cytochalasin D to initiate cytoskeletal alterations independently of a specific external stimulus. Our results point to JNK as a transcriptional regulator of MMP-2 and MT1-MMP in response to reorganization of the actin cytoskeleton. JNK regulates transcription through activation of members of the AP-1 protein family including c-Jun, Jun B, Jun D, NFAT, egr-1, and ATF-2 (14, 37). Utilizing c-Jun siRNA, we identified c-Jun as an important transcriptional regulator of MMP-2 expression in SMEC.

A key role for JNK in mediating numerous steps of the angiogenic process is gaining recognition. JNK has been implicated in endothelial cell migration and proliferation as well as matrix invasion and network formation (38, 60). Recently, Zhang et al. (60), using DNAzymes to inhibit c-Jun, were able to inhibit aspects of the angiogenic response following VEGF stimulation, including migration, chemoinvasion, and tubule formation. We have shown in the present study that inhibition of JNK or c-Jun attenuates VEGF-induced MMP-2 mRNA expression, thus extending our knowledge of the roles played by JNK in these angiogenesis assays and physiological angiogenesis.

PI3K activates the Akt signaling pathway but also is reported to be upstream of JNK in a number of cellular processes, including endothelial cell migration (49). Cytochalasin D has been shown to either increase or decrease phospho-Akt levels in different cell lines (32, 51). Our results suggest that PI3K regulates MMP-2 and MT1-MMP protein independently of mRNA synthesis in response to both Cytochalasin D and VEGF. Both total and active MMP-2 protein were partially, but significantly, decreased with LY-294002 without a concurrent inhibition of MMP-2 mRNA. Likewise, MT1-MMP protein decreased independently of changes in MT1-MMP mRNA, and similar effects of LY-294002 on MT1-MMP translation were observed in smooth muscle cells in response to balloon injury (59). Several possibilities exist to explain the results observed with PI3K inhibition: 1) inhibition of MMP-2 and MT1-MMP mRNA translation, 2) inhibition of glycogen synthase kinase-3beta (GSK-3beta)-mediated effects, and 3) PI3K-dependent protein trafficking.

PI3K modulates mRNA translation by modifying the 70-kDa ribosomal S6 kinase (S6K) through phosphoinositide-dependent kinase 1 (PDK) or 4E-binding protein 1 (4E-BP1) via the mTOR (mammalian target of rapamycin) pathway (57). Activation of S6K by PI3K has been shown to be required for MMP-2 translation, whereas the translation factor e1F-4B (and its inhibitory binding protein, 4E-BP1) regulates MT1-MMP translation in smooth muscle cells (10). These multiple effects on mRNA translation by PI3K may explain the effects observed in MMP-2 and MT1-MMP protein in response to PI3K inhibition.

A second possible explanation for the effect of PI3K inhibition is that a decrease in PI3K-dependent phosphorylation of GSK-3beta alters GSK-3beta signals within the cell. GSK-3beta has a broad range of cellular functions, including regulation of protein transport, mRNA translation (through the activation of eIF-2B) (46), and suppression of c-Jun (44). It is possible that altered GSK-3beta activity is responsible for the observed changes in active MMP-2 by altering MT1-MMP translation and its localization within the cell. Under quiescent conditions, much of the cellular MT1-MMP is localized to endosomal compartments and, upon stimulation, rapidly moves to the cell surface to areas of active angiogenesis (18). PI3K, through the activation of PIKfyve, causes redistribution of the glucose transporter GLUT-4 receptor from endosomes in response to insulin, thereby coordinating its trafficking and sorting (6, 50). It is possible that a similar PI3K-dependent transport mechanism exists for MT1-MMP trafficking. This would be consistent with our results, because we measured a decrease in the amount of active MMP-2 with PI3K inhibition without a decrease in MT1-MMP mRNA, suggesting that fewer MT1-MMP molecules were on the cell surface after treatment with LY-294009. Ultimately, our data support differing roles of JNK and PI3K in the regulation of MMP-2 and MT1-MMP gene products, through transcriptional regulation and posttranscriptional protein processing, respectively, rather than the two acting in series through a direct signal cascade as originally hypothesized.

