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Molecular Cancer Research 4:361-370 (2006)
© 2006 American Association for Cancer Research

Angiogenesis, Metastasis, and the Cellular Microenvironment

Interplay between Epidermal Growth Factor Receptor and Janus Kinase 3 Regulates Polychlorinated Biphenyl–Induced Matrix Metalloproteinase-3 Expression and Transendothelial Migration of Tumor Cells

Sung Yong Eum1, Yong Woo Lee1, Bernhard Hennig2 and Michal Toborek1

1 Molecular Neuroscience and Vascular Biology Laboratory, Department of Surgery and 2 College of Agriculture, University of Kentucky, Lexington, Kentucky

Requests for reprints: Michal Toborek, Molecular Neuroscience and Vascular Biology Laboratory, Department of Surgery, Division of Neurosurgery, University of Kentucky Medical Center, 593 Wethington Building, 900 South Limestone, Lexington, KY 40536. Phone: 859-323-4094; Fax: 859-323-2705. E-mail: mjtobo00{at}uky.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
We hypothesize that environmental toxicants, such as polychlorinated biphenyl congeners, can activate vascular endothelial cells and thus increase formation of blood-borne metastases. This study indicates that exposure of human microvascular endothelial cells to 2,2',4,6,6'-pentachlorobiphenyl can stimulate transendothelial migration of tumor cells through up-regulation of matrix metalloproteinase (MMP)-3. In a series of experiments with specific small interfering RNA and pharmacologic inhibitors, we provide evidence that 2,2',4,6,6'-pentachlorobiphenyl can activate epidermal growth factor receptor (EGFR) and Janus kinase 3 (JAK3) in a closely coordinated and cross-dependent fashion. Activated EGFR and JAK3 stimulate in concert c-Jun NH2-terminal kinase and extracellular signal-regulated kinase 1/2 as well as increase DNA-binding activity of transcription factors activator protein-1 and polyomavirus enhancer activator protein 3, leading to transcriptional up-regulation of MMP-3 expression. These results indicate that the interplay among EGFR, JAK3, and mitogen-activated protein kinases, such as c-Jun NH2-terminal kinase and extracellular signal-regulated kinase 1/2, is critical for polychlorinated biphenyl–induced MMP-3 expression and accelerated transendothelial migration of tumor cells. (Mol Cancer Res 2006;4(6):361–70)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Degradation and the consequent rearrangement of extracellular matrix (ECM) are involved in multiple steps of tumor metastasis, such as invasion of the basement membrane at both primary and metastatic sites, invasion through the stroma, and tumor cell extravasation (1, 2). Many proteinases are capable of degrading ECM components, but one family of enzymes that seems to be particularly important for matrix degradation is the matrix metalloproteinases (MMP). The MMPs comprise a large family of >20 structurally and functionally related zinc endopeptidases that together can degrade all known components of the ECM and the basement membranes (3, 4). Thus, MMPs are capable of disrupting the integrity of the basement membranes and mediating tumor cell extravasation with their subsequent migration into underlying tissues (1, 5). Although experimental studies initially suggested that cancer cells themselves produce the MMPs required for degradation of the ECM, more recent studies have indicated that most MMPs during tumor invasion are produced by host cells, including fibroblasts and endothelial cells (6, 7). Therefore, the locally activated host microenvironment, including vascular endothelial cells, may modify the adjacent ECM and promote the metastasis of cancer cells (8-11).

Epidermal growth factor (EGF) receptor (EGFR) is a transmembrane protein receptor with tyrosine kinase activity that results in the generation of several intracellular signals, which culminate in not only cell proliferation but also other processes that are crucial to cancer progression, including angiogenesis and metastatic spread (12, 13). The vascular endothelial cells in several tumor types are known to express EGFR. Blockade of EGFR can reduce the microvessel density of tumor and survival of tumor-associated endothelial cells. In addition, EGFR activation was reported to induce various downstream effectors, such as vascular endothelial growth factor, interleukin-8, and MMPs, which are involved in the processes of metastasis (14). For example, overexpression and activation of EGFR in primary esophageal keratinocytes resulted in enhanced cell migration through up-regulation of MMP-1 (13). Up-regulation of MMP-3 in EGFR-overexpressed oral squamous cell carcinoma was associated with an advanced pathologic stage, a diffuse invasive mode, and a high incidence of neck metastasis (15). Moreover, exposure of human skin in vivo to UV-activated EGFR stimulated mitogen-activated protein kinases (MAPK) and resulted in up-regulation of activator protein-1 (AP-1), a transcription factor that is required for transcription of MMPs (16).

Vascular endothelial cells form a continuous monolayer, which functions as a selective barrier to the passage of cancer cells from bloodstream to the underlying tissues. Therefore, activation of endothelial cells has a significant influence on the fate of circulating cancer cells in the blood vessel (9, 17, 18). We have reported previously that environmental toxicants, such as polychlorinated biphenyls (PCB), a class of widespread polychlorinated aromatic hydrocarbons, can activate vascular endothelial cells resulting in disruption of endothelial barrier function (19-21). Among different PCBs, our recent studies indicated that 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), a highly ortho-chlorinated PCBs congener, can increase the adhesion of human leukemia cells to human endothelial cells through the induction of proinflammatory molecules and increase endothelial permeability via up-regulation of vascular endothelial growth factor (20, 21). However, the detailed mechanisms of PCB 104–induced transendothelial migration of tumor cells remain unclear.

Activation of MMPs and the degradation of the ECM seem to be necessary steps in tumor cell extravasation. Therefore, we hypothesize that ortho-substituted noncoplanar PCBs can facilitate MMP expression in vascular endothelial cells and thus accelerate transendothelial migration of tumor cells. Results of the present study indicate that exposure of human microvascular endothelial cells (HMEC) to PCB 104 can increase transendothelial migration of tumor cells through overexpression of MMP-3. In addition, we identified that EGFR, Janus kinase 3 (JAK3), and the MAPK signaling pathways play the critical role in PCB-induced up-regulation of MMP-3.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
PCB 104 Stimulates MMP-3 Expression in Microvascular Endothelial Cells
During metastasis formation, cancer cells cross several ECM barriers, including the endothelial basement membrane, for the entry and exit through the blood vessels. Degradation of ECM by MMPs is believed to be a prerequisite for tumor cells to migrate to the secondary metastatic sites. Therefore, overexpression of MMPs by activated endothelial cells may accelerate transmigration of cancer cells.

