Biomedical subjects

V M Dixit

Publications and source records attributed to V M Dixit.

At least 109 records · Page 6Linked to original sources

PubMed1995

CrmA-inhibitable cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein during Fas- and tumor necrosis factor-induced apoptosis.

Fas and the type I tumor necrosis factor receptor (TNF-R) are two cell surface receptors that, when stimulated with ligand or cross-linking antibody, trigger apoptotic cell death by a mechanism that has yet to be elucidated. The CrmA protein is a serpin family protease inhibitor than can inhibit interleukin-1 beta converting enzyme (ICE) and ICE-like proteases. We showed previously that expression of CrmA potently blocks apoptosis induced by activation of either Fas or TNF-R, implicating protease involvement in these death pathways (Tewari, M., and Dixit, V.M. (1995) J. Biol. Chem. 270, 3255-3260). Here we report that the 70-kDa component of the U1 small ribonucleoprotein (U1-70 kDa) is a proteolytic substrate rapidly cleaved during both Fas- and TNF-R-induced apoptosis. This cleavage was inhibited by expression of CrmA, but not by expression of an inactive point mutant of CrmA, confirming the involvement of an ICE-like protease. These data for the first time identify U1-70 kDa as a death substrate cleaved during Fas- and TNF-R-induced apoptosis and emphasize the importance of protease activation in the cell death pathway.

Antigens, Surface↗

PubMed1995

The baculovirus p35 protein inhibits Fas- and tumor necrosis factor-induced apoptosis.

The baculovirus p35 gene product inhibits virally induced apoptosis, developmental cell death in Caenorhabditis elegans and Drosophila, and neuronal cell death in mammalian systems. Therefore, p35 likely inhibits a component of the death machinery that is both ubiquitous and highly conserved in evolution. We now show for the first time that p35 also inhibits Fas- and tumor necrosis factor (TNF)-induced apoptosis. Additionally, p35 blocks TNF- and Fas-induced proteolytic cleavage of the death substrate poly(ADP-ribose) polymerase from its native 116-kDa form to the characteristic 85-kDa form. This cleavage is thought to be catalyzed by an aspartate-specific protease of the interleukin 1 beta-converting enzyme family designated prICE (Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346-347). Our data suggest that p35 must directly or indirectly inhibit prICE. Given that p35 inhibits both TNF and Fas killing, along with previous reports of its ability to block developmental, viral, and x-irradiation-induced cell death, the present results indicate that TNF- and Fas-mediated apoptotic pathways must have components in common with these highly conserved death programs.

Antigens, Surface↗

PubMed1995

Bcl-x and Bcl-2 inhibit TNF and Fas-induced apoptosis and activation of phospholipase A2 in breast carcinoma cells.

Tumor necrosis factor (TNF) induces cel death in several tumor cell lines by undefined mechanisms. Using a cDNA expression cloning strategy we identified two cDNAs that completely inhibit the TNF-induced death pathway in MCF7 breast carcinoma cells. These cDNAs encoded for Bcl-2 and Bcl-x. To compare the cytotoxic signal transduction pathway induced by the TNF receptor versus that induced by Fas, we transfected MCF7 cells with a Fas expression construct. The resulting cell line, MCF-Fas, was highly sensitive to cytotoxicity induced by TNF or anti-Fas. Expression of either bcl-2 or bcl-x in these cells rendered them completely resistant to lysis induced by either TNF or Fas. Interestingly, exposure of MCF-Fas cells to anti-Fas or TNF induced activation of phospholipase A2 (PLA2), while only TNF activated NF-kappa B. Activation of PLA2 was completely blocked whereas activation of NF-kappa B was unaffected by overexpression of either bcl-x or bcl-2. Moreover, PLA2-inhibitors, quinacrine and dexamethasone, partially inhibited cytotoxicity induced by either TNF or anti-Fas. These data suggest an involvement of PLA2 in both TNF- and Fas-mediated cytotoxicity and a novel mechanism of action for bcl-2 and bcl-x, i.e. inhibition of arachidonic acid metabolism, by which they may, in addition of apoptosis, modulate inflammation.

