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

AbstractThe 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 RARβ 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 RARβ, suggesting that the receptor is involved in the regulation of the transporter gene

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

<p>Abstract</p> <p>Background</p> <p>In the recessive aminoaciduria Lysinuric Protein Intolerance (LPI), mutations of <it>SLC7A7</it>/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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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 <it>SLC7A7</it>/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 <it>SLC7A6</it>/y+LAT2 isoform; 3) in both normal and LPI monocytes, GM-CSF induces the expression of <it>SLC7A7</it>, 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.</p> <p>Conclusions</p> <p>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 <it>SLC7A7 </it>and <it>SLC7A6</it>. Moreover, the expression of <it>SLC7A7 </it>is regulated by GM-CSF in monocytes, pointing to a role of y+LAT1 in the pathogenesis of LPI associated PAP.</p

The regulation of sodium-dependent transport of anionic amino acids in cultured human fibroblasts

AbstractIn cultured human fibroblasts the transport of anionic amino acids through the sodium-dependent system X−AG is stimulated rapidly and transiently by phorbol 12,13-dibutyrate. Transport stimulation is consistent with an effect due to the activation of protein kinase C. Bradykinin (1 μM) and PDGF-AA (100 ng/ml) also stimulate the activity of system X−AG. The bradykinin effect appears to be fully dependent upon PKC activation whereas the stimulation of aspartate transport by PDGF-AA is also due to PKC-independent mechanisms

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 si- RNAs 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