Treatment with 10 µmol/L PCB 104 increased MMP-3 mRNA expression in a time-dependent pattern as determined by Northern blot and real-time reverse transcription-PCR (RT-PCR; Fig. 1A, top and bottom , respectively). A statistically significant increase in MMP-3 mRNA expression was observed after a 3-hour exposure to PCB 104 and remained at the same level for at least 26 hours. The effects of PCB 104 on induction of MMP-3 mRNA were also dose dependent (Fig. 1B). Using real-time RT-PCR, a statistically significant increase in MMP-3 mRNA levels was observed in cells treated with 10 µmol/L PCB 104. However, the maximum MMP-3 mRNA expression was observed in endothelial cell cultures treated with 15 µmol/L PCB. In addition, the secreted MMP-3 protein into cell culture medium was markedly increased in endothelial cell cultures treated with 10 µmol/L PCB 104 for 24 hours (Fig. 1C).


Figure 1
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  		FIGURE 1. PCB 104 increases MMP-3 expression and release in HMEC-1. Confluent HMEC-1 cultures were exposed to 10 µmol/L PCB 104 for the indicated time points (A) or exposed to the indicated concentrations of PCB 104 for 12 hours (B). mRNA levels of MMP-3 and tissue inhibitor of MMP-1 (TIMP 1) were determined using Northern blotting hybridization (A and B, top) or real-time RT-PCR (A and B, bottom). Western blotting analysis was used to assess the levels of MMP-3 proteins released to the cell culture medium after 24-hour exposure to the indicated concentrations of PCB 104 (C). Phorbol 12-myristate 13-acetate (PMA) was used as a positive control at the concentration of 10 nmol/L. All experiments were repeated thrice. Representative blots. *, statistically different compared with controls. Columns, mean; bars, SD.

 
PCB 104 at 10 µmol/L was selected for further experiments that were focused on the mechanisms of PCB 104–induced MMP-3 up-regulation. It should be noted that exposure to PCB 104 did not affect mRNA levels of tissue inhibitor of MMP-1, a natural intracellular inhibitor of MMP-3 (Fig. 1A and B, top).

MMP-3 Mediates PCB 104–Induced Transendothelial Migration of Tumor Cells but Not Tumor Cell Adhesion or Endothelial Permeability
Activated MMPs are the critical enzymes in transendothelial migration of tumor cells. Therefore, the effects of MMP-3 on migration of the MDA-MB-231 cells across endothelial monolayers were studied in the present study. As indicated in Fig. 2A , N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH; a specific inhibitor of MMP-3) completely prevented PCB 104–induced transmigration of the tumor cells. Similar protection against PCB-stimulated migration of the MDA-MB-231 cells was exerted by a general inhibitor of MMPs, GM1498 (Fig. 2A).


Figure 2
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  		FIGURE 2. MMP-3 is involved in PCB 104–induced transendothelial migration of tumor cells but not in endothelial permeability and tumor cell adhesion. A. For transmigration assay, HMEC-1 were grown to confluence on fibronectin-coated Transwell membranes (8-µm pores) and pretreated with GM1489 (general MMP inhibitor, 10 µmol/L) or NNGH (specific MMP-3 inhibitor, 10 µmol/L) for 30 minutes before 10 µmol/L PCB 104 exposure for 22 hours. Transmigration of calcein-labeled MDA-MB-231 cells was quantified as described in Materials and Methods. B. For permeability assay, HMEC-1 were grown on fibronectin-coated Transwell membranes with 0.4-µm pores. Cultures were pretreated with NNGH (specific MMP-3 inhibitor, 1 or 10 µmol/L) for 30 minutes before 10 µmol/L PCB 104 exposure for 22 hours. Endothelial permeability was determined using FITC-dextran 40. C. For cell adhesion assay, HMEC-1 were grown on 24-well cell culture plates. Cultures were treated as described in B and adhesion of calcein-labeled MDA-MB-231 cells was quantified as described in Materials and Methods. *, statistically different compared with control cultures; {dagger}, levels in the groups PCB 104 + GM1489 or NNGH are statistically different compared with those in cultures treated with PCB 104 alone. All experiments were repeated at least thrice. Columns, mean; bars, SD.

 
Increased transendothelial migration can be affected by enhanced cell adhesion and/or endothelial permeability. Although treatment with PCB 104 elevated endothelial permeability and tumor cell adhesion, inhibition of MMP-3 by NNGH did not alter these events (Fig. 2B and C, respectively). Thus, it seems that PCB 104–induced overexpression of MMP-3 selectively increases transendothelial migration of tumor cells without the influence on PCB-mediated elevated cell adhesion and endothelial permeability.

PCB 104–Induced Overexpression of MMP-3 and Transendothelial Migration Are Mediated by Activation of EGFR and JAK3
Vascular endothelial cells are known to express EGFR that can influence several other signaling pathways, including JAK3 and/or the MAPK signaling. Therefore, we examined whether these pathways can participate in PCB 104–triggered overexpression of MMP-3 and accelerated transmigration of tumor cells. Both gene silencing techniques and pharmacologic inhibition of JAK3 and EGFR were employed in these experiments.

The effectiveness of JAK3 and EGFR silencing is illustrated in Fig. 3A . Transfections with specific JAK3 small interfering RNA (siRNA) and EGFR siRNA remarkably reduced expression of respective proteins in HMEC-1. In these experiments, we used three different commercially available JAK3 siRNAs [JAK3 D (Dharmacon, Lafayette, CO) and JAK3 A and B (Ambion, Inc. Austin, TX)] and two different commercially available EGFR siRNAs [EGFR A and B (Ambion)]. In contrast to specific siRNAs, scrambled siRNA did not affect JAK3 or EGFR protein expression.


