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Age-related T cell dysfunction can lead to failure of immune tolerance mechanisms, resulting in aberrant T cell-driven cytokine and cytotoxic responses that ultimately cause tissue damage. In this Review, we discuss the role of T cells in the onset and progression of age-associated conditions, focusing on cardiovascular disorders, metabolic dysfunction, neuroinflammation and defective tissue repair and regeneration. We present different mechanisms by which T cells contribute to inflammageing and might act as modulators of age-associated diseases, including through enhanced pro-inflammatory and cytotoxic activity, defective clearance of senescent cells or regulation of the gut microbiota. Finally, we propose that 'resetting' immune system tolerance or targeting pathogenic T cells could open up new therapeutic opportunities to boost resilience to age-related diseases.

SUMMARY Exquisite regulation of energy homeostasis protects from nutrient deprivation but causes metabolic dysfunction upon nutrient excess. In human and murine adipose tissue, the accumulation of ligands of the receptor for advanced glycation end products (RAGE) accompanies obesity, implicating this receptor in energy metabolism. Here, we demonstrate that mice bearing global- or adipocyte-specific deletion of Ager, the gene encoding RAGE, display superior metabolic recovery after fasting, a cold challenge, or high-fat feeding. The RAGE-dependent mechanisms were traced to suppression of protein kinase A (PKA)-mediated phosphorylation of its key targets, hormone-sensitive lipase and p38 mitogen-activated protein kinase, upon β-adrenergic receptor stimulation—processes that dampen the expression and activity of uncoupling protein 1 (UCP1) and thermogenic programs. This work identifies the innate role of RAGE as a key node in the immunometabolic networks that control responses to nutrient supply and cold challenges, and it unveils opportunities to harness energy expenditure in environmental and metabolic stress., Graphical Abstract, In Brief Hurtado del Pozo et al. show that the deletion of adipocyte RAGE, whose ligands accumulate in metabolic stress, protects from obesity and cold challenges through the modulation of protein kinase A activities. This work adds RAGE to the immunometabolic networks that regulate energy expenditure in environmental and metabolic stress.

The biochemical, ionic, and signaling changes that occur within cardiomyocytes subjected to ischemia are exacerbated by reperfusion; however, the precise mechanisms mediating myocardial ischemia/reperfusion (I/R) injury have not been fully elucidated. The receptor for advanced glycation end-products (RAGE) regulates the cellular response to cardiac tissue damage in I/R, an effect potentially mediated by the binding of the RAGE cytoplasmic domain to the diaphanous-related formin, DIAPH1. The aim of this study was to investigate the role of DIAPH1 in the physiological response to experimental myocardial I/R in mice. After subjecting wild-type mice to experimental I/R, myocardial DIAPH1 expression was increased, an effect that was echoed following hypoxia/reoxygenation (H/R) in H9C2 and AC16 cells. Further, compared to wild-type mice, genetic deletion of Diaph1 reduced infarct size and improved contractile function after I/R. Silencing Diaph1 in H9C2 cells subjected to H/R downregulated actin polymerization and serum response factor-regulated gene expression. Importantly, these changes led to increased expression of sarcoplasmic reticulum Ca2 + ATPase and reduced expression of the sodium calcium exchanger. This work demonstrates that DIAPH1 is required for the myocardial response to I/R, and that targeting DIAPH1 may represent an adjunctive approach for myocardial salvage after acute infarction., Graphical Abstract Image 1, Highlights • DIAPH1 is a key mediator of ischemia/reperfusion injury in the heart. • Diaph1 deletion regulates the transcription factor, serum response factor, leading to modulation of calcium transporters in cardiomyocytes. • The effects of Diaph1 deletion are associated with improved contractile function after short-term ischemia/reperfusion injury, making it a potential adjunctive therapeutic target for treatment of evolving myocardial infarction. Although reperfusion therapy, using thrombolytic agents, has become the mainstay for the treatment of patients with myocardial infarction, benefits of this therapy are reduced by multiple factors including reperfusion injury. The precise mechanisms for this effect are yet to be fully delineated. In this study we identify the formin DIAPH1 as a novel mediator of ischemia/reperfusion (I/R) injury in the heart. We show that Diaph1 deletion regulates the transcription factor, serum response factor, leading to modulation of calcium transporters in cardiomyocytes. We show that deletion of Diaph1 improved contractile function after I/R in the mouse heart, making DIAPH1 a potential adjunctive therapeutic target for the treatment of evolving myocardial infarction.

Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice.Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50-fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10-20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups.The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels.

Retrograde transport (RT) allows cells to retrieve receptors and other cellular cargoes for delivery to the Golgi apparatus, contributing to the maintenance of cellular homeostasis. This transport route is also commonly used by several bacterial toxins to exert their deleterious actions on eukaryotic cells. While the retrograde transport process has been well characterized, the contribution of microRNAs (miRNAs) in regulating this cellular transport mechanism remains unknown. Here, we determined that mir-199a and mir-199b, members of the intronic miRNA family, coordinate genes regulating RT and endosome trafficking. We demonstrate that miR-199a-5p attenuates the expression of Vps26A, Rab9B, and M6PR, thereby controlling RT from endosomes to the trans-Golgi network (TGN). Importantly, we found that overexpression of a Vps26A construct resistant to the inhibitory action of miR-199a-5p abrogates the effect of miR-199a-5p on RT. Finally, we demonstrate that miR-199-5p overexpression attenuates Shiga toxin type 1 (Stx1)-mediated inhibition of protein biosynthesis. In summary, our work identifies the first noncoding RNA that influences RT and reduces the inhibition of protein biosynthesis caused by bacterial toxins.

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element-binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease.

Scope The increased prevalence of cardiovascular diseases (CVDs) has been hypothesized to be the result of an increased exposure to a host of atherogenic environmental factors, paramount among them being unhealthy dietary habits. Long-chain n-3 polyunsaturated fatty acids (PUFAs) have been shown to have cardio protective effects, partially due to their ability to regulate gene expression. In this regard, increasing attention has been devoted to the role of miRNAs as regulators of multiple metabolic pathways whose deregulation has been associated with CVD risk. In this work we investigated whether miRNA expression was regulated by docosahexanoic acid, conjugated linoleic acid and cholesterol in Caco-2 cells. Results Among the modulated miRNAs, miR-107 was differentially expressed by all treatments and this modulation was independent of its hosting gene, panthothenate kinase 1, possibly through its own promoter, which contains binding sites for metabolically relevant transcription factors. Among the putative target genes of miR-107, we found some genes with key roles in circadian rhythm. Specifically, we demonstrated that binding of miR-107 to the circadian locomotor output cycles kaput gene results in the deregulation of the circadian rhythm of the cells. Conclusion Since chronodisruption has been linked to metabolic disorders such as type 2 diabetes, atherosclerosis, obesity, and CVD, our findings suggest that miR-107 could represent a new approach for pharmacological treatment of these diseases.

PURPOSE OF REVIEW The objective of this review article is to summarize the recent findings about the importance of microRNAs (miRNAs) in regulating lipoprotein metabolism. We highlight the recent findings that uncover the importance of miRNAs in controlling plasma LDL-cholesterol (LDL-C) levels. RECENT FINDINGS In 2013, several studies reported a number of miRNAs that regulate plasma LDL-C levels, including miR-30c. In this review article, we discuss those miRNAs that modulate LDL-C levels and lipoprotein secretion. We also discuss the numerous studies that demonstrate the critical role of miRNAs in governing the many facets of HDL metabolism, such as the ATP transporters, ABCA1, and ABCG1, and the scavenger receptor, SRB1. SUMMARY The understanding of how these miRNAs modulate lipoprotein metabolism promises to reveal new therapeutic targets to treat dyslipidemias and related cardiovascular disorders.

Hair follicle stem cells (HF-SCs) alternate between periods of quiescence and proliferation, to finally differentiate into all the cell types that constitute the hair follicle. Also, they have been recently identified as cells of origin in skin cancer. HF-SCs localize in a precise region of the hair follicle, the bulge, and molecular markers for this population have been established. Thus, HF-SCs are good model to study the potential role of oncogenic activations on SC physiology. Expression of a permanently active form of Akt (myrAkt) in basal cells leads to Akt hyperactivation specifically in the CD34+Itga6H population. This activation causes bulge stem cells to exit from quiescence increasing their response to proliferative stimuli and affecting some functions such as cell migration. HF-SC identity upon Akt activation is preserved; in this sense, increased proliferation does not result in stem cell exhaustion with age suggesting that Akt activation does not affect self-renewal an important aspect for normal tissue maintenance and cancer development. Genome-wide transcriptome analysis of HF-SC isolated from myrAkt and wild-type epidermis underscores changes in metabolic pathways characteristic of cancer cells. These differences manifest during a two-step carcinogenesis protocol in which Akt activation in HF-SCs results in increased tumor development and malignant transformation. Stem Cells 2014;32:1917–1928

