Transforming Growth Factor-β1 Stimulates l-Arginine Transport and Metabolism in Vascular Smooth Muscle Cells
Role in Polyamine and Collagen Synthesis
Background—Transforming growth factor-β1 (TGF-β1) contributes to arterial remodeling by stimulating vascular smooth muscle cell (VSMC) growth and collagen synthesis at sites of vascular injury. Because l-arginine is metabolized to growth-stimulatory polyamines and to the essential collagen precursor l-proline, we examined whether TGF-β1 regulates the transcellular transport and metabolism of l-arginine by VSMCs.
Methods and Results—TGF-β1 increased l-arginine uptake, and this was associated with a selective increase in cationic amino acid transporter-1 (CAT-1) mRNA. In addition, TGF-β1 stimulated l-arginine metabolism by inducing arginase I mRNA and arginase activity. TGF-β1 also stimulated l-ornithine catabolism by elevating ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) activity. TGF-β1 markedly increased the capacity of VSMCs to generate the polyamine putrescine and l-proline from extracellular l-arginine. The TGF-β1–mediated increase in putrescine and l-proline production was reversed by methyl-l-arginine, a competitive inhibitor of cationic amino acid transport, or by hydroxy-l-arginine, an arginase inhibitor. Furthermore, the formation of putrescine was inhibited by the ODC inhibitor α-difluoromethylornithine, and l-proline generation was blocked by the OAT inhibitor l-canaline. l-Canaline also inhibited TGF-β1–stimulated type I collagen synthesis.
Conclusions—These results demonstrate that TGF-β1 stimulates polyamine and l-proline synthesis by inducing the genes that regulate the transport and metabolism of l-arginine. In addition, they show that TGF-β1–stimulated collagen production is dependent on l-proline formation. The ability of TGF-β1 to upregulate l-arginine transport and direct its metabolism to polyamines and l-proline may contribute to arterial remodeling at sites of vascular damage.
l-Arginine is a dibasic, semiessential amino acid involved in a variety of physiological processes. It is a necessary precursor for protein, creatine, polyamine, l-proline, and nitric oxide (NO) biosynthesis and serves as an intermediate in the urea cycle.1 Although l-arginine can be obtained by endogenous synthesis, most cells rely on extracellular transport for their l-arginine requirements. In vascular smooth muscle cells (VSMCs), the uptake of l-arginine is mediated by the system y+ carrier.2 3 This particular transport system is characterized by its high affinity for cationic amino acids, its independence of Na+, and the ability of substrate on the opposite (trans) side of the membrane to increase transport activity.4 Recently, the genes encoding the proteins responsible for the activity of the system y+ carrier have been cloned and designated as cationic amino acid transporter-1 (CAT-1), CAT-2, CAT-2A, and CAT-3. Whereas CAT-1, CAT-2, and CAT-3 are high-affinity (Km 100 μmol/L) transporters for l-arginine, CAT-2A is an alternative splice variant of CAT-2 that possesses low affinity (Km=1 to 2 mmol/L) forl-arginine.5 6 7 8 9 Previous studies in our laboratory and others have demonstrated that VSMCs express only CAT-1 and CAT-2 mRNA and that specific humoral mediators induce gene expression of both of these transporters.10 11 12 13 14
Once transported into VSMCs, l-arginine is metabolized to important regulatory molecules. Inducible NO synthase converts l-arginine to NO, which is a potent inhibitor of VSMC growth and collagen synthesis.15 16 Alternatively, l-arginine is metabolized to l-ornithine by arginase.1 There are 2 distinct isoenzymes of mammalian arginase that are encoded by separate genes.17 18 Although these isozymes possess similar enzymatic properties, they differ with regard to their tissue distribution, subcellular localization, and immunological reactivity.19 Type I arginase is a cytosolic enzyme that is highly expressed in the liver and constitutes a majority of total body arginase activity.20 In contrast, arginase II is a mitochondrial enzyme that is found predominantly in extrahepatic tissues.20
The arginase metabolite l-ornithine is metabolized by ornithine decarboxylase (ODC) to the polyamine putrescine, which forms the successive polyamines spermine and spermidine via the sequential transfer of a propylamine moiety from S-adenosylmethionine.21 Polyamines play an integral role in the mitogenic response of VSMCs. VSMC proliferation is preceded by increases in ODC activity and polyamine synthesis, and inhibition of polyamine formation inhibits VSMC growth.10 14 22 l-Ornithine is also converted by the mitochondrial enzyme ornithine aminotransferase (OAT) to pyrroline-5-carboxylate, which is further metabolized to l-proline, which is essential for the synthesis of many structural proteins, including collagen.22
Transforming growth factor-β1 (TGF-β1) plays an important role in development, differentiation, immune function, and tissue repair. In the vasculature, TGF-β1 plays a critical role in arterial remodeling by stimulating VSMC proliferation and collagen deposition.23 24 25 Because l-arginine can be metabolized to important growth-regulatory molecules and to the essential matrix component l-proline, the present study examined the effect of TGF-β1 on l-arginine transport and metabolism in VSMCs. We now report that TGF-β1 stimulates the transport of l-arginine and directs its metabolism to l-ornithine by coordinately inducing CAT-1 and arginase I gene expression. In addition, we demonstrate that TGF-β1 stimulates l-ornithine metabolism in VSMCs, resulting in the production of growth-stimulatory polyamines and of the integral collagen constituent l-proline.
