Nerve growth factor activation of Erk-1 and Erk-2 induces matrix metalloproteinase-9 expression in vascular smooth muscle cells.

In response to vascular injury, smooth muscle cells migrate from the media into the intima, where they contribute to the development of neointimal lesions. Increased matrix metalloproteinase (MMP) expression contributes to the migratory response of smooth muscle cells by releasing them from their surrounding extracellular matrix. MMPs may also participate in the remodeling of extracellular matrix in vascular lesions that could lead to plaque weakening and subsequent rupture. Neurotrophins and their receptors, the Trk family of receptor tyrosine kinases, are expressed in neointimal lesions, where they induce smooth muscle cell migration. We now report that nerve growth factor (NGF)-induced activation of the TrkA receptor tyrosine kinase induces MMP-9 expression in both primary cultured rat aortic smooth muscle cells and in a smooth muscle cell line genetically manipulated to express TrkA. The response to NGF was specific for MMP-9 expression, as the expression of MMP-2, MMP-3, or the tissue inhibitor of metalloproteinase-2 was not changed. Activation of the Shc/mitogen-activated protein kinase pathway mediates the induction of MMP-9 in response to NGF, as this response is abrogated in cells expressing a mutant TrkA receptor that does not bind Shc and by pretreatment of cells with the MEK-1 inhibitor, U0126. Thus, these results indicate that the neurotrophin/Trk receptor system, by virtue of its potent chemotactic activity for smooth muscle cells and its ability to induce MMP-9 expression, is a critical mediator in the remodeling that occurs in the vascular wall in response to injury.

The neurotrophins, which include nerve growth factor (NGF), 1 brain-derived growth factor, and neurotrophins 3, 4/5, and 6 (NT-3, NT-4/5, and NT-6), are a family of highly conserved proteins best characterized for their critical roles in the differentiation and survival of neurons (1)(2)(3). These activities are mediated, for the most part, by the binding of neurotro-phins to the Trk family of receptor tyrosine kinases. This receptor family includes TrkA, TrkB, and TrkC, which bind selectively to distinct neurotrophins, with NGF binding TrkA, brain-derived growth factor binding TrkB, and NT-3 binding TrkC.
Recent studies demonstrate that, in addition to their differentiation and survival activities in the nervous system, neurotrophins and Trk receptors are also expressed in the cardiovascular system, where they mediate cardiac (4) and intramyocardial vessel development (5). They are also expressed by neointimal smooth muscle cells in lesions that develop after experimentally induced injury of the rat thoracic aorta and in advanced human atherosclerotic lesions (6). Moreover, NGF is a potent chemotactic agent for smooth muscle cells expressing Trk receptors, eliciting a response comparable with PDGF-BB (6,7). The high level of expression of neurotrophins and their receptors in advanced neointimal lesions, however, suggest that in addition to their chemotactic activity on smooth muscle cells, there may be other mechanisms by which NGF and neurotrophins can contribute to lesion development. One potential mechanism is the regulation of proteolytic activity in developing neointimal lesions.
Increased proteolytic activity in the vessel wall mediates the degradation of the extracellular matrix surrounding smooth muscle cells in response to injury (8), a necessary step to allow medial smooth muscle cells to migrate into the intimal space. Of the proteolytic enzymes expressed by vascular smooth muscle cells, matrix metalloproteinases (MMPs), a large family of neutral proteases, are thought to be one of the principal regulators of matrix degradation in response to vascular injury. First, although MMPs are expressed at low levels by smooth muscle cells in noninjured vessels, their expression is induced in the rat aorta following balloon de-endothelialization (9 -11). Second, treatment with MMP inhibitors reduces smooth muscle cell accumulation in the intima and reduces lesion formation following balloon injury of the rat thoracic aorta (9,12). Finally, MMPs are expressed in human atherosclerotic lesions, particularly at sites of plaque instability, where their expression may predispose the plaque to rupture (13). Thus, expression of MMPs appears to play a critical role in the pathogenesis of neointimal lesions.