SummaryThe miR-143/145 cluster regulates VSMC specific gene expression, thus controlling differentiation, plasticity and contractile function, and promoting the VSMC phenotypic switch from a contractile/non-proliferative to a migrating/proliferative state. More recently increased miR-145 expression was observed in human carotid atherosclerotic plaques from symptomatic patients. The goal of this study was to investigate the contribution of miR-143/145 during atherogenesis by generating mice lacking miR-143/145 on an Ldlr-deficient background. Ldlr-/- and Ldlr-/--miR-143/145-/- (DKO) were fed a Western diet (WD) for 16 weeks. At the end of the treatment, the lipid profile and the atherosclerotic lesions were assessed in both groups of mice. Absence of miR-143/145 significantly reduced atherosclerotic plaque size and macrophage infiltration. Plasma total cholesterol levels were lower in DKO and FLPC analysis showed decreased cholesterol content in VLDL and LDL fractions. Interestingly miR-143/145 deficiency per se resulted in increased hepatic and vascular ABCA1 expression. We further confirmed the direct regulation of miR-145 on ABCA1 expression by qRT-PCR, Western blotting and 3′UTR-luciferase reporter assays. In summary, miR-143/145 deficiency significantly reduces atherosclerosis in mice. Therapeutic inhibition of miR-145 might be useful for treating atherosclerotic vascular disease.

The regulation of the metabolism of cholesterol has been one of the most studied biological processes since its first isolation from gallstones in 1784. High levels of plasma low-density lipoprotein (LDL) cholesterol and reduced levels of plasma high-density lipoprotein (HDL) cholesterol are widely recognized as major risk factors of cardiovascular disease. An imbalance in the production of reactive oxygen species can oxidize LDL particles, increasing the levels of the highly proatherogenic oxidized LDL. Furthermore, under pathological scenarios, numerous molecules can function as pro-oxidants, such as iron or (high levels of) glucose. In addition to the classical mechanisms regulating lipid homeostasis, recent studies have demonstrated the important role of microRNAs (miRNAs) as regulators of lipoprotein metabolism, oxidative derivatives of lipoprotein, and redox balance. Here, we summarize recent findings in the field, highlighting the contributions of some miRNAs to lipid- and oxidative-associated pathologies. We also discuss how therapeutic intervention of miRNAs may be a promising strategy to decrease LDL, increase HDL, and ameliorate lipid- and oxidative-related disorders, including atherosclerosis, nonalcoholic fatty liver disease, and metabolic syndrome.

Summary Transcytosis is a widespread pathway for apical targeting in epithelial cells. MAL2, an essential protein of the machinery for apical transcytosis, functions by shuttling in vesicular carriers between the apical zone and the cell periphery. We have identified INF2, an atypical formin with actin polymerization and depolymerization activities, which is a binding partner of MAL2. MAL2-positive vesicular carriers associate with short actin filaments during transcytosis in a process requiring INF2. INF2 binds Cdc42 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are necessary for apical transcytosis and the formation of lateral lumens in hepatoma HepG2 cells. INF2 and MAL2 are also essential for the formation of the central lumen in organotypic cultures of epithelial MDCK cells. Our results reveal a functional mechanism whereby Cdc42, INF2, and MAL2 are sequentially ordered in a pathway dedicated to the regulation of transcytosis and lumen formation.

In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane “raft” proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n = 22) or immunoblotting ( n = 6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.

Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

Polarized transport of newly synthesized proteins to the apical surface of epithelial cells takes place by a direct pathway from the Golgi or by an indirect route involving the delivery of the protein to the basolateral surface, followed by its endocytosis and transport across the cell. The indirect pathway, named transcytosis, is also used to translocate external material across the cell. MAL, a raft-associated integral membrane protein required for the direct apical route, is known to be expressed in the thyroid epithelium. MAL2, a member of the MAL protein family, has been recently identified as an essential component of the machinery for the transcytotic route in human hepatoma cells. Herein, we have investigated the expression and distribution of MAL2 in the human thyroid. MAL2 mRNA species were detected in the thyroid. Immunohistochemical analysis of thyroid follicles indicated that, in contrast to MAL, which predominantly distributed to the Golgi region, MAL2 distributed to the apical membrane. Biochemical analysis in primary thyrocyte cultures indicated that MAL2 exclusively resides in raft membranes. Confocal immunofluorescence analysis of thyrocyte cultures revealed that MAL2 predominantly localized in a subapical endosome compartment that was positive for Rab11a. Alterations in MAL2 expression, distribution, and appearance were found in specific types of follicular cell-derived carcinomas. Although the role of MAL2 has not been directly addressed in this study, the simultaneous expression of MAL and MAL2 suggests that traffic to the apical membrane in thyrocytes may rely on MAL for the direct route and on MAL2 for the transcytotic pathway.

MAL is an integral protein component of the machinery for apical transport in epithelial Madin–Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.

Atherosclerosis is the major cause of death and disability in diabetic and obese subjects with insulin resistance. Akt2, a phosphoinositide-dependent serine-threonine protein kinase, is highly express in insulin-responsive tissues; however, its role during the progression of atherosclerosis remains unknown. Thus, we aimed to investigate the contribution of Akt2 during the progression of atherosclerosis. We found that germ-line Akt2-deficient mice develop similar atherosclerotic plaques as wild-type mice despite higher plasma lipids and glucose levels. It is noteworthy that transplantation of bone marrow cells isolated from Akt2−/− mice to Ldlr−/− mice results in marked reduction of the progression of atherosclerosis compared with Ldlr−/− mice transplanted with wild-type bone marrow cells. In vitro studies indicate that Akt2 is required for macrophage migration in response to proatherogenic cytokines (monocyte chemotactic protein-1 and macrophage colony-stimulating factor). Moreover, Akt2−/− macrophages accumulate less cholesterol and have an alternative activated or M2-type phenotype when stimulated with proinflammatory cytokines. Together, these results provide evidence that macrophage Akt2 regulates migration, the inflammatory response and cholesterol metabolism and suggest that targeting Akt2 in macrophages might be beneficial for treating atherosclerosis.—Rotllan, N., Chamorro-Jorganes, A., Araldi, E., Wanschel, A. C., Aryal, B., Aranda, J. F., Goedeke, L., Salerno, A. G., Ramírez, C. M., Sessa,W. C., Suárez, Y., Fernández-Hernando, C. Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis.

MicroRNAs (miRNAs) are small (~22nucleotide) sequences of RNA that regulate gene expression at posttranscriptional level. MiRNA/mRNA base pairing complementarity provokes mRNA decay and consequent gene silencing. These endogenous gene expression inhibitors were primarily described in cancer but recent exciting findings have also demonstrated a key role in cardiovascular diseases (CVDs) including atherosclerosis. MiRNAs controls endothelial cell (EC), vascular smooth muscle cell (VSMC) and macrophage functions, and thereby regulate the progression of atherosclerosis. MiRNAs expression is modulated by different stimuli involved in every stage of atherosclerosis and conversely miRNAs modulates several pathways implicated in plaque development such as cholesterol metabolism. In the present review, we focus on the importance of miRNAs in atherosclerosis and we further discuss their potential use as biomarkers and therapeutic targets in CVDs.