l-Arginine, FCS, minimum essential media, SDS, EDTA, methyl-l-arginine (L-NMA), l-canaline, ammonium hydroxide, chloroform, formamide, urea, β-mercaptoethanol, Tris, HEPES, ninhydrin spray, and thin-layer chromatography plates were purchased from Sigma Chemical Co; TGF-β1 was from R & D Systems Inc; Dowex resin (50W-X8, 100- to 200-mesh), DDT, and nitrocellulose were from Bio-Rad Laboratories; GAPDH cDNA and RNA molecular weight markers were from Ambion Inc; hydroxy-l-arginine (L-NOHA) was from Alexis Corp; rabbit anti-rat collagen type I polyclonal antibody was from Biodesign International; α-difluoromethylornithine (DFMO) was generously provided by Dr Ekkhardt H.W. Bohme (Hoechst Marion Roussel, Cincinnati, Ohio); [3H]l-arginine (58 Ci/mmol) was from American Radiolabeled Chemicals; and [1-14C]l-ornithine (55 mCi/mmol), [guanido-14C]l-arginine (52 Ci/mmol), and [α-32P]UTP (400 Ci/mmol) were from Amersham Life Sciences.
VSMCs were isolated from rat thoracic aorta and cultured in minimum essential medium.14 When cells reached confluence, the culture media were replaced with serum-free media containing BSA (0.1%) for 24 hours and then exposed to the various treatment regimens.
VSMC lysates were prepared by sonication in Tris buffer (10 mmol/L Tris, 0.4% Triton X-100, 10 mg/mL leupeptin, and 10 mg/mL aprotinin, pH 7.4), and arginase activity was determined by monitoring the formation of [14C]urea from [guanido-14C]l-arginine.13
VSMCs were harvested in ice-cold Tris buffer (mmol/L: Tris 20, EDTA 0.1, DTT 2, and pyridoxal-5-phosphate 0.1, pH 7.4), sonicated, and centrifuged at 14 000g for 20 minutes at 4°C. The supernatant was collected, and ODC activity was determined by measuring the release of [14C]CO2 from [1-14C]l-ornithine, as previously described.13
VSMC lysates were prepared by sonication in KPO4 buffer (0.1 mol/L KPO4, 50 μmol/L pyridoxal-5-phosphate, 10 mg/mL leupeptin, and 10 mg/mL aprotinin, pH 7.4), and OAT activity was determined by measuring the conversion of [3H]l-ornithine to [3H]pyrroline-5-carboxylate.22
Polyamine and l-Proline Production
Polyamine and l-proline formation were determined by incubating VSMCs with [3H]l-arginine (20 μCi/mmol) for 24 hours and measuring the intracellular formation of radiolabeled putrescine and l-proline by thin-layer chromatography, as previously described.13 14
Generation of CAT and Arginase Probes
CAT and arginase cDNA fragments were amplified from VSMCs by reverse transcriptase–polymerase chain reaction (RT-PCR).10 Primers were designed according to the published sequence of the rat CAT and arginase gene products. The forward 5′-TGGCACTCTCC- TGGCTTACT-3′ and the reverse 5′-ACTTCTCGGCTGCTGGTA- AA-3′ primers were used to amplify a 182-bp CAT-1 transcript, and the forward 5′-GGGTGTCTTTCCTCATCGCTG-3′ and the reverse 5′-CAAAGGTGCCACTCCATGCTC-3′ primers were used to amplify a 210-bp CAT-2 transcript.6 7 The forward 5′-TAGAGAAA- GGTCCCGCAGCAT-3′ and reverse 5′-TGCTTCCAATTGCCAT- ACTGTG-3′ primers were used to amplify a 252-bp arginase I transcript, and the forward 5′-CCTAGTGAAGCTGCGAAC- GTG-3′ and reverse 5′-AGAGAAAGGGGCTCCGACTACA-3′ were used to amplify a 197-bp arginase II transcript.17 18 The PCR fragments were then subcloned into pCRII plasmids (Invitrogen) and sequenced to confirm their identity and orientation.
