Impaired phagocytosis in macrophages from patients affected by lysinuric protein intolerance.

Lysinuric Protein Intolerance (LPI, MIM 222700) is a recessive aminoaciduria caused by defective cationic amino acid transport in epithelial cells of intestine and kidney. SLC7A7, the gene mutated in LPI, codifies for the y+LAT1 subunit of system y(+)L amino acid transporter. LPI patients frequently display severe complications, such as pulmonary disease, haematological abnormalities and disorders of the immune response. The transport defect may explain only a part of the clinical aspects of the disease, while the mechanisms linking the genetic defect to the clinical features of the patients remain thus far obscure. The aim of the study is to investigate the consequences of SLC7A7 mutations on specific macrophage functions, so as to evaluate if a macrophage dysfunction may have a role in the development of pulmonary and immunological complications of LPI. The results presented 1) confirm previous data obtained in one LPI patient, demonstrating that arginine influx through system y(+)L is markedly compromised in LPI macrophages; 2) demonstrate that also system y(+)L-mediated arginine efflux is significantly lower in LPI macrophages than in normal cells and 3) demonstrate that the phagocytic activity of LPI macrophages is severely impaired. In conclusion, SLC7A7/y+LAT1 mutations lead to a defective phenotype of macrophages, supporting the pathogenetic role of these cells in the development of LPI-associated complications.

Arginine transport in human monocytic leukemia THP-1 cells during macrophage differentiation.

L-arginine metabolism in myeloid cells plays a central role in the processes of macrophage activation and in the regulation of immune responses. In this study, we investigated arginine transport activity and the expression of the related transporter genes during the differentiation of monocytes to macrophages. We show here that the induction of THP-1 monocyte differentiation by PMA markedly increases the expression of SLC7A7 mRNA and of y(+)LAT1 protein and consequently, the activity of system y(+)L-mediated arginine transport. Conversely, the activity of system y(+) decreases during macrophage differentiation as a result of a reduction in CAT1 protein expression. The PMA-induced, macrophage-differentiated phenotype and the increased activity of system y(+)L through the induction of SLC7A7 gene are mediated by the specific activation of PKCß. SLC7A7 gene silencing causes a significant reduction of system y(+)L activity and a subsequent, marked increase of arginine and lysine cell content, thus suggesting that in macrophagic cells, system y(+)L activity is mainly directed outwardly. Differentiating agents other than PMA, i.e., VD3 and ATRA, are equally effective in the stimulation of system y(+)L transport activity through the increased expression of SLC7A7 mRNA and y(+)LAT1 protein. Moreover, we found that also during differentiation of human monocytes from peripheral blood SLC7A7 mRNA and system y(+)L activity are increased. These findings point to SLC7A7 gene as a marker of macrophage differentiation.

In Lysinuric Protein Intolerance system y+L activity is defective in monocytes and in GM-CSF-differentiated macrophages.

BACKGROUND: In the recessive aminoaciduria Lysinuric Protein Intolerance (LPI), mutations of SLC7A7/y+LAT1 impair system y+L transport activity for cationic amino acids. A severe complication of LPI is a form of Pulmonary Alveolar Proteinosis (PAP), in which alveolar spaces are filled with lipoproteinaceous material because of the impaired surfactant clearance by resident macrophages. The pathogenesis of LPI-associated PAP remains still obscure. The present study investigates for the first time the expression and function of y+LAT1 in monocytes and macrophages isolated from a patient affected by LPI-associated PAP. A comparison with mesenchymal cells from the same subject has been also performed. METHODS: Monocytes from peripheral blood were isolated from a 21-year-old patient with LPI. Alveolar macrophages and fibroblastic-like mesenchymal cells were obtained from a whole lung lavage (WLL) performed on the same patient. System y+L activity was determined measuring the 1-min uptake of [3H]-arginine under discriminating conditions. Gene expression was evaluated through qRT-PCR. RESULTS: We have found that: 1) system y+L activity is markedly lowered in monocytes and alveolar macrophages from the LPI patient, because of the prevailing expression of SLC7A7/y+LAT1 in these cells; 2) on the contrary, fibroblasts isolated from the same patient do not display the transport defect due to compensation by the SLC7A6/y+LAT2 isoform; 3) in both normal and LPI monocytes, GM-CSF induces the expression of SLC7A7, suggesting that the gene is a target of the cytokine; 4) GM-CSF-induced differentiation of LPI monocytes is comparable to that of normal cells, demonstrating that GM-CSF signalling is unaltered; 5) general and respiratory conditions of the patient, along with PAP-associated parameters, markedly improved after GM-CSF therapy through aerosolization. CONCLUSIONS: Monocytes and macrophages, but not fibroblasts, derived from a LPI patient clearly display the defect in system y+L-mediated arginine transport. The different transport phenotypes are referable to the relative levels of expression of SLC7A7 and SLC7A6. Moreover, the expression of SLC7A7 is regulated by GM-CSF in monocytes, pointing to a role of y+LAT1 in the pathogenesis of LPI associated PAP.