The observed increase in percent active MMP-2 protein independent of an increase in MT-MMP mRNA when JNK was inhibited (Fig. 1, B and D) is consistent with previous reports that actin cytoskeleton depolymerization increases the number of MT1-MMP molecules on the cell surface (63). Increased MMP-2 activation was detected in cell extracts as early as 2 h after cytochalasin D treatment (data not shown). Endocytosis of cell surface localized MT1-MMP occurs by both clathrin- and caveolae-dependent internalization (3, 19, 28, 52). Both of these processes rely on intact actin scaffolding; thus disruption of the actin cytoskeleton may result in an increase in MT1-MMP molecules on the cell surface. Zucker et al. (63) showed that actin depolymerization increases the number of cell surface receptors for tissue inhibitor of MMP (TIMP)-2 (i.e., MT1-MMP) without altering the binding affinity for TIMP-2. In our study, VEGF-induced activation of MMP-2 without a concurrent increase in MT1-MMP mRNA correlates with an increased amount of cell surface MT1-MMP. This finding is in line with recent observations by Labrecque et al. (34), who showed that VEGF increases cell surface MT1-MMP through a Src-dependent mechanism.

Our laboratory (48) previously reported that VEGF did not increase MMP-2 protein production in rat endothelial cells isolated from the epididymal fat pad. We have observed several differences in MAPK signaling that may underlie this differential responsiveness to VEGF. Most notably, in rat endothelial cells, the MMP-2 and MT1-MMP response to three-dimensional collagen occurs via ERK1/2 and is not affected by JNK inhibition (9). This suggests that endothelial cells of different origin utilize unique combinations of signal molecules, which may account for the differences in VEGF responsiveness. Notably, VEGF-induced MMP-2 secretion has been observed in human cells, implying that the signal pathways are conserved across species (25, 36).

Upstream activators of PI3K and JNK may include Rac1, which is involved in actin cytoskeleton signaling through stimulation of lamellipodia and regulation of cell-cell junction during migration (12, 40). VEGF activates Rac1 (15), and Rac1 has been linked to increased MMP-2 production and activation in fibrocarcinoma cells (62). Future work is needed to further elucidate the VEGF-dependent signals that mediate MMP-2 production in endothelial cells.

In summary, endothelial cell production of both MMP-2 and MT1-MMP is induced by actin cytoskeleton destabilization. MMP-2 and MT1-MMP mRNA expression requires activation of JNK, whereas MMP-2 protein production and activation are dependent on PI3K. The dependence of VEGF-induced MMP-2 mRNA expression on JNK/c-Jun activity demonstrates that this signal pathway may be a component of physiological and pathological angiogenesis. Together, these data illustrate that initial remodeling of the actin cytoskeleton may coordinate the angiogenesis process, linking early angiogenic responses to subsequent stages that require the production of proteases responsible for the degradation of the basement membrane and interstitial matrix.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Funding for this project was granted to T. L. Haas from the National Sciences and Engineering Research Council and the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
We thank G. Dhanota and S. Pallan for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. L. Haas, School of Kinesiology and Health Sciences, York Univ., 4700 Keele St., Toronto, Ontario, Canada M3J 1P3 (e-mail: thaas{at}yorku.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Ailenberg M and Silverman M. Cytochalasin D disruption of actin filaments in 3T3 cells produces an anti-apoptotic response by activating gelatinase A extracellularly and initiating intracellular survival signals. Biochim Biophys Acta 1593: 249–258, 2003.[Medline]

2. Aimes RT and Quigley JP. Matrix metalloproteinase-2 is an interstitial collagenase. J Biol Chem 270: 5872–5876, 1995.[Abstract/Free Full Text]

3. Annabi B, Lachambre M, Bousquet-Gagnon N, Page M, Gingras D, and Beliveau R. Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains. Biochem J 353: 547–553, 2001.[CrossRef][ISI][Medline]

