Mónica Yunta

Tetraspanin proteins as organisers of membrane microdomains and signalling complexes

Tetraspanins are a group of hydrophobic proteins with four transmembrane domains and two extracellular loops, both with conserved residues. Some tetraspanins are cell specific and others are very ubiquitous. Tetraspanins interact with very different types of proteins such as integrins, membrane receptors, as well as intracellular signalling molecules. Tetraspanins can interact with other tetraspanins to form a larger complex, whose core is formed by six tetraspanins, surrounded several tetraspanin-associated proteins. These complexes can further aggregate and behave as a membrane microdomain. The great heterogeneity in their composition and the dynamics of tetraspanin complexes confers great flexibility on these proteins to participate in many different biological roles.

Aberrant expression of tetraspanin molecules in B-cell chronic lymphoproliferative disorders and its correlation with normal B-cell maturation

Tetraspanin proteins form signaling complexes between them and with other membrane proteins and modulate cell adhesion and migration properties. The surface expression of several tetraspanin antigens (CD9, CD37, CD53, CD63, and CD81), and their interacting proteins (CD19, CD21, and HLA-DR) were analyzed during normal B-cell maturation and compared to a group of 67 B-cell neoplasias. Three patterns of tetraspanin expression were identified in normal B cells. The first corresponded to bone marrow CD10+ B-cell precursors (BCP) which showed high expression of CD81 and CD9, low reactivity for CD53 and negativity for CD37. CD10− B-lymphocytes showed downregulation of CD9/CD81 and upregulation of CD53/CD37. Plasma cells showed re-expressed CD9 and downregulated CD37. Hierarchical clustering analysis of flow cytometry immunophenotypic data showed a good correlation between the tumor differentiation stage and the pattern of tetraspanin expression, with all analyzed individual samples classified into three major groups, independently of their normal or neoplastic origin. Despite this, neoplastic B-cells frequently showed aberrantly high/low expression of the different markers analyzed. Interestingly, in B-cell chronic lymphocytic leukemia, abnormal expression of CD53 and CD9 were associated with different patterns of disease infiltration, which would support the role of these molecules on modulating adhesion and migration of neoplastic B cells.

MicroRNA Dysregulation in the Spinal Cord following Traumatic Injury

Spinal cord injury (SCI) triggers a multitude of pathophysiological events that are tightly regulated by the expression levels of specific genes. Recent studies suggest that changes in gene expression following neural injury can result from the dysregulation of microRNAs, short non-coding RNA molecules that repress the translation of target mRNA. To understand the mechanisms underlying gene alterations following SCI, we analyzed the microRNA expression patterns at different time points following rat spinal cord injury. The microarray data reveal the induction of a specific microRNA expression pattern following moderate contusive SCI that is characterized by a marked increase in the number of down-regulated microRNAs, especially at 7 days after injury. MicroRNA downregulation is paralleled by mRNA upregulation, strongly suggesting that microRNAs regulate transcriptional changes following injury. Bioinformatic analyses indicate that changes in microRNA expression affect key processes in SCI physiopathology, including inflammation and apoptosis. MicroRNA expression changes appear to be influenced by an invasion of immune cells at the injury area and, more importantly, by changes in microRNA expression specific to spinal cord cells. Comparisons with previous data suggest that although microRNA expression patterns in the spinal cord are broadly similar among vertebrates, the results of studies assessing SCI are much less congruent and may depend on injury severity. The results of the present study demonstrate that moderate spinal cord injury induces an extended microRNA downregulation paralleled by an increase in mRNA expression that affects key processes in the pathophysiology of this injury.

MicroRNA dysregulation in spinal cord injury: causes, consequences and therapeutics

Trauma to the spinal cord causes permanent disability to more than 180,000 people every year worldwide. The initial mechanical damage triggers a complex set of secondary events involving the neural, vascular, and immune systems that largely determine the functional outcome of the spinal cord injury (SCI). Cellular and biochemical mechanisms responsible for this secondary injury largely depend on activation and inactivation of specific gene programs. Recent studies indicate that microRNAs function as gene expression switches in key processes of the SCI. Microarray data from rodent contusion models reveal that SCI induces changes in the global microRNA expression patterns. Variations in microRNA abundance largely result from alterations in the expression of the cells at the damaged spinal cord. However, microRNA expression levels after SCI are also influenced by the infiltration of immune cells to the injury site and the death and migration of specific neural cells after injury. Evidences on the role of microRNAs in the SCI pathophysiology have come from different sources. Bioinformatic analysis of microarray data has been used to identify specific variations in microRNA expression underlying transcriptional changes in target genes, which are involved in key processes in the SCI. Direct evidences on the role of microRNAs in SCI are scarcer, although recent studies have identified several microRNAs (miR-21, miR-486, miR-20) involved in key mechanisms of the SCI such as cell death or astrogliosis, among others. From a clinical perspective, different evidences make clear that microRNAs can be potent therapeutic tools to manipulate cell state and molecular processes in order to enhance functional recovery. The present article reviews the actual knowledge on how injury affects microRNA expression and the meaning of these changes in the SCI pathophysiology, to finally explore the clinical potential of microRNAs in the SCI.