Figure 3
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  		FIGURE 3. EGFR and JAK3 modulate PCB 104–induced MMP-3 mRNA expression and transendothelial migration of tumor cells. A. HMEC-1 were transfected with 40 nmol/L EGFR or JAK3 siRNA and cultured for 3 days until reaching confluence. JAK3 and EGFR protein levels were measured by Western blot to assess the effectiveness of gene silencing. Commercially available siRNA was purchased from Ambion (JAK3 A, JAK3 B, EGFR A, and EGFR B) or Dharmacon (JAK3 D). Scrambled siRNA was obtained from Dharmacon and used as negative control. All Western blotting were repeated at least thrice. Representative blots. Akt1/2 was determined as internal control to show the specificity of responses. B. HMEC-1 were transfected with siRNA as described in A and treated with 10 µmol/L PCB 104 for 25 hours. MMP-3 protein levels were determined by Western blot (top). In separate experiments, confluent HMEC-1 cultures were pretreated for 30 minutes with 5 µmol/L WHI-P154, 5 µg/mL WHI-P131 (both specific inhibitors of JAK3), or 2 µmol/L PD168393 (specific inhibitor of EGFR tyrosine kinase) before exposure to 10 µmol/L PCB 104 for 9 hours. MMP-3 mRNA levels were determined by real-time RT-PCR (bottom). C. HMEC-1 were transfected with siRNA as described in A and treated with 10 µmol/L PCB 104 for 24 hours. Transendothelial migration was quantified using the calcein-labeled MDA-MB-231 cells as described in Materials and Methods. *, statistically different compared with control cultures; {dagger}, levels in the groups PCB 104 + kinase inhibitors or siRNA-transfected cultures are statistically different compared with those in cultures treated with PCB 104 alone. All experiments were repeated at least thrice. Columns, mean; bars, SD.

 
As illustrated in Fig. 3B, inhibition of JAK3 and EGFR by gene silencing (top) or pharmacologic inhibitors (bottom) markedly blocked MMP-3 expression in HMEC-1. In gene silencing experiments, MMP-3 protein levels were assessed by Western blot, and in pharmacologic inhibition studies, real-time RT-PCR was used to determine MMP-3 mRNA levels. In addition, transmigration of MDA-MB-231 cells across endothelial monolayers was significantly inhibited in cultures transfected with JAK3 or EGFR siRNA (Fig. 3C). These results indicate that activation of EGFR and JAK3 is involved in PCB 104–induced MMP-3 overexpression and the accelerated transendothelial migration of tumor cells.

PCB-Mediated Phosphorylation of EGFR and JAK3 Is Highly Coordinated and Involved in AP-1 and Polyomavirus Enhancer Activator Protein 3 Activation
A series of experiments was done to examine the cross-talk between EGFR and JAK3 phosphorylation. As illustrated in Fig. 4A , tyrosine phosphorylation of both EGFR and JAK3 was induced at the same time (i.e., 2 minutes after treatment with PCB 104). Moreover, phosphorylation of EGFR and JAK3 returned to basal level simultaneously (i.e., 20 minutes after PCB exposure; Fig. 4A). Most interesting, transfection with JAK3 siRNA or EGFR siRNA cross-inhibited each other's phosphorylation without affecting the total protein levels in PCB-treated HMEC-1 (Fig. 4B). Similar effects were also observed when pharmacologic inhibitors were employed. Figure 4C indicates that cotreatment with both WHI-P154 (JAK3 inhibitor) and the general JAK inhibitor markedly blocked phosphorylation of EGFR. In addition, inhibition of EGFR kinase by PD168393 completely prevented tyrosine phosphorylation of JAK3 (Fig. 4C).


Figure 4
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  		FIGURE 4. Interrelationship between EGFR and JAK3 in PCB 104–treated HMEC-1. A. Cultures were treated with 10 µmol/L PCB 104 for the indicated time. To assess activation of JAK3, cell lysate was immunoprecipitated with anti-JAK3 antibody and blotted with anti-phosphotyrosine antibody. Activation of EGFR was determined by Western blot using antibody against phosphorylated EGFR at Tyr1068. Total Akt expression was determined as internal standard. B. Cultures were transfected with 40 nmol/L specific siRNA and treated with PCB 104 for 3 minutes. Phosphorylated JAK3 and EGFR were determined as described in A. Total levels of JAK3 and EGFR were assessed as internal standards. C. Cultures were pretreated for 30 minutes with PD168393 (EGFR inhibitor, 0.2 or 2 µmol/L), WHI-P154 (JAK3 inhibitor, 1 or 5 µmol/L), or the general JAK inhibitor (0.1 or 0.5 µmol/L) before treatment with 10 µmol/L PCB 104 for 3 minutes. Phosphorylated JAK3 and EGFR were determined as described in A. Total levels of JAK3 and EGFR were assessed as internal standards.

 
Inducible MMPs, including MMP-3, possess a conserved AP-1 cis-regulatory element in the proximal promoter. In addition, the promoter region of MMP3 contains the polyomavirus enhancer activator protein 3 (PEA3) site, which binds members of the ETS family of transcription factors and cooperates with the AP-1 motif for optimal activation of the MMP-3 promoter (22). Therefore, the effects of PCB 104 on AP-1 and PEA3 DNA-binding activity also were examined in the present study. As shown in Fig. 5 , treatment with PCB markedly activated DNA-binding activity of AP-1 and PEA3. In addition, these effects were markedly attenuated by silencing JAK3 and EGFR by transfection with specific siRNAs (Fig. 5A and B, top) or by pretreatment with pharmacologic inhibitors of JAK3 and EGFR kinases (Fig. 5A and B, bottom). The specificity of AP-1 and PEA3 DNA-binding assays was confirmed using specific antibodies. Indeed, the addition of anti-c-Jun or anti-Jun family antibody markedly reduced the AP-1-specific band (Fig. 5A, top). Anti-PEA3 antibody and mutated oligonucleotide of the PEA3-binding sequence completely diminished the PEA3-specific band (Fig. 5B).


