HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 290/3/C883    most recent 00359.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Moshal, K. S. Articles by Tyagi, S. C. Search for Related Content PubMed PubMed Citation Articles by Moshal, K. S. Articles by Tyagi, S. C.

RECEPTORS AND SIGNAL TRANSDUCTION

Regulation of homocysteine-induced MMP-9 by ERK1/2 pathway

Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky

Submitted 18 July 2005 ; accepted in final form 20 October 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Homocysteine (Hcy) induces matrix metalloproteinase (MMP)-9 in microvascular endothelial cells (MVECs). We hypothesized that the ERK1/2 signaling pathway is involved in Hcy-mediated MMP-9 expression. In cultured MVECs, Hcy induced activation of ERK, which was blocked by PD-98059 and U0126 (MEK inhibitors). Pretreatment with BAPTA-AM, staurosporine (PKC inhibitor), or Gö6976 (specific inhibitor for Ca²⁺-dependent PKC) abrogated ERK phosphorylation, suggesting the role of Ca²⁺ and Ca²⁺-dependent PKC in Hcy-induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G protein-coupled receptors (GPCRs) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVECs with genistein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting the involvement of the PTK pathway in Hcy-induced ERK activation, which was mediated by intracellular Ca²⁺ pool depletion. ERK activation was attenuated by preincubation with N-acetylcysteine (NAC) and SOD, suggesting the role of oxidation in Hcy-induced ERK activation. Pretreatment with an ERK1/2 blocker (PD-98059), staurosporine, folate, or NAC modulated Hcy-induced MMP-9 activation as measured using zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVECs.

calcium signaling; protein kinase C; Src; G protein-coupled receptor; nonreceptor tyrosine kinase; protein G_i; protein G_q; protein tyrosine kinase 2; microvascular endothelial cell; cardiovascular remodeling

Elevated plasma levels of homocysteine (Hcy) are an important independent risk factor for the development of cardiovascular diseases, stroke, thrombosis, and dementia (14). Hcy induces endocardial endothelial cell dysfunction (28, 29) and impairs microvascular endothelial cell (MVEC) function in vivo (40). Because these effects are mediated in part by Hcy-induced MMP-9 expression (17), it is possible that Hcy might alter the expression of tissue inhibitors of metalloproteinase (TIMPs), which results in abnormal MMP activity (31). MMP-9 has been implicated as a key effector in Hcy-induced vascular remodeling and atherosclerosis. For example, increased MMP-9 activity has been reported in macrophages isolated from patients with hyperhomocysteinemia leading to atherosclerosis (16). Furthermore, Hcy induces the activation of MMP-9, which is regulated in part by TIMP-1 in cultured MVECs (37).

Activation of MMP-9 is important in the process of vascular remodeling, but the upstream regulatory pathways that control the expression and secretion of MMP-9 are less well understood. MMP-9 contains activator protein (AP)-1 sites in its gene promoter region (13, 41). Because AP-1 transcriptional activity is specifically regulated by MAPK, we hypothesized that MAPK is involved as an upstream trigger in MMP-9 regulation.

MAPKs are a family of serine/threonine kinases that regulate the diversity of cellular activities. Three major classes have been described: ERKs, JNKs, and p38 MAPK (12). One of the ubiquitous intracellular signaling mechanisms is the ERK pathway. Two major isoforms of ERK, p44 (ERK1) and p42 (ERK2), have been identified in mammalian systems. ERK activation is reported to be involved in the receptor activation of multiple heteromeric G proteins, including both pertussis toxin (PTX)-sensitive protein G_i/o and PTX-insensitive protein G_q (15, 39). The major pathway involved in ERK1/2 phosphorylation in a variety of cell types requires the activation of all isoforms of Raf by binding to the GTP-bound form of Ras, and MEK is the only commonly recognized substrate (21, 25).

The present work was conducted to characterize the Hcy-induced ERK signaling pathway and its possible involvement in MMP-9 regulation in cultured MVECs. We report herein that Hcy triggered G protein-coupled receptors (GPCRs), which instigated signal transduction involving PTKs, Ca²⁺-dependent PKC, and oxidative stress transduction pathways, leading ultimately to ERK activation. Furthermore, we present evidence that Hcy-induced MMP-9 was regulated by the ERK1/2 signaling pathway.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Chemicals. The antibody against phosphorylated ERK1/2 (p44/42) was obtained from Cell Signaling Technology (Beverly, MA). ERK2, MMP-9, PTK2, SRC2, and -actin, and PKC- MAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated antibody was obtained from Cappel Laboratories (Durham, NC). MEK inhibitors PD-98059 and U0126, phorbol 12,13-dibutyrate (PDBu), PMA, thapsigargin (Tg), Gö6976, and PTX B were purchased from Calbiochem (La Jolla, CA). ATP, N-acetylcysteine (NAC), SOD, D,L-homocysteine, L-cysteine, folate, fura-2 AM, ionomycin, BAPTA-AM, genistein, staurosporine, and gelatin were obtained from Sigma (St. Louis, MO).

