A novel role for nuclear factor of activated T cells in receptor tyrosine kinase and G protein-coupled receptor agonist-induced vascular smooth muscle cell motility.

In addition to their role in cytokine gene regulation in T cells, nuclear factors of activated T cells (NFATs) have been shown to be involved in cardiac development and hypertrophy. We have reported previously that NFATs play an important role in the regulation of vascular smooth muscle cell (VSMC) proliferation by receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists, platelet-derived growth factor-BB (PDGF-BB) and thrombin, respectively. To understand the role of NFATs in vascular disease and development, we have now studied the role of these transcriptional factors in VSMC motility. PDGF-BB and thrombin induced VSMC motility in a dose-dependent manner. Blockade of NFAT activation resulted in substantial reduction in PDGF-BB- and thrombin-induced VSMC motility. PDGF-BB and thrombin also induced interleukin-6 (IL-6) expression in NFAT-dependent manner. Furthermore, IL-6 dose-dependently caused VSMC motility. A neutralizing anti-rat IL-6 antibody inhibited VSMC motility induced by IL-6, PDGF-BB, and thrombin. In addition, exogenous addition of IL-6 rescued both PDGF-BB- and thrombin-induced VSMC motility from inhibition by the blockade of NFAT activation. Together, these results for the first time demonstrate that NFATs mediate both RTK and GPCR agonist-induced VSMC motility via induction of expression of IL-6.


In addition to their role in cytokine gene regulation in T cells, nuclear factors of activated T cells (NFATs) have been shown to be involved in cardiac development and hypertrophy.
We have reported previously that NFATs play an important role in the regulation of vascular smooth muscle cell (VSMC) proliferation by receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists, platelet-derived growth factor-BB (PDGF-BB) and thrombin, respectively. To understand the role of NFATs in vascular disease and development, we have now studied the role of these transcriptional factors in VSMC motility. PDGF-BB and thrombin induced VSMC motility in a dose-dependent manner. Blockade of NFAT activation resulted in substantial reduction in PDGF-BB-and thrombin-induced VSMC motility. PDGF-BB and thrombin also induced interleukin-6 (IL-6) expression in NFAT-dependent manner. Furthermore, IL-6 dose-dependently caused VSMC motility. A neutralizing anti-rat IL-6 antibody inhibited VSMC motility induced by IL-6, PDGF-BB, and thrombin. In addition, exogenous addition of IL-6 rescued both PDGF-BB-and thrombin-induced VSMC motility from inhibition by the blockade of NFAT activation. Together, these results for the first time demonstrate that NFATs mediate both RTK and GPCR agonist-induced VSMC motility via induction of expression of IL-6.
Sustained inflammation at the site of vascular injury is now believed to be a triggering event in the pathogenesis of vessel wall diseases (1,2). Both the dysfunctional endothelial cells and inflammatory cells at the site of vascular injury produce a large number of molecules with a broad spectrum of biological activities (1)(2)(3). A majority of these molecules are either mitogenic, motogenic, or both to vascular smooth muscle cells (VSMC) 1 (4 -8). The availability of these bioactive molecules at the site of vascular injury, therefore, provides a permissive milieu for VSMC dedifferentiation (9). The dedifferentiated VSMC acquire their embryonic non-contractile and synthetic state phenotype and via their migration from media to intima and multiplication in intima contribute to the progression of lesions such as restenosis after angioplasty (10). Several studies (11,12) have reported that inhibition of expression or bioactivity of growth factors or cytokines that are produced at the site of vascular injury ameliorates lesion progression. Since a large number of molecules are involved in arterial wall lesions, identifying the unifying mechanisms of these molecules may eventually lead to development of better therapeutics against these vascular diseases.