Regulation of arginine transport and metabolism by protein kinase Calpha in endothelial cells: stimulation of CAT2 transporters and arginase activity.

Endothelial metabolism of arginine plays a key role in vascular homeostasis. While it is documented that the availability of extracellular arginine is critical for nitric oxide synthesis by eNOS, little is known about the relationships existing between arginine transport and the activity of arginase, the enzyme responsible for the production of ornithine and urea. The present study aims to characterize the role of PKC in the regulation of arginine transport and metabolism by human umbilical vein (HUVEC) and aortic (HAEC) endothelial cells. The results obtained demonstrate that the activation of PKCalpha by phorbol esters or thymeleatoxin causes a transient increase of arginine transport through system y(+), referable to the induction of SLC7A2 mRNAs and to the increased expression of CAT2 transporters. PKCalpha-dependent stimulation of arginine transport requires the activation of MEK/ERK1/2 cascade, which leads to the stimulation of AP-1 and to the consequent induction of CAT2 expression. In parallel, PKCalpha activation also increases arginase expression and activity and promotes eNOS phosphorylation, resulting in decreased NO production. It is concluded that the activation of PKCalpha stimulates arginine entry in human endothelial cells and shifts the metabolism of the cationic amino acid from NO synthesis to arginase-dependent production of ornithine and urea. This metabolic deviation may contribute to the endothelial dysfunction associated with conditions of PKC overactivity.

Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles.

Since arginine metabolites, such as nitric oxide and polyamines, influence the expression of genes involved in erythroid differentiation, the transport of the cationic amino acid may play an important role in erythroid cells. However, available data only concern the presence in these cells of CAT1 transporter (system y(+)), while no information exists on the role of the heterodimeric transporters of system y(+)L (4F2hc/y(+)LAT1 and 4F2hc/y(+)LAT2) which operates transmembrane arginine fluxes cis-inhibited by neutral amino acids in the presence of sodium. Using erythroleukemia K562 cells and normal erythroid precursors, we demonstrate here that arginine transport in human erythroid cells is due to the additive contributions of a leucine-sensitive and leucine-insensitive component. In both cell types, leucine inhibition of arginine influx is much less evident in the absence of sodium, a hallmark of system y(+)L. In K562 cells, N-ethylmaleimide, a known inhibitor of CAT transporters (system y(+)), suppresses only a fraction of arginine influx corresponding to leucine-insensitive uptake. Moreover, in Xenopus oocytes coexpressing 4F2hc and y(+)LAT2, leucine exerts a marked inhibition of arginine transport, partially dependent on sodium, while no inhibition is seen in oocytes expressing CAT1. Lastly, silencing of SLC7A6, the gene for y(+)LAT2, lowers arginine transport and doubles the intracellular content of the cationic amino acid in K562 cells. We conclude that arginine transport in human erythroid cells is due to both system y(+) (CAT1 transporter) and system y(+)L (4F2hc/y(+)LAT2 isoform), which mainly contribute, respectively, to the influx and to the efflux of the cationic amino acid.

The ATRA-dependent overexpression of the glutamate transporter EAAC1 requires RARbeta induction.

The mechanisms underlying trafficking and membrane targeting of EAAC1, the rodent counterpart of the human EAAT3 carrier for anionic amino acids, are well characterized. In contrast, much less is known on the regulation of Slc1a1, the gene that encodes for the transporter. We have recently found that all-trans retinoic acid (ATRA) stimulates EAAC1 expression and anionic amino acid transport in C6 rat glioma cells. We report here that the ATRA effect on EAAC1 activity was inhibited by the specific RAR antagonist LE540 and mimicked by Am80, a RAR agonist, but not by the RXR agonist HX630. Moreover, the ATRA-dependent induction of Slc1a1 mRNA required the synthesis of a protein intermediate and was not associated with changes in the messenger half-life. ATRA treatment induced the expression of both Rarb mRNA and RARbeta protein several hours before the induction of Slc1a1, while the mRNA for RFX1, a transcription factor recently involved in Slc1a1 transcription, was unchanged. In addition, Rarb silencing markedly inhibited the ATRA-dependent increase of both Rarb and Slc1a1 mRNAs. We conclude that in C6 glioma cells the induction of Slc1a1 by ATRA requires the synthesis of RARbeta, suggesting that the receptor is involved in the regulation of the transporter gene.