Cell culture and treatments. Human heart MVECs were procured from Cambrex (Walkersville, MD) and cultured in endothelial basal medium 2 supplemented with growth factors as described in the supplier's protocol and 12% (vol/vol) heat-inactivated FCS maintained at 37°C in a 95% O₂-5% CO₂ humidified atmosphere. MVECs were used at passages 8–13, grown to near confluence, and serum starved overnight before being treated with the indicated reagents for Western blot analysis, cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured, and gelatin zymography was performed. Serum-starved cells were pretreated for 30 min with 50 µM PD-98059, 40 µM U0126, 1 µM staurosporine, 1 µM Gö6976, 1 µg/ml PTX B, 200 µM genistein, and 1 µM Tg before 30-min treatment with 10 µM Hcy, unless otherwise indicated. Pretreatment (24 h) with 100 nM PDBu was used to downregulate PKC, whereas 30-min pretreatment with PDBu was used to activate PKC. Where indicated, serum-starved cells were pretreated for 30 min with folate (100 µM), PD-98059, U0126, NAC (50 µM), SOD (2,500 U/ml), ionomycin (1 µM), ATP, and BAPTA-AM (30 µM) before Hcy treatment.

Assay of MMP-9 activity using gelatin zymography. MMP-9 activity in cultured endothelial cells was measured as described previously (39) with some modifications. After treatment, the culture medium was collected and centrifuged at 14,000 rpm for 20 min at 4°C to remove cellular debris. The supernatant was concentrated 40-fold using a Minicon filter (Millipore, Billerica, MA) with a 15-kDa cutoff pore diameter. Conditioned medium was used to access MMP-9 activity. Protein concentration was assayed using the Bradford method (5), and the samples were mixed with equal volumes of 2x SDS sample buffer. The samples were incubated at room temperature for 5 min and electrophoresed on 7.5% SDS-PAGE gels containing 2 mg/ml gelatin as a substrate at constant voltage. After electrophoresis, the gels were rinsed in renaturation buffer (2.5% Triton X-100) on a shaker for 30 min to remove SDS and then incubated overnight at 37°C in a water bath in activation buffer composed of (in mM) 50 Tris·HCl, pH 7.4, and 5 CaCl₂. Gels were stained using 0.5% Coomassie blue R-250 for 2 h, followed by appropriate destaining. To confirm that the digested bands were caused by Ca²⁺-dependent protease, replicate gels were developed in Ca²⁺-free buffer containing 25 mM EDTA. MMP activity was detected as a white band on a dark blue background and quantitated densitometrically using Un-Scan-It software (Silk Scientific, Orem, UT).

Measurement of [Ca²⁺]_i. [Ca²⁺]_i levels were measured as described previously (30). Human MVECs were cultured on glass coverslips until they reached 80% confluence. The cells were then washed with PBS, preincubated in fura-2 AM loading buffer (in mM: 110 NaCl, 5.5 KCl, 25 NaHCO₃, 0.8 MgCl₂, 0.4 KH₂PO₄, 0.33 Na₂HPO₄, 20 HEPES, and 1.2 CaCl₂) for 10 min, and then loaded with 5 µM fura-2 AM dye for 30 min in the dark at 37°C. Fura-2 AM-loaded cells were washed to remove unbound fura-2 AM before treatment. Fluorometric reading was conducted using a Fluorolog-2 spectrofluorometer (Spex Industries, Edison, NJ) at 37°C with fluorescence (F) measured at 340/380-nm excitation and 510-nm emission. [Ca²⁺]_i was calculated using the following equation: [Ca²⁺]_i = K_d(F – F_min)/(F_max – F). F_max was obtained by treating cells with ionomycin, and F_min was achieved by addition of EGTA. K_d is the dissociation constant of the Ca²⁺-fura-2 complex. A known value (115 nM) of K_d was used to calculate [Ca²⁺]_i. At the end of each experiment, the functional integrity of the cells was checked by treating them with ionomycin (2 µM) and the traces were calibrated in terms of [Ca²⁺]_i.