4. Ara T, Deyama Y, Yoshimura Y, Higashino F, Shindoh M, Matsumoto A, and Fukuda H. Membrane type 1-matrix metalloproteinase expression is regulated by E-cadherin through the suppression of mitogen-activated protein kinase cascade. Cancer Lett 157: 115–121, 2000.[CrossRef][ISI][Medline]

5. Barberis L, Wary KK, Fiucci G, Liu F, Hirsch E, Brancaccio M, Altruda F, Tarone G, and Giancotti FG. Distinct roles of the adaptor protein Shc and focal adhesion kinase in integrin signaling to ERK. J Biol Chem 275: 36532–36540, 2000.[Abstract/Free Full Text]

6. Berwick DC, Dell GC, Welsh GI, Heesom KJ, Hers I, Fletcher LM, Cooke FT, and Tavare JM. Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles. J Cell Sci 117: 5985–5993, 2004.[Abstract/Free Full Text]

7. Birkedal-Hansen H. Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol 7: 728–735, 1995.[CrossRef][ISI][Medline]

8. Birukov KG, Birukova AA, Dudek SM, Verin AD, Crow MT, Zhan X, DePaola N, and Garcia JGN. Shear stress-mediated cytoskeletal remodeling and cortactin translocation in pulmonary endothelial cells. Am J Respir Cell Mol Biol 26: 453–464, 2002.[Abstract/Free Full Text]

9. Boyd PJ, Doyle J, Gee E, Pallan S, and Haas TL. Mitogen-activated protein kinase signaling regulates endothelial cell assembly into networks and the expression of MT1-MMP and MMP-2. Am J Physiol Cell Physiol 288: C659–C668, 2005.[Abstract/Free Full Text]

10. Bradley JM, Kelley MJ, Rose A, and Acott TS. Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch. Invest Ophthalmol Vis Sci 44: 5174–5181, 2003.[Abstract/Free Full Text]

11. Civelekoglu G, Tardy Y, and Meister JJ. Modeling actin filament reorganization in endothelial cells subjected to cyclic stretch. Bull Math Biol 60: 1017–1037, 1998.[CrossRef][ISI][Medline]

12. Connolly JO, Simpson N, Hewlett L, and Hall A. Rac regulates endothelial morphogenesis and capillary assembly. Mol Biol Cell 13: 2474–2485, 2002.[Abstract/Free Full Text]

13. D'Angelo G, Struman I, Martial J, and Weiner RI. Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin. Proc Natl Acad Sci USA 92: 6374–6378, 1995.[Abstract/Free Full Text]

14. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 103: 239–252, 2000.[CrossRef][ISI][Medline]

15. Eriksson A, Cao RH, Roy J, Tritsaris K, Wahlestedt C, Dissing S, Thyberg J, and Cao YH. Small GTP-binding protein Rac is an essential mediator of vascular endothelial growth factor-induced endothelial fenestrations and vascular permeability. Circulation 107: I532–I538, 2003.

16. Fang J, Shing Y, Wiederschain D, Yan L, Butterfield C, Jackson G, Harper J, Tamvakopoulos G, and Moses MA. Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model. Proc Natl Acad Sci USA 97: 3884–3889, 2000.[Abstract/Free Full Text]

17. Franke RP, Grafe M, Schnittler H, Seiffge D, Mittermayer C, and Drenckhahn D. Induction of human vascular endothelial stress fibers by fluid shear-stress. Nature 307: 648–649, 1984.[CrossRef][Medline]

18. Galvez BG, Matias-Roman S, Yanez-Mo M, Sanchez-Madrid F, and Arroyo AG. ECM regulates MT1-MMP localization with beta1 or {alpha}vbeta3 integrins at distinct cell compartments modulating its internalization and activity on human endothelial cells. J Cell Biol 159: 509–521, 2002.[Abstract/Free Full Text]