One class of MMPs that has been implicated as mediators of lesion development in response to vascular injury are the gelatinases, MMP-2 (72 kDa) and MMP-9 (92 kDa). Both are expressed in the balloon-injured rat thoracic aorta (9 -11) and in human atherosclerotic lesions (13)(14)(15). Although MMP-2 and MMP-9 have similar substrate specificities (16,17), there are differences in the regulation of their expression. MMP-2 is constitutively expressed by several cell types, including smooth muscle cells, and its expression is not induced by cytokines or growth factors (18,19). In contrast, the basal levels of MMP-9 are usually low, and its expression can be induced by treatment of cells with tumor necrosis factor ␣ (TNF-␣) and interleukin-1, but not platelet-derived growth factor (PDGF; Refs. 18 and 19). Moreover, in the balloon-injured rat thoracic aorta, MMP-9 expression, both protein and mRNA, is induced early after injury (9 -11). In contrast, the expression of the MMP-2 is not changed following vascular injury (9 -11). These data demonstrate that the expression of MMP-9 and MMP-2 may be differentially regulated in response to vascular injury.
Studies examining the signal transduction pathways that regulate MMP-9 expression have demonstrated that activation of the mitogen-activated protein kinases, Erk-1 and Erk-2, is critical for the increased expression of MMP-9 in response to different agonists (20,21). Previous studies from our laboratory demonstrated that NGF is a potent activator of Erk-1 and Erk-2 in vascular smooth muscle cells expressing TrkA receptors (7). This suggests that, in addition to its potent chemotactic activity, NGF could also potentially regulate MMP-9 expression in smooth muscle cells. Thus, the aim of the present study was to determine whether NGF regulates MMP expression by vascular smooth muscle cells. In studies reported here, we demonstrate that NGF treatment of smooth muscle cells expressing TrkA induces MMP-9 expression. Moreover, NGFinduced MMP-9 expression is dependent on the activation of the MAP kinases, Erk-1 and Erk-2. These studies further support a role for the neurotrophins in vascular remodeling and lesion development following vascular injury.

MATERIALS AND METHODS
Cell Culture-Temperature-sensitive mouse smooth muscle cells (TsTmSMC), grown from aortic explants of a transgenic mouse line expressing a temperature sensitive SV40-T antigen under the control of the promoter for smooth muscle cell ␣-actin (22), and a previously established clone of TsTmSMC stably expressing TrkA (TrkA-TsT-mSMC; previously referred to as mTrkA48) were cultured as described (7). Rat aortic smooth muscle cells purchased from Cell Applications (San Diego, CA) were cultured as directed and were used at passages 4 -7.
Stable clones expressing TrkA mutated at Tyr-490 (Y490F-TrkA; Shc binding mutant; Tyr 3 Phe; Ref. 23) or at Lys-538, the ATP binding site in the kinase domain of Trk (kinase-dead Trk (KD-TrkA) receptor (Lys 3 Asn); Refs. 23 and 24) were generated by transfecting TsTmSMC with a pLNCX5 vector containing the cDNA for the TrkA mutant and encoding neomycin (G418) resistance (kindly provided by David R. Kaplan, McGill University, Montreal, Canada). After selection in G418 (1 mg/ml; Invitrogen), colonies were subcloned, expanded and tested for stable expression of full-length Trk receptors by Western blot analysis, using a polyclonal antiserum directed against the carboxyl tail of Trk (rabbit polyclonal, C-14; 200 ng/ml; Santa Cruz Biotechnology, Santa Cruz, CA). In some experiments, cell lysates were subjected to immunoprecipitation with polyclonal antisera directed against the carboxyl tail of TrkA as described previously (␣203; 1:250 dilution; Refs. 7 and 25). Immunoreactive proteins were precipitated by incubation with protein A-Sepharose and separated by SDS-PAGE, and Western blot analysis was performed using the anti-Trk antibody described above. Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham Biosciences, Inc.) with anti-rabbit IgG conjugated to horseradish peroxidase (1:2000 dilution; Chemicon, El Segundo, CA).
SDS-PAGE Zymography-Rat aortic smooth muscle cells were plated in 100-mm tissue culture plates at 37°C in media containing 10% FCS. Native TsTmSMC and TsTmSMC expressing either wild type or mutant TrkA receptors were plated in 100-mm dishes in medium containing 10% FCS at 39.5°C for 2 days. The cells were then cultured for 24 h in serum-free medium in the absence (control) or presence of murine NGF (Harlan Bioproducts for Science, 10 -100 ng/ml), recombinant murine TNF (10 -50 ng/ml; R&D, Minneapolis, MN), or recombinant human PDGF-BB (10 -30 ng/ml; R&D). The conditioned media were collected at 18, 24, 36, and 48 h and clarified by centrifugation. In some experiments, where indicated, conditioned media were concentrated 3-fold by centrifugation through a Centricon Filter (10,000 molecular weight cutoff, Millipore, Beverly, MA). MMP activity in cellular conditioned media was analyzed by zymography, as described previously (26). Briefly, conditioned medium was mixed with SDS sample buffer without ␤-mercaptoethanol and heated for 30 min at 37°C. The volume of conditioned medium to be loaded on the zymography gel was normalized to cellular protein as assessed by a Bio-Rad protein assay, using bovine serum albumin equivalent as a standard. Samples and molecular weight markers were electrophoresed in a 10% polyacrylamide gel containing 0.1% gelatin. Recombinant human latent (92 kDa) and active (83 kDa) MMP-9 (Calbiochem, La Jolla, CA) were loaded as positive controls. The gel was washed in 2.5% Triton X-100 to remove SDS, incubated at 37°C for 48 h in 200 mM NaCl containing 40 mM Tris-HCl and 10 mM CaCl 2 , pH 7.5, and stained with Coomassie Blue. The presence of gelatinolytic activity was identified as clear bands on a uniform blue background following destaining.