Preparation of samples, Western blot analysis, and immunodetection. After treatment, medium was aspirated from six-well culture dishes and MVECs were washed twice with ice-cold 1x PBS. Ice-cold lysis buffer (in mM: 50 Tris·HCl, pH 7.4, 150 NaCl, 1% Triton X-100, and 1 EGTA) and freshly prepared inhibitors (1 mM PMSF, 1 µg/ml leupeptin, 200 µM sodium orthovandate, and 1 µg/ml aprotinin) were added to each well. Plates were scraped on ice, and the supernatant containing cytosolic protein was collected and centrifuged at 14,000 g for 20 min at 4°C, and resolved by SDS-PAGE on 10% polyacrylamide gels and blotted onto a polyvinylidene difluoride membrane. After being transferred, blots were washed with Tris-buffered saline (TBS) for 5 min at room temperature and incubated in blocking buffer for 1 h at room temperature. The blots were then incubated with the indicated primary antibodies [appropriate dilutions in 5% BSA-1x TBS with 0.1% Tween 20 (TBST)] overnight at 4°C using gentle agitation. The blots were washed three times (8-min wash each time) with TBST and incubated with HRP-conjugated secondary antibody (1:3,000 dilution in 5% BSA-TBST). After being washed, the proteins of interest were detected using an ECL plus kit (Amersham Biosciences, Piscataway, NJ). The membranes were then stripped using 0.2 M NaOH solution for 30 min at room temperature and reprobed for total proteins. Band densities were normalized to the untreated controls and presented as percentage changes as indicated.

Statistical analysis. Statistical analysis was performed using an unpaired Student's t-test for comparison between control, Hcy, and other pharmacological treatment groups. P < 0.05 was considered statistically significant. All results are averages ± SE of at least three different experiments.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Hcy activates ERK1/2. To determine time- and dose-dependent responses of Hcy-induced ERK activation, we cultured MVECs with different doses of Hcy for different intervals. ERK activation was dose- and time-dependent (Fig. 1, A and B). Maximal ERK phosphorylation was observed at 30 min with 10 µM Hcy supplementation. In subsequent experiments, we used one concentration (10 µM) and one time point (30 min).

View larger version (29K): [in this window] [in a new window]   Fig. 1. Concentration- and time-dependent activation of ERK1/2 by homocysteine (Hcy). A: serum-starved microvascular endothelial cells (MVECs) were left untreated or were treated for 1 h with different concentrations of Hcy. Bottom gel: ERK activation was measured in cell lysates using Western blot analysis for phosphorylated ERK1/ERK2, i.e., p44 and p42, respectively (pERK1/2); top gel: membranes were stripped and reprobed with ERK2 MAb for total ERK2. Density of the bands was determined and quantified for ERK1/2 activation as shown in histograms (increase relative to control). B: serum-starved MVECs were treated with 10 µM Hcy for the indicated times. Bottom gel: ERK1/2 phosphorylation was measured in cell lysates; top gel: membranes were reprobed for total ERK2. Density of the bands was determined and quantified for ERK1/2 activation (increase relative to control) as shown in histograms. Bars represent averages ± SE from 3 different experiments. #P < 0.05 vs. control.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Activation of ERK by Hcy requires MEK. Serum-starved MVECs received no pretreatment or were pretreated for 30 min with 50 µM PD-98059 and 40 µM U0126, respectively, followed by 30-min treatment with 10 µM Hcy. Bottom gel: ERK1/2 phosphorylation was measured in cell lysates using Western blot analysis; top gel: membranes were reprobed for total ERK2. Band density is indicated by bars representing ERK1/2 activation (increase relative to control). Bars represent averages ± SE from 3 different experiments. #P < 0.05 vs. control. *P < 0.05 vs. Hcy treatment.

View larger version (30K): [in this window] [in a new window]   Fig. 3. A: activation of ERK by Hcy requires involvement of G protein-coupled receptors (GPCRs) and PTKs. Serum-starved MVECs either were left untreated or were pretreated for 20 min with 1 µg/ml PTX and 200 µM genistein, followed by 30-min treatment with 10 µM Hcy. Bottom gel: ERK1/2 phosphorylation was measured in cell lysates using Western blot analysis; top gel: membranes were reprobed for total ERK2. Band density was determined and quantified for ERK1/2 activation (increase relative to control) and is indicated by bars. Bars represent averages ± SE from 3 different experiments. #P < 0.05 vs. control. *P < 0.05 vs. Hcy treatment. B: Western blot analysis of Hcy-induced PKC-, PTK2, and SRC2 in the presence and absence of 50 µM antioxidant N-acetyl cysteine (NAC).