Figure 5
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  		FIGURE 5. PCB 104–induced activation of AP-1 and PEA3 DNA binding is mediated by JAK3 and EGFR kinases. Cultures were transfected with specific siRNA (A and B, top) or pretreated for 30 minutes with PD168393 (EGFR inhibitor, 0.2 or 2 µmol/L), WHI-P154 (JAK3 inhibitor, 1 or 5 µmol/L), or the general JAK inhibitor (0.1 or 0.5 µmol/L; A and B, bottom) before treatment with 10 µmol/L PCB 104 for 1.5 hours. Isolation of nuclear extracts and EMSA for DNA-binding activities of AP-1 and PEA3 were done as described in Materials and Methods. Competition study was done by the addition of excess of unlabeled AP-1 or PEA3 consensus oligonucleotides (cold). In addition, specificity of DNA-protein binding was confirmed using antibodies against c-Jun, c-Jun family, c-Fos, and PEA3 as well mutated PEA3 oligonucleotides.

 
Extracellular Signal-Regulated Kinase 1/2 and c-Jun NH2-Terminal Kinase Mediate EGFR/JAK3-Dependent MMP-3 Expression in PCB 104–Treated Microvascular Endothelial Cells
To further evaluate downstream signaling pathways of PCB 104–induced MMP-3 overexpression, we assessed the influence of EGFR and JAK3 on activation of the MAPK pathway. Endothelial cells were pretreated with the EGFR tyrosine kinase inhibitor, PD168393, or with JAK3 inhibitor, WHI-P154, and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK) was assessed by Western blotting. As shown in Fig. 6A , PCB 104 markedly increased phosphorylation of ERK1/2 and phosphorylated JNK; however, pretreatment with the inhibitors of EGFR kinase or JAK3 dose-dependently blocked these effects.


Figure 6
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  		FIGURE 6. ERK1/2 and JNK kinases are involved in PCB 104–stimulated EGFR/JAK3 signaling, MMP-3 expression, and transendothelial migration of tumor cells. A. Cultures were pretreated for 30 minutes with PD168393 (EGFR inhibitor, 0.2 or 2 µmol/L), WHI-P154 (JAK3 inhibitor, 1 or 5 µmol/L), or the general JAK inhibitor (0.1 or 0.5 µmol/L) before coincubation with 10 µmol/L PCB 104 for 20 minutes. Phosphorylated ERK1/2 and JNK and total ERK1/2 (internal standard) were determined by Western blot. B. Cultures were pretreated for 30 minutes with PD98059 (ERK1/2 inhibitor, 5 µmol/L) or SP600125 (JNK inhibitor, 5 µmol/L) before treatment with PCB 104 for 1.5 hours. Isolation of nuclear extracts and EMSA for DNA-binding activities of AP-1 and PEA3 were done as described in Materials and Methods. C. Cultures were pretreated with PD98059 (1 or 5 µmol/L) or SP600125 (1 or 5 µmol/L) for 30 minutes before exposure to 10 µmol/L PCB 104 for 12 hours. MMP-3 mRNA was determined using real-time RT-PCR. *, statistically different compared with control cultures; {dagger}, levels in the groups PCB 104 + kinase inhibitor are statistically different compared with those in cultures treated with PCB 104 alone. All experiments were repeated at least thrice. Columns, mean; bars, SD. D. Confluent HMEC-1 cultures on Transwell membranes were pretreated with PD98059 (5 µmol/L) or SP600125 (5 µmol/L) for 30 minutes before exposure to 10 µmol/L PCB 104 for 22 hours. Transendothelial migration of the calcein-labeled MDA-MB-231 cells was quantified as described in Materials and Methods. Statistical analysis as in C.

 
To confirm the importance of the MAPK signaling in PCB-mediated MMP-3 expression, AP-1 and PEA3 activation as well as MMP-3 mRNA levels were evaluated in cells pretreated with ERK1/2 inhibitor, PD98059, or JNK inhibitor, SP600125. As illustrated in Fig. 6B, blockage of ERK1/2 or JNK markedly reduced DNA-binding activity of both AP-1 and PEA3. In addition, PCB 104–induced MMP-3 expression and transendothelial migration of MDA-MB-231 cells were significantly reduced by treatment with these inhibitors (Fig. 6C and D, respectively). Thus, it seems that PCB 104–mediated activation of EGFR and JAK3 results in an increase in MMP-3 expression via the ERK1/2- and JNK-dependent pathways.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
One of the key events in cancer metastasis is transendothelial migration of tumor cells. Intravascular circulating tumor cells attach to endothelium and the junctions between vascular cells are retracted (9, 18). Thus, it seems that tumor cells can be captured by the vascular cells during entry and exit from the circulation and the locally activated endothelial cells can modify metastatic behavior of circulating tumor cells. Despite significant progress on the biology of tumor metastasis, it is still not fully understood how environmental contaminants, such as PCBs, can influence this process.

The novel observations of the present study include the following findings: (a) ortho-chlorinated PCB, such as PCB 104, can induce MMP-3 expression in human endothelial cells through the interplay between JAK3 and EGFR; (b) PCB 104–meditated activation of JAK3 and EGFR is highly coordinated and cross-regulated; and (c) JAK3 and EGFR stimulate in concert PCB-induced activation of JNK and ERK1/2 followed by increased DNA binding of AP-1 and PEA3 and transcriptional up-regulation of MMP-3 expression.

A variety of vascular mechanisms can be responsible for facilitation of extravasation and dissemination of tumor cells. For example, up-regulation of specific adhesion molecules on the endothelial surface can increase transendothelial migration of tumor cells through the increased adhesive properties of endothelial cells (17). In addition, transmigration of tumor cells can be augmented by disruption of endothelial cell junctions and hyperpermeability across the endothelial barrier (23). Finally, MMP overexpression by activated endothelial cells can induce the degradation of the basement membrane components and thus directly enhance penetration of tumor cells as well as facilitate establishment of cancer metastasis (9). MMP activity can be regulated at least at three levels: transcription, proteolytic activation of the zymogen, and inhibition of the active enzyme. MMPs are expressed in tissues at various stages of the development but are typically absent in normal cells of the adult organism and their basal expression is low in most cultured cells. However, a variety of external stimuli, including cytokines and growth factors, can rapidly induce MMPs by their influence on transcriptional regulation (2).