In experiments in which specific enzyme inhibitors were used, TrkA-TsTmSMC were cultured as above and then preincubated with either the PI 3-kinase inhibitor wortmannin (100 nM to 1 M; Sigma) or the MEK inhibitor U0126 (Refs. 27 and 28; 10 nM to 100 M, Promega, Madison, WI) for 30 min prior to treatment with NGF (50 -100 ng/ml). After 24 h, conditioned media were collected and zymography was performed as above.
Isolation of RNA and Northern Blot Analysis for MMPs-RNA was isolated from growth factor-or cytokine-treated TrkA-TsTmSMC by phenol chloroform extraction (29,30). For Northern blot analysis for detection of mRNA for MMP-3, 20 g of total RNA was electrophoresed in 1% denaturing agarose gels containing formaldehyde and ethidium bromide and transferred to Zetaprobe followed by cross-linking using a UV Stratalinker (Stratagene, La Jolla, CA). For Northern blot analysis for MMP-9, poly(A) mRNA was first purified from total RNA, using the poly(A)tract isolation kit from Promega. After purification, mRNA was electrophoresed and transferred to a Zetaprobe filter as above. The filters were hybridized with a 32 P-radiolabeled MMP-3 cDNA (American Type Culture Collection, Manassas, VA) or MMP-9 cDNA, (kindly provided by Ghislain Opdenakker, Rega Institute for Medical Research, University of Leuven, Belgium; Refs. 31 and 32) in Hybrisol solution for 16 -24 h at 43°C in a hybridization oven (Appligene, Pleasanton, CA). The filter was then washed under high stringency conditions and exposed to Fuji x-ray film with intensifying screens at Ϫ70°C. The amounts of mRNA for MMP-3 and MMP-9 was quantified by NIH image software and normalized by comparison with the mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (American Type Culture Collection) or ␤-actin. The ␤-actin probe was generated by reverse transcriptase-PCR of RNA from the murine RAW264.7 macrophage cell line and was a gift from Dr. Jihong Han (Weill Medical College, New York, NY).
Western Blot Analysis for TIMPs-Cells were plated and treated with growth factors and cytokines as above. After 24 h, conditioned media were collected and volumes of conditioned media normalized to cell protein were separated by 12% SDS-PAGE and blotted onto nitrocellulose. Western blot analysis was performed using either monoclonal anti-TIMP-1 IgG (Ab-2; 1 g/ml; Calbiochem) or monoclonal anti-TIMP-2 IgG (Ab-2; 5 g/ml; Calbiochem) using recombinant TIMP-1 or TIMP-2 (Calbiochem) as positive controls. Immunoreactive proteins were detected using enhanced chemiluminescence as above.

NGF Induces MMP-9 Expression by Rat Smooth Muscle
Cells-The effect of NGF on MMP expression was first examined in cultured rat aortic smooth muscle cells. Previous studies from our laboratory demonstrated that these cells express TrkA receptors at low passage (6). Rat smooth muscle cells were cultured in serum-free media in the absence (control) or presence of NGF (50 and 100 ng/ml), TNF (10 ng/ml), or PDGF-BB (10 ng/ml). After 24 h, conditioned media were collected and concentrated and MMP activity determined by zymography ( Fig. 1). Media from control smooth muscle cells did not demonstrate proteolytic activity at 92 kDa, corresponding to MMP-9. In contrast, treatment with 100 ng/ml NGF induced the expression of a band of proteolytic activity that co-migrated with recombinant human latent MMP-9. These data indicate that NGF induces the synthesis of MMP-9. Media from control smooth muscle cells did contain two bands of proteolytic activity at 72 and 66 kDa (results not shown), corresponding to the latent and active forms of MMP-2, respectively. Importantly, the expression of MMP-2 was not changed by treatment of the cells with NGF. These data suggest that the effect of NGF on vascular smooth muscle cells is specific for MMP-9. The observed response of smooth muscle cells to NGF was similar to that observed after treatment with TNF, which had been shown previously to induce MMP-9 expression in human, rabbit, and rat cultured smooth muscle cells (18,19,21). As reported by others (18,19,21), PDGF-BB had no effect on the expression of either MMP-9 ( Fig. 1) or MMP-2 (results not shown) in rat smooth muscle cells.