View larger version (40K): [in this window] [in a new window]   Fig. 4. Folic acid and oxidative stress in Hcy-induced activation of ERK. Serum-starved MVECs either were left untreated or were pretreated for 30 min with 100 µM folate, folate + PD-98059, 50 µM NAC, and 2,500 U/ml SOD, followed by 30-min treatment with 10 µM Hcy. Bottom gel: ERK1/2 phosphorylation was measured in cell lysates by Western blot analysis; top gel: membranes were reprobed for total ERK2. Bars represent ERK1/2 activation (increase relative to control). Data are averages ± SE from 3 different experiments. #P < 0.05 vs. control. *P < 0.05 vs. Hcy treatment.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Serum-starved MVECs were cultured on coverslips and loaded with fura-2 AM Ca2+ dye. A: representative confocal image showing increase in intracellular Ca2+ concentration ([Ca2+]i) induced by Hcy (10 µM). B: changes in Ca2+ transients ([Ca2+]i) were monitored in fura-2 AM-loaded MVECs using spectrofluorometry in response to different concentrations of Hcy (downward arrows indicating 0, 1, 10, 50, and 100 µM from left to right, respectively). C: [Ca2+]i in cells in serum-free medium (control) treated with 1 mM ATP, 10 µM Hcy, and ATP + Hcy. #P < 0.05 vs. control. *P < 0.05 vs. Hcy treatment.

View larger version (23K): [in this window] [in a new window]   Fig. 6. A: serum-starved MVECs either were left untreated or were pretreated for 30 min with 1 µM ionomycin and 30 µM BAPTA-AM, followed by 30-min treatment with 10 µM Hcy. Bottom gel: ERK1/2 phosphorylation was measured in cell lysates by Western blot analysis; top gel: membranes were reprobed for total ERK2. Density of the bands was determined and quantified for ERK1/2 activation (increase relative to control), with the results presented in the bar graph below Western blots. Data are averages ± SE from 3 different experiments. B: serum-starved MVECs either were left untreated or were pretreated for 15 min with 1 µM thapsigargin (Tg), 200 µM genistein (Gen), genistein + Tg, and 30 µM BAPTA-AM + genistein + Tg, followed by 30-min treatment with 10 µM Hcy. Top: Western blot analysis showing that ERK1/2 phosphorylation measurement in cell lysates. An equal amount of protein was loaded in each lane. Density of bands was determined and quantified for ERK1/2 activation (increase relative to control). Hcy-mediated ERK activation induced by Ca2+ store depletion occurred through a PTK-sensitive pathway. Data are averages ± SE from 3 different experiments. #P < 0.05 vs. control. *P < 0.05 vs. Hcy treatment.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Activation of the ERK by Hcy involves PKC. A: serum-starved MVECs either were left untreated or were pretreated for 30 min with 0.1% DMSO; 1 µM staurosporine; and 100 nM PMA, an agonist for PKC; followed by 30-min treatment with 10 µM Hcy. Top gel: ERK1/2 phosphorylation was measured using Western blot analysis of cell lysates; bottom gel: membranes were reprobed for total ERK2. Bar graphs below gels demonstrate ERK1/2 activation (increase relative to control). Ca2+-dependent PKC was involved in Hcy-induced ERK activation. B: serum-starved MVECs either were left untreated or were pretreated for 24 h with 100 nM PBDu for 30 min with 1 µM Gö6976, an inhibitor of Ca2+-dependent PKC, and for 30 min with PD-98059, followed by 30-min treatment with 10 µM Hcy Bottom gel: ERK1/2 phosphorylation was measured using Western blot analysis of cell lysates; top gel: membranes were reprobed for total ERK2. The density of the bands was determined and quantified for the ERK1/2 activation (increase relative to control). C: cells treated with 100 nM PBDu for 24 h. Bottom gel: PKC- expression was measured using Western blot analysis of cell lysates (MATERIALS AND METHODS); top gel: membranes were reprobed with -actin MAb as a loading control. Bar graph represents PKC- expression (change compared with control). Data in bar graph are averages ± SE from 3 different experiments. #P < 0.05 vs. control. *P < 0.05 vs. Hcy treatment.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Hcy-induced matrix metalloproteinase (MMP)-9 is regulated by ERK1/2, which involves PKC and oxidative stress transduction pathways. A: serum-starved MVECs either were left untreated or were pretreated with 50 µM MEK inhibitor PD-98059 or 50 µM NAC. B: treatment with 1 µM staurosporine for 30 min was followed by 30-min treatment with 10 µM Hcy. MMP-9 expression in conditioned was examined using Western blot analysis. C and D: MMP-9 activity was assayed using gelatin zymography. For Western blot analysis, the membranes were reprobed with -actin and treated as a loading control. Purified MMP-9 was used as a standard in performing zymography. MMP-9 expression and activity are indicated by bars (change compared with control). A–D: data shown in bars are averages ± SE from 3 different experiments. #P < 0.05 vs. control; *P < 0.05 vs. Hcy treatment.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