PMCID: PMC3929583.-- et al., [Rationale]: Foam cell formation because of excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis, the major cause of morbidity and mortality in Western societies. Liver X nuclear receptors (LXRs) regulate the expression of the adenosine triphosphate-binding cassette (ABC) transporters, including adenosine triphosphate-binding cassette transporter A1 (ABCA1) and adenosine triphosphate-binding cassette transporter G1 (ABCG1). ABCA1 and ABCG1 facilitate the efflux of cholesterol from macrophages and regulate high-density lipoprotein (HDL) biogenesis. Increasing evidence supports the role of microRNA (miRNAs) in regulating cholesterol metabolism through ABC transporters. [Objective]: We aimed to identify novel miRNAs that regulate cholesterol metabolism in macrophages stimulated with LXR agonists. [Methods and Results]: To map the miRNA expression signature of macrophages stimulated with LXR agonists, we performed an miRNA profiling microarray analysis in primary mouse peritoneal macrophages stimulated with LXR ligands. We report that LXR ligands increase miR-144 expression in macrophages and mouse livers. Overexpression of miR-144 reduces ABCA1 expression and attenuates cholesterol efflux to apolipoproteinA1 in macrophages. Delivery of miR-144 oligonucleotides to mice attenuates ABCA1 expression in the liver, reducing HDL levels. Conversely, silencing of miR-144 in mice increases the expression of ABCA1 and plasma HDL levels. Thus, miR-144 seems to regulate both macrophage cholesterol efflux and HDL biogenesis in the liver. [Conclusions]: miR-144 regulates cholesterol metabolism via suppressing ABCA1 expression and modulation of miRNAs may represent a potential therapeutical intervention for treating dyslipidemia and atherosclerotic vascular disease. © 2013 American Heart Association, Inc., This work was supported by grants from the National Institutes of Health (R01HL107953 and R01HL106063 to C. Fernández-Hernando, R01HL105945 to Y. Suárez, and P30NS069329 to J. Kim) and the Spanish Ministry of I+D (grant SAF2008-00057 and SAF2011-29244 to A. Castrillo). C.M. Ramírez was supported by a postdoctoral fellowship from the American Heart Association (12POST9780016), D. Cirera-Salinas was supported by the Deutsche Forschunqsqemeinschaft, and A. Wanschel was supported by Capes Foundation, Ministry of Education of Brazil, Brazil. N. Rotllan was supported by a postdoctoral fellowship from Spanish Ministry of I+D. E. Araldi is a Howard Hughes Institute International Student Research Fellow.

Mutations in the TP53 gene are very common in human cancers, and are associated with poor clinical outcome. Transgenic mouse models lacking the Trp53 gene or that express mutant Trp53 transgenes produce tumours with malignant features in many organs. We previously showed the transcriptome of a p53-deficient mouse skin carcinoma model to be similar to those of human cancers with TP53 mutations and associated with poor clinical outcomes. This report shows that much of the 682-gene signature of this murine skin carcinoma transcriptome is also present in breast and lung cancer mouse models in which p53 is inhibited. Further, we report validated gene-expression-based tests for predicting the clinical outcome of human breast and lung adenocarcinoma. It was found that human patients with cancer could be stratified based on the similarity of their transcriptome with the mouse skin carcinoma 682-gene signature. The results also provide new targets for the treatment of p53-defective tumours.

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes., This work was supported by grants BFU2009–07886 (to M.A.A.), SAF-2008–01936 and SAF2008–01158-E (to J.M.), BFU2008–02460 (to I.C.), BFU2009–10335 (to C.E.), CONSOLIDER COAT CSD2009–00016 (to M.A.A. and C.E.), all grants from the Ministerio de Ciencia e Innovación, and grants GEN-0166/2006 from the Comunidad de Madrid (to I.C.), CSIC-200920I016 from Consejo Superior de Investigaciones Científicas (to L.K.), and PI040236 from Fundació Marató TV3 (to C.E.).

Oligodendrocytes (OLs), the myelin-producing cells of the central nervous system, segregate different surface subdomains at the plasma membrane as do other differentiated cells such as polarized epithelia and neurons. To generate the complex membrane system that characterizes myelinating OLs, large amounts of membrane proteins and lipids need to be synthesized and correctly targeted. In polarized epithelia, a considerable fraction of apical proteins are transported by an indirect pathway involving a detour to the basolateral membrane before being internalized and transported across the cell to the apical membrane by a process known as transcytosis. The apical recycling endosome (ARE) or its equivalent, the subapical compartment (SAC), of hepatocytes is an intracellular trafficking station involved in the transcytotic pathway. MAL2, an essential component of the machinery for basolateral-to-apical transcytosis, is an ARE/SAC resident protein. Here, we show that, after differentiation, murine oligodendrocyte precursor and human oligodendroglioma derived cell lines, Oli-neu and HOG, respectively, up-regulate the expression of MAL2 and accumulate it in an intracellular compartment, exhibiting a peri-centrosomal localization. In these oligodendrocytic cell lines, this compartment shares some of the main features of the ARE/SAC, such as colocalization with Rab11a, sensitivity to disruption of the microtubule cytoskeleton with nocodazole, and lack of internalized transferrin. Therefore, we suggest that the MAL2-positive compartment in oligodendrocytic cells could be a structure analogous to the ARE/SAC and might have an important role in the sorting of proteins and lipids for myelin assembly during oligodendrocyte differentiation. © 2009 Elsevier B.V. All rights reserved.

