IRF1 Mediates Growth Arrest and the Induction of a Secretory Phenotype in Alveolar Epithelial Cells in Response to Inflammatory Cytokines IFNγ/TNFα.

In COVID-19, cytokine release syndrome can cause severe lung tissue damage leading to acute respiratory distress syndrome (ARDS). Here, we address the effects of IFNγ, TNFα, IL-1ß and IL-6 on the growth arrest of alveolar A549 cells, focusing on the role of the IFN regulatory factor 1 (IRF1) transcription factor. The efficacy of JAK1/2 inhibitor baricitinib has also been tested. A549 WT and IRF1 KO cells were exposed to cytokines for up to 72 h. Cell proliferation and death were evaluated with the resazurin assay, analysis of cell cycle and cycle-regulator proteins, LDH release and Annexin-V positivity; the induction of senescence and senescence-associated secretory phenotype (SASP) was evaluated through ß-galactosidase staining and the quantitation of secreted inflammatory mediators. While IL-1 and IL-6 proved ineffective, IFNγ plus TNFα caused a proliferative arrest in A549 WT cells with alterations in cell morphology, along with the acquisition of a secretory phenotype. These effects were STAT and IRF1-dependent since they were prevented by baricitinib and much less evident in IRF1 KO than in WT cells. In alveolar cells, STATs/IRF1 axis is required for cytokine-induced proliferative arrest and the induction of a secretory phenotype. Hence, baricitininb is a promising therapeutic strategy for the attenuation of senescence-associated inflammation.

Osmolarity modulates the de-differentiation of horse articular chondrocytes during cell expansion in vitro: implications for tissue engineering in cartilage repair.

Due to the importance of joint disease and ostearthritis (OA) in equine athletes, new regenerative treatments to improve articular cartilage repair after damage are gaining relevance. Chondrocyte de-differentiation, an important pathogenetic mechanism in OA, is a limiting factor when differentiated articular chondrocytes are used for cell-based therapies. Current research focuses on the prevention of this de-differentiation and/or on the re-differentiation of chondrocytes by employing different strategies in vitro and in vivo. Articular chondrocytes normally live in a condition of higher osmolarity (350-450 mOsm/L) compared to normal physiological fluids (~ 300 mOsm/L) and some studies have demonstrated that osmolarity has a chondroprotective effect in vitro and in vivo. Therefore, the response of horse articular chondrocytes to osmolarity changes (280, 380, and 480 mOsm/L) was studied both in proliferating, de-differentiated chondrocytes grown in adhesion, and in differentiated chondrocytes grown in a 3D culture system. To this aim, cell proliferation (cell counting), morphology (optical microscopy), and differentiation (gene expression of specific markers) were monitored along with the expression of osmolyte transporters involved in volume regulation [betaine-GABA transporter (BGT-1), taurine transporter (SLC6A6), and neutral amino acid transporter (SNAT)] real-time qPCR. Proliferating chondrocytes cultured under hyperosmolar conditions showed low proliferation, spheroidal morphology, a significant reduction of de-differentiation markers [collagen type I (Col1) and RUNX2] and an increase of differentiation markers [collagen type II (Col2) and aggrecan]. Notably, a persistently high level of BGT-1 gene expression was maintained in chondrocyte cultures at 380 mOsm/L, and particularly at 480 mOsm/L both in proliferating and differentiated chondrocytes. These preliminary data encourage the study of osmolarity as a microenvironmental co-factor to promote/maintain chondrocyte differentiation in both 2D and 3D in vitro culture systems.

Expression and Function of ABC Transporters in Human Alveolar Epithelial Cells.

ATP-binding cassette (ABC) transporters are a large superfamily of membrane transporters that facilitate the translocation of different substrates. While ABC transporters are clearly expressed in various tumor cells where they can play a role in drug extrusion, the presence of these transporters in normal lung tissues is still controversial. Here, we performed an analysis of ABC transporters in EpiAlveolarTM, a recently developed model of human alveoli, by defining the expression and activity of MDR1, BCRP, and MRPs. Immortalized primary epithelial cells hAELVi (human alveolar epithelial lentivirus-immortalized cells) were employed for comparison. Our data underline a close homology between these two models, where none of the ABC transporters here studied are expressed on the apical membrane and only MRP1 is clearly detectable and functional at the basolateral side. According to these findings, we can conclude that other thus-far-unidentified transporter/s involved in drug efflux from alveolar epithelium deserve investigations.

