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V M Dixit

Publications and source records attributed to V M Dixit.

At least 127 records · Page 7Linked to original sources
PubMed1994

Modulation of thrombospondin receptor expression during HL-60 cell differentiation.

Thrombospondin (TSP), a multifunctional homotrimeric glycoprotein of approximately 450,000 M(r), is a component of the extracellular matrix that mediates the adhesive interactions of several different cell types including hematopoietic progenitor cells. We have used the promyelocytic leukemia HL-60 cell line to examine TSP receptor expression during differentiation of leukocytes along either the monocyte/macrophage or the polymorphonuclear leukocyte (PMN) pathway. 125I-labeled TSP binding to undifferentiated or differentiated HL-60 cells was time-dependent reaching saturation by 45 min. Undifferentiated HL-60 cells expressed a single class of heparin-inhibitable TSP receptors. Treating HL-60 cells with PMA induced their differentiation to macrophage-like cells and resulted in a concomitant 10-fold increase in TSP receptor expression. As with undifferentiated cells, a single class of heparin-inhibitable receptors was observed. Treating HL-60 cells with DMSO induced their differentiation to PMN-like cells and resulted in a fivefold increase in TSP receptor expression. However, in this case two classes of binding sites were apparent on PMN-like cells, only 40% of which were heparin inhibitable. This is reminiscent of TSP binding to normal peripheral blood PMN (S.J. Suchard, L.A. Boxer, and V.M. Dixit. 1991. J. Immunol. 147:651). In parallel studies, we also examined TSP synthesis during HL-60 cell differentiation. Undifferentiated HL-60 cells synthesized and secreted TSP as assessed by immunoprecipitation. TSP synthesis increased about fourfold when cells were differentiated toward PMN-like cells. In contrast, TSP was not detected in macrophage-like cells. RNase protection assays showed that TSP transcript levels paralleled TSP protein expression during differentiation. These findings suggest that expression of both TSP and TSP receptors are differentially regulated during blood cell maturation.

CD36 Antigens↗
PubMed1994

Thrombospondin 3 is a developmentally regulated heparin binding protein.

The thrombospondins (TSPs) are a growing family of cell surface and extracellular matrix molecules composed of multiple repeating elements. Thrombospondin 3 is a recently described member that possesses the calcium binding Type 3 repeats, has 4 epidermal growth factor receptor-like Type 2 repeats, a complete absence of the complement-like Type 1 repeats, and a distinct N terminus that has no significant homology to the other TSPs. Metabolic labeling and immunoprecipitation analysis of cells transfected with a TSP3 expression vector revealed it to be an oligomeric heparin binding protein present in both the cell layer and medium. Finally, a combination of in situ hybridization and immunocytochemistry demonstrated TSP3 to be expressed in a temporal and spatial manner during murine embryogenesis, especially in the gut, cartilage, lung, and central nervous system.

Amino Acid Sequence↗
PubMed1994

Aberrant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis.

Psoriasis is a common inherited skin disease that is characterized by hyperproliferation of epidermal keratinocytes and excessive dermal angiogenesis. A growing body of evidence supports a key pathogenetic role for activated keratinocytes in the angiogenic response that accompanies psoriasis. We investigated the role of psoriatic epidermis in the aberrant expression of angiogenesis by examining the ability of pure populations of multipassaged keratinocytes obtained from the skin of normal individuals and psoriatic patients to induce angiogenesis in vivo in the rat corneal bioassay and endothelial cell chemotaxis in vitro. Media conditioned by keratinocytes from psoriatic patients, including both symptomless skin and psoriatic plaques, induced vigorous angiogenic responses in over 90% of corneas tested and potently stimulated directional migration of capillary endothelial cells in vitro. In contrast, conditioned medium from normal keratinocyte cultures was weakly positive in less than 10% of corneas assayed and failed to stimulate endothelial cell chemotaxis. Furthermore, keratinocytes from psoriatic skin exhibited a 10- to 20-fold increase in interleukin-8 production and a seven-fold reduction in thrombospondin-1 production. The angiogenic activity present in keratinocyte-conditioned media from psoriatic patients was suppressed by adding either highly purified thrombospondin-1 (125 ng) or following the addition of either normal keratinocyte-conditioned media or neutralizing interleukin-8 antibody. We conclude that psoriatic keratinocytes are phenotypically different from normal keratinocytes with respect to their angiogenic capacity and that this aberrant phenotype is attributable to a defect in the overproduction of interleukin-8 and a deficiency in the production of the angiogenesis inhibitor thrombospondin-1.

Adult↗
PubMed1993

Activated T-cell adhesion to thrombospondin is mediated by the alpha 4 beta 1 (VLA-4) and alpha 5 beta 1 (VLA-5) integrins.