19. Galvez BG, Matias-Roman S, Yanez-Mo M, Vicente-Manzanares M, Sanchez-Madrid F, and Arroyo AG. Caveolae are a novel pathway for membrane-type 1 matrix metalloproteinase traffic in human endothelial cells. Mol Biol Cell 15: 678–687, 2004.[Abstract/Free Full Text]

20. Haas TL, Davis SJ, and Madri JA. Three dimensional type I collagen lattices induce coordinate expression of matrix metalloproteinases MT1-MMP and MMP-2 in microvascular endothelial cells. J Biol Chem 273: 3604–3610, 1998.[Abstract/Free Full Text]

21. Han XY, Boyd PJ, Colgan S, Madri JA, and Haas TL. Transcriptional up-regulation of endothelial cell matrix metalloproteinase-2 in response to extracellular cues involves GATA-2. J Biol Chem 278: 47785–47791, 2003.[Abstract/Free Full Text]

22. Haralabopoulos GC, Grant DS, Kleinman HK, and Maragoudakis ME. Thrombin promotes endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo. Am J Physiol Cell Physiol 273: C239–C245, 1997.[Abstract/Free Full Text]

23. Herren B, Levkau B, Raines EW, and Ross R. Cleavage of beta-catenin and plakoglobin and shedding of VE-cadherin during endothelial apoptosis: evidence for a role for caspases and metalloproteinases. Mol Biol Cell 9: 1589–1601, 1998.[Abstract/Free Full Text]

24. Hoeben A, Landuyt B, Highley MS, Wildiers H, van Oosterom AT, and De Bruijn EA. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 56: 549–580, 2004.[Abstract/Free Full Text]

25. Hoffmann S, He S, Ehren M, Ryan SJ, Wiedemann P, and Hinton DR. MMP-2 and MMP-9 secretion by RPE is stimulated by angiogenic molecules found in choroidal neovascular membranes. Retina 26: 454–461, 2006.[CrossRef][Medline]

26. Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robey PG, Poole AR, Pidoux I, Ward JM, and Birkedal-Hansen H. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99: 81–92, 1999.[CrossRef][ISI][Medline]

27. Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, and Itohara S. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res 58: 1048–1051, 1998.[Abstract/Free Full Text]

28. Jiang A, Lehti K, Wang X, Weiss SJ, Keski-Oja J, and Pei D. Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis. Proc Natl Acad Sci USA 98: 13693–13698, 2001.[Abstract/Free Full Text]

29. Kato T, Kure T, Chang JH, Gabison EE, Itoh T, Itohara S, and Azar DT. Diminished corneal angiogenesis in gelatinase A-deficient mice. FEBS Lett 508: 187–190, 2001.[CrossRef][ISI][Medline]

30. Keely PJ, Westwick JK, Whitehead IP, Der CJ, and Parise LV. Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature 390: 632–636, 1997.[CrossRef][Medline]

31. Kilarski WW, Jura N, and Gerwins P. Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton. Exp Cell Res 291: 70–82, 2003.[CrossRef][ISI][Medline]

32. Kim SJ, Hwang SG, Kim IC, and Chun JS. Actin cytoskeletal architecture regulates nitric oxide-induced apoptosis, dedifferentiation, and cyclooxygenase-2 expression in articular chondrocytes via mitogen-activated protein kinase and protein kinase C pathways. J Biol Chem 278: 42448–42456, 2003.[Abstract/Free Full Text]

33. Kiosses WB, Cheresh DA, Yang S, Gerritsen ME, Cheresh DA, Alderson N, and Schwartz MA. A dominant negative p65 PAK peptide inhibits angiogenesis. Circ Res 90: 697–702, 2002.[Abstract/Free Full Text]

34. Labrecque L, Nyalendo C, Langlois S, Durocher Y, Roghi C, Murphy G, Gingras D, and Beliveau R. Src-mediated tyrosine phosphorylation of caveolin-1 induces its association with membrane type 1 matrix metalloproteinase. J Biol Chem 279: 52132–52140, 2004.[Abstract/Free Full Text]