In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function.

AIM: Drug-eluting stents are widely used to prevent restenosis but are associated with late endothelial damage. To understand the basis for this effect, we have studied the consequences of a prolonged incubation with rapamycin on the viability and functions of endothelial cells. METHODS AND RESULTS: Human umbilical vein or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor alpha (TNFalpha). After a 24 h-incubation, rapamycin (100 nM) caused a significant cell loss associated with the increase of both apoptosis and necrosis, as quantified by propidium iodide staining, caspase 3 activity, and lactate dehydrogenase release. Rapamycin also impaired cell mobility, as assessed by a wound test, and promoted the formation of actin stress fibres, as determined with confocal microscopy. Moreover, the inhibitor prolonged TNFalpha-dependent E-selectin induction, inhibited endothelial nitric oxide synthase expression at both mRNA (quantitative real-time polymerase chain reaction) and protein level (enzyme-linked immunosorbent assay and western blot), and lowered bioactive nitric oxide output (RFL-6 reporter cell assay). Under the conditions adopted, rapamycin inhibited both mammalian target-of-rapamycin complexes (mTORC1 and mTORC2), as indicated by the reduced amount of raptor and rictor bound to mTOR in immunoprecipitates and by the marked hypophosphorylation of protein S6 kinase I (p70S6K) and Akt, determined by western blotting. The selective inhibition of mTORC1 by AICAR did not affect endothelial viability. CONCLUSION: A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.

Rapamycin stimulates arginine influx through CAT2 transporters in human endothelial cells.

In endothelial cells Tumor Necrosis Factor-alpha (TNFalpha) stimulates arginine transport through the increased expression of SLC7A2/CAT2 transcripts. Here we show that also rapamycin, an inhibitor of mTOR kinase, stimulates system y(+)-mediated arginine uptake in human endothelial cells derived from either saphenous (HSVECs) or umbilical veins (HUVECs). When used together with TNFalpha, rapamycin produces an additive stimulation of arginine transport in both cell models. These effects are observed also upon incubation with AICAR, a stimulator of Adenosine-Monophosphate-dependent-Protein Kinase (AMPK) that produces a rapamycin-independent inhibition of the mTOR pathway. Rapamycin increases the V(max) of high affinity arginine transport and causes the appearance of a low affinity component that is particularly evident if the treatment is carried out in the presence of TNFalpha. RT-qPCR studies have demonstrated that these kinetic changes correspond to the induction of both the high affinity transporter CAT2B and the low affinity isoform CAT2A. Western blot and immunocytochemical analyses indicate that, consistently, the expression of CAT2 proteins is also stimulated under the same conditions. These changes are associated with an increase of the intracellular arginine concentration but with a decrease of NO production. Thus, our data suggest that mTOR activity is associated with the repression of CAT2 expression at mRNA and protein level.

Alveolar macrophages from normal subjects lack the NOS-related system y+ for arginine transport.

Systems y+ and y+L represent the main routes for arginine transport in mammalian cells. While system y+ activity is needed for the stimulated NO production in rodent alveolar macrophages (AM), no information is yet available about arginine transport in human AM. We study here arginine influx and genes for arginine transporters in AM from bronchoalveolar lavage of normal subjects. These cells express the y+ -related genes SLC7A1/CAT1 and SLC7A2/CAT2B, as well as the y+L genes SLC7A7/y+LAT1 and SLC7A6/y+LAT2. However, compared with human endothelial cells, AM express much less SLC7A2 mRNA and higher levels of SLC7A7 mRNA. Granulocyte macrophage colony-stimulating factor or IFN-gamma do not change the expression of any transporter gene, while lipopolysaccharide induces SLC7A2/CAT2B. Under all the conditions tested, leucine inhibits most of the arginine transport in the presence of Na+ and N-ethylmaleimide, an inhibitor of system y+, is completely ineffective, indicating that system y+L operates most of the arginine influx. Comparable results are obtained in AM from patients with interstitial lung disease, such as Nonspecific Interstitial Pneumonia (NSIP), although these cells have a higher SLC7A1 and a lower SLC7A7 expression than AM from normal subjects. It is concluded that AM from normal subjects or patients with NSIP lack a functional transport system y+, a situation that may limit arginine availability for NO synthesis. Moreover, since mutations of SLC7A7/y+LAT1 cause Lysinuric Protein Intolerance, a disease often associated with AM impairment and alveolar proteinosis, the high SLC7A7 expression observed in human AM suggests that y+LAT1 activity is important for the function of these cells.