To determine whether ERK is activated via the Raf-MEK-MAPK pathway, we used a specific inhibitor for MEK. PD-98059 and U0126 significantly reduced ERK activation, suggesting that Hcy-mediated ERK activation occurred in a MEK-dependent manner. MAPK (ERK) is a key point of convergence in signaling for many cell surface receptors, including receptor PTKs and GPCRs (2, 8). Hcy may activate the growth factor signal transduction pathway, resulting in atherosclerosis (18). Various growth factor and G protein-linked agonists initiate the release of Ca²⁺ from intracellular stores. Recently, capacitative Ca²⁺ entry, which is also applicable to Hcy-mediated Ca²⁺ transients, has been suggested to occur via activation of one or more PTKs (23). Receptor and nonreceptor PTKs constitute the initiators of the ERK signaling pathway (1, 35). Others have suggested that Ca²⁺ is involved in ERK activation induced by hormones acting on GPCRs in hepatocytes (27). Hcy and its metabolites are known to interact with molecular targets such as neurotransmitter receptors, channels, or transporters and are potent and effective agonists at several rat metabotropic glutamate receptors (mGluRs). Moreover, mGluRs are candidate GPCRs for intracellular signal transduction by Hcy (38). G_q has also been implicated in the regulation of Ca²⁺, PKC, and PTKs. However, the mechanisms by which Hcy affects GPCR coupling to G proteins are incompletely understood. We evaluated the role of PTKs and GPCRs in Hcy-induced ERK activation in MVECs. We found that the inhibitors for PTKs and GPCRs, i.e., genistein and PTX, abrogated Hcy-induced ERK activation. PTX data suggested the involvement of G_i, although G_q also has been shown to regulate Ca²⁺, PKC, and PTKs. Previously, we demonstrated that Hcy induced an increase in [Ca²⁺]_i in VSMCs that was ameliorated by genistein (30). This suggests the involvement of protein G_i-sensitive GPCRs in Hcy-induced ERK activation, which involves Ca²⁺, PKC, and PTKs. These findings imply that Hcy-stimulated cell surface receptors initiate a signal transduction pathway and lead to ERK activation. We sought to validate the role played by Ca²⁺ in Hcy-induced ERK activation.

We observed that Hcy-induced ERK activation was repressed by incubating the cells with a cell-permeable intracellular Ca²⁺ chelator (BAPTA-AM) and exposure of cells to ionomycin-activated ERK, which imply the involvement of Ca²⁺ in ERK activation (Fig. 6). This finding is in accordance with the results reported by Ingram et al. (18), which showed the involvement of Ca²⁺ in Hcy-induced ERK activation in mesangial cells. Furthermore, we noticed that Tg, a sarco(endo)plasmic reticulum Ca²⁺-ATPase blocker, activated ERK, which was abrogated by treatment with BAPTA-AM and genistein. These findings suggested that the PTK pathway is involved in Hcy-induced ERK activation, which is mediated by intracellular Ca²⁺ pool depletion. These observations are in agreement with the finding in rat liver epithelial WB cells that Ca²⁺ store depletion increases ERK phosphorylation via the PTK pathway (24).

In cardiac myocytes, activation of ERK (MAPK) was blocked by NAC, implicating a role for oxidative stress in signaling events (4). In previous studies of mesangial cells, oxidative stress was not involved in Hcy-induced ERK activation (6). We found that treatment of the cells with NAC and SOD reduced Hcy-mediated ERK phosphorylation (Fig. 4). This disparity might be attributed to differences in cell type, doses of Hcy used, and the mode of treatment applied.

Folic acid administration reduces the risk of atherosclerosis caused by elevated levels of Hcy. We sought to determine the effect of folate supplementation on Hcy-induced ERK activation. This report is the first to describe abrogation of Hcy-induced ERK phosphorylation, which was found to be MEK dependent, upon treatment of MVECs with folate (Fig. 4). Few reports have suggested a role of folate in ERK activation. Folate acts as a chemoattractant and induces ERK2 activation in Dictyostelium discoideum (20).

Hcy induced the activation of PKC and stimulated c-Fos in murine macrophages (3). Several lines of evidence indicate that PKC activates the MAPK signal transduction pathway. PKC played an important role in mediating ERK activation and subsequent DNA synthesis in intestinal epithelial cells in response to ANG II (9). PKC is a multigene family composed of at least 12 distinct isoforms and is an important constituent of the signaling pathway triggered by mitogen through cell surface receptors. In the present study, we observed that treatment with staurosporine reduced Hcy-mediated ERK activation (Fig. 7), which suggests that Hcy-stimulated cell surface receptors initiated signal transduction involving PKC and led to ERK activation. Because Hcy induced alterations in Ca²⁺ dynamics, we suspected the involvement of the Ca²⁺-dependent PKC isoform. We have shown that Gö6976, a specific inhibitor of PKC-, abrogated Hcy-induced ERK activation in MVECs (Fig. 7C). Furthermore, to validate the involvement of Ca²⁺-dependent PKC, we downregulated PKC by treating cells with PBDu for 24 h and evaluated the Hcy-induced ERK phosphorylation and PKC- expression, which was found to be suppressed (Fig. 7C). Additional information is required to confirm which other isoforms of PKC are induced by Hcy treatment and are involved in the ERK signaling pathway.