35. Labrecque L, Royal I, Surprenant DS, Patterson C, Gingras D, and Beliveau R. Regulation of vascular endothelial growth factor receptor-2 activity by caveolin-1 and plasma membrane cholesterol. Mol Biol Cell 14: 334–347, 2003.[Abstract/Free Full Text]

36. Lamoreaux WJ, Fitzgerald ME, Reiner A, Hasty KA, and Charles ST. Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro. Microvasc Res 55: 29–42, 1998.[CrossRef][ISI][Medline]

37. Lim CP, Jain N, and Cao X. Stress-induced immediate-early gene, egr-1, involves activation of p38/JNK1. Oncogene 16: 2915–2926, 1998.[CrossRef][ISI][Medline]

38. Meadows KN, Bryant P, Vincent PA, and Pumiglia KM. Activated Ras induces a proangiogenic phenotype in primary endothelial cells. Oncogene 23: 192–200, 2004.[CrossRef][ISI][Medline]

39. Milkiewicz M and Haas TL. Effect of mechanical stretch on HIF-1{alpha} and MMP-2 expression in capillaries isolated from overloaded skeletal muscles: laser capture microdissection study. Am J Physiol Heart Circ Physiol 289: H1315–H1320, 2005.[Abstract/Free Full Text]

40. Minden A, Lin AN, Claret FX, Abo A, and Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81: 1147–1157, 1995.[CrossRef][ISI][Medline]

41. Murphy G, Nguyen Q, Cockett MI, Atkinson SJ, Allan JA, Knight CG, Willenbrock F, and Docherty AJP. Assessment of the role of the fibronectin-like domain of gelatinase-A by analysis of a deletion mutant. J Biol Chem 269: 6632–6636, 1994.[Abstract/Free Full Text]

42. Oh J, Takahashi R, Adachi E, Kondo S, Kuratomi S, Noma A, Alexander DB, Motoda H, Okada A, Seiki M, Itoh T, Itohara S, Takahashi C, and Noda M. Mutations in two matrix metalloproteinase genes, MMP-2 and MT1-MMP, are synthetic lethal in mice. Oncogene 23: 5041–5048, 2004.[CrossRef][ISI][Medline]

43. Ohno-Matsui K, Uetama T, Yoshida T, Hayano M, Itoh T, Morita I, and Mochizuki M. Reduced retinal angiogenesis in MMP-2-deficient mice. Invest Ophthalmol Vis Sci 44: 5370–5375, 2003.[Abstract/Free Full Text]

44. Oudit GY, Sun H, Kerfant BG, Crackower MA, Penninger JM, and Backx PH. The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease. J Mol Cell Cardiol 37: 449–471, 2004.[CrossRef][ISI][Medline]

45. Pan MR and Hung WC. Nonsteroidal anti-inflammatory drugs inhibit matrix metalloproteinase-2 via suppression of the ERK/Sp1-mediated transcription. J Biol Chem 277: 32775–32780, 2002.[Abstract/Free Full Text]

46. Pap M and Cooper GM. Role of translation initiation factor 2B in control of cell survival by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta signaling pathway. Mol Cell Biol 22: 578–586, 2002.[Abstract/Free Full Text]

47. Pedram A, Razandi M, and Levin ER. Deciphering vascular endothelial cell growth factor/vascular permeability factor signaling to vascular permeability—Inhibition by atrial natriuretic peptide. J Biol Chem 277: 44385–44398, 2002.[Abstract/Free Full Text]

48. Rivilis I, Milkiewicz M, Boyd P, Goldstein J, Brown MD, Egginton S, Hansen FM, Hudlicka O, and Haas TL. Differential involvement of MMP-2 and VEGF during muscle stretch- versus shear stress-induced angiogenesis. Am J Physiol Heart Circ Physiol 283: H1430–H1438, 2002.[Abstract/Free Full Text]