PKC-dependent stimulation of EAAT3 glutamate transporter does not require the integrity of actin cytoskeleton.

The activity and the membrane expression of EAAT3 glutamate transporter are stimulated upon PKC activation by phorbol esters in C6 rat glioma cells. To investigate the role of cytoskeleton in these effects, we have employed actin-perturbing toxins and found that the perturbation of actin cytoskeleton inhibits basal but not phorbol-stimulated EAAT3 activity and membrane trafficking. In the absence of phorbols, latrunculin A, a toxin that disassembles actin cytoskeleton, produced a rapid inhibition of EAAT3 activity, due to a decrease in transport V(max). The inhibitory effect was fully reversible and was not detected for other sodium dependent transport systems for amino acids. However, latrunculin did not prevent the increase in transport caused by phorbol esters and, moreover, cells pre-treated with phorbols were resistant to the inhibitory effect of the toxin on EAAT3 activity. Biotinylation experiments indicated that the inhibitory effect of latrunculin was attributable to a decreased expression of the carrier on the membrane, while the toxin did not suppress the PKC-dependent increase in EAAT3 membrane abundance. Latrunculin A effects on EAAT3 were shared by cytochalasin D, a toxin that disorganizes actin filaments with a distinct mechanism of action. On the contrary, a small, but significant, increase of EAAT3 activity was observed upon incubation with jasplakinolide, a drug that stabilizes actin microfilaments. Also jasplakinolide, however, did not hinder phorbol-dependent stimulation of aspartate transport. Colchicine, a toxin that disrupts microtubules, also lowered EAAT3 activity without preventing transport stimulation by phorbols, while microtubule stabilization by paclitaxel led to an increase in aspartate transport. It is concluded that, in C6 cells, the PKC-mediated stimulatory effects on EAAT3 are cytoskeleton-independent, while in the absence of phorbols, the transporter is partially inhibited by the disorganization of either actin microfilaments or microtubules. These results suggest that EAAT3 trafficking in C6 cells involves different pools of transporters.

Inhibition of glutamine synthetase triggers apoptosis in asparaginase-resistant cells.

The resistance to L-asparaginase (ASNase) has been associated to the overexpression of asparagine synthetase (AS), although the role played by other metabolic adaptations has not been yet defined. Both in ASNase-sensitive Jensen rat sarcoma cells and in ARJ cells, their ASNase-resistant counterparts endowed with a five-fold increased AS activity, ASNase treatment rapidly depletes intracellular asparagine. Under these conditions, cell glutamine is also severely reduced and the activity of glutamine synthetase (GS) is very low. After 24 h of treatment, while sensitive cells have undergone massive apoptosis, ARJ cells exhibit a marked increase in GS activity, associated with overexpression of GS protein but not of GS mRNA, and a partial restoration of glutamine and asparagine. However, when ARJ cells are treated with both ASNase and L-methionine-sulfoximine (MSO), an inhibitor of GS, no restoration of cell amino acids occurs and the cell population undergoes a typical apoptosis. No toxicity is observed upon MSO treatment in the absence of ASNase. The effects of MSO are not referable to depletion of cell glutathione or inhibition of AS. These findings indicate that, in the presence of ASNase, the inhibition of GS triggers apoptosis. GS may thus constitute a target for the suppression of ASNase-resistant phenotypes.

SNAT2 silencing prevents the osmotic induction of transport system A and hinders cell recovery from hypertonic stress.

Under hypertonic conditions the induction of SLC38A2/SNAT2 leads to the stimulation of transport system A and to the increase in the cell content of amino acids. In hypertonically stressed human fibroblasts transfection with two siRNAs for SNAT2 suppressed the increase in SNAT2 mRNA and the stimulation of system A transport activity. Under the same condition, the expansion of the intracellular amino acid pool was significantly lowered and cell volume recovery markedly delayed. It is concluded that the up-regulation of SNAT2 is essential for the rapid restoration of cell volume after hypertonic stress.

The transport of cationic amino acids in human airway cells: expression of system y+L activity and transepithelial delivery of NOS inhibitors.

