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

Pathways of L-glutamic acid transport in cultured human fibroblasts.

The transport of L-glutamic acid has been studied in skin-derived diploid human fibroblasts. Competition analysis in the presence and absence of Na+ and mathematical discrimination by nonlinear regression indicated that L-glutamic acid enters the cell by at least three transport systems: 1) a high affinity Na+-dependent system which has been found to be identical to the previously described system for anionic amino acids (Gazzola, G. C., Dall'Asta, V., Bussolati, O., Makowske, M., and Christensen, H. N. (1981) J. Biol. Chem. 256, 6054-6059) and which is provisionally designated as System X-AG; this route was shared by L-aspartic acid; 2) a low affinity Na+-dependent system resembling the ASC System for neutral amino acids (Franchi-Gazzola, R., Gazzola, G. C., Dall'Asta, V., and Guidotti, G. G. (1982) J. Biol. Chem. 257, 9582-9587); its reactivity toward L-glutamic acid was strongly inhibited by L-serine, but not by 2-(methyl-amino)isobutyric acid; and 3) a Na+-independent system similar to System XC- described in fetal human lung fibroblasts (Bannai, S., and Kitamura, E. (1980) J. Biol. Chem. 255, 2372-2376). The XC- system served for L-glutamic acid and L-cystine, the latter amino acid behaving as a potent inhibitor of L-glutamic acid uptake. Amino acid starvation did not change the uptake of L-glutamic acid by the two Na+-dependent systems, but enhanced the activity of System XC- by increasing its Vmax. L-Glutamic acid transport was also affected by the density of the culture. An increased cell density lowered the uptake of the amino acid by Systems ASC and XC- and promoted the uptake by System X-AG. All these variations were dependent upon changes in Vmax

Mesenchymal stem cell-derived extracellular vesicles protect human corneal endothelial cells from endoplasmic reticulum stress-mediated apoptosis

Corneal endothelial dystrophy is a relevant cause of vision loss and corneal transplantation worldwide. In the present study, we analyzed the effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in an in vitro model of corneal dystrophy, characterized by endoplasmic reticulum stress. The effects of MSC-EVs were compared with those of serum-derived EVs, reported to display a pro-angiogenic activity. MSC-EVs were able to induce a significant down-regulation of the large majority of endoplasmic reticulum stress-related genes in human corneal endothelial cells after exposure to serum deprivation and tunicamycin. In parallel, they upregulated the Akt pathway and limited caspase-3 activation and apoptosis. At variance, the effect of the serum EVs was mainly limited to Akt phosphorylation, with minimal or absent effects on endoplasmic reticulum stress modulation and apoptosis prevention. The effects of MSC-EVs were correlated to the transfer of numerous endoplasmic reticulum (ER)-stress targeting miRNAs to corneal endothelial cells. These data suggest a potential therapeutic effect of MSC-EVs for corneal endothelial endoplasmic reticulum stress, a major player in corneal endothelial dystrophy

Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized rats

Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MVs) derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MVs in the hepatocytes by an α4-integrin-dependent mechanism. However, MVs pre-treated with RNase, even if internalized, were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA-dependent effect. Microarray analysis and quantitative RT-PCR demonstrated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MVs accelerated the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre-treatment of MVs. Using human AGO2, as a reporter gene present in MVs, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MVs. These data suggested a translation of the MV shuttled mRNA into hepatocytes of treated rats. In conclusion, these results suggest that MVs derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets

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

Differences and homologies of chromosomal alterations within and between breast cancer cell lines: A clustering analysis

BACKGROUND: The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism. RESULTS: A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines. CONCLUSIONS: Our study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines. The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level

ALL blasts drive primary mesenchymal stromal cells to increase asparagine availability during asparaginase treatment

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to L-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to L-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid tradeoff. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to L-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P &lt;.05), secrete more asparagine (P &lt;.05), and protect leukemic blasts (P &lt;.05) better than MSCs isolated from healthy donors. In conclusion, ALL blasts arrange a pro-leukemic amino acid trade-off with bone marrow mesenchymal cells, which depends on GS and SNAT5 and promotes leukemic cell survival during L-asparaginase treatment