Nuclear factors of activated T cells (NFATs) are members of a multigene family of transcription factors that belong to the Rel group (13). These are named as NFATc1 (also known as NFATc or NFAT2), NFATc2 (also known as NFATp or NFAT1), NFATc3 (also known as NFAT4 or NFATx), and NFATc4 (also known as NFAT3), and each of these molecules appeared to be expressed as several isoforms by alternative splicing (13)(14)(15)). An additional member of the NFAT family of transcription factors is NFAT5, which was cloned and characterized in recent years (16). Although all five NFATs exhibit a similar DNA binding specificity, NFAT5 differs from the rest of the four members in its lack of co-operativity with Fos or Jun proteins (16). In regard to the functional role of these transcription factors, earlier studies (17) have reported the presence of NFATc1, NFATc2, and NFATc3 mainly in T cells regulating the expression of cytokine genes such as interleukin (IL)-2. However, later studies (13,18,19) have demonstrated the presence of all five NFAT proteins in non-immune cells as well. Studies with knock-out mice also established a role for NFATs in non-immune cells as observed from: 1) knock-out mice for NFATc1 failed to develop normal cardiac valves (20,21); 2) knock-out mice for NFATc2 and NFATc3 exhibited reduced skeletal muscle size; and 3) mice with disruption of NFATc3/c4 genes died around E11 with defects in vessel wall assembly (22)(23)(24). Interestingly, one of the members of the NFAT family transcription factors, namely NFATc4, was reported to play a role in cardiac hypertrophy (25). In contrast to NFATc1 to NFATc4, NFAT5 was shown to be involved in volume regulation (26). NFATc1 to NFATc4 exist in the cytoplasm as phosphoproteins in resting state, and their activation requires dephosphorylation. A calcium/calmodulin-dependent serine/ threonine phosphatase known as calcineurin has been reported to specifically dephosphorylate and activate these NFATs (17). In regard to NFAT5, both calcineurin-dependent and -independent mechanisms were reported to be involved in its regulation (27,28). Activated NFATs translocate to the nucleus, bind to consensus DNA sequence GGAAAAT present in the promoter regions of genes as monomers or dimers via their Rel homology domain, and activate transcription (13). However, other than the phenotypic observations made from the knock-out mice models (20 -24), the functional aspects of NFATs in non-immune cells are largely unclear. Toward understanding the functional role of NFATs in non-immune cells, we have reported previously that their activation is required for RTK and GPCR agonist-induced VSMC proliferation (29). Here, we tested their role in RTK and GPCR agonist-induced VSMC motility. Both PDGF-BB and thrombin, the RTK and GPCR agonists, respectively, stimulated VSMC motility in a dose-and NFAT-dependent manner. PDGF-BB and thrombin also induced the expression of IL-6 in NFAT-dependent manner. In addition, IL-6 dose-dependently caused VSMC motility and a neutralizing anti-rat IL-6 antibody suppressed VSMC motility induced by IL-6, PDGF-BB, and thrombin. Furthermore, exogenous addition of IL-6 rescued PDGF-BB-and thrombin-induced VSMC motility from inhibition by the blockade of NFAT activation. Together, these findings for the first time show that NFATs mediate both RTK and GPCR agonist-induced VSMC motility via induction of expression of IL-6.

PDGF-BB and thrombin transactivate NFATs in VSMC.
A, quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) in the presence and absence of CsA (10 M) for 2 h, and nuclear extracts were prepared. Wherever pGFP or pGF-PVIVIT are used, cells were transfected first with these plasmid DNAs and quiesced before they were subjected to the indicated treatments. Nuclear extracts containing an equal amount of protein from control and each treatment were analyzed for NFAT-DNA binding activity using 32 P-labeled consensus NFAT oligonucleotide probe. B, VSMC that were transfected with pT8NFAT-Luc plasmid DNA and quiesced were treated with and without PDGF-BB (20 ng/ml) or throm- bin (0.5 unit/ml) for 6 h, and cell extracts were prepared. Cell extracts containing an equal amount of protein from control and each treatment were assayed for luciferase activity. C, conditions were the same as described for B except that cells were co-transfected with pGFP or pGFPVIVIT in combination with pT8NFAT-Luc and quiesced before they were subjected to treatments. *, p Ͻ 0.01 versus control; **, p Ͻ 0.05 versus PDGF-BB; ‫,ءءء‬ p Ͻ 0.01 versus thrombin treatment alone.