Ribonuclease Protection Assay
Total RNA (15 μg) was hybridized with ≈1×106 cpm of [32P]UTP-labeled antisense CAT, arginase, and GAPDH (316-bp) riboprobes. Protected RNA was analyzed by electrophoresis using 6% acrylamide/8 mmol/L urea gels. The size of the protected nucleotide fragments was confirmed by use of a 32P-labeled RNA ladder. Relative mRNA levels were quantified by scanning densitometry (LKB 2222-020 Ultrascan laser densitometer) and normalized with respect to GAPDH mRNA.
Culture media were collected and concentrated with Centricon YM30 filters (Amicon Inc), and proteins were solubilized with electrophoresis buffer [125 mmol/L Tris-HCl (pH 6.8), 2% SDS, 12.5% glycerol, 1% β-mercaptoethanol, and trace bromphenol blue]. Samples were boiled and proteins separated by SDS-PAGE using 6% gels. Gels were electrophoretically transferred to nitrocellulose membranes, blocked for 1 hour at room temperature in Tris buffer (50 mmol/L, pH 7.4) containing 1% BSA, and incubated with rabbit anti–collagen type I antibody (1 μg/mL) for 1 hour. Membranes were then washed in Tris buffer, incubated for 1 hour with anti-rabbit horseradish peroxidase–conjugated antibody (1:5000 dilution), and incubated with chemoluminescence reagents. Type I collagen levels were quantified by scanning densitometry.
Results are expressed as mean±SEM. Significant differences between multiple groups were evaluated by ANOVA with post hoc Bonferroni’s t test. A Student’s 2-tailed t test was used to examine significant differences between 2 groups. Values of P<0.05 were considered to be statistically significant.
Treatment of VSMCs with TGF-β1 for 24 hours stimulated the transport of l-arginine (Figure 1A⇓). Time-course studies demonstrated that TGF-β1 had a biphasic effect on l-arginine transport (Figure 1B⇓). Initially, TGF-β1 inhibited the transport of l-arginine, but by 24 hours of TGF-β1 treatment, a significant increase in transport was observed. Figure 2⇓ shows a representative Eadie-Hofstee plot demonstrating that saturable, high-affinity uptake of l-arginine by VSMCs was mediated by a single carrier. Data from several experiments (n=5) indicated that this transporter had a Michaelis constant (Km) of 81.2±7.2 μmol/L and a maximum transport velocity (Vmax) of 742±65 pmol · mg protein−1 · 45 s−1. Pretreatment of VSMCs with TGF-β1 (10 ng/mL) for 24 hours significantly (P<0.05) increased both the Km (148.6±10.8 μmol/L) and Vmax (1955±202 pmol · mg protein−1 · 45 s−1) of l-arginine transport. The increase in Vmax of l-arginine transport by TGF-β1 was completely abolished by cycloheximide (5 μg/mL) (data not shown). Ribonuclease protection assays demonstrated that treatment of VSMCs with TGF-β1 for 24 hours significantly stimulated the expression of CAT-1 mRNA (6.0±1.2-fold) but had no effect on CAT-2 message (0.9±0.2-fold) (Figure 3⇓).
TGF-β1 also stimulated arginase activity (Figure 4A⇓). An increase in arginase activity was first detected after 8 hours of TGF-β1 exposure, and arginase activity remained elevated after 24 hours (Figure 4B⇓). The TGF-β1–mediated increase in arginase activity was blocked by cycloheximide (5 μg/mL) (data not shown). Ribonuclease protection analysis revealed that TGF-β1 induced arginase I mRNA expression (1.7±0.2-fold) (Figure 5⇓); however, it failed to detect arginase II mRNA expression either in control cells or in cells exposed to TGF-β1 (data not shown).
