(1/29) Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis.
Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione. (+info)
(2/29) Increased expression of the tetraspanins CD53 and CD63 on apoptotic human neutrophils.
The recently discovered tetraspanin superfamily comprises a group of cell-surface proteins that are suggested to be involved in cell activation and signal transduction as well as in cell adhesion, motility, and metastasis. In this study, we have assessed the expression of two tetraspanins, CD53 and CD63, and two principal leukocyte adhesion molecules, CD11b and CD62L, on human apoptotic neutrophils. After aging of human neutrophils for 20 and 40 h in vitro, apoptosis was analyzed by light microscopy and flow cytometry. The binding of monoclonal antibodies directed against CD11b, CD62L, CD53, and CD63 on apoptotic and nonapoptotic cells was determined by dual-color flow cytometry. Aging of neutrophils in vitro resulted in a significant (P < 0.05) down-regulation of expression of the selectin CD62L, and a significantly increased expression of the two tetraspanins CD53 and CD63. The selective analysis of apoptotic versus nonapoptotic cells proved that both the increased expression of the tetraspanins and the loss of CD62L were restricted to the apoptotic subpopulation. An identical pattern of surface molecule expression was detected at 12 h after induction of apoptosis by an agonistic anti-Fas IgM monoclonal antibody. Further studies are required to clarify whether tetraspanins participate in the recognition of apoptotic circulating or extravasated neutrophils by macrophages. (+info)
(3/29) Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins.
Translocation of conventional protein kinases C (PKCs) to the plasma membrane leads to their specific association with transmembrane-4 superfamily (TM4SF; tetraspanin) proteins (CD9, CD53, CD81, CD82, and CD151), as demonstrated by reciprocal co-immunoprecipitation and covalent cross-linking experiments. Although formation and maintenance of TM4SF-PKC complexes are not dependent on integrins, TM4SF proteins can act as linker molecules, recruiting PKC into proximity with specific integrins. Previous studies showed that the extracellular large loop of TM4SF proteins determines integrin associations. In contrast, specificity for PKC association probably resides within cytoplasmic tails or the first two transmembrane domains of TM4SF proteins, as seen from studies with chimeric CD9 molecules. Consistent with a TM4SF linker function, only those integrins (alpha(3)beta(1), alpha(6)beta(1), and a chimeric "X3TC5" alpha(3) mutant) that associated strongly with tetraspanins were found in association with PKC. We propose that PKC-TM4SF-integrin structures represent a novel type of signaling complex. The simultaneous binding of TM4SF proteins to the extracellular domains of the integrin alpha(3) subunit and to intracellular PKC helps to explain why the integrin alpha3 extracellular domain is needed for both intracellular PKC recruitment and PKC-dependent phosphorylation of the alpha(3) integrin cytoplasmic tail. (+info)
(4/29) 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 human CD53 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 affect CD53. 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. (+info)
(5/29) 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. (+info)
(6/29) CD53, a thymocyte selection marker whose induction requires a lower affinity TCR-MHC interaction than CD69, but is up-regulated with slower kinetics.
The molecular mechanisms that govern the survival, maturation and export of thymocytes are the subject of intense study, and candidates for involvement in these processes might be identified by their differential expression during thymocyte selection. One such molecule is the tetraspanin CD53, which is not expressed on most CD4(+)CD8(+) double-positive (DP) cells in the normal mouse. We have examined CD53 expression on DP from several class I- and class II-restricted TCR transgenic (Tg) mice, and have found a strong correlation between CD53 expression and positive selection. CD53 expression in DP was formally demonstrated to be dependent upon MHC recognition as evidenced by studying DP from MHC-deficient mice which totally lack expression of this molecule. This link between selection and CD53 expression was reminiscent of CD69, and indeed the majority of selected DP from normal mice that express CD53 also express CD69. We compared CD53 and CD69 induction in vitro using pre-selected thymocytes from TCR-Tg mice that were stimulated either with mAb against TCR or with antigen-presenting cells (APC) pulsed with peptides. The data shows that with either stimulus, CD69 is induced rapidly on the thymocyte surface with expression detected in as little as 2 h. CD53 induction is slower with maximal expression taking up to 20 h. We also stimulated pre-selected thymocytes from the OT-1 TCR-Tg strain with APC pulsed with peptides of varying affinities for the TCR. Here low-affinity peptides which induce CD69 expression poorly were able to induce significant levels of CD53 expression. These data demonstrate that the induction of CD53 and CD69 upon selection is not identical. Thus a combination of the CD69 and CD53 selection markers may be a powerful tool to isolate thymocytes that have either been very recently selected or have arisen from differing MHC--TCR affinity interactions during selection. (+info)
(7/29) Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation.
The tetraspanins associate with various surface molecules and with each other to build a network of molecular interactions, the tetraspanin web. The interaction of tetraspanins with each other seems to be central for the assembly of the tetraspanin web. All tetraspanins studied, CD9, CD37, CD53, CD63, CD81, CD82 and CD151, were found to incorporate [3H]palmitate. By site-directed mutagenesis, CD9 was found to be palmitoylated at any of the four internal juxtamembrane regions. The palmitoylation of CD9 did not influence the partition in detergent-resistant membranes but contributed to the interaction with CD81 and CD53. In particular, the resistance of the CD9/CD81 interaction to EDTA, which disrupts other tetraspanin/tetraspanin interactions, was entirely dependent on palmitoylation. (+info)
(8/29) Identification of three genes up-regulated in PU.1 rescued monocytic precursor cells.
The requirement of the transcription factor PU.1 for macrophage development has been well documented. However, the target genes regulated by PU.1 controlling macrophage maturation are not known. A granulocyte macrophage colony stimulating factor (GM-CSF)-dependent PU.1 null monocytic precursor cell was stably transduced with a PU.1-expressing retrovirus. The expression of PU.1 altered the surface expression of a few proteins expressed on monocytes; these cells, however, remained GM-CSF dependent and maintained an immature phenotype. In contrast to the PU.1 null cells, the cells expressing PU.1 responded to macrophage colony stimulating factor (M-CSF) with subsequent development into mature macrophages. Using suppressive subtractive hybridization between the PU.1 null and immature PU.1 rescued cells, three genes, MRP-14, Dap12 and CD53, were found expressed in the rescued cells, but not in the PU.1 null cells. In addition, these genes were modulated during M-CSF-induced maturation of the PU.1 rescued cells. The PU.1 null and rescued early monocytic cells provide a useful model to study the role of PU.1 in macrophage development. (+info)