The transport of arginine has been characterized in human airway Calu-3 cells. As assessed with RT-PCR, Calu-3 cells express the genes for several transporters, such as the system y+-related SLC7A1, SLC7A2, and SLC7A4; the system y+L-related SLC7A6, SLC7A7, and SLC3A2; and the system B0,+-related SLC6A14. In polarized Calu-3 cell monolayers, apical arginine influx has a leucine-sensitive, sodium-dependent component and a leucine- and lysine-resistant sodium-independent fraction. At the basolateral membrane, arginine transport was fully sodium-independent and partially inhibited by leucine provided that sodium was present in the extracellular medium. Moreover, extracellular leucine trans-stimulated arginine efflux from the basolateral membrane in the presence, but not in the absence, of sodium. The transepithelial, apical to basolateral, arginine transport strictly depended on the presence of sodium and was markedly inhibited by apical leucine, but significantly trans-stimulated by the neutral amino acid added at the basolateral side. When added at the apical side, the NOS-inhibitors NMMA and NIL, CAA analogs with a free carboxyl group, markedly inhibited the apical arginine influx and the transepithelial flux of the cationic amino acid. The same compounds trans-stimulated basolateral arginine efflux. None of these effects were observed in the presence of the methyl ester analog NAME. The basolateral medium of Calu-3 cell monolayers, obtained after incubation in the presence of the three inhibitors at the apical side, inhibited the production of NO by activated murine macrophages. The inhibitory effect of the Calu-3 cell conditioned medium was time-dependent and markedly higher with NMMA and NIL than with NAME. Moreover, the NOS-inhibitory effect of the medium was significantly enhanced if NMMA and NIL, at the apical side, and basolateral leucine were simultaneously present during the conditioning procedure. These results indicate that 1) human airway epithelial cells express a functional system y+L at the basolateral membrane; 2) in this model, transepithelial arginine transport involves apical influx through system B0,+ and basolateral efflux through system y+L, and 3) the same transporters also perform an efficient transepithelial transport of amino acid-like NOS inhibitors.

The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

INFgamma stimulates arginine transport through system y+L in human monocytes.

Freshly isolated human monocytes transport L-arginine mostly through a sodium independent, NEM insensitive pathway inhibited by L-leucine in the presence, but not in the absence of sodium. Interferon-gamma (IFNgamma) stimulates this pathway, identifiable with system y+L, and markedly enhances the expression of SLC7A7, the gene that encodes for system y+L subunit y+LAT1, but not of SLC7A6, that codes for the alternative subunit y+LAT2. System y+ plays a minor role in arginine uptake by monocytes and the expression of system y+-related genes, SLC7A1 and SLC7A2, is not changed by IFNgamma. These results demonstrate that system y+L is sensitive to IFNgamma.

The stimulation of arginine transport by TNFalpha in human endothelial cells depends on NF-kappaB activation.

In human saphenous vein endothelial cells (HSVECs), tumor necrosis factor-alpha (TNFalpha) and bacterial lipopolysaccharide (LPS), but neither interferon gamma (IFNgamma) nor interleukin 1beta (IL-1beta), stimulate arginine transport. The effects of TNFalpha and LPS are due solely to the enhancement of system y+ activity, whereas system y+L is substantially unaffected. TNFalpha causes an increased expression of SLC7A2/CAT-2B gene while SLC7A1/CAT-1 expression is not altered by the cytokine. The suppression of PKC-dependent transduction pathways, obtained with the inhibitor chelerytrhine, the inhibitor peptide of PKCzeta isoform, or chronic exposure to phorbol esters, does not prevent TNFalpha effect on arginine transport. Likewise, ERK, JNK, and p38 MAP kinases are not involved in the cytokine effect, since arginine transport stimulation is unaffected by their specific inhibitors. On the contrary, inhibitors of NF-kappaB pathway hinder the increase in CAT2B mRNA and the stimulation of arginine uptake. These results indicate that in human endothelial cells the activation of NF-kappaB pathway mediates the TNFalpha effects on arginine transport.

Two-way arginine transport in human endothelial cells: TNF-alpha stimulation is restricted to system y(+).

Human umbilical vein endothelial cells transport arginine through two Na(+)-independent systems. System y(+)L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na(+), and referable to the expression of SLC7A6/y(+)LAT2, SLC7A7/y(+)LAT1, and SLC3A2/4F2hc. System y(+) is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y(+). Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y(+)L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-alpha, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-alpha or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production.