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Platelet-derived growth factor stimulates LAT1 gene expression in vascular smooth muscle: role in cell growth¹

Michael E. DeBakey VA Medical Center and Departments of Medicine and
^* Pharmacology, Baylor College of Medicine, Houston, Texas, USA

² Correspondence: VA Medical Center, Bldg. 109, Room 130, 2002 Holcome Blvd., Houston, TX 77030, USA. E-mail: wdurante{at}bcm.tmc.edu

SPECIFIC AIM

System L amino acid transport mediates the sodium-independent uptake of nonpolar branched-chain or aromatic neutral amino acids and is the major route by which mammalian cells take up nutritionally essential amino acids from extracellular fluids. Genes encoding the proteins responsible for system L amino acid transport have been cloned and designated LAT1 and LAT2. Both proteins require covalent association with the heavy chain of 4F2 cell surface antigen (4F2hc) for their functional expression in the plasma membrane. Since essential amino acids are required for cell growth, the present study examined the effect of the vascular smooth muscle cell (SMC) mitogen, platelet-derived growth factor (PDGF), on system L-amino acid transport.

PRINCIPAL FINDINGS

1. Vascular SMC express system L amino acid transport activity
We measured system L amino acid transport in cultured rat aortic SMCs by characterizing the uptake of one of its substrates, L-leucine. The specific transport of L-leucine by SMCs was time dependent and linear for 2 min. Substitution of sodium in the uptake buffer with choline at equimolar concentration had no effect on the uptake of L-leucine, indicating that transport into these cells was independent of extracellular sodium. Incubation of SMCs with the model substrate for the system L amino acid transporter 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) inhibited the uptake of L-leucine. In contrast, preloading SMCs with L-leucine or L-phenylalanine for 3 h stimulated the rate of uptake by twofold. The sodium independence, inhibition by BCH, and trans-stimulation confirms that system L amino acid transport mediates the uptake of L-leucine by vascular SMCs.

2. PDGF stimulates system L amino acid transport
Treatment of vascular SMCs with PDGF for 24 h stimulated transport of L-leucine in a concentration-dependent manner with an twofold increase in transport at a concentration of 30 ng/mL. Time course studies demonstrated that PDGF had a biphasic effect on L-leucine transport. PDGF initially inhibited transport of L-leucine but by 6 h of PDGF treatment a significant rise in L-leucine transport was seen; this increased further after 24 h. Other vascular mitogens also evoked a significant increase in L-leucine transport, serum being the most potent inducer of L-leucine uptake. Kinetic studies indicated that L-leucine transport is saturable and mediated by a single high-affinity (K_m=98±5 µM) carrier with a maximum transport velocity (V_max) of 4028 ± 522 pmol·mg protein^–1·min^–1. PDGF had no effect on the K_m but increased the V_max by >twofold. The increase in V_max by PDGF was completely abolished by cycloheximide.

3. PDGF stimulates LAT1 gene expression via mTOR
RT-PCR identified cDNA encoding LAT1, LAT2, and 4F2hc, but the LAT2 band was extremely faint (Fig. 1 A). Northern blots demonstrated that PDGF stimulated expression of LAT1 mRNA in a time-dependent fashion (Fig. 1B, C ). An increase in LAT1 message was seen after 2 h of PDGF exposure, which persisted for 8 h before returning back to basal levels at 24 h. In contrast, PDGF failed to stimulate the expression of 4F2hc mRNA (Fig. 1B, C ). Northern blots failed to detect LAT2 mRNA expression in control cells or in cells exposed to PDGF. Western blots revealed that PDGF selectively stimulated the expression of LAT1 protein but had no effect on the expression of 4F2hc (Fig. 1D ). Serum, thrombin, and angiotensin II likewise selectively stimulated LAT1 expression; the magnitudeof LAT1 induction paralleled the increase in L-leucine transport.

View larger version (26K): [in this window] [in a new window]   Figure 1. Expression of LAT and 4F2hc mRNA by vascular SMCs. A) Ethidium-stained agarose electrophoresis gel showing PCR amplified LAT and 4F2hc cDNA from vascular SMCs. Similar findings were made in 3 separate experiments. B) Northern blot of LAT1 and 4F2hc mRNA after treatment of SMCs with PDGF (30 ng/mL). C) Relative LAT1 and 4F2hc mRNA levels derived from laser densitometric analysis of transcripts expressed after PDGF treatment compared with control SMCs. Data shown are representative of 3 separate experiments. D) Western blot of LAT1 and 4F2hc protein after treatment of SMCs with PDGF (30 ng/mL) for 24 h. Data shown are representative of 3 separate experiments.

Induction of LAT1 mRNA by PDGF was dependent on de novo protein and DNA synthesis since it was prevented by cycloheximide and actinomycin D. However, PDGF-mediated elevation of LAT1 message was unaffected by the ERK kinase inhibitor PD98059, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, or the JNK kinase inhibitor SP600125. Phosphatidlyinositol-3-kinase inhibitors wortmannin and LY2942002 similarly failed to block the induction of LAT1. The specific mTOR inhibitor rapamycin potently inhibited LAT1 mRNA expression.

4. LAT1 promotes SMC growth and survival
Treatment of quiescent vascular SMCs with PDGF or serum for 4 days stimulated a significant increase in cell number that was inhibited by BCH (Fig. 2 A, B). BCH had no effect on the number of quiescent cells (Fig. 2A, B ). BCH failed to modulate DNA synthesis in either the presence or absence of growth factors. However, BCH induced rounding and blebbing of PDGF- or serum-treated SMCs associated with pronounced DNA fragmentation (Fig. 2C ), and an twofold increase in caspase-3 activity (Fig. 2D ). Induction of DNA laddering and caspase-3 activation by BCH was concentration dependent and was not observed in the absence of growth factors (Fig. 2C, D ).