MMPs play an important role in the degradation of the ECM, leading to vascular pathology. Previously, we showed that Hcy induces the activation of MMP-9 in MVECs; however, the regulatory mechanisms remain to be elucidated fully. The promoter region of the MMP-9 gene contains AP-1 sites, thus implying a role for the MAPK signaling pathway in the regulation of MMP-9. Different studies of various cell types have suggested a role for ERK activation in MMP-9 induction (26, 34, 42).

Our data demonstrate that Hcy increased the activity of MMP-9 in MVECs. Treatment with the ERK1/2 pathway inhibitor PD-98059 resulted in inhibition of MMP-9 induction (Fig. 8), which suggested that Hcy-induced MMP-9 is regulated by the ERK pathway. It is still possible that other members of the MAPK family, i.e., JNK/SAPK and p38, are involved in MMP-9 regulation. In fact, Hcy is capable of activating JNK (7) and p38 (10).

In the present study, we have shown that the Hcy-induced ERK activation involved PKC and oxidative stress transduction pathways. Our aim was to investigate whether these transduction pathways are involved in Hcy-induced MMP-9 upregulation. Evidence from different cell systems showed that oxidative stress is involved in the induction of MMP-9. We observed that inhibiting ROS generation by NAC treatment significantly inhibited the activation of MMP-9 in MVECs. These observations suggest that the oxidative transduction pathway is important for Hcy-mediated MMP-9 induction. Hcy-induced MMP-9 activation was blocked by treating the cells with staurosporine, a PKC inhibitor, which suggested the role of the PKC transduction pathway in MMP-9 regulation. LPS induced MMP-9 secretion in part via sequential activation of PKC and the ERK1/2-dependent pathway in rat primary astrocytes (22).

In conclusion, we have demonstrated that Hcy triggers PTX-sensitive GPCRs, which initiate signal transduction involving PTKs, Ca²⁺-dependent PKC, and oxidative stress, leading to ERK1/2 activation. Furthermore, we have presented evidence that Hcy-mediated MMP-9 induction is regulated by the ERK1/2 signaling pathway in cultured MVECs (Fig. 9).

View larger version (23K): [in this window] [in a new window]   Fig. 9. Schematic of possible Hcy-induced MAPK ERK1/2 signaling pathways and MMP-9 regulation in MVECs. Hcy-induced MMP-9 was regulated by a pertussis toxin (PTX)-sensitive ERK1/2 signaling pathway involving PKC and oxidative stress transduction pathways. AP-1, activator protein (AP)-1.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. C. Tyagi, Dept. of Physiology and Biophysics, Univ. of Louisville School of Medicine, A-1115, 500 S. Preston St., Louisville, KY 40202 (e-mail: s0tyag01{at}louisville.edu)

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

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Adachi T, Nakashima S, Saji S, Nakamura T, and Nozawa Y. Mitogen-activated protein kinase activation in hepatocyte growth factor-stimulated rat hepatocytes: involvement of protein tyrosine kinase and protein kinase C. Hepatology 23: 1244–1253, 1996.[CrossRef][ISI][Medline]

2. Alblas J, van Corven EJ, Hordijk PL, Milligan G, and Moolenaar WH. G_i-mediated activation of the p21^ras-mitogen-activated protein kinase pathway by ₂-adrenergic receptors expressed in fibroblasts. J Biol Chem 268: 22235–22238, 1993.[Abstract/Free Full Text]

3. Beauchamp MC and Renier G. Homocysteine induces protein kinase C activation and stimulates c-Fos and lipoprotein lipase expression in macrophages. Diabetes 51: 1180–1187, 2002.[Abstract/Free Full Text]

4. Bogoyevitch MA, Ng DCH, Court NW, Draper KA, Dhillon A, and Abas L. Intact mitochondrial electron transport function is essential for signalling by hydrogen peroxide in cardiac myocytes. J Mol Cell Cardiol 32: 1469–1480, 2000.[CrossRef][ISI][Medline]

5. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]

6. Brown JC III, Rosenquist TH, and Monaghan DT. ERK2 activation by homocysteine in vascular smooth muscle cells. Biochem Biophys Res Commun 251: 669–676, 1998.[CrossRef][ISI][Medline]

7. Cai Y, Zhang C, Nawa T, Aso T, Tanaka M, Oshiro S, Ichijo H, and Kitajima S. Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activation of c-Jun NH₂-terminal kinase and promoter response element. Blood 96: 2140–2148, 2000.[Abstract/Free Full Text]

8. Carraway KL and Carraway CA. Signaling, mitogenesis and the cytoskeleton: where the action is. Bioessays 17: 171–175, 1995.[CrossRef][ISI][Medline]

