PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability.

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

XIAP Interacts with and Regulates the Activity of FAF1.

Cell death depends on the balance between the activities of pro- and anti-apoptotic factors. X-linked inhibitor of apoptosis protein (XIAP) plays an important role in the cytoprotective process by inhibiting the caspase cascade and regulating pro-survival signaling pathways. While searching for novel interacting partners of XIAP, we identified Fas-associated factor 1 (FAF1). Contrary to XIAP, FAF1 is a pro-apoptotic factor that also regulates several signaling pathways in which XIAP is involved. However, the functional relationship between FAF1 and XIAP is unknown. Here, we describe a new interaction between XIAP and FAF1 and describe the functional implications of their opposing roles in cell death and NF-κB signaling. Our results clearly demonstrate the interaction of XIAP with FAF1 and define the specific region of the interaction. We observed that XIAP is able to block FAF1-mediated cell death by interfering with the caspase cascade and directly interferes in NF-κB pathway inhibition by FAF1. Furthermore, we show that XIAP promotes ubiquitination of FAF1. Conversely, FAF1 does not interfere with the anti-apoptotic activity of XIAP, despite binding to the BIR domains of XIAP; however, FAF1 does attenuate XIAP-mediated NF-κB activation. Altered expression of both factors has been implicated in degenerative and cancerous processes; therefore, studying the balance between XIAP and FAF1 in these pathologies will aid in the development of novel therapies.

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.

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.

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.

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 deer's normal and pedicle skin.

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.

Induction of DNA synthesis by ligation of the CD53 tetraspanin antigen in primary cultures of mesangial cells.

BACKGROUND: The interaction of mesangial cells with the extracellular matrix plays a major role in kidney biology. Tetraspanin proteins modulate cell interaction with the extracellular matrix. Tetraspanins form supramolecular structures on the cell membrane that send signals after engagement by unknown ligands, modulate different signaling processes, and regulate cell adhesion and motility. METHODS: CD53 was determined by immunohistochemistry, and on the cell surface of cultured rat mesangial cells by flow cytometry. Mesangial cell cultures were stimulated with MRC OX-44 antibody. DNA synthesis was measured by thymidine incorporation. Extracellular signal-regulated kinase (ERK) activation was determined by Western blot. RESULTS: CD53 was present in mesangial cells in vivo and in culture. Ligation of CD53 antigen with a monoclonal antibody triggered the induction of DNA synthesis, which was not sensitive to inhibitors of signaling pathways that use phosphatidylinositol 3-kinase (PI3K) and protein kinase C, or to calcium channel inhibitors, such as thapsigargin and verapamil. The DNA synthesis was inhibited by PD98059, a specific inhibitor of MEK that prevents ERK1/ERK2 activation. In addition, ERK1 and ERK2 activation by phosphorylation occurred following CD53 antigen ligation. The DNA synthesis was due to de novo synthesis and not to DNA repair as a consequence of the initiation of apoptosis, determined by flow cytometry, and lack of proteolytic activation of PARP by caspase 3. CD53 antigen ligation also induced an increase in mitochondrial activity. CONCLUSIONS: To our knowledge this is the first identification of a tetraspanin protein in mesangial cells. CD53 antigen delivers a signal that initiates DNA synthesis. This signal is mediated by ERK1/ERK2 activation, but it is not sufficient to complete the cell cycle.

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-X(L) 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.

Transient activation of the c-Jun N-terminal kinase (JNK) activity by ligation of the tetraspan CD53 antigen in different cell types.

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. We have studied the implication of CD53 antigen in signal transduction by determining the effect of its ligation on the c-Jun N-terminal kinase (JNK) in different cell types. Ligation of the rat or human CD53 antigen induces a three- to fourfold transient activation of JNK activity that peaks at 3-5 min. The effect was detected by assaying the endogenous or exogenous (transfected) JNK activity. The JNK response was detected in IR938F cells, a rat B-cell lymphoma, and in Jurkat cells derived from a human T-cell lymphoma. This JNK activation was not mediated by the vav oncogene, and CD53 does not cooperate with CD3 for vav activation. A similar JNK activation was also detected in a human renal carcinoma cell line that was transiently transfected with the human CD53 cDNA to mimic the CD53 ectopic expression in carcinomas. In stable CD53-transfected cells it stimulated Jun-dependent transcriptional activity. We conclude that parts of the cell responses modulated by the CD53 are mediated by JNK activation, and this activation is independent of the different protein interactions that the CD53 protein has on specific cell types.