Among the MMPs, the stromelysin subgroup, including MMP-3 (stromelysin-1), is known to degrade basement membrane components, such as type IV collagen, nidogen, and fibronectin (1, 3). Therefore, it is important that PCB 104 can increase expression of MMP-3 in HMEC-1, leading to the enhanced transendothelial migration of tumor cells. Exposure to PCB 104 increased MMP-3 expression at both mRNA and protein levels in dose- and time-dependent patterns (Fig. 1). In addition, inhibition of MMP-3 significantly reduced PCB 104–induced transendothelial migration of tumor cells (Fig. 2A). These effects seem to be highly specific for tumor cell migration, because the blockage of MMP-3 activity did not affect PCB 104–induced adhesion of the MDA-MB-231 cells to HMEC-1 or hyperpermeability of HMEC-1 monolayers (Fig. 2B and C). These results are in line with our previous studies, which indicated that PCB-mediated production of vascular endothelial growth factor is the critical factor that controls endothelial hyperpermeability (20, 21). To support the role of MMPs in cell motility, it was indicated that MMP-knockout mice exert alterations in migration-related processes, such as pathologic inflammatory reactions, reduced angiogenesis, and delayed tumor progression (24).

Results of the present study with specific siRNAs and pharmacologic inhibitors identified JAK3 and EGFR as upstream molecules that control the signaling pathways, leading to PCB 104–mediated overexpression of MMP-3. JAK3 is expressed constitutively at high levels in hematopoietic cells; however, it was reported that it also is expressed in human endothelial cells, including HMEC-1 (25, 26). The role of JAK3 in endothelial cells is not fully understood; however, it was shown that JAK3 can contribute to interleukin-4-induced up-regulation of vascular cell adhesion molecule-1 expression, leading to the recruitment of lymphocytes and eosinophils (26). The expression levels of JAK3 in endothelial cells were also shown to be significantly increased by stimulation with interleukin-1ß, tumor necrosis factor-{alpha}, and lipopolysaccharide (25). In addition to these reports, our present studies indicate that JAK3 can mediate the disruption of endothelial barrier function resulting in the recruitment and transendothelial migration of tumor cells. These findings are supported by recent literature data that the JAK family kinase cascade can regulate MMP expression at transcriptional level (13, 27, 28). For example, oncostatin M, a member of interleukin-6 superfamily, induced MMP-1, MMP-3, and MMP-9 via cross-talk between MAPK and JAK pathways (13, 27). Borrelia burgdorferi–stimulated MMP-3 induction in human primary chondrocytes has also been shown to be mediated by the MAPK and JAK3 signaling (29). These reports indicated also that inhibition of JAK3 kinase can block both MMP-3 expression and activation of the signal transducers and activators of transcription family of transcription factors. Although signal transducers and activators of transcription can bind to the AP-1/ETS sequence on the tissue inhibitor of MMP-1 promoter, similar sequences have not been identified on the human MMP-3 promoter (30, 31). Therefore, the present study focused on the mechanisms leading to MMP-3 expression via stimulation of JAK3 and EGFR with the subsequent activation of AP-1 and PEA3 transcription factor.

Although the ligand-mediated EGFR activation can usually trigger JAK and MAPK signaling cascade as downstream targets, our present data clearly indicate that EGFR and JAK3 are indispensable signaling molecules that cross-stimulate their phosphorylation. This notion is supported by the following evidence: (a) PCB 104–induced phosphorylation of EGFR and JAK3 occurs at the same time (Fig. 5A); (b) knockdown of JAK3 by siRNA or pharmacologic inhibition of JAK3 kinase results in a complete inhibition of PCB-mediated phosphorylation of EGFR; and (c) siRNA or pharmacologic inhibition of EGFR exerts inhibitory effects on JAK3 phosphorylation (Fig. 5B and C). In line with our present finding that EGFR is required for PCB 104–mediated MMP-3 induction, it was reported that activation of EGFR can enhance cell migration through up-regulation of MMP expression in primary keratinocytes and ovarian cancer cells (13, 32, 33). It also was shown that MMP expression was not induced in EGFR-deficient mice (34).

EGFR activation is known to trigger the MAPK signaling (16). However, our novel results indicate that JNK and ERK1/2 activation is dependent on both JAK3 and EGFR in PCB 104–stimulated endothelial cells. In addition, both MAPKs, ERK1/2 and JNK, seemed to cooperate in concert to activate AP-1 and PEA3 DNA-binding activity and induce both MMP-3 expression and transendothelial migration of the MDA-MB-231 cells. To support these results, it was reported that the members of the MAPK family are the signaling molecules that can regulate expression of inducible MMPs via activation of AP-1- and PEA3-binding elements (1, 3). The members of the MAPK family can be involved in the regulation of AP-1 activity through several different mechanisms. For example, the transcriptional activity and stability of c-Jun, a component of AP-1 dimer, can be increased through phosphorylation by JNK. Activation of ERK1/2 was recently shown to induce c-Jun expression and phosphorylation, indicating cross-talk between ERK1/2 and JNK pathways in the regulation of c-Jun activity. In addition to activation of AP-1, the results of the present study indicated that JNK and ERK1/2 are involved in concert in stimulation of the PEA3 DNA-binding activity. Conserved PEA3 elements that bind members of ETS transcription factors are located adjacent to at least one AP-1 element in most inducible MMP promoters. It has reported that ETS proteins do not usually dimerize and bind to DNA alone but prefer to form complexes with other transcription factors, such as AP-1, for which they function as coactivators (22, 35). Thus, the functional interplay between AP-1 and ETS factors enhances the activity of full-length MMP-3.

In conclusion, our present study indicates that exposure of HMEC-1 to ortho-chlorinated PCBs, such as PCB 104, can augment expression of MMP-3, leading to the accelerated transendothelial migration of tumor cells. It seems that activation of the EGFR/JAK3/MAPK signaling is responsible for transcriptional activation of MMP-3. These results suggest that highly ortho-substituted noncoplanar PCB congeners can induce vascular endothelial barrier disruption that may promote the development of blood-borne metastasis.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Reagents
PCB 104 (>99% pure) was purchased from AccuStandard (New Haven, CT) and dissolved in DMSO. Levels of DMSO in the experimental medium were <0.1% and did not affect endothelial cell. NNGH, GM1489, PD98059, SP600125, PD168393, WHI-P154, and WHI-P131 were purchased from Calbiochem (La Jolla, CA). Anti-Akt antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antibodies against phosphorylated ERK1/2, phosphorylated JNK, and phosphorylated EGFR were obtained from Cell Signaling (Beverly, MA). Anti-JAK3 antibody was a generous gift from Dr. John J. O'Shea (National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD). Phosphotyrosine (4G10 clone) antibody was purchased from Upstate (Waltham, MA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO).