Cell Culture-VSMC were isolated from the thoracic aortae of 100 -150-g male Sprague-Dawley rats by enzymatic dissociation as described earlier (29). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat inactivated fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Cultures were maintained at 37°C in a humidified 95% air and 5% CO 2 atmosphere. Cells were quiesced by incubating in DMEM containing 0.1% calf serum for 72 h and used to perform the experiments unless otherwise stated.

FIG. 3. Blockade of NFAT activation inhibits PDGF-BB-and thrombin-induced VSMC motility.
A, a cell-free gap was made in a monolayer of VSMC and treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) in the presence and absence of CsA (10 M) for 24 h, and cell motility was measured using the NIH image 1.62 program. B, VSMC were transfected with pGFP or pGFPVIVIT and quiesced before they were subjected to PDGF-BB-or thrombin-induced cell motility assay. *, p Ͻ 0.01 versus control; **, p Ͻ 0.05 versus PDGF-BB or thrombin treatment alone.

FIG. 4. PDGF-BB and thrombin induce the expression of IL-6 mRNA in VSMC.
Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) (A) or thrombin (0.5 unit/ml) (B) for various times, and total cellular RNA was isolated. One microgram of RNA from control and each treatment was subjected to RT-PCR using rat IL-6 cDNA-specific primers, and the products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, and quantified by densitometry.
Cell Motility-VSMC motility was measured by cell wounding assay (30). Quiescent confluent monolayers of VSMC were wounded with a sterile pipette tip to generate a cell-free gap of ϳ1-mm width and the wound location in the culture dish marked. Cells were washed and fresh serum-free DMEM was added and photographed to record the wound width at 0 h. Cells were then treated with and without the indicated concentrations of PDGF-BB or thrombin in the presence and absence of the indicated inhibitors or antibodies for 24 h. To prevent replicative DNA synthesis, hydroxyurea was added to the medium to a final concentration of 5 mM just before the addition of agonist. Photographs were taken again at the end of the 24-h incubation period at the marked wound location. Cell migration was measured using the NIH image 1.62 program, and the cell motility was expressed as distance migrated in micrometer units. Wherever pGFP or pGFPVIVIT are used, cells were first transfected with these plasmid DNAs for 48 h and then quiesced before they were subjected to agonist-induced motility.
Electrophoretic Mobility Shift Assay-Quiescent VSMC were treated with and without PGDF-BB (20 ng/ml) or thrombin (0.5 unit/ml) in the presence and absence of CsA (10 M) for 2 h, and nuclear extracts were prepared according to the procedure described by Dignam et al. (31). Protein-DNA complexes were formed by incubating 5 g of nuclear protein in a total volume of 20 l consisting of 15 mM HEPES, pH 7.9, 3 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4.5 g of bovine serum albumin, 2 g of poly(dI-dC), 15% glycerol, and 100,000 counts/min of 32 P-labeled consensus NFAT oligonucleotide probe for 30 min on ice. Protein-DNA complexes were resolved on a 4% polyacrylamide gel using 1ϫ Trisglycine-EDTA buffer (25 mM Tris-HCl, pH 8.5, 200 mM glycine, 0.1 mM EDTA). Double-stranded oligonucleotides (NFATc, 5Ј-CGC CCA AAG AGG AAA ATT TGTTTCATA-3Ј) were labeled with [␥-32 P]ATP using the T4 polynucleotide kinase kit as per the supplier's protocol (Promega). To test the effect of VIVIT on PDGF-BB-and thrombin-induced NFAT-DNA binding activity, cells were first transfected with either pGFP or pGFPVIVIT plasmids for 48 h and quiesced before they were subjected to treatments with agonists and nuclear extract preparation.
IL-6 ELISA-After appropriate treatments, cell culture medium was collected, and IL-6 released into the medium was measured using an ELISA kit following the manufacturer's instructions (Pierce).