Antigens, Surface↗

PubMed1995

Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase.

Although the mechanism of mammalian apoptosis has not been elucidated, a protease of the CED-3/ICE family is anticipated to be a component of the death machinery. Several lines of evidence predict that this protease cleaves the death substrate poly(ADP-ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis, is inhibitable by the CrmA protein, and is distinct from ICE. We cloned a ced-3/ICE-related gene, designated Yama, that encodes a protein identical to CPP32 beta. Purified Yama was a zymogen that, when activated, cleaved PARP to generate the 85 kDa apoptotic fragment. Cleavage of PARP by Yama was inhibited by CrmA but not by an inactive point mutant of CrmA. Furthermore, CrmA blocked cleavage of PARP in cells undergoing apoptosis. We propose that Yama may represent an effector component of the mammalian cell death pathway and suggest that CrmA blocks apoptosis by inhibiting Yama.

Apoptosis↗

PubMed1995

Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis.

CD40 activation is critical for B-cell function, leading to activation and expression of cell surface markers, proliferation, immunoglobulin class switching and inhibition of programmed cell death (PCD). Germinal center B-cells, for example, can be prevented from undergoing PCD by CD40 activation. The mechanism by which PCD is inhibited has been an enigma. A potential role for A20, a novel zinc finger protein, in inhibiting B-cell apoptosis was suggested by our previous finding that it is induced by the Epstein-Barr virus LMP-1 gene product, a potent cell death inhibitor. We now show that CD40 activation induces A20 and that expression of A20 renders B-cell lines resistant to PCD. Additionally, we show that CD40 activation of A20 expression is mediated by inducible binding of NF-kappa B complexes to the A20 promoter and provide evidence for a critical role for Thr234 (in the CD40 cytoplasmic domain) in activating NF-kappa B.

Antigens, CD↗

PubMed1995

FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis.

Using the cytoplasmic domain of Fas in the yeast two-hybrid system, we have identified a novel interacting protein, FADD, which binds Fas and Fas-FD5, a mutant of Fas possessing enhanced killing activity, but not the functionally inactive mutants Fas-LPR and Fas-FD8. FADD contains a death domain homologous to the death domains of Fas and TNFR-1. A point mutation in FADD, analogous to the lpr mutation of Fas, abolishes its ability to bind Fas, suggesting a death domain to death domain interaction. Overexpression of FADD in MCF7 and BJAB cells induces apoptosis, which, like Fas-induced apoptosis, is blocked by CrmA, a specific inhibitor of the interleukin-1 beta-converting enzyme. These findings suggest that FADD may play an important role in the proximal signal transduction of Fas.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha-induced angiogenesis.

B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera showed that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK was activated by tumor necrosis factor-alpha (TNF-alpha) through induction of B61, and an antibody to B61 attenuated angiogenesis induced by TNF-alpha but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation.

Animals↗

PubMed1995

Characterization of B61, the ligand for the Eck receptor protein-tyrosine kinase.

B61 was originally described as a novel secreted tumor necrosis factor-alpha-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M. (1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (Bartley, T.D., Hunt, R. W., Welcher, A. A., Boyle, W. J., Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R. L., Guthrie, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M. (1994) Nature 368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidyl-inositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situ hybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific back-cross analysis.

Animals↗

PubMed1995

cDNA cloning and characterization of a Cek7 receptor protein-tyrosine kinase ligand that is identical to the ligand (ELF-1) for the Mek-4 and Sek receptor protein-tyrosine kinases.