Apoptosis protection and survival signal by the CD53 tetraspanin antigen

The CD53 antigen is a tetraspanin protein of the lymphoid–myeloid lineage, but its implication in biological effects is hardly known. Radioresistant tumor cells express very high levels of this antigen. We have studied the effect of CD53 antigen ligation on the survival response of tumor cells to serum deprivation, a well-known stimulator of cell death that may mimic the tumor environment; for this aim IR938F and Jurkat cells, a B- and T-cell lymphoma, were used. Ligation of CD53 triggers a survival response and reduces the number of cells that enter apoptosis. In CD53- stimulated cells there is a significant reduction in caspase activation, measured by caspase processing of poly ADP-ribose polymerase, as well as a reduction in the fragmentation of DNA. CD53- stimulated cells also have an increase in the level of bcl-XL and a reduction of bax protein, two components of the mitochondrial apoptotic pathway, changing their ratio by 24-fold in the direction of survival. This survival signal appears to be mediated by activation of the AKT, as detected by its phosphorylation in Ser473 upon CD53 ligation. The CD53 antigen interactions might contribute to cell survival in poorly vascularized regions of the tumor mass.

Differential cooperation between regulatory sequences required for human CD53 gene expression

CD53 is a tetraspanin protein mostly expressed in to the lymphoid-myeloid lineage. We have characterized the humanCD53 gene regulatory region. Within the proximal 2 kilobases, and with opposite transcriptional orientation, is located the promoter-enhancer of a second gene, which does not affectCD53. Twenty-four copies of a CA dinucleotide repeat separate these two gene promoters. The proximal enhanceosome of the human CD53 gene is comprised between residues −266 and +84, and can be subdivided into four major subregions, two of them within exon 1. Mutational analysis identified several cooperating sequences. An Sp1 and an ets-1 site, at positions −115 and +62, respectively, are essential for transcriptional competence in all cell lines. Five other regulatory sequences have a dual role, activator or down-regulator, depending on the cell line. At the end of the non-coding exon 1, +64 to +83, there is a second ets-1 regulatory element, which is required for high level of transcription, in cooperation with the Sp1 site, in K562 and Molt-4, but not in Namalwa cells, where it functions as a repressor. This Sp1 site also cooperates with another ets-1/PU.1 site at −172. Different cell types use different regulatory sequences in the enhanceosome for the expression of the same gene.

Gene expression of axon growth promoting factors in the deer antler.

The annual regeneration cycle of deer (Cervidae, Artiodactyla) antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus). Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deers normal and pedicle skin.

Pattern of expression of tetraspanin antigen genes in Burkitt lymphoma cell lines

Tetraspanin antigens are implicated in the prognosis of different types of tumours. In this study we determine by semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) the level of 13 tetraspan messages in 21 Burkitt lymphoma (BL) cell lines. All tumour cell lines have a common pattern of tetraspanin gene expression. There are five antigens which are detected in 90% of cell lines at high levels, CD53, CD81, CD63, SAS and CD82. Another two, CD9 and CD37, were detected in 60% of cell lines, and have a very variable level of expression. The remaining antigens, A15, CoO29, KRAG, L6, TI‐1 and il‐TMP, are expressed at low levels in very few cell lines without any specific pattern. The level of gene expression corresponds with the level of cell surface antigen determined by flow cytometry. The average number of tetraspan proteins expressed per cell line is six. These proteins may form subunits of an oligomeric structure with 24 transmembrane domains. There are no major differences in tetraspan expression pattern among sporadic or endemic tumours, type of translocation or Epstein–Barr virus status, suggesting the original cell of these tumours is the same, probably a late pre‐B cell, at the CD9 to CD37 transition point. Tetraspanin gene expression is consistent with BL being a single entity, despite variations in other parameters.

Deer antler innervation and regeneration.

Nervous system injuries are a major cause of impairment in the human society. Up to now, clinical approaches have failed to adequately restore function following nervous system damage. The regenerative cycle of deer antlers may provide basic information on mechanisms underlying nervous system regeneration. The present contribution reviews the actual knowledge on the antler innervation and the factors responsible for its regeneration and fast growth. Growing antlers are profusely innervated by sensory fibers from the trigeminal nerve, which regenerate every year reaching elongation rates up to 2 cm a day. Antler nerves grow through the velvet in close association to blood vessels. This environment is rich in growth promoting molecules capable of inducing and guiding neurite outgrowth of rat sensory neurons in vitro. Conversely, endocrine regulation failed to show effects on neurite outgrowth in vitro, in spite of including hormones of known promoting effects on axon growth. Additional studies are needed to analyze unexplored factors promoting on growth in antlers such as electric potentials or mechanical stretch, as well as on the survival of antler innervating neurons.

Discrimination of biclonal B-cell chronic lymphoproliferative neoplasias by tetraspanin antigen expression

This work was supported by Grants from MEC SAF2004-02900 (PAL) and SAF 2002-03096 (AO), Fondo de Investigacion Sanitaria PI02-0585 (PAL), and Fundacion Memoria Samuel Solorzano Barruso (PAL).