RT-PCR Analysis of IL-6 mRNA-Total cellular RNA was isolated with Trizol reagent (Invitrogen) according to the manufacturer's instructions. The RNA was quantified, and 1 g of it from control and each treatment was used for the RT-PCR reaction. The RT-PCR was performed using the SuperScript III one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). The reaction mix in a total volume of 50 l consists of 1ϫ reaction buffer, 1 g of RNA, a 0.5 M concentration each of forward (5Ј-ATGAAGTTTCTCTCCGCA-3Ј) and reverse (5Ј-GGGGTAGGAAGGACTATT-3Ј) rat IL-6 specific primers, and 2 l of SuperScript III RT/platinum Taq polymerase. RT-PCR amplification was carried out in a Gene Amp 2400 system (PerkinElmer Life Sciences) with an initial first strand cDNA synthesis reaction at 55°C for 30 min. The PCR reaction was performed at 94°C for 4 min followed by 25 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The final extension reaction was carried out at 72°C for 10 min. Fifteen microliters of the PCR products were then separated by electrophoresis on a 2% agarose gel. The PCR products were visualized by ethidium bromide staining, photographed, and quantified by densitometry.
Transient Transfection and Luciferase Assay-VSMC were plated evenly onto 100-mm dishes and grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. At 50 -80% confluence, medium was replaced, and cells were transfected with 3 g/dish of pT8NFAT-Luc plasmid (32) using FuGENE 6 transfection reagent according to the manufacturer's instructions (Roche Applied Science). Forty-eight hours after transfection, VSMC were quiesced and treated with and without PGDF-BB (20 ng/ml) or thrombin (0.5 unit/ml) in the presence and absence of CsA (10 M) for 16 h, and cell lysates were prepared. VSMC lysates were normalized for protein and assayed for luciferase activity using the Luciferase Assay System (Promega) and a Turner Luminometer (TD-20/20). To test the effect of VIVIT on PDGF-BB-and thrombininduced NFAT-Luc activity, cells were co-transfected with either pGFP or pGFPVIVIT along with pT8NFAT-Luc plasmid for 48 h and quiesced before they were subjected to treatments with agonists and measuring luciferase activity.
Statistics-All the experiments were repeated at least three times with similar pattern of results. Data are presented as mean Ϯ S.D., and the treatment effects were analyzed by Student's t test.

FIG. 5. CsA inhibits PDGF-BB-and thrombin-induced IL-6 mRNA expression in VSMC.
Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) (A) or thrombin (0.5 unit/ml) (B) in the presence and absence of CsA (10 M) for 2 h, and total cellular RNA was isolated. One microgram of RNA from control and each treatment was subjected to RT-PCR using rat IL-6 cDNA-specific primers, and the products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, and quantified by densitometry.

RESULTS AND DISCUSSION
NFATs are phosphoproteins and exist in the cytoplasm in an inactive state. These transcription factors are activated via dephosphorylation. Calcineurin, a calcium/calmodulin-dependent serine/threonine phosphatase, specifically dephosphorylates NFATs leading to their translocation from the cytoplasm to the nucleus (12). In the nucleus, they bind as monomers or dimers via their Rel homology domain to a consensus DNA sequence GGAAAAT present in the promoter region of genes and induce transcription (24). Previously we have reported that the RTK and GPCR agonists, PDGF-BB and thrombin, respectively, cause translocation of NFATs, particularly NFATc1, from the cytoplasm to the nucleus in VSMC (29). In addition, we have demonstrated that NFATs are involved in PDGF-BBand thrombin-induced VSMC proliferation. To understand the role of NFATs in vascular wall remodeling, we have now tested their involvement in VSMC motility. Toward testing this hypothesis, we first measured their activity in response to PDGF-BB and thrombin in VSMC. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5

FIG. 6. Forced expression of pGFPVIVIT inhibits PDGF-BB-and thrombin-induced IL-6 mRNA expression in VSMC.