We have isolated a murine cDNA encoding a ligand for the Cek7 receptor protein-tyrosine kinase (RPTK), a member of the Eph/Eck RPTK subfamily. Sequence analysis predicts an open reading frame of 209 amino acids with a predicted molecular mass of 24 kDa. The Cek7 ligand shows a 48% sequence identity at the protein level to B61, a ligand for the related Eck RPTK, 30% to the Cek5 ligand, 59% to the recently cloned Ehk1-L, and identity to ELF-1, a recently described ligand for the Mek4 and Sek RPTKs. The expressed Cek7 ligand is functionally active as it induces autophosphorylation of the Cek7 RPTK.

Amino Acid Sequence↗

PubMed1995

Fas- and tumor necrosis factor-induced apoptosis is inhibited by the poxvirus crmA gene product.

crmA is a cowpox virus gene that encodes a protease inhibitor of the serpin family. The only reported target for the CrmA protein is the cysteine protease interleukin-1 beta converting enzyme (ICE). ICE, by virtue of its homology to the Caenorhabditis elegans cell death protein Ced-3, has been suggested to play a fundamentally important role in mammalian apoptosis. We hypothesized that a function of crmA may be to inhibit cell death, since a major mechanism of viral clearance is the immune system-mediated induction of apoptosis of infected cells. The tumor necrosis factor receptor and the Fas antigen are two cytokine receptors which, by engaging and activating the death pathway, can eliminate virus-infected cells. Remarkably, crmA was found to be an exceptionally potent inhibitor of apoptosis induced by both these receptors, capable of blocking the cell death program even at pharmacological doses of the death stimulus. Therefore, an important new function for crmA is the inhibition of cytokine-induced apoptosis. Further, the data suggest that a protease, either ICE or a related crmA-inhibitable protein, is a component of the Fas- and tumor necrosis factor-induced cell death pathways.

Animals↗

PubMed1995

Lymphoid expression and regulation of A20, an inhibitor of programmed cell death.

A20 is a cytokine-inducible primary response gene that encodes a protein that inhibits apoptosis. We cloned and characterized a murine A20 cDNA and mapped the gene to mouse chromosome 10, 3.5 centimorgans proximal to the c-myb locus. Using the murine cDNA, we conducted in situ hybridization studies to examine patterns of A20 expression in mouse embryos and postnatal tissues. Both temporally restricted and tissue-specific patterns of A20 expression were observed, with strikingly high levels in lymphoid organs, including the thymus, spleen, and gut-associated lymphoid tissue. Northern analysis using purified human lymphocyte populations demonstrated that A20 is constitutively expressed in both immature and mature thymocyte subpopulations, as well as in resting peripheral T cells. Activation leads to a down-regulation of A20 expression in both mature thymocytes and peripheral T cells. This pattern of expression and regulation differs markedly from that observed in prior studies with cell lines in which stimulation led to an induction of A20 expression. Additionally, this is the first demonstration of constitutive expression of A20 in primary cells. The data suggest a role for A20 in the function of the lymphoid system.

Amino Acid Sequence↗

PubMed1995

Age-dependent injury in human umbilical vein endothelial cells: relationship to apoptosis and correlation with a lack of A20 expression.

BACKGROUND: It has recently been shown that human umbilical vein endothelial cells (HUVEC) become increasingly sensitive to growth factor deprivation, resulting in cell death, as a function of age in culture. The overall goal of the present study was to investigate the mechanism of lethal injury in these cells and compare the injury process to other known mechanisms of injury in the same cells. EXPERIMENTAL DESIGN: HUVEC were established in culture and maintained for four passages. Injury to first-passage cells and fourth-passage cells were examined for injury in the presence of agents that are known to confer resistance to apoptosis. Ultrastructural features of injury and DNA fragmentation patterns were assessed. Expression of factors that are known to be associated with resistance to apoptosis in other models were assessed. RESULTS: Fourth-passage HUVEC undergoing injury exhibited morphologic features characteristic of apoptosis and DNA fragmentation. Agents known to inhibit apoptotic cell injury in other models inhibited injury. A20 expression was correlated with resistance to injury in fourth-passage HUVEC, but there was no correlation between bcl-2 and bcl-x expression and resistance to injury. CONCLUSIONS: HUVEC injury resulting from growth factor deprivation increases as a function of age in vitro and appears to be a form of apoptosis. A20 expression may confer resistance to cell injury through this pathway.