NGF Induces MMP-9 Expression in TrkA TsTmSMC-To further characterize NGF-induced MMP-9 expression in smooth muscle cells, we utilized an established, conditionally immortalized temperature-sensitive mouse smooth muscle cell line (TsTmSMC) in which we could express various Trk receptor isoforms. The cells were derived as aortic explants from a transgenic mouse line expressing a temperature-sensitive Tantigen (22). Culturing of cells expressing the temperaturesensitive T-antigen at the nonpermissive temperature of 39.5°C reduces the expression of T-antigen and induces a nontransformed phenotype (7,33). Previous studies demonstrated that TsTmSMC express PDGF-␤ receptors and demonstrate both chemotactic and proliferative responses to PDGF-BB (7). TsTmSMC, however, do not express full-length Trk receptors, but clones stably expressing TrkA have been established by using gene transfection techniques (7). Using a previously established clone of TsTmSMC stably expressing TrkA receptors (TrkA-TsTmSMC; Ref. 7), the effect of NGF binding to TrkA on MMP-9 expression was assessed. TrkA-TsTmSMC cultured for 3 days at 39.5°C were treated with NGF (10 -100 ng/ml), TNF (100 ng/ml), or PDGF-BB (10 -50 ng/ml) in serum-free media. Conditioned media collected after 24 h were concentrated and MMP activity determined utilizing zymography. Media derived from TrkA-TsTmSMC contained both active and latent MMP-2, as indicated by the proteolytic activity at 66 and 72 kDa, respectively (Fig. 2, panel A). Cells also expressed proteolytic activity at a slightly higher molecular weight than the recombinant human latent 92-kDa MMP-9 (see Fig. 7). This band corresponds to the murine latent MMP-9, which has a molecular mass of 105-110 kDa (31). NGF induces a dose-dependent increase in MMP-9 activity, where the induction of MMP-9 by 100 ng/ml NGF was comparable with that induced by TNF. In addition to latent MMP-9, a proteolytic band which corresponds to active murine MMP-9 ( Fig. 2, panel A) was observed in zymographs utilizing concentrated media derived from cells incubated with NGF. These data indicate that, in murine smooth muscle cells expressing TrkA, NGF induces both the synthesis and activation of MMP-9. The increase in MMP-9 activity is detectable within 18 h after NGF treatment and remains increased for up to 48 h after the addition of NGF (Fig. 2, panel B, nonconcentrated media). In contrast to its effect on TrkA-TsTmSMC, NGF did not induce MMP-9 expression in native TsTmSMC (Fig. 2, panel C, nonconcentrated media), confirming that the response to NGF is mediated through its binding to TrkA. As was observed in rat smooth muscle cells, NGF did not increase the expression of MMP-2 in TsTmSMC (Fig. 2, panels A and B). PDGF-BB treatment did not effect expression of either MMP-2 or MMP-9 (Fig. 2, panels A-C).
NGF Increases mRNA Levels of MMP-9 -To determine whether the NGF-induced increase in MMP-9 activity was because of increased gene expression, Northern blot analysis for steady state levels of mRNA for MMP-9 was performed (Fig.  3). NGF induced a dose-dependent increase in the steady state MMP-9 mRNA levels in doses ranging from 25 to 100 ng/ml. The response to NGF was observed within 18 h after treatment and was sustained for at least 24 h (results not shown). Treatment of TrkA-TsTmSMC with TNF (Fig. 3), but not PDGF-BB (results not shown), also increased mRNA levels for MMP-9, consistent with the results obtained by zymography. The increases in mRNA levels in response to 50 ng/ml NGF were ϳ50% of the response observed with TNF treatment. These results confirm that NGF induces MMP-9 gene expression in smooth muscle cells.