VSMC that were transfected with either pGFP or pGFPVIVIT were quiesced and treated with and without PDGF-BB (20 ng/ml) (A) or thrombin (0.5 unit/ml) (B) for 2 h, and total cellular RNA was isolated. One microgram of RNA from control and each treatment was subjected to RT-PCR using rat IL-6 cDNA-specific primers, and the products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, and quantified by densitometry.

FIG. 7. Blockade of NFAT activation inhibits PDGF-BB-and thrombin-induced release of IL-6 into the culture medium.
A, quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) in the presence and absence of CsA (10 M) for 24 h, and IL-6 release into the culture medium was measured by ELISA. B, VSMC were transfected with pGFP or pGFPVIVIT, quiesced, and treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) for 24 h, and IL-6 release into the culture medium was measured by ELISA. *, p Ͻ 0.01 versus control; **, p Ͻ 0.05 versus PDGF-BB or thrombin treatment alone. unit/ml) in the presence and absence of CsA (10 M), a potent inhibitor of calcineurin (17), for 2 h, and nuclear extracts were prepared. Nuclear extracts containing an equal amount of protein from control and PDGF-BB or thrombin-treated VSMC were incubated with 100,000 counts/min of 32 P-labeled consensus NFAT oligonucleotide probe, and the protein-DNA complexes were separated by electrophoresis on polyacrylamide gel. PDGF-BB and thrombin increased NFAT-DNA binding activity by 2-3-fold as compared with control (Fig. 1A). CsA significantly blocked the NFAT-DNA binding activity induced by both PDGF-BB and thrombin. NFATc1 to NFATc4 possess a highly conserved calcineurin-binding site PxIxIT in their regulatory domain at the NH 2 terminus. Based on this information a peptide, namely, VIVIT that specifically competes with NFATs for binding to calcineurin, was developed (33). Expression of GFPVIVIT in T cells inhibited only calcineurin-sensitive NFAT-dependent, but not calcineurin-sensitive, NFAT-independent inducible expression of IL-2, IL-3, IL-13, tumor necrosis factor-␣ (TNF-␣), granulocyte-macrophage colony stimulating factor (GM-CSF), and macrophage inflammatory protein-1␣ (MIP-1␣) by phorbol 12-myristate 13-acetate and ionomycin (33). To confirm the effect of CsA on PDGF-BB-and thrombin-induced NFAT-DNA binding activity, VSMC were transfected with pGFP or pGFPVIVIT, growth-arrested for 48 h, and treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) for 2 h, and nuclear extracts were prepared. An equal amount of nuclear protein from control and each treatment was analyzed for NFAT-DNA binding activity as described above. Consistent with the effect of CsA, GF-PVIVIT but not GFP reduced PDGF-BB-and thrombin-induced NFAT-DNA binding activity (Fig. 1A). To find whether the increased NFAT-DNA binding activity leads to a corresponding up-regulation in NFAT-dependent transcription, VSMC were transfected with a plasmid (pT8NFAT-Luc) in which a luciferase reporter gene expression is controlled by NFAT-dependent IL-2 promoter and quiesced (33). Cells were then treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) for 6 h in the presence and absence of CsA (10 M), and cell extracts were prepared. Cell extracts from control and agonist-treated VSMC normalized for protein were assayed for luciferase activity. Consistent with NFAT-DNA binding activity, luciferase activity was also increased by about 2-fold in PDGF-BB-and thrombin-treated VSMC versus control (Fig. 1B). CsA blocked both PDGF-BB-and thrombin-induced increase in luciferase activity (Fig. 1B). CsA alone had no effect on basal luciferase activity. Similarly, forced expression of GF-PVIVIT but not GFP inhibited both PDGF-BB-and thrombininduced luciferase activity (Fig. 1C). These results are consistent with our previously published findings (29).