9. Chiu T, Santiskulvong C, and Rozengurt E. ANG II stimulates PKC-dependent ERK activation, DNA synthesis, and cell division in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 285: G1–G11, 2003.[Abstract/Free Full Text]

10. Danese S, Sgambato A, Papa A, Scaldaferri F, Pola R, Sans M, Lovecchio M, Gasbarrini G, Cittadini A, and Gasbarrini A. Homocysteine triggers mucosal microvascular activation in inflammatory bowel disease. Am J Gastroenterol 100: 886–895, 2005.[CrossRef][ISI][Medline]

11. Davies B, Brown PD, East N, Crimmin MJ, and Balkwill FR. A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts. Cancer Res 53: 2087–2091, 1993.[Abstract/Free Full Text]

12. Davis RJ. Transcriptional regulation by MAP kinases. Mol Reprod Dev 42: 459–467, 1995.[CrossRef][ISI][Medline]

13. Fini ME, Cook JR, Mohan R, and Brinckerhoff CE. Regulation of matrix metalloproteinase gene expression. In: Matrix Metalloproteinases, edited by Parks WC and Mecham RP. San Diego, CA: Academic, 1998, p. 300–356.

14. Hashimoto T, Wen G, Lawton MT, Boudreau NJ, Bollen AW, Yang GY, Barbaro NM, Higashida RT, Dowd CF, Halbach VV, and Young WL; University of California, San Francisco BAVM Study Group. Abnormal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in brain arteriovenous malformations. Stroke 34: 925–931, 2003.[Abstract/Free Full Text]

15. Hawes BE, van Biesen T, Koch WJ, Luttrell LM, and Lefkowitz RJ. Distinct pathways of G_i- and G_q-mediated mitogen-activated protein kinase activation. J Biol Chem 270: 17148–17153, 1995.[Abstract/Free Full Text]

16. Holven KB, Halvorsen B, Schulz H, Aukrust P, Ose L, and Nenseter MS. Expression of matrix metalloproteinase-9 in mononuclear cells of hyperhomocysteinaemic subjects. Eur J Clin Invest 33: 555–560, 2003.[CrossRef][ISI][Medline]

17. Hunt MJ and Tyagi SC. Peroxisome proliferators compete and ameliorate Hcy-mediated endocardial endothelial cell activation. Am J Physiol Cell Physiol 283: C1073–C1079, 2002.[Abstract/Free Full Text]

18. Ingram AJ, Krepinsky JC, James L, Austin RC, Tang D, Salapatek AM, Thai K, and Scholey JW. Activation of mesangial cell MAPK in response to homocysteine. Kidney Int 66: 733–745, 2004.[CrossRef][ISI][Medline]

19. Kieseier BC, Seifert T, Giovannoni G, and Hartung HP. Matrix metalloproteinases in inflammatory demyelination: targets for treatment. Neurology 53: 20–25, 1999.[Abstract/Free Full Text]

20. Kosaka C and Pears CJ. Chemoattractants induce tyrosine phosphorylation of ERK2 in Dictyostelium discoideum by diverse signalling pathways. Biochem J 324: 347–352, 1997.[Medline]

21. Kyriakis JM, App H, Zhang X, Banerjee P, Brautigan DL, Rapp UR, and Avruch J. Raf-1 activates MAP kinase-kinase. Nature 358: 417–421, 1992.[CrossRef][Medline]

22. Lee JW, Shin CY, Yoo BK, Ryu RJ, Choi EY, Cheong JH, Ryu JH, and Ko KH. Induction of matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide-stimulated primary astrocytes is mediated by extracellular signal-regulated protein kinase 1/2 (Erk1/2). Glia 41: 15–24, 2003.[CrossRef][ISI][Medline]

23. Lee KM, Toscas K, and Villereal ML. Inhibition of bradykinin- and thapsigargin-induced Ca²⁺ entry by tyrosine kinase inhibitors. J Biol Chem 268: 9945–9948, 1993.[Abstract/Free Full Text]

24. Maloney JA, Tsygankova OM, Yang L, Li Q, Szot A, Baysal K, and Williamson JR. Activation of ERK by Ca²⁺ store depletion in rat liver epithelial cells. Am J Physiol Cell Physiol 276: C221–C230, 1999.[Abstract/Free Full Text]

25. Macdonald SG, Crews CM, Wu L, Driller J, Clark R, Erikson RL, and McCormick F. Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. Mol Cell Biol 13: 6615–6620, 1993.[Abstract/Free Full Text]

26. McCawley LJ, Li S, Wattenberg EV, and Hudson LG. Sustained activation of the mitogen-activated protein kinase pathway: a mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration. J Biol Chem 274: 4347–4353, 1999.[Abstract/Free Full Text]