Monocytes from infliximab-resistant patients with Crohn's disease exhibit a disordered cytokine profile.

Crohn's disease (CD) is a chronic inflammatory disorder characterized by immune response dysregulation. Tumor necrosis factor-α (TNFα) is a key cytokine in the pathogenesis of CD, as indicated by the efficacy of anti-TNF-α therapy with infliximab (IFX). However, approximately 30-40% of CD patients fail to respond to IFX with still unclear underlying mechanisms. This study compares the inflammatory phenotype of monocytes from CD patients, who respond or non-respond to IFX. Under basal conditions, the mRNA for the cytokines TNFα, IL-23, IL-1ß and the chemokines CXCL8/IL-8, CCL5/RANTES and CCL2/MCP-1 was up-regulated in monocytes from non-responders than responders. The expression of the same cytokines and CCL2/MCP-1 was higher in non-responders also upon LPS treatment. Moreover, higher secretion of TNFα, IL-1ß, IFNγ and IL-2 proteins occurred in the supernatants of LPS-treated non-responders cells. Resistance to IFX in CD may result from a transcriptional dysregulation of circulating monocytes, leading to hyperactivation of pro-inflammatory pathways. Monocytes' cytokine profile may thus represent a predictive marker of response to IFX. Monocytes were isolated from blood samples of 19 CD patients (11 responders, 8 non-responders) and incubated with or without LPS. Cytokine profiles were assessed by RT-qPCR and, in the supernatants, by ELISA assay.

Characterization of ABC Transporters in EpiAirway™, a Cellular Model of Normal Human Bronchial Epithelium.

The ATP-binding cassette (ABC) transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) play a crucial role in the translocation of a broad range of drugs; data about their expression and activity in lung tissue are controversial. Here, we address their expression, localization and function in EpiAirway™, a three-dimensional (3D)-model of human airways; Calu-3 cells, a representative in vitro model of bronchial epithelium, are used for comparison. Transporter expression has been evaluated with RT-qPCR and Western blot, the localization with immunocytochemistry, and the activity by measuring the apical-to-basolateral and basolateral-to-apical fluxes of specific substrates in the presence of inhibitors. EpiAirway™ and Calu-3 cells express high levels of MRP1 on the basolateral membrane, while they profoundly differ in terms of BCRP and MDR1: BCRP is detected in EpiAirway™, but not in Calu-3 cells, while MDR1 is expressed and functional only in fully-differentiated Calu-3; in EpiAirway™, MDR1 expression and activity are undetectable, consistently with the absence of the protein in specimens from human healthy bronchi. In summary, EpiAirway™ appears to be a promising tool to study the mechanisms of drug delivery in the bronchial epithelium and to clarify the role of ABC transporters in the modulation of the bioavailability of administered drugs.

Functional analysis of OCTN2 and ATB0,+ in normal human airway epithelial cells.

In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by ß-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery.

y+LAT1 and y+LAT2 contribution to arginine uptake in different human cell models: Implications in the pathophysiology of Lysinuric Protein Intolerance.