Having demonstrated the activation of NFATs by both RTK and GPCR agonists, we now examined their role in VSMC motility. A cell-free gap was generated in a monolayer of quiescent VSMC as described under "Materials and Methods." Cells were then treated with and without various doses of PDGF-BB or thrombin for 24 h, and cell motility was measured. As shown in Fig. 2, A and B, both PDGF-BB and thrombin induced VSMC motility in a dose-dependent manner. Maximum VSMC motility was observed in response to 10 ng/ml PDGF-BB and 0.1 unit/ml thrombin. To address the role of NFATs in PDGF-BB-and thrombin-induced VSMC motility, we now studied the effect of CsA and GFPVIVIT. As shown in Fig. 3, A and B 9. Neutralizing anti-rat IL-6 antibodies block IL-6-, PDGF-BB-, and thrombin-induced VSMC motility. A cell-free gap was made in a monolayer of quiescent VSMC and treated with and without IL-6 (20 ng/ml) (A), PDGF-BB (20 ng/ml) (B), or thrombin (0.5 unit/ml) (C) in the presence and absence of 1 g/ml of neutralizing anti-rat IL-6 antibodies, and cell motility was measured using the NIH image 1.62 program. *, p Ͻ 0.01 versus control; **, p Ͻ 0.05 versus IL-6, PDGF-BB, or thrombin treatment alone. and thrombin-induced VSMC motility. Earlier studies from other laboratories (34) have indicated that NFATs are involved in the regulation of expression of IL-6 in response to PDGF-BB in VSMC. To understand the mechanisms by which NFATs mediate PDGF-BB-and thrombin-induced VSMC motility, we tested the effect of PDGF-BB and thrombin on IL-6 expression. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) for various times, and total cellular RNA was isolated. An equal amount of RNA from control and each treatment was analyzed by RT-PCR for IL-6 mRNA using its specific primers. As shown in Fig. 4, A and B, both PDGF-BB and thrombin induced the expression of IL-6 mRNA in a time-dependent manner with a maximum response at 2-h treatment. Next we tested the effect of CsA and VIVIT on PDGF-BB-and thrombin-induced IL-6 mRNA expression. Both CsA and VIVIT significantly reduced IL-6 mRNA expression induced by PDGF-BB and thrombin (Figs. 5, A and B, and 6, A  and B). To find whether CsA also blocks the IL-6 expression at protein level, quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) in the presence and absence of CsA (10 M) for 24 h, and IL-6 release into the culture medium was determined using an ELISA kit specific for IL-6. Both PDGF-BB and thrombin treatments caused the release of IL-6 into the culture medium, and CsA significantly FIG. 10. IL-6 rescues PDGF-BB-and thrombin-induced motility from inhibition by CsA or pGFPVIVIT. A cell-free gap was made in a monolayer of quiescent VSMC and treated with and without PDGF-BB (20 ng/ml) (A) or thrombin (0.5 unit/ml) (B) in the presence and absence of CsA (10 M) for 24 h, and cell migration was measured. IL-6 was added at the indicated doses after the addition of agonists. Wherever pGFP or pGFPVIVIT were used (C and D), cells were transfected first with these plasmid DNAs and quiesced before they were subjected to cell motility assay in response to treatments with PDGF-BB (C) or thrombin (D) in the presence and absence of the indicated doses of IL-6. *, p Ͻ 0.01 versus control; **, p Ͻ 0.05 versus PDGF-BB or thrombin treatment alone; ***, p Ͻ 0.01 versus CsA ϩ PDGF-BB, pGFPVIVIT ϩ PDGF-BB, CsA ϩ thrombin, or pGFPVIVIT ϩ thrombin treatments. blocked this effect (Fig. 7A). Forced expression of GFPVIVIT also blocked PDGF-BB-and thrombin-induced IL-6 release into the culture medium (Fig. 7B).