27. Melien Ø, Nilssen LS, Dajani OF, Larsen Sand K, Iversen JG, Sandnes DL, and Christoferssen C. Ca²⁺-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F₂: a role of calmodulin and Src kinases. BMC Cell Biol 3: 5, 2002. doi:10.1186/1471-2121-3-5.[CrossRef][Medline]

28. Miller A, Mujumdar V, Palmer L, Bower JD, and Tyagi SC. Reversal of endocardial endothelial dysfunction by folic acid in homocysteinemic hypertensive rats. Am J Hypertens 15: 157–163, 2002.[CrossRef][ISI][Medline]

29. Miller A, Mujumdar V, Shek E, Guillot J, Angelo M, Palmer L, and Tyagi SC. Hyperhomocyst(e)inemia induces multiorgan damage. Heart Vessels 15: 135–143, 2000.[CrossRef][ISI][Medline]

30. Mujumdar VS, Hayden MR, and Tyagi SC. Homocyst(e)ine induces calcium second messenger in vascular smooth muscle cells. J Cell Physiol 183: 28–36, 2000.[CrossRef][ISI][Medline]

31. Mujumdar VS, Tummalapalli CM, Aru GM, and Tyagi SC. Mechanism of constrictive vascular remodeling by homocysteine: role of PPAR. Am J Physiol Cell Physiol 282: C1009–C1015, 2002.[Abstract/Free Full Text]

32. Parks WC and Mecham RP. Matrix Metalloproteinases. London: Academic, 1998.

33. Rao JS, Steck PA, Mohanam S, Stetler-Stevenson WG, Liotta LA, and Sawaya R. Elevated levels of M_r 92,000 type IV collagenase in human brain tumors. Cancer Res 53, Suppl 10: 2208–2211, 1993.[Abstract/Free Full Text]

34. Reddy KB, Krueger JS, Kondapaka SB, and Diglio CA. Mitogen-activated protein kinase (MAPK) regulates the expression of progelatinase B (MMP-9) in breast epithelial cells. Int J Cancer 82: 268–273, 1999.[CrossRef][ISI][Medline]

35. Sargeant P, Farndale RW, and Sage SO. ADP- and thapsigargin-evoked Ca²⁺ entry and protein-tyrosine phosphorylation are inhibited by the tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate in fura-2-loaded human platelets. J Biol Chem 268: 18151–18156, 1993.[Abstract/Free Full Text]

36. Sato H and Seiki M. Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells. Oncogene 8: 395–405, 1993.[ISI][Medline]

37. Shastry S and Tyagi SC. Homocysteine induces metalloproteinase and shedding of -1 integrin in microvessel endothelial cells. J Cell Biochem 93: 207–213, 2004.[CrossRef][ISI][Medline]

38. Shi Q, Savage JE, Hufeisen SJ, Rauser L, Grajkowska E, Ernsberger P, Wroblewski JT, Nadeau JH, and Roth BL. L-Homocysteine sulfinic acid and other acidic homocysteine derivatives are potent and selective metabotropic glutamate receptor agonists. J Pharmacol Exp Ther 305: 131–142, 2003.[Abstract/Free Full Text]

39. Sugden PH and Clerk A. Regulation of the ERK subgroup of MAP kinase pathways through G protein-coupled receptors. Cell Signal 9: 337–351, 1997.[CrossRef][ISI][Medline]

40. Ungvari Z, Csiszar A, Bagi Z, and Koller A. Impaired nitric oxide-mediated flow induced coronary dilation in hyperhomocysteinemia: morphological and functional evidence for increased peroxynitrite formation. Am J Pathol 161: 145–153, 2002.[Abstract/Free Full Text]

41. Vu TH and Werb J. Gelatinase B: structure, regulation, and function. In: Matrix Metalloproteinases, edited by Parks WC and Mecham RP. New York: Academic, 1998, p. 115–148.

42. Zeigler ME, Chi Y, Schmidt T, and Varani J. Role of ERK and JNK pathways in regulating cell motility and matrix metalloproteinase 9 production in growth factor-stimulated human epidermal keratinocytes. J Cell Physiol 180: 271–284, 1999.[CrossRef][ISI][Medline]

This article has been cited by other articles:

				U. Sen, N. Tyagi, M. Kumar, K. S. Moshal, W. E. Rodriguez, and S. C. Tyagi Cystathionine- -synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1779 - C1787. [Abstract] [Full Text] [PDF]

				K. S. Moshal, M. Singh, U. Sen, D. S. E. Rosenberger, B. Henderson, N. Tyagi, H. Zhang, and S. C. Tyagi Homocysteine-mediated activation and mitochondrial translocation of calpain regulates MMP-9 in MVEC Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2825 - H2835. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 290/3/C883    most recent 00359.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Moshal, K. S. Articles by Tyagi, S. C. Search for Related Content PubMed PubMed Citation Articles by Moshal, K. S. Articles by Tyagi, S. C.