In addition to stimulating l-arginine transport and l-arginine metabolism to l-ornithine, TGF-β1 stimulated the metabolism of l-ornithine to polyamines and l-proline. Treatment of VSMCs with TGF-β1 (10 ng/mL) induced an ≈8-fold increase in ODC activity and a >2-fold rise in OAT activity (Figure 6⇓). In addition, TGF-β1 increased the capacity of VSMCs to generate the polyamine putrescine and l-proline from extracellular l-arginine in a concentration-dependent manner (Figures 7A⇓ and 8A⇓). The latter experiments were based on the rationale that l-arginine must be converted to l-ornithine by arginase and then metabolized to putrescine and l-proline by the enzymes ODC and OAT, respectively. Therefore, the stimulation of arginase, ODC, and OAT by TGF-β1 would result in increased synthesis of [3H]putrescine and [3H]l-proline from [3H]l-arginine. The stimulatory effect of TGF-β1 on putrescine and l-proline synthesis was inhibited by the cationic amino acid transport inhibitor L-NMA (10 mmol/L)3 and by the arginase inhibitor L-NOHA (1 mmol/L)26 (Figures 7B⇓ and 8B⇓). Furthermore, the TGF-β1–mediated putrescine formation was blocked by the ODC inhibitor DFMO (2 mmol/L)27 (Figure 7B⇓), whereas l-proline generation was reversed by the specific OAT inhibitor l-canaline (100 μmol/L)28 (Figure 8B⇓).
Treatment of VSMCs with TGF-β1 (10 ng/mL) stimulated the secretion of type I collagen by nearly 3-fold (Figure 9⇓). The addition of l-canaline (100 μmol/L) to VSMCs abolished the TGF-β1–induced production of type I collagen without affecting the synthesis of collagen from untreated control cells (Figure 9⇓). Finally, the TGF-β1–stimulated increase in l-arginine transport and collagen production occurred independently of any effects on NO synthesis. Neither control SMCs nor TGF-β1–treated VSMCs generated NO (data not shown).
The present study demonstrates that TGF-β1 stimulates polyamine and l-proline synthesis in VSMCs by inducing the expression of genes that modulate the transport and metabolism of l-arginine. In particular, TGF-β1 increases the transcellular transport of l-arginine and the intracellular metabolism of l-arginine to l-ornithine by specifically stimulating the expression of the genes for CAT-1 and arginase I. Furthermore, TGF-β1 directs the intracellular metabolism of l-ornithine to polyamines and l-proline by stimulating ODC and OAT activity, respectively. Moreover, the TGF-β1–mediated production of collagen by VSMCs is dependent on l-proline synthesis.
TGF-β1 stimulates the transport of l-arginine in a time-dependent manner. Kinetic experiments indicate that high-affinity l-arginine (Km ≈80 μmol/L) transport is mediated by a single carrier and that TGF-β1 increases both the Vmax and Km of this transporter. These kinetic data suggest that the TGF-β1–induced decrease in l-arginine uptake observed at early time points probably arises from the TGF-β1–mediated decrease in the affinity of the transporter. In contrast, the increase in l-arginine transport observed after 24 hours probably arises from the de novo synthesis of additional transport proteins. Consistent with this, we found that cycloheximide blocks TGF-β1–induced transport. Moreover, we observed that TGF-β1 stimulates CAT-1 gene expression. The selective induction of CAT-1 mRNA by TGF-β1 contrasts with the coinduction of CAT-1 and CAT-2 mRNA observed after the administration of other growth factors.10 11 13 14
In addition to stimulating transcellular l-arginine transport, TGF-β1 stimulates the intracellular metabolism of l-arginine to l-ornithine in VSMCs. TGF-β1 induces a time-dependent increase in arginase activity. This increase in arginase activity is dependent on de novo protein synthesis and is paralleled by a selective increase in arginase I mRNA, suggesting that arginase I mediates the TGF-β1 effect. Our finding that TGF-β1 stimulates arginase activity in VSMCs complements an earlier study showing that TGF-β increases arginase activity in macrophages,29 indicating a role for this protein in regulating extrahepatic arginase activity. The selective expression of arginase isozymes has also been reported in other tissues and may serve to regulate l-ornithine metabolism by preferentially directing l-ornithine to the cytosol for polyamine synthesis via ODC or to the mitochondria for l-proline production by OAT.30 31 l-Ornithine transporters have been identified in mitochondria, however, suggesting that l-ornithine may rapidly equilibrate between the cytosolic and mitochondrial compartments.32 The coinduction of CAT-1 and arginase I by TGF-β1 found in our study may provide a mechanism by which increased levels of substrate (l-arginine) are provided to VSMCs during activation of the arginase enzyme.