Since PDGF-BB and thrombin induced IL-6 expression in a NFAT-dependent manner, we next examined whether IL-6 causes VSMC motility. A cell-free gap was made in a monolayer of quiescent VSMC and treated with and without various doses of IL-6 for 24 h, and cell motility was determined. IL-6 induced VSMC motility in a dose-dependent manner with a maximum effect at 10 ng/ml (Fig. 8). To find whether IL-6 mediates PDGF-BB-and thrombin-induced motility, we next studied the effect of a neutralizing anti-rat IL-6 antibody on VSMC motility induced by IL-6 as well as PDGF-BB and thrombin. Neutralizing anti-rat IL-6 antibodies significantly blocked VSMC motility induced by all three agonists (Fig. 9, A and B). If NFATs mediate PDGF-BB-and thrombin-induced VSMC motility via induction of expression of IL-6, then one would expect that exogenous addition of IL-6 rescues PDGF-BB-and thrombininduced VSMC motility from inhibition by blockade of NFAT activation. To test this, a cell-free gap was made in a monolayer of quiescent VSMC and treated with and without PDGF-BB (20 ng/ml) or thrombin (0.5 unit/ml) in the presence and absence of CsA (10 M) with and without a combination of the indicated doses of IL-6 for 24 h, and cell motility was determined. As shown in Fig. 10, A and B, IL-6 rescued both PDGF-BB-and thrombin-induced VSMC motility from inhibition by CsA in a dose-dependent manner. Similarly, IL-6 negated the inhibitory effect of GFPVIVIT on PDGF-BB-and thrombin-induced VSMC motility (Fig. 10, C and D).
The important findings of the present study are as follows: 1) inhibition of NFAT activation blocked both RTK and GPCR agonist-induced VSMC motility; 2) both RTK and GPCR agonists induced the expression of IL-6 in NFAT-dependent manner; 3) IL-6 caused VSMC motility in a dose-dependent manner; 4) a neutralizing anti-rat IL-6 antibody prevented VSMC motility induced by IL-6, PDGF-BB, and thrombin; and 5) exogenous addition of IL-6 rescued PDGF-BB-and thrombininduced VSMC motility from inhibition by blockade of NFAT activation. These results demonstrate that NFATs mediate PDGF-BB-and thrombin-induced VSMC motility via induction of expression of IL-6. A role for NFATs in cell migration was previously suggested by the findings that the levels of NFATc2 and NFAT5 are increased in human breast carcinomas and their up-regulation correlated with increased carcinoma invasiveness (35). In this regard, the present study provides direct and mechanistic evidence for the role of NFATs in cell migration. Earlier work from our laboratory as well as others (19,29) showed that NFATc1 is highly expressed in VSMC compared with other members of the NFAT family of transcription factors and is translocated from the cytoplasm to the nucleus in response to RTK and GPCR agonists. Furthermore, overexpression of NFATc1 enhanced PDGF-BB-induced expression of IL-6 in VSMC (34). Based on these findings, it is likely that NFATc1 is involved in the mediation of VSMC motility in response to RTK and GPCR agonists.
Several studies (25, 36 -38) using genetic approaches have shown that NFATs play an important role in cardiac development and hypertrophy. A requirement for NFAT function was also demonstrated in vessel wall assembly (22)(23)(24). These findings clearly reveal the involvement of NFATs in cardiovascular development. Migration of VSMC from media to intima and their proliferation in intima play a contributing role in the formation of neointima after angioplasty (4 -8). We have reported previously (29) that NFATs are involved in the regulation of VSMC proliferation. A role for NFATs in the regulation of cell proliferation was also suggested by the finding that forced expression of a constitutively active NFATc1 in murine 3T3-L1 preadipocyte fibroblasts leads to their transformation (39). NFATs via their transcriptional involvement in the regulation of pro-inflammatory cytokine genes were even thought to be important in the propagation of inflammation (40). Since inflammation and VSMC migration and proliferation are key events in vessel wall diseases (1)(2)(3)(4)(5)(6)(7)(8), and the present and previous studies from our as well as other laboratories (13,17,29,40) provide evidence for a role of NFATs in the regulation of these events, it is possible that these transcriptional factors also play an essential role in vascular diseases. Future studies are required to test the involvement of NFATs in vessel wall diseases such as restenosis after angioplasty.
In summary, the present study demonstrates for the first time that NFATs mediate RTK and GPCR agonist-stimulated motility via induction of expression of IL-6 in VSMC.