y+LAT1 (encoded by SLC7A7), together with y+LAT2 (encoded by SLC7A6), is the alternative light subunits composing the heterodimeric transport system y+L for cationic and neutral amino acids. SLC7A7 mutations cause lysinuric protein intolerance (LPI), an inherited multisystem disease characterized by low plasma levels of arginine and lysine, protein-rich food intolerance, failure to thrive, hepatosplenomegaly, osteoporosis, lung involvement, kidney failure, haematologic and immunological disorders. The reason for the heterogeneity of LPI symptoms is thus far only poorly understood. Here, we aimed to quantitatively compare the expression of SLC7A7 and SLC7A6 among different human cell types and evaluate y+LAT1 and y+LAT2 contribution to arginine transport. We demonstrate that system y+L-mediated arginine transport is mainly accounted for by y+LAT1 in monocyte-derived macrophages (MDM) and y+LAT2 in fibroblasts. The kinetic analysis of arginine transport indicates that y+LAT1 and y+LAT2 share a comparable affinity for the substrate. Differences have been highlighted in the expression of SLC7A6 and SLC7A7 mRNA among different cell models: while SLC7A6 is almost equally expressed, SLC7A7 is particularly abundant in MDM, intestinal Caco-2 cells and human renal proximal tubular epithelial cells (HRPTEpC). The characterization of arginine uptake demonstrates that system y+L is operative in renal cells and in Caco-2 where, at the basolateral side, it mediates arginine efflux in exchange with leucine plus sodium. These findings explain the defective absorption/reabsorption of arginine in LPI. Moreover, y+LAT1 is the prevailing transporter in MDM sustaining a pivotal role in the pathogenesis of immunological complications associated with the disease.

Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells.

Lysinuric protein intolerance (LPI) is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA), a significant induction of the expression and release of the inflammatory mediators IL1ß and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1ß has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.

Gluten peptides drive healthy and celiac monocytes toward an M2-like polarization.

Celiac disease (CD) is an immune-mediated enteropathy triggered by ingested gluten in genetically susceptible individuals and sustained by both adaptive and innate immune responses. Recent studies in murine macrophages demonstrated that the activation of arginase (ARG) metabolic pathway by gluten peptides contributes to the modulation of intestinal permeability in vitro. Here we characterize the effects of gluten on arginine metabolism and cell polarization in human monocytes from both healthy and CD subjects; both a simplified enzymatic digestion of gliadin and a physiological digestion of whole wheat have been tested. Results indicate that gluten digests induce the onset of an M2-like phenotype in activated macrophages; more precisely, both isoforms of arginase, ARG1 and ARG2, are induced likely due to the inhibition of mTOR and the consequent induction of C/EBPß transcription factor. These effects are independent from the origin of gluten as well as from the digestive protocol employed; moreover, no statistical difference can be evidenced between healthy and CD patients, excluding a diverse predisposition of CD monocytes to gluten-triggered polarization with respect to healthy immune cells. Overall, the present findings sustain a role for arginase pathway in the immune response elicited by human monocytes toward ingested gluten that, hence, deserves particular attention when addressing the pathogenesis of CD.

Human macrophage differentiation induces OCTN2-mediated L-carnitine transport through stimulation of mTOR-STAT3 axis.

l-Carnitine, in addition to playing a fundamental role in the ß-oxidation of fatty acids, has been recently identified as a modulator of immune function, although the mechanisms that underlie this role remain to be clarified. In this study, we addressed the modulation of l-carnitine transport and expression of related transporters during differentiation of human monocytes to macrophages. Whereas monocytes display a modest uptake of l-carnitine, GM-CSF-induced differentiation massively increased the saturable Na+-dependent uptake of l-carnitine. Kinetic and inhibition analyses demonstrate that in macrophage l-carnitine transport is mediated by a high-affinity component (Km ∼4 µM) that is identifiable with the operation of OCTN2 transporter and a low-affinity component (Km > 10 mM) that is identifiable with system A for neutral amino acids. Consistently, both SLC22A5/OCTN2 and SLC38A2/SNAT2 are induced during the differentiation of monocytes to macrophages at gene and protein levels. Elucidation of GM-CSF signaling demonstrates that the cytokine causes the activation of mTOR kinase, leading to the phosphorylation and activation of STAT3, which, in turn, is responsible for OCTN2 transcription. SLC22A5/OCTN2 therefore emerges as a novel member of the set of genes markers of macrophage differentiation.

Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target.

The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

Functional activity of L-carnitine transporters in human airway epithelial cells.

Carnitine plays a physiologically important role in the ß-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km<2 µM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 µM) and a low affinity (Km>1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects.

Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50µM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20µM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation.

Gliadin activates arginase pathway in RAW264.7 cells and in human monocytes.

Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. Recent studies have demonstrated that macrophages play a key role in the pathogenesis of CD through the release of inflammatory mediators such as cytokines and nitric oxide (NO). Since arginine is the obliged substrate of iNOS (inducible nitric oxide synthase), the enzyme that produces large amount of NO, the aim of this work is to investigate arginine metabolic pathways in RAW264.7 murine macrophages after treatment with PT-gliadin (PTG) in the absence and in the presence of IFNγ. Our results demonstrate that, besides strengthening the IFNγ-dependent activation of iNOS, gliadin is also an inducer of arginase, the enzyme that transforms arginine into ornithine and urea. Gliadin treatment increases, indeed, the expression and the activity of arginase, leading to the production of polyamines through the subsequent induction of ornithine decarboxylase. This effect is strengthened by IFNγ. The activation of these pathways takes advantage of the increased availability of arginine due to a decreased system y(+)l-mediated efflux, likely ascribable to a reduced expression of Slc7a6 transporter. A significant induction of arginase expression is also observed in human monocytes from healthy subject upon treatment with gliadin, thus demonstrating that gluten components trigger changes in arginine metabolism in monocyte/macrophage cells.

Oxidative stress induced by copper and iron complexes with 8-hydroxyquinoline derivatives causes paraptotic death of HeLa cancer cells.

Here, we report the antiproliferative/cytotoxic properties of 8-hydroxyquinoline (8-HQ) derivatives on HeLa cells in the presence of transition metal ions (Cu(2+), Fe(3+), Co(2+), Ni(2+)). Two series of ligands were tested, the arylvinylquinolinic L1-L8 and the arylethylenequinolinic L9-L16, which can all interact with metal ions by virtue of the N,O donor set of 8-HQ; however, only L9-L16 are flexible enough to bind the metal in a multidentate fashion, thus exploiting the additional donor functions. L1-L16 were tested for their cytotoxicity on HeLa cancer cells, both in the absence and in the presence of copper. Among them, the symmetric L14 exhibits the highest differential activity between the ligand alone (IC50 = 23.7 µM) and its copper complex (IC50 = 1.8 µM). This latter, besides causing a significant reduction of cell viability, is associated with a considerable accumulation of the metal inside the cells. Metal accumulation is also observed when the cells are incubated with L14 complexed with other late transition metal ions (Fe(3+), Co(2+), Ni(2+)), although the biological response of HeLa cells is different. In fact, while Ni/L14 and Co/L14 exert a cytostatic effect, both Cu/L14 and Fe/L14 trigger a caspase-independent paraptotic process, which results from the induction of a severe oxidative stress and the unfolded protein response.

Glutamine stimulates mTORC1 independent of the cell content of essential amino acids.

Glutamine and leucine are important mTORC1 modulators, although their roles are not precisely defined. In HepG2 and HeLa cells glutamine-free incubation lowers mTORC1 activity, although cell leucine is not decreased. mTORC1 activity, suppressed by amino acid-free incubation, is completely rescued only if essential amino acids (EAA) and glutamine are simultaneously restored, although cell leucine is higher in the absence than in the presence of glutamine. Thus, glutamine stimulates mTORC1 independent of cell leucine, suggesting the existence of two distinct stimulatory signals from either glutamine or EAA.

Radiochemical high-performance liquid chromatography detection of arginine metabolism in human endothelial cells.

Arginine is a semi-essential amino acid that plays an important role in the regulation of metabolic processes associated with several pathological/physiological conditions. In the vasculature, it mainly exerts its biological functions as a substrate of two alternative pathways: the conversion to nitric oxide (NO) by nitric oxide synthase (NOS) and the breakdown to urea and ornithine by arginase. To determine arginine metabolism, in the current study we propose an original radiochemical technique that allows the simultaneous monitoring of NOS and arginase activation within intact cells. Taking advantage of this method, we show here the consequences of different experimental conditions known to modulate endothelial homeostasis on arginine metabolism.

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.

The non-proteinogenic amino acids L-methionine sulfoximine and DL-phosphinothricin activate mTOR.

L-Methionine sulfoximine (MSO) and DL-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators.

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.