Cell Cultures and PCB Treatment
HMEC were a generous gift from Dr. Eric Smart (University of Kentucky Medical Center, Lexington, KY). HMEC-1 is an immortalized cell line obtained by transformation of HMEC with the SV40 large T antigen. These cells retain endothelial cell phenotype and functional characteristics (36). HMEC-1 were cultured in MCDB 131 enriched with 10% fetal bovine serum, 2 mmol/L L-glutamine, 50 units/mL penicillin, 50 µg/mL streptomycin, 1 µg/mL hydrocortisone, and 0.01 µg/mL EGF in 5% CO2 atmosphere at 37°C. Before each experiment, the cells were serum starved in experimental medium containing 1% fetal bovine serum without EGF for at least 12 hours. The MDA-MB-231 cells (a metastatic breast cancer cell line) were purchased from the American Type Culture Collection (Manassas, VA) and cultured in suspension in RPMI 1640 supplemented with 10% fetal bovine serum, 50 units/mL penicillin, and 50 units/mL streptomycin. Serum concentration of PCBs can reach ~3 µmol/L in people exposed to these toxicants (37, 38); however, local microenvironmental levels of PCBs in extracellular space are not known. Therefore, in the present study, cells were treated with the range of PCB 104 concentrations, such as 2, 5, 10, or 15 µmol/L. In selected experiment, HMEC-1 were pretreated with inhibitors of specific signaling pathways for 30 minutes before adding PCB 104. The inhibitors were then maintained in the medium throughout the PCB 104 exposure. Stock solution of PCB 104 was prepared in DMSO and the same amounts of DMSO as in PCB-treated cells were added to control cultures.

Silencing of EGFR and JAK3 mRNA
Transfection of HMEC-1 with siRNA was done using the GeneSilencer transfection reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's protocol. siRNA was used at a final concentration of 40 or 100 nmol/L, and transfection reagent was used at the dilution of 1:250 (v/v). JAK3 siRNAs were purchased from Ambion (JAK3 A, ID214436; JAK3 B, ID32) or Dharmacon (JAK3 D, JAK3 siGENOME SMARTpool). EGFR siRNAs were obtained from Ambion (EGFR A, ID644; EGFR B, ID42833). Scrambled, nonspecific siRNA was obtained from Dharmacon and used as negative control. Transfections were done for 5 hours. Cells were allowed to recover for 2 to 3 days in MCDB 131 with 10% serum before PCB 104 treatment.

Northern Blot Analysis
Total cellular RNA was purified from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA). For Northern blot analysis, 15 µg RNA was electrophoresed on 1% agarose gels containing 37% formaldehyde and transferred to Hybond-N membranes (Amersham Pharmacia Biotech, Piscataway, NJ) by capillary transfer. Prehybridization and hybridization were done at 68°C in ExpressHyb hybridization solution (Clontech, Mountain View, CA). cDNA probes for MMP-3, tissue inhibitor of MMP-1, and actin were labeled with [32P]dCTP (3,000 Ci/mmol, Amersham Pharmacia Biotech) using a Random Primer Kit (Takara, Shiga, Japan).

Real-time RT-PCR
Total RNA was prepared from PCB 104–treated HMEC-1 using RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. First-strand cDNA was generated from 2 µg total RNA using the Reverse Transcription System kit (Promega, Madison, WI) with random hexamer primers. MMP-3 mRNA expression was determined by real-time RT-PCR using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). PCR amplification was done using Taqman Universal PCR Master Mix (Applied Biosystems) and commercially available predeveloped primer pair and Taqman probe (Applied Biosystems) according to the manufacturer's instructions. PCR cycles consisted of an initial denaturation step at 95°C for 10 minutes followed by 95°C for 15 seconds and 60°C for 60 seconds (for up to 45 cycles). PCR amplification of actin (a housekeeping gene) was done for each sample to normalize MMP-3 mRNA levels. All assays were done at least in triplicate.

Immunoprecipitation and Immunoblotting
Confluent HMEC-1 were washed with cold PBS and lysed with lysis buffer [1.0% NP40, 20 mmol/L Tris-HCl (pH 7.6), 1 mmol/L EDTA, 0.5 mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L Na3VO4, 2 mmol/L DTT, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride]. Immunoprecipitation was done using 100 µg whole-cell lysate and 1 µg JAK3-specific mouse monoclonal antibody (Upstate). Antigen-antibody complexes were isolated with protein A/G-agarose bead. Protein samples were electrophoresed in SDS-PAGE and transferred onto a Hybond-ECL membrane (Amersham Biosciences, Piscataway, NJ). Immunoreactive protein bands were visualized with the enhanced chemiluminescence system (Amersham Biosciences).

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described earlier (39). In gene silencing experiments, cultures were incubated with 10 µmol/L PCB 104 for 1.5 hours on the third day following siRNA transfections. Double-stranded oligonucleotides containing the consensus sequences of the binding sites for transcription factors AP-1 and PEA3 were end labeled with [32P]ATP using bacteriophage T4 polynucleotide kinase (Promega) according to the manufacturer's instructions. Binding reactions were done with 2 µg nuclear protein extracts in a 20 µL volume of reaction mixture [10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 1 mmol/L EDTA, 0.1 mmol/L DTT, 10% glycerol, 2 µg poly(deoxyinosinic-deoxycytidylic acid)]. For gel supershift assay, 1 µg antibody against c-Jun, c-Fos, PEA3 (Santa Cruz Biotechnology), or Jun family (BD PharMingen, San Jose, CA) was added to the reaction mixture. Protein-DNA complexes were analyzed on a nondenaturing 5% polyacrylamide gel using 0.25x Tris-borate EDTA buffer [50 mmol/L Tris-HCl, 45 mmol/L boric acid, 0.5 mmol/L EDTA (pH 8.4)].