Apoptosis↗

PubMed1994

Properties of recombinant mouse thrombospondin 2 expressed in Spodoptera cells.

A baculovirus system was used to express full-length recombinant mouse thrombospondin 2 (rTSP2) as a disulfide-bonded homotrimer with an NH2 terminus beginning with Asp20.rTSP2, like TSP1, was more sensitive to trypsin digestion if depleted of calcium ion. The trypsin digestion pattern of rTSP2 and TSP1 differed in that trypsin cut between the first and second type 1 modules of rTSP2. For bovine aortic endothelial cells adhering to TSP-coated polystyrene plates, reduction after coating caused both TSPs to be much more adhesive; these adhesions were blocked completely by RGDS peptide or antibody to alpha v beta 3 integrin.rTSP2 and TSP1 also mediated the adhesion of HT-29 human colon adenocarcinoma cells that carry alpha v beta 5 but not alpha v beta 3 integrin. Antibody to alpha v beta 5 did not inhibit adhesion of HT-29 cells to TSP1 or rTSP2. Rather, adhesion of HT-29 cells was decreased by treatment of TSPs with EDTA, abolished by reduction of the TSPs, and, in the case of rTSP2, blocked by heparin. Adhesion of MG63 cells to both TSPs was complex. Treatment with EDTA enhanced the adhesive activity of rTSP2 but decreased the adhesive activity of TSP1. These results show that TSP2 can be processed and secreted when overexpressed using baculovirus, TSP1 and rTSP2 differ in protease susceptibility in the type 1 module region, and TSP1 and rTSP2 mediate cell adhesion by complex and similar but not identical mechanisms.

Amino Acid Sequence↗

PubMed1994

A novel RING finger protein interacts with the cytoplasmic domain of CD40.

CD40 is a member of the tumor necrosis factor receptor family and, like other members, it appears to possess no intrinsic signaling capacity (e.g. kinase activity), suggesting that signal transduction is likely mediated by associating molecules. To identify such molecules, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the CD40 cytoplasmic domain. One such interacting protein, designated CD40-binding protein, has a N-terminal RING finger motif that is found in a number of DNA-binding proteins, including the V(D)J recombination activating gene RAG1. In addition, it contains a prominent central coiled-coil segment that may allow homo- or hetero-oligomerization. The C terminus possesses substantial homology to the tumor necrosis factor receptor-associated factor (TRAF) domain that is found in two proteins (TRAF1 and TRAF2) that associate with the cytoplasmic domain of the related 75-kDa tumor necrosis factor receptor. This is the first identification of a molecule that interacts with CD40 and whose sequence suggests a potential role in signaling.

Amino Acid Sequence↗

PubMed1994

Activation of the Eck receptor protein tyrosine kinase stimulates phosphatidylinositol 3-kinase activity.

The Eph/Eck subfamily of receptor protein tyrosine kinases is currently the largest subfamily of receptor protein tyrosine kinases with a dozen members (Van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. Rev. Cell Biol. 10, 251-337). Using the cytoplasmic domain of Eck as bait in a yeast two-hybrid screen of mouse embryonic and T-cell cDNA libraries, it was discovered that the p85 subunit of phosphatidylinositol 3-kinase bound Eck. Further, using glutathione S-transferase fusion proteins, it was found that the C-terminal src homology 2 domain of the p85 subunit specifically interacted with Eck. Additionally, Eck coimmunoprecipitated with p85 in ligand activated cells confirming their interaction in vivo. In keeping with the above observations, activation of Eck by its ligand, B61, increased phosphatidylinositol 3-kinase activity. This is the first description of a signal transduction pathway initiated by any member of the Eph/Eck family.