View larger version (36K): [in this window] [in a new window]   Figure 2. Effect of BCH on vascular SMC proliferation and apoptosis. A) SMC proliferation was monitored in cells treated with PDGF (30 ng/mL) for 4 days in the presence or absence of BCH (1–20 mM). B) SMC proliferation was monitored in cells treated with serum (5%) for 4 days in the presence or absence of BCH (1–20 mM). C) DNA laddering in SMCs treated with serum (5%) for 4 days in the presence or absence of BCH (1–20 mM). D) Caspase-3 activity in SMCs treated with serum (5%) for 4 days in the presence or absence of BCH (20 mM). Results are means ± SE of 3 separate experiments or are representative of 3 separate experiments. *Statistically significant effect of growth factors. Statistically significant effect of BCH.

CONCLUSIONS

In the present study, we demonstrated that L-leucine transport by vascular SMCs is mediated via the classical system L amino acid transporter. RT-PCR identified mRNA for LAT1, LAT2, and 4F2hc in vascular SMCs, but the expression of LAT2 was faint and could not be detected by Northern blot. Kinetic studies revealed that the uptake of L-leucine by SMCs is mediated by a single high-affinity (K_m 100 µM) carrier. This suggests that SMC L-leucine transport is primarily mediated by the LAT1-4F2hc complex.

Treatment of vascular SMCs with growth factors (including PDGF, serum, thrombin or angiotensin II) stimulates L-leucine transport. Kinetic studies indicate that PDGF selectively increases the V_max without affecting the K_m of this transport system. These kinetic data suggest that growth factor-induced increases in L-leucine transport probably arise from de novo synthesis of additional transport proteins. We found that cycloheximide blocks the PDGF-induced increase in V_max. We also observed that PDGF stimulates LAT1 mRNA and protein expression. The induction of LAT1 by PDGF is dependent on de novo protein and DNA synthesis and on the activity of mTOR. The latter finding is consistent with recent studies demonstrating that mTOR plays a central role in controlling amino acid transport.

We found that the magnitude of LAT1 induction parallels the increase in L-leucine transport. However, the increase in LAT1 message precedes the increase in L-leucine transport and LAT1 transcripts decay to near basal levels by 24 h when transport activity is maximally increased. This dissociation between LAT1 mRNA levels and transport activity likely reflects the time required for the translation and trafficking of the LAT1 protein to the plasma membrane to yield a functional transporter.

System L amino acid transport requires the association of LAT with 4F2hc. In T cells and trophoblasts, increases in system L amino acid transport involve the coordinate induction of both LAT1 and 4F2hc mRNA expression. In contrast, the adaptive increase in system L amino acid transport in response to L-arginine depletion is associated with the selective up-regulation of message for LAT1 in hepatocytes. Cell-specific effects are also observed after gene transfer of LAT1. It is likely that the cellular ratio of LAT1/4F2hc determines whether induction of one or both proteins is required to elicit an increase in transport activity. We found that the protein level of LAT1 was substantially lower than 4F2hc in SMCs, which may explain why an increase in only LAT1 expression is sufficient to elevate transport activity in these cells.

We found that growth factors are potent inducers of LAT1 in vascular SMCs. The relative induction of LAT1 by growth factors correlates with their proliferative capacity, serum being the most potent LAT1 inducer. We observed that inhibition of LAT1 activity by BCH markedly inhibits the proliferation of vascular SMCs. The decrease in SMC proliferation by BCH is independent of any effects on DNA synthesis but is associated with a significant increase in the rate of SMC apoptosis, as reflected by cell morphology, DNA laddering, and caspase-3 activation. These results indicate that BCH blocks the proliferation of vascular SMCs by stimulating programmed cell death. Thus, LAT1 may play a fundamental role in promoting SMC growth and survival by providing cells with the necessary levels of essential amino acids. The induction of apoptosis after LAT1 inhibition is specific for proliferating vascular SMCs. BCH has no effect on the viability of quiescent SMCs. Since SMC proliferation is extremely low or absent in healthy blood vessels but prevalent at sites of vascular injury, inhibition of LAT1 activity may provide a highly selective approach in preventing the accumulation of SMCs in vascular lesions. Thus, pharmacologic or genetic strategies that target specific amino acid transport proteins may offer a promising novel therapeutic modality in treating occlusive vascular disease as well as other proliferative disorders.

In conclusion, these studies demonstrate that system L amino acid transport by vascular SMCs is mediated via the LAT1-4F2hc complex and that PDGF stimulates system L amino acid transport by selectively inducing the expression of LAT1 via an mTOR-dependent pathway (see Fig. 3 ). They show that LAT1 plays a critical role in promoting SMC growth and survival. LAT1 represents a potentially new therapeutic target for treating vasculoproliferative disorders.

View larger version (12K): [in this window] [in a new window]   Figure 3. Model for the regulation and function of system L amino acid transport in vascular SMC. PDGF stimulates system L amino acid transport by selectively inducing LAT1 expression via an mTOR-dependent pathway. Induction of LAT1 promotes SMC growth and survival by providing cells with necessary levels of essential amino acids.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0886fje; doi: 10.1096/fj.03-0886fje

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