Transendothelial Cell Migration Assay
HMEC-1 were grown to confluence on fibronectin-coated Transwell polycarbonate filters (6.5 mm diameter, 8.0 µm pore size, Corning Costar, Corning, NY) and all experiments were done 5 to 6 days after seeding. In gene silencing experiments, siRNA transfections were done with cells cultured on six-well plates. Six hours after transfections, HMEC-1 were seeded on inserts of the Transwell system and cultured to reach confluence for 3 days. PCB 104 was added to both the lower and the upper compartments of Transwell system with or without pretreatment of inhibitors and incubated for 22 hours. HMEC-1 were then washed twice with migration medium (serum-free MCDB 131 containing 1% bovine serum albumin) and the calcein-labeled MDA-MB-231 cells (100,000) suspended in 100 µL of the same medium were added to the monolayer of HMEC-1 (i.e., to the upper chamber of the Transwell system). After incubation for 9 hours, cells were fixed with 4% formaldehyde and washed extensively with PBS. To remove nonmigrating cells, cells on the upper face of the filter were gently scraped using a cotton swab and the migrating tumor cells were observed under fluorescent microscope (Nikon Eclipse E600, Nikon, Melville, NY). Migrating cells were counted from five random fields using the x200 magnification. All assays were done in triplicate.

Permeability Assay
HMEC-1 were seeded on fibronectin-coated Transwell polycarbonate filters (12 mm diameter, 0.4 µm pore size, Corning Costar) and allowed to grow to confluence. PCB 104 with or without pretreatment of inhibitors for 30 minutes was added for 22 hours to both the lower and the upper compartments of Transwell system. After incubation, 0.5 mL FITC-dextran 40 (1 mg/mL in KRG solution) was loaded into the upper chambers for 1 hour. Fluorescence of FITC-dextran 40 in 0.5 mL medium of lower chamber was determined with a microplate spectrofluorometer (Spectramax GeminiXS) using 483 nm as excitation and 517 nm as emission wavelengths. Relative permeability was expressed by the ratio of FITC-dextran 40 transported into lower chamber compared with untreated control group. All assays were done at least in triplicate.

Cell Adhesion Assay
Adhesion of MDA-MB-231 cells to HMEC-1 was assessed as described previously (21). Briefly, the cultures were treated with PCB 104 for 24 hours before the adhesion assay, and then were washed thrice with HBSS containing 1% bovine serum albumin. The MDA-MB-231 cells labeled with 5 µmol/L calcein (Calbiochem) were incubated with PCB 104–treated HMEC-1 for 30 minutes at 37°C. The adherence was quantified by fluorescence measurements of the attached calcein-labeled MDA-MB-231 cells using excitation of 490 nm and emission of 517 nm. The results are expressed as the percentage of adhesion values determined in control cultures. All assays were done at least in triplicate.

Statistical Analysis
Results are expressed as mean ± SD. Data were statistically analyzed using one-way ANOVA followed by Student's t test. P < 0.05 was considered significant.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. John J. O'Shea for the kind gift of anti-JAK3 antibody.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: NIH/National Institute of Environmental Health Sciences grant P42 ES 07380 and Kentucky Lung Cancer Research Program.