The ability of TGF-β1 to stimulate arginase activity may function to direct l-arginine to l-ornithine metabolism to generate biologically relevant polyamines and l-proline. In support of this proposal, we found that TGF-β1 increased both ODC and OAT activity. Moreover, TGF-β1 treatment resulted in a prominent increase in the capacity of VSMCs to generate polyamines and l-proline from extracellular l-arginine. This TGF-β1–mediated effect is blocked by the cationic amino acid transport inhibitor L-NMA and by the arginase inhibitor L-NOHA, indicating that both the transcellular transport of l-arginine and intracellular arginase activity are limiting factors that govern the ability of TGF-β1 to generate polyamines and l-proline. In addition, the formation of polyamines was blocked by the ODC inhibitor DFMO, whereas l-proline generation was inhibited by the OAT inhibitor l-canaline. These findings suggest that TGF-β1–mediated increases in both intracellular l-ornithine synthesis and metabolism are coordinated to maximize the cellular capacity for polyamine and l-proline synthesis.
The concentration of TGF-β1 (1 to 30 ng/mL) necessary to stimulate l-arginine transport and metabolism in our study is physiologically relevant. Circulating levels of TGF-β1 range from 2 to 10 ng/mL in healthy individuals.33 Moreover, at sites of vascular injury, where vessel wall TGF-β1 synthesis is induced and activated platelets release TGF-β1, the local concentration of TGF-β1 may be significantly higher.24
The biological role of ODC and polyamines in stimulating VSMC growth is well established. VSMC proliferation is associated with a striking increase in ODC activity and polyamine formation.10 14 Moreover, inhibition of ODC activity inhibits VSMC proliferation.14 In contrast, the physiological function of OAT in vascular cells is not known. In the present study, we are the first to demonstrate that VSMCs express OAT activity and suggest that OAT plays a critical role in collagen synthesis. As previously reported,34 we found that TGF-β1 increases the formation of type I collagen, which is the predominant form of collagen in blood vessels. Moreover, we observed that the OAT inhibitor l-canaline blocks TGF-β1–mediated collagen production, suggesting that endogenous l-proline synthesis is necessary for collagen synthesis by TGF-β1. Thus, OAT may play an essential role in providing VSMCs with the necessary intracellular levels of l-proline required during periods of elevated collagen synthesis.
Considerable evidence indicates that TGF-β1 plays an important role in stimulating intimal thickening and collagen synthesis at sites of vascular injury. Carotid artery injury results in a 5- to 7-fold increase in TGF-β1 mRNA that persists during neointima formation and correlates with increases in both type I and type III collagen mRNA expression.24 Furthermore, infusion of recombinant TGF-β124 or overexpression of TGF-β1 in the vessel wall25 leads to neointima formation. In contrast, inhibition of TGF-β1 activity by injecting neutralizing antibodies to TGF-β135 or a soluble TGF-β1 type II receptor36 significantly diminishes intimal thickening and collagen formation after vascular injury. Thus, our finding that physiologically relevant concentrations of TGF-β1 stimulate l-arginine transport and direct its metabolism to growth-stimulatory polyamines and to the collagen precursor l-proline may provide an important mechanism by which TGF-β1 stimulates VSMC growth and collagen synthesis at sites of vascular trauma. Moreover, these TGF-β1 actions that promote VSMC growth and collagen formation may be further amplified in vivo by the ability of TGF-β1 to suppress the inducible NO synthase–mediated synthesis of NO,37 which is an established inhibitor of VSMC proliferation and collagen synthesis.15 16
In conclusion, these studies demonstrate that TGF-β1 stimulates VSMC polyamine and l-proline synthesis by stimulating the transport and metabolism of l-arginine. In addition, they show that TGF-β1–stimulated collagen synthesis is dependent on l-proline synthesis. The ability of TGF-β1 to upregulate l-arginine transport and direct its metabolism to polyamines and l-proline may contribute to arterial remodeling at sites of vascular damage by stimulating VSMC growth and collagen deposition.
This study was supported in part by National Heart, Lung, and Blood Institute grants HL-59976, HL-62467, and HL-36045 and a Grant-in-Aid from the American Heart Association. W. Durante is an Established Investigator of the American Heart Association.
Guest Editor for this article was Joseph Loscalzo, MD, PhD, Boston University School of Medicine, Boston, Mass.
- Received June 26, 2000.
- Revision received August 24, 2000.
- Accepted September 8, 2000.
- Copyright © 2001 by American Heart Association
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