Previous studies demonstrated that NGF induces MMP-3 FIG. 1. Zymographic analysis of conditioned media obtained from rat smooth muscle cells. Rat smooth muscle cells were cultured for 24 h at 37°C in serum-free media in the absence (C) or presence of NGF (50 and 100 ng/ml), TNF (10 ng/ml), or PDGF (10 ng/ml). Conditioned media were collected, concentrated by centrifugation through a Centricon filter, and zymography performed as described under "Materials and Methods." Standards shown are recombinant human latent MMP-9 and recombinant human active MMP-9. synthesis in neuronal cell lines (34). Faint amounts of proteolytic activity, corresponding to either MMP-1 or MMP-3 (at 48 -57 kDa) were detected in media from control cells, but the activity was not increased in media obtained from NGF-treated TrkA-TsTmSMC (results not shown). Northern blot analysis for steady state levels of mRNA for MMP-3 confirmed that NGF treatment of smooth muscle cells did not induce the expression of MMP-3 (Fig. 4). NGF, at doses ranging from 25 to 100 ng/ml did not increase the steady state mRNA levels of MMP-3 when examined at 6 (results not shown) or 24 h (Fig. 4), whereas treatment with TNF results in a 5-fold increase in mRNA levels. Thus, these results further support our conclusion that NGF selectively induces the expression of MMP-9 in vascular smooth muscle cells expressing TrkA receptors.
TIMP-2 Release from TsTmSMC-The enzymatic activity of the MMPs is regulated by their equimolar association with TIMPs. To assess NGF-induced regulation of TIMP synthesis, Western blot analysis for TIMPs 1 and 2 were performed using conditioned media from NGF-, PDGF-BB-, or TNF-treated TrkA-TsTmSMC (Fig. 5). TrkA-TsTmSMC, cultured in serumfree media, released TIMP-2 into the conditioned media, and treatment with either NGF or TNF did not increase its expression. Treatment with PDGF-BB caused a small, ϳ2-fold-increase in TIMP-2 expression. TIMP-1 was not present at detectable levels in media from either control or treated TrkA TsTmSMC. Thus, NGF does not induce TIMP expression by smooth muscle cells.
Signal Transduction Pathways Regulating NGF-induced MMP-9 Synthesis-The biological responses initiated by neurotrophins binding to Trk receptors are mediated through the integration of several signaling pathways, including Erk-1 and Erk-2, phospholipase C␥, and phosphatidylinositol 3-kinase (PI 3-kinase). Ligand-induced autophosphorylation of specific cytoplasmic tyrosine residues within the Trk receptor initiates the signaling activity. For example, phosphorylation of Tyr-785 induces binding and activation of phospholipase C␥, whereas phosphorylation of Tyr-490 mediates the activation of both Erk-1 and -2 and PI 3-kinase, via the adapter protein Shc (35,36). Previous studies from our laboratory demonstrated that laminin-induced MMP-9 expression by macrophages is dependent on the activation of the Erk signaling pathway (20). Moreover, TNF-induced MMP-9 expression is partially mediated by this signaling pathway (21). Because NGF binding to TrkA in smooth muscle cells can activate both Erk-1 and Erk-2 (7), we assessed whether this signaling pathway mediates NGF-induced MMP-9 expression in vascular smooth muscle cells.
To begin to dissect the signal transduction pathways regu- . Moreover, Western blot analysis using an antibody that recognizes phosphorylated, and hence activated, Erk-1 and Erk-2 (Fig. 6, panel C) confirm that only TsTmSMC expressing wild type TrkA, but not Y490F-TrkA, show NGF-induced Erk-1/Erk-2 activation. The ability of NGF to induce MMP-9 expression in TsT-mSMC expressing mutant TrkA receptors was assessed using cells cultured for 2 days at 39.5°C, followed by treatment with either NGF (50 -100 ng/ml), PDGF-BB (10 ng/ml), or TNF (50 ng/ml). After 24 h, MMP activity in conditioned media was analyzed by zymography, as above (Fig. 7). In contrast to TsT-mSMC expressing wild type TrkA, TsTmSMC expressing the kinase-dead mutant TrkA receptors do not demonstrate NGFinduced MMP-9 expression (Fig. 7, panel A). This indicates that activation of the TrkA tyrosine kinase domain in response to NGF binding is necessary for the induction of MMP-9 expression. Moreover, in cells expressing the Shc-binding TrkA mu- FIG. 3. Analysis of MMP-9 mRNA levels. Northern blot analysis for steady state mRNA levels for MMP-9. Cells were cultured for 2 days at 39.5°C in media containing 10% FCS. Poly(A) mRNA purified from total RNA from NGF (10 -100 ng/ml) or TNF (T; 10 ng/ml) treated TrkA-TsTmSMC for 24 h in serum-free media. Control cells were cultured in the presence of media alone (C). RNA was separated by gel electrophoresis and transferred to Zeta-Probe membrane, and Northern blot analysis was performed as described under "Materials and Methods." Steady state mRNA levels for MMP-9 was normalized to the housekeeping gene ␤-actin. The amounts of mRNA for the 3.2-kb band of MMP-9 and the 2.5-kb band of MMP-9 were quantified by NIH Image analysis, and the total amounts of both MMP-9 mRNAs were compared with the amount of ␤-actin. The ratios of mRNA for total MMP-9/␤-actin are as follows: control, 0.75; NGF (10 ng/ml), 1.  