Animals↗

PubMed1994

cDNA cloning and characterization of a ligand for the Cek5 receptor protein-tyrosine kinase.

We have isolated a murine cDNA encoding a ligand for the Cek5 receptor protein-tyrosine kinase (RPTK), a member of the Eph/Eck RPTK subfamily. Sequence analysis predicts an open reading frame of 345 amino acids with a predicted molecular mass of 38 kDa. Metabolic labeling and immunoprecipitation of cells transfected with a cDNA encoding the Cek5 ligand revealed the mature protein to have an apparent molecular mass of 45 kDa. The extracellular domain of the Cek5 ligand shows a 27% sequence identity at the protein level to B61, a ligand for the related Eck RPTK (Bartley, T. D., et al. (1994) Nature 368, 558-560). Consistent with the presence of a transmembrane domain, flow cytometry analysis revealed the Cek5 ligand to be expressed on the cell surface. The expressed Cek5 ligand is functionally active as it induces autophosphorylation of the Cek5 RPTK.

Amino Acid Sequence↗

PubMed1994

B94, a primary response gene inducible by tumor necrosis factor-alpha, is expressed in developing hematopoietic tissues and the sperm acrosome.

B94 was originally described as a novel tumor necrosis factor-alpha-inducible primary response gene in endothelial cells which was also induced in an in vitro model of angiogenesis. To further characterize its expression, we cloned the mouse homologue and mapped its developmental and tissue specific expression. The predicted amino acid sequence of mouse B94 was found to be 83% similar to its human homologue. The gene was localized to mouse chromosome 12 just centromeric to the immunoglobulin heavy chain locus, in a region that is often rearranged in T-cell neoplasms. To explore the possibility that B94 is expressed during vasculogenesis and other developmental processes, the expression of its transcript was determined during mouse development by in situ hybridization. In 10-day embryos B94 was expressed prominently in the myocardium and in the aortic arch. By the 15th day of gestation, expression was restricted largely to the liver, the bone forming regions of the jaw, the aortic endothelium, and the nasopharynx: a pattern that was maintained until just prior to birth. Postnatally, expression shifted to the red pulp of the spleen and the thymic medulla. B94 expression was extinguished in most adult tissues but was detectable in lymphopoietic tissues including the spleen, tonsil, and lymphatic aggregates in the gut. Consistent with this was the finding that mononuclear progenitor cells in bone marrow and mature peripheral blood monocytes expressed B94. A truncated testis-specific transcript previously identified by Northern blot analysis was determined to result from the use of an alternate polyadenylation signal which was surprisingly located within the open reading frame. This shorter transcript was expressed at high levels exclusively in late stage spermatids. Immunostaining with an affinity-purified polyclonal antiserum revealed B94 to be localized to the acrosomal compartment of mature sperm. These studies demonstrate that B94 expression is tightly regulated during development and suggests distinct roles for B94 in myelopoiesis and spermatogenesis.

Acrosome↗

PubMed1994

Expression of thrombospondin in the adult nervous system.

Thrombospondin (TSP) is an extracellular matrix molecule that has been previously associated with neural development and neurite outgrowth in vitro. Little is known, however, about the expression of TSP in the adult nervous system. In this study, TSP localization was examined in nervous tissue from adult mouse, goldfish, newt, and adult and juvenile Xenopus. TSP was associated with neurons in the brains of all species examined. TSP was present in central nerve tracts capable of regeneration, such as the goldfish, Xenopus, and newt optic nerves, but was absent from tracts not capable of regeneration, such as the mouse optic nerve. TSP was also present in the neuropil of goldfish and newt spinal cord, but was restricted to motor neurons in mice and adult Xenopus. In addition, TSP was observed in sciatic nerves of mice, Xenopus, and newt. These results indicate a correlation between the presence of TSP and the potential for successful nerve regeneration across a wide range of animal classes.

Animals↗