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

Received 8/ 2/05; revised 3/14/06; accepted 4/11/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Nabeshima K, Inoue T, Shimao Y, Sameshima T. Matrix metalloproteinases in tumor invasion: role for cell migration. Pathol Int 2002;52:255–64.[CrossRef][Medline]
  2. Stamenkovic I. Matrix metalloproteinases in tumor invasion and metastasis. Semin Cancer Biol 2000;10:415–33.[CrossRef][Medline]
  3. Massova I, Kotra LP, Fridman R, Mobashery S. Matrix metalloproteinases: structures, evolution, and diversification. FASEB J 1998;12:1075–95.[Abstract/Free Full Text]
  4. Stetler-Stevenson WG, Hewitt R, Corcoran M. Matrix metalloproteinases and tumor invasion: from correlation and causality to the clinic. Semin Cancer Biol 1996;7:147–54.[CrossRef][Medline]
  5. Bode W, Fernandez-Catalan C, Grams F, et al. Insights into MMP-TIMP interactions. Ann N Y Acad Sci 1999;878:73–91.[CrossRef][Medline]
  6. Zucker S, Hymowitz M, Conner C, et al. Measurement of matrix metalloproteinases and tissue inhibitors of metalloproteinases in blood and tissues. Clinical and experimental applications. Ann N Y Acad Sci 1999;878:212–27.[CrossRef][Medline]
  7. Sternlicht MD, Lochter A, Sympson CJ, et al. The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 1999;98:137–46.[CrossRef][Medline]
  8. Brown LF, Guidi AJ, Schnitt SJ, et al. Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast. Clin Cancer Res 1999;5:1041–56.[Abstract/Free Full Text]
  9. Liotta LA, Kohn EC. The microenvironment of the tumour-host interface. Nature 2001;411:375–9.[CrossRef][Medline]
  10. Vaccariello M, Javaherian A, Wang Y, Fusenig NE, Garlick JA. Cell interactions control the fate of malignant keratinocytes in an organotypic model of early neoplasia. J Invest Dermatol 1999;113:384–91.[CrossRef][Medline]
  11. Park CC, Bissell MJ, Barcellos-Hoff MH. The influence of the microenvironment on the malignant phenotype. Mol Med Today 2000;6:324–9.[CrossRef][Medline]
  12. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–25.[CrossRef][Medline]
  13. Andl CD, Mizushima T, Oyama K, Bowser M, Nakagawa H, Rustgi AK. EGFR induced cell migration is mediated predominantly by the JAK/Stat pathway in primary esophageal keratinocytes. Am J Physiol Gastrointest Liver Physiol 2004;287:G1227–37.[Abstract/Free Full Text]
  14. Menashi S, Serova M, Ma L, Vignot S, Mourah S, Calvo F. Regulation of extracellular matrix metalloproteinase inducer and matrix metalloproteinase expression by amphiregulin in transformed human breast epithelial cells. Cancer Res 2003;63:7575–80.[Abstract/Free Full Text]
  15. Kusukawa J, Harada H, Shima I, Sasaguri Y, Kameyama T, Morimatsu M. The significance of epidermal growth factor receptor and matrix metalloproteinase-3 in squamous cell carcinoma of the oral cavity. Eur J Cancer B Oral Oncol 1996;32B:217–21.
  16. Fisher GJ, Talwar HS, Lin J, et al. Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo. J Clin Invest 1998;101:1432–40.[Medline]
  17. Orr FW, Wang HH, Lafrenie RM, Scherbarth S, Nance DM. Interactions between cancer cells and the endothelium in metastasis. J Pathol 2000;190:310–29.[CrossRef][Medline]
  18. Al-Mehdi AB, Tozawa K, Fisher AB, Shientag L, Lee A, Muschel RJ. Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat Med 2000;6:100–2.[CrossRef][Medline]
  19. Hennig B, Meerarani P, Slim R, et al. Proinflammatory properties of coplanar PCBs: in vitro and in vivo evidence. Toxicol Appl Pharmacol 2002;181:174–83.[CrossRef][Medline]
  20. Choi W, Eum SY, Lee YW, Hennig B, Robertson LW, Toborek M. PCB 104-induced proinflammatory reactions in human vascular endothelial cells: relationship to cancer metastasis and atherogenesis. Toxicol Sci 2003;75:47–56.[Abstract/Free Full Text]
  21. Eum SY, Lee YW, Hennig B, Toborek M. VEGF regulates PCB 104-mediated stimulation of permeability and transmigration of breast cancer cells in human microvascular endothelial cells. Exp Cell Res 2004;296:231–44.[CrossRef][Medline]
  22. Westermarck J, Kahari VM. Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 1999;13:781–92.[Abstract/Free Full Text]
  23. Lee TH, Avraham HK, Jiang S, Avraham S. Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability. J Biol Chem 2003;278:5277–84.[Abstract/Free Full Text]
  24. Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res 1998;58:1048–51.[Abstract/Free Full Text]
  25. Verbsky JW, Bach EA, Fang YF, Yang L, Randolph DA, Fields LE. Expression of Janus kinase 3 in human endothelial and other non-lymphoid and non-myeloid cells. J Biol Chem 1996;271:13976–80.[Abstract/Free Full Text]
  26. Verbsky JW, Randolph DA, Shornick LP, Chaplin DD. Nonhematopoietic expression of Janus kinase 3 is required for efficient recruitment of Th2 lymphocytes and eosinophils in OVA-induced airway inflammation. J Immunol 2002;168:2475–82.[Abstract/Free Full Text]
  27. Li WQ, Dehnade F, Zafarullah M. Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway. J Immunol 2001;166:3491–8.[Abstract/Free Full Text]
  28. Bergers G, Coussens LM. Extrinsic regulators of epithelial tumor progression: metalloproteinases. Curr Opin Genet Dev 2000;10:120–7.[CrossRef][Medline]
  29. Behera AK, Thorpe CM, Kidder JM, Smith W, Hildebrand E, Hu LT. Borrelia burgdorferi-induced expression of matrix metalloproteinases from human chondrocytes requires mitogen-activated protein kinase and Janus kinase/signal transducer and activator of transcription signaling pathways. Infect Immun 2004;72:2864–71.[Abstract/Free Full Text]
  30. Bugno M, Graeve L, Gatsios P, et al. Identification of the interleukin-6/oncostatin M response element in the rat tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. Nucleic Acids Res 1995;23:5041–7.[Abstract/Free Full Text]
  31. Korzus E, Nagase H, Rydell R, Travis J. The mitogen-activated protein kinase and JAK-STAT signaling pathways are required for an oncostatin M-responsive element-mediated activation of matrix metalloproteinase 1 gene expression. J Biol Chem 1997;272:1188–96.[Abstract/Free Full Text]
  32. Alper O, Bergmann-Leitner ES, Bennett TA, Hacker NF, Stromberg K, Stetler-Stevenson WG. Epidermal growth factor receptor signaling and the invasive phenotype of ovarian carcinoma cells. J Natl Cancer Inst 2001;93:1375–84.[Abstract/Free Full Text]
  33. O-charoenrat P, Modjtahedi H, Rhys-Evans P, Court WJ, Box GM, Eccles SA. Epidermal growth factor-like ligands differentially up-regulate matrix metalloproteinase 9 in head and neck squamous carcinoma cells. Cancer Res 2000;60:1121–8.[Abstract/Free Full Text]
  34. Kheradmand F, Rishi K, Werb Z. Signaling through the EGF receptor controls lung morphogenesis in part by regulating MT1-MMP-mediated activation of gelatinase A/MMP2. J Cell Sci 2002;115:839–48.[Abstract/Free Full Text]
  35. Sharrocks AD, Brown AL, Ling Y, Yates PR. The ETS-domain transcription factor family. Int J Biochem Cell Biol 1997;29:1371–87.[CrossRef][Medline]
  36. True AL, Rahman A, Malik AB. Activation of NF-{kappa}B induced by H(2)O(2) and TNF-{alpha} and its effects on ICAM-1 expression in endothelial cells. Am J Physiol Lung Cell Mol Physiol 2000;279:L302–11.[Abstract/Free Full Text]
  37. Wassermann M, Wassermann D, Cucos S, Miller HJ. World PCBs map: storage and effects in man and his biologic environment in the 1970s. Ann N Y Acad Sci 1979;320:69–124.[Medline]
  38. Jensen AA. Background levels in humans, in halogenated biphenyls, terphenyls, naphthalenes, dibenzodioxines and related products. San Diego (CA): Elsevier Science Publishers; 1989. p. 345–64.
  39. Lee YW, Park HJ, Son KW, Hennig B, Robertson LW, Toborek M. 2,2',4,6,6'-pentachlorobiphenyl (PCB 104) induces apoptosis of human microvascular endothelial cells through the caspase-dependent activation of CREB. Toxicol Appl Pharmacol 2003;189:1–10.[CrossRef][Medline]



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