4. Northern blot analysis for steady state mRNA for MMP-3. RNA was harvested from TrkA-TsTmSMC cultured for 2 days at 39.5°C in media containing 10% FCS then for 24 h in serum-free media in the absence (C) or presence of NGF at the indicated doses or TNF (T; 50 ng/ml). Northern blot analysis was performed as described in Fig. 3. Steady state mRNA levels for MMP-3 was normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and the ratio of mRNA for MMP-3/glyceraldehyde-3-phosphate dehydrogenase is as follows: control, 0.7; NGF (50 ng/ml), 0.9; NGF (75 ng/ml), 0.9; TNF (3.9). tant, NGF also fails to induce MMP-9 expression (Fig. 7, panel  B), whereas the response to TNF was comparable with that observed in cells expressing wild type receptors. These results suggest that NGF-induced MMP-9 expression is mediated through phosphorylation of tyrosine 490.
As mentioned above, two distinct signaling pathways are activated from NGF-induced phosphorylation of tyrosine 490: the MAP kinases Erk-1 and Erk-2 and PI 3-kinase (36). To determine which of these two signaling pathways regulates NGF-induced MMP-9 activity, TsTmSMC expressing wild type TrkA were treated with the pharmacological inhibitors of either PI 3-kinase (Wortmannin) or the MAP kinase kinase (MEK-1) inhibitor (U0126), 30 min prior to treatment with NGF. Treatment of TrkA-TsTmSMC with U0126 resulted in a dose-dependent inhibition of both NGF and PDGF-␤␤-induced Erk-1/Erk-2 activation, as assessed by Western blot analysis using the anti-phospho-Erk antibody described above (Fig. 8,  panel A). The doses of U0126 that inhibited NGF-induced phosphorylation of Erk-1 and Erk-2 were similar to those that inhibited MEK-1 activity and subsequent activation of Erk-1 and Erk-2 in other cell systems (20,21,27,28). 24 h after treatment with NGF, conditioned media were collected and zymography was performed as above. Inhibition of MEK-1 activity by U0126 inhibits NGF-induced MMP-9 expression in a dose-dependent manner (Fig. 8, panel C) in doses comparable with those inhibiting NGF-induced phosphorylation of Erk-1 and Erk-2. U0126 (10 M) completely inhibited the NGF-induced phosphorylation of Erk-1 and Erk-2, reflecting completion inhibition of MEK activity, concomitant with the complete inhibition in the increase in MMP-9 expression in response to NGF. Similar to what was observed with NGF-induced phosphorylation of Erk-1 and Erk-2, the doses of U0126 inhibiting NGF-induced MMP-9 expression are similar to those inhibiting MMP-9 expression in response to other agonists (20,21). In contrast, the PI 3-kinase inhibitor, Wortmannin, had no effect on NGF-induced MMP-9 expression (Fig. 8, panel B) at doses that completely inhibited NGF and PDGF-BB-induced phosphorylation of a downstream effector of PI 3-kinase, Akt (Fig. 8,  panel B (panels A and B), KD-TrkA  (panel A), or Y490F-TrkA (panel B). Cells were cultured for 2 days at 39.5°C. The cells were then cultured for 24 h in serum-free media in the absence (C) or presence of NGF (50 -100 ng/ml), TNF (T; 50 ng/ml), or PDGF-BB (P; 10 ng/ml). Conditioned medium was collected and zymography was performed as described under "Materials and Methods." Std., human recombinant latent and active MMP-9. were treated for 5 min with either NGF (50 ng/ml) or PDGF-BB (10 ng/ml). Whole cell lysate Western blot analysis was then performed using an antibody that recognizes phosphorylated Erk-1 and Erk-2. Panel B, inhibition of NGF and induced phosphorylation of the PI 3-kinase effector, Akt. TrkA-TsTmSMC preincubated with wortmannin were treated for 10 min with either NGF or PDGF as in panel A. Whole cell lysate Western blot analysis was then performed using an antibody that recognized phosphorylated Akt. Panel C, zymographic analysis of U0126 inhibition of NGF-induced MMP-9 activity. After pretreatment with either wortmannin or U0126, the cells were treated with either NGF (N; 100 ng/ml) or TNF (T, 50 ng/ml). After 24 h, conditioned media were collected and zymographic analysis was performed as above. Arrow indicates area where latent MMP-9 migrated in the gel. This represents one of three similar experiments. that activation of the MAP kinase signaling pathway and not PI 3 kinase mediates NGF-induced MMP-9 expression in smooth muscle cells. DISCUSSION Evidence is emerging that the neurotrophin/Trk receptor system plays a role in the biological responses of vascular smooth muscle cells to injury (6,7). The high level of expression of both the ligand and receptor in acute and chronic forms of vascular injury (6) and the multiple signaling pathways activated by NGF binding to TrkA expressed by smooth muscle cells (7) suggest that, in addition to their chemotactic activities, the neurotrophins may mediate other biological activities of smooth muscle cells in the injured vascular wall. Because NGF has been shown to regulate neuronal MMP expression (34), we explored whether NGF could regulate MMP expression by vascular smooth muscle cells.