49. Steinle JJ, Meininger CJ, Chowdhury U, Wu G, and Granger HJ. Role of ephrin B2 in human retinal cell proliferation and migration. Cell Signal 15: 1011–1017, 2003.[CrossRef][ISI][Medline]

50. Tanti JF, Grillo S, Gremeaux T, Coffer PJ, Van Obberghen E, and Marchand-Brustel Y. Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes. Endocrinology 138: 2005–2010, 1997.[Abstract/Free Full Text]

51. Tran NL, Adams DG, Vaillancourt RR, and Heimark RL. Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization. J Biol Chem 277: 32905–32914, 2002.[Abstract/Free Full Text]

52. Uekita T, Itoh Y, Yana I, Ohno H, and Seiki M. Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity. J Cell Biol 155: 1345–1356, 2001.[Abstract/Free Full Text]

53. Werb Z. ECM and cell surface proteolysis: regulating cellular ecology. Cell 91: 439–442, 1997.[CrossRef][ISI][Medline]

54. Woessner JF Jr. The family of matrix metalloproteinases. Ann NY Acad Sci 732: 11–21, 1994.[ISI][Medline]

55. Wojciak-Stothard B, Potempa S, Eichholtz T, and Ridley AJ. Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci 114: 1343–1355, 2001.[Abstract]

56. Wojciak-Stothard B and Ridley AJ. Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases. J Cell Biol 161: 429–439, 2003.[Abstract/Free Full Text]

57. Wymann MP and Marone R. Phosphoinositide 3-kinase in disease: timing, location, and scaffolding. Curr Opin Cell Biol 17: 141–149, 2005.[CrossRef][ISI][Medline]

58. Zachary I. VEGF signalling: integration and multi-tasking in endothelial cell biology. Biochem Soc Trans 31: 1171–1177, 2003.[ISI][Medline]

59. Zahradka P, Harding G, Litchie B, Thomas S, Werner JP, Wilson DP, and Yurkova N. Activation of MMP-2 in response to vascular injury is mediated by phosphatidylinositol 3-kinase-dependent expression of MT1-MMP. Am J Physiol Heart Circ Physiol 287: H2861–H2870, 2004.[Abstract/Free Full Text]

60. Zhang GS, Dass CR, Sumithran E, Di Girolamo N, Sun LQ, and Khachigian LM. Effect of deoxyribozymes targeting c-Jun on solid tumor growth and angiogenesis in rodents. J Natl Cancer Inst 96: 683–696, 2004.[Abstract/Free Full Text]

61. Zhou Z, Apte SS, Soininen R, Cao R, Baaklini GY, Rauser RW, Wang J, Cao Y, and Tryggvason K. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc Natl Acad Sci USA 97: 4052–4057, 2000.[Abstract/Free Full Text]

62. Zhuge Y and Xu J. Rac1 mediates type I collagen-dependent MMP-2 activation. Role in cell invasion across collagen barrier. J Biol Chem 276: 16248–16256, 2001.[Abstract/Free Full Text]

63. Zucker S, Hymowitz M, Conner CE, DiYanni EA, and Cao J. Rapid trafficking of membrane type 1-matrix metalloproteinase to the cell surface regulates progelatinase a activation. Lab Invest 82: 1673–1684, 2002.[ISI][Medline]




This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. L. Haas, J. L. Doyle, M. R. Distasi, L. E. Norton, K. M. Sheridan, and J. L. Unthank
Involvement of MMPs in the outward remodeling of collateral mesenteric arteries
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2429 - H2437.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
M. Milkiewicz, J. L. Doyle, T. Fudalewski, E. Ispanovic, M. Aghasi, and T. L. Haas
HIF-1{alpha} and HIF-2{alpha} play a central role in stretch-induced but not shear-stress-induced angiogenesis in rat skeletal muscle
J. Physiol., September 1, 2007; 583(2): 753 - 766.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/4/C579    most recent
00300.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ispanovic, E.
Right arrow Articles by Haas, T. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ispanovic, E.
Right arrow Articles by Haas, T. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.