Material suplementario: 1 archivo PDF 5 Videos, MAL, a compact hydrophobic, four-transmembrane-domain apical protein that copurifies with detergent-resistant membranes is obligatory for the machinery that sorts glycophosphatidylinositol (GPI)-anchored proteins and others to the apical membrane in epithelia. The mechanism of MAL function in lipid-raft–mediated apical sorting is unknown. We report that MAL clusters formed by two independent procedures—spontaneous clustering of MAL tagged with the tandem dimer DiHcRED (DiHcRED-MAL) in the plasma membrane of COS7 cells and antibody-mediated cross-linking of FLAG-tagged MAL—laterally concentrate markers of sphingolipid rafts and exclude a fluorescent analogue of phosphatidylethanolamine. Site-directed mutagenesis and bimolecular fluorescence complementation analysis demonstrate that MAL forms oligomers via xx intramembrane protein–protein binding motifs. Furthermore, results from membrane modulation by using exogenously added cholesterol or ceramides support the hypothesis that MAL-mediated association with raft lipids is driven at least in part by positive hydrophobic mismatch between the lengths of the transmembrane helices of MAL and membrane lipids. These data place MAL as a key component in the organization of membrane domains that could potentially serve as membrane sorting platforms., the United States-Israel Binational Science foundation grant 281/05 (to K. H.). This work was supported in part by grants from the Ministerio de Educacio´n y Ciencia (BMC2003-03297; BFU2006-01925; GEN2003-20662-C07-02) and the Comunidad de Madrid (GR/SAL/0096/2004) (to M.A.A.).

Plitidepsin (aplidin) is a marine cyclic depsipeptide in phase II clinical development against several neoplasias. Plitidepsin is a potent inducer of apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We have reported that this activation depends on the early induction of oxidative stress, activation of Rac1 small GTPase, and the later down-regulation of MKP-1 phosphatase. Using Scatchard and saturation binding analyses, we have found that 14C-labeled plitidepsin binds to a moderately high-affinity receptor (Kd of 44.8 ± 3.1 and 35.5 ± 4.8 nM, respectively) in MDA-MB-231 breast cancer cells. Two minutes after addition to cells, half of the drug was membrane-bound and was subsequently found in the cytosolic fraction. At 4°C, plitidepsin cellular binding was around 10-fold lower than at 37°C but sufficed to induce cell death, suggesting that this process is triggered from the membrane. Depletion of plasma membrane cholesterol by short treatment with methyl-β-cyclodextrin diminished plitidepsin binding and Rac1 and JNK activation. Rac1 is targeted to the plasma membrane by plitidepsin as shown by subcellular fractioning and immunofluorescence analysis followed by confocal microscopy. Methyl-β-cyclodextrin blocked this effect. A subline of HeLa cells (HeLa-R), partially resistant to plitidepsin, showed similar affinity (Kd of 79.5 ± 2.5 versus 37.7 ± 8.2 nM) but 7.5-fold lower binding capacity than wild-type HeLa cells. Moreover, HeLa-R cells had lower total (71%) and membrane (67%) cholesterol content and membrane-bound Rac1, and showed no Rac1 activation upon plitidepsin treatment. In conclusion, cellular plitidepsin uptake and induction of apoptosis via activation of the Rac1-JNK pathway is membrane-cholesterol dependent. Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics., This study was supported in part by grant SAF04-01015 (to A.M.), SAF03-02604 (to J.M.R.) and BMC2003-03297 and GEN2003-20662-C07-02 (to M.A.A.) from Ministerio de Educación y Ciencia and FIS03-C03/10 (to A.M.) and Intramural ISCIII (03/ESP27) (to J.M.R.) from Instituto de Salud Carlos III. An institutional grant from the Fundación Ramón Areces to Centro de Biologı´a Molecular “Severo Ochoa” is also acknowledged.