Our results identify the neurotrophin/Trk receptor system as a regulator of MMP-9 expression by vascular smooth muscle cells. Physiological concentrations of NGF induced MMP-9 expression in TrkA-expressing smooth muscle cells by both primary cultures constitutively expressing TrkA and a smooth muscle cell line genetically manipulated to express high levels of TrkA. Moreover, ligand-induced activation of the TrkA kinase domain was necessary for this response, as cells expressing a kinase-inactive Trk receptor failed to demonstrate NGFinduced MMP-9 expression.
Although MMP-9 expression was increased in response to NGF, PDGF-BB was without effect, similar to what has been observed by other laboratories (18,19). Previous studies from our laboratory demonstrated that, although NGF and PDGF-BB activate similar signaling pathways in vascular smooth muscle cells, there are differences in the extent and duration of activation of these signaling molecules (7). In particular, NGF induces a more prolonged activation of Erk-1 and Erk-2 (greater than 1 h), whereas the response to PDGF-BB is more transient, peaking within 5 min and returning to control levels within 20 min. The current study demonstrates that activation of the Erk-1 and Erk-2 mediates NGF-induced MMP-9 expression. Thus, the ability of NGF to induce prolonged activation of Erk-1 and Erk-2 in vascular smooth muscle cells (7) as opposed to the transient response to PDGF-BB would explain the differences in their ability to mediate induction of MMP activity by smooth muscle cells.
The role of prolonged activation of Erk-1 and Erk-2 in mediating NGF-induced MMP-9 expression in smooth muscle cells is in agreement with previous studies performed in keratinocytes, where prolonged activation of the Erk signaling pathway by hepatocyte growth factor and epidermal growth factor correlated with increased expression of MMP-9. In contrast, keratinocyte growth factor and insulin-like growth factor induced transient activation of Erk-1 and Erk-2 and had no effect on MMP-9 expression (38). Activation of Erk-1 and Erk-2 causes their translocation to the nucleus, where they remain until their activity decreases (39). The retention of activated Erk-1 and Erk-2 in the nucleus could lead to the continued expression and/or activation of specific transcription factors regulating gene expression in response to NGF that are distinct from those induced by PDGF-BB. For example, prolonged activation of Erk-1 and Erk-2 is associated with continued expression and phosphorylation of the transcription factor, Ets-2 (40), which can promote MMP-9 expression in tumor cell lines (41). Activation of NFB and AP-1 also contribute to MMP-9 expression in tumor cells (42), as well as in primary cultured foreskin fibroblasts (43).
Studies on the differential expression of MMPs suggest that the transcriptional regulation of MMPs is both cell-and agonist-dependent. For example, whereas both epidermal growth factor and PDGF-BB induce MMP-3 expression in fibroblasts (44), they have no effect on its expression in the pheochromocytoma neuronal cell line, PC12. In contrast, NGF is a potent inducer of MMP-3 expression in PC12 cells (34) but did not induce its expression in smooth muscle cells (current study). Although NGF activates similar signaling pathways in smooth muscle cells as observed in neuronal cell lines, including Erk-1 and Erk-2, phospholipase C␥, and PI 3-kinase, the cell type-specific responses elicited by NGF may depend on the intracellular environment and downstream signaling molecules that are activated in a specific cell type. NGF regulation of MMP-3 gene expression in PC12 cells involves different promoter elements and possibly different transcription factors that are cell typespecific and are not involved in the regulation of MMP-3 gene expression by other growth factors (45). Thus, the ability of an agonist to induce specific MMP expression may depend on the cell-specific transcription factors that are induced, which will ultimately depend on the integration of specific signal transduction pathways that are activated in response to the agonist.
In contrast to its effect on MMP-9 expression, NGF treatment of TrkA-expressing smooth muscle cells had no effect on the expression of TIMP-2, one of the endogenous inhibitors of MMP-9 (46). TIMPs bind MMPs in a 1:1 stoichiometry to inhibit their activity. Thus, an increase in MMP-9 expression without a concomitant increase in TIMPs would tip the balance toward increased proteolytic activity. In the uninjured vessel, normally quiescent smooth muscle cells are surrounded by an extracellular matrix. The composition of the extracellular matrix is maintained by a balance between its synthesis and breakdown. An increase in proteolytic activity in the injured vascular wall disrupts this balance and results in increased degradation of connective tissue components. Smooth muscle cells then enter an activated state, where they begin to proliferate and eventually migrate into the intimal space (46). As both neurotrophins and Trk receptors are expressed by neointimal smooth muscle cells in the balloon-injured rat aorta and in human atherosclerotic lesions, our current data support a role for neurotrophin-induced Trk activation as an important regulator of proteolytic activity in the injured vascular wall.
NGF treatment of smooth muscle cells also did not effect the expression of the other gelatinase MMP-2. Both MMP-9 and MMP-2 are expressed in atherosclerotic lesions. They may, however, play different roles in the development and progression of vascular disease because of their differential expression in atherosclerotic lesions and their potential proteolytic activity. For example, whereas MMP-2 is expressed in both normal arteries and atherosclerotic lesions, its expression is not altered by cytokines or growth factors (18,19). Moreover, its proteolytic activity is thought to be limited by the colocalization of its endogenous inhibitor, TIMP-2 (13). In contrast, increased matrix degrading activity in atherosclerotic lesions colocalizes with increased expression of immunoreactive MMP-9, in particular in the shoulder regions of the plaques, areas vulnerable to rupture (13). In support of a role for MMP-9 in plaque instability, increased expression of MMP-9 is also observed in atherosclerotic lesions from patients that had undergone acute ischemic coronary syndrome, a condition often caused by plaque rupture (14). In addition to the differential expression of MMP-9 versus MMP-2, there may also be differences in the extracellular matrix components that they can degrade. For example, both MMP-2 and MMP-9 can degrade types IV and V collagen (16), as well as elastin (17). However, only MMP-9 can degrade type I and III collagens (16), whereas MMP-2 specifically degrades fibronectin and laminin (47). These data further support the hypothesis that MMP-9 and MMP-2 play different roles in the remodeling of the extracellular matrix, and thus the pathogenesis of atherosclerotic lesions.
The ability of NGF to induce MMP-9 expression demonstrates an important mechanistic difference in NGF-and PDGF-BB-induced migration of medial smooth muscle cells in response to vascular injury. For smooth muscle cells to migrate, they must be released from the complex extracellular matrix which surrounds them. As neurotrophin and Trk receptor expression is increased in response to vascular injury, NGF could act in an autocrine manner to induce MMP-9 expression and initiate detachment of medial smooth muscle cells from their surrounding matrix. In contrast, PDGF-BB induction of MMP activity requires co-stimulation with other agonists, such as interleukin-1 (19). Thus, it would appear that PDGF-BB alone cannot mediate detachment of the medial smooth muscle cells from their extracellular matrix to initiate migration. In the injured vascular wall, smooth muscle cells will simultaneously be exposed to multiple growth factors that contribute to the responses of medial smooth muscle cells to the injurious stimulus. NGF, through increased MMP-9 expression, can also promote extracellular matrix degradation to permit the full migratory activity of smooth muscle cells, in response to itself as well as to other chemotactic factors, including PDGF-BB. Moreover, in advanced atherosclerotic lesions, NGF-induced MMP expression could result in the degradation of plaque matrix, weakening of the plaque, and subsequent plaque rupture. Thus, our data demonstrate that the neurotrophins promote multiple biological responses in smooth muscle cells that can contribute to the pathogenesis of vascular lesions.