T lymphocytes utilize adhesion receptors in a regulated manner to interact with other cells and with components of the extracellular matrix. These cell-cell and cell-matrix interactions serve a critical role in T cell recognition of foreign Ag and in the migration of T cells to various anatomic sites in vivo. Thrombospondin is an extracellular matrix protein that is transiently expressed at high concentration in damaged and inflamed tissue. Given recent evidence implicating a role for the extracellular matrix in modulating T-cell migration and function, we analyzed T-lymphocyte interactions with thrombospondin. We show here that CD4+ T cells specifically adhere to thrombospondin predominantly via the 70 kDa core region of the thrombospondin molecule. Antibody blocking and affinity chromatography analysis suggest that T-cell adhesion to thrombospondin involves three distinct receptors: an activation-independent receptor that mediates adhesion of resting T cells, and the alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate a rapid increase in adhesion to thrombospondin upon activation. These three molecules appear to be novel thrombospondin receptors, as other receptors previously implicated in the adhesion of nonlymphoid cells to thrombospondin appear not to be involved in T-cell/thrombospondin interactions. The up-regulation of alpha 4 beta 1 and alpha 5 beta 1 functional activity upon activation is associated with the preferential adhesion of memory T cells to thrombospondin. Our results thus define three novel thrombospondin receptors, and provide additional evidence that extracellular matrix proteins play an important role in lymphocyte migration into, and retention in, inflammatory sites.

CD4-Positive T-Lymphocytes↗
PubMed1993

Human T cell leukemia virus type I Tax and phorbol 12-myristate 13-acetate induce expression of the A20 zinc finger protein by distinct mechanisms involving nuclear factor kappa B.

A20 was originally identified as a primary tumor necrosis factor alpha (TNF)-responsive gene, which encodes a 790-amino acid zinc finger protein. A20 is expressed in a wide variety of cell lines, including fibroblasts, in which A20 expression protects cells from TNF cytotoxicity. An analysis of A20 expression in lymphocytic and monocytic cells lines revealed that A20 protein expression correlates with lymphocyte activation and monocyte differentiation. A20 expression was also induced in Jurkat T cells expressing the human T cell leukemia virus type I Tax protein. Transient transfection studies demonstrated that stimulation of A20 transcription by TNF, phorbol 12-myristate 13-acetate (PMA), and Tax was mediated by two kappa B elements within the A20 promoter. Accordingly, DNA electrophoretic mobility shift assays confirmed inducible binding of nuclear factor kappa B (NF-kappa B) to a promoter fragment containing both A20 kappa B elements. Analysis of individual A20 kappa B sites revealed that both kappa B sites were required for TNF or PMA activation of the A20 promoter; however, Tax activation required only one kappa B site. Overexpression of NF-kappa B subunits activated the wild type A20 promoter, but did not activate mutated forms containing single kappa B sites. Thus, Tax activation of A20 transcription occurs through a mechanism distinct from PMA and TNF, possibly due to differential activation of NF-kappa B complexes or transcriptional cofactors.

Base Sequence↗
PubMed1993

High level thrombospondin 1 expression in two NIH 3T3 cloned lines confers serum- and anchorage-independent growth.

Thrombospondin (TSP) is a trimeric molecule synthesized by a variety of normal and transformed cells and secreted into the extracellular matrix. A number of studies have shown TSP to be intimately involved in the regulation of cellular proliferation. These include the findings that TSP is a mitogen-inducible immediate-early response gene; it localizes to areas of cellular proliferation in the developing mouse embryo; it augments the proliferative response to epidermal growth factor; anti-TSP monoclonal antibodies block cell proliferation; and TSP levels correlate directly with the growth and invasive phenotype and inversely with the degree of differentiation of squamous carcinoma cells. To determine whether TSP could behave as an oncogene, conferring serum and anchorage independence, NIH 3T3 cells were stably transfected with a TSP expression vector. Clones producing high levels of TSP displayed enhanced viability and a proliferative advantage in serum-reduced media. Moreover, this growth advantage could be specifically negated by treatment of the transfected cells with anti-TSP monoclonal antibodies. While the TSP overexpressing clones were capable of anchorage-independent growth in soft agar, surprisingly, they were incapable of forming tumors in nude mice, possibly due to the in vivo antiangiogenic activity of TSP. Regardless, these studies demonstrate that overexpression of thrombospondin, a constituent of the extracellular matrix, results in serum and anchorage independent growth, attributes normally associated with the transformed phenotype.

3T3 Cells↗
PubMed1993

Motility of human carcinoma cells in response to thrombospondin: relationship to metastatic potential and thrombospondin structural domains.

The interactions of tumorigenic cells with the extracellular matrix play a critical role in the establishment of metastases. Thrombospondin (TSP) is prominent at sites of tissue injury and promotes the attachment, spreading, and motility of several cell types. We have investigated the relationship between human carcinoma cell metastatic potential and TSP-mediated cell motility by comparing highly metastatic 11B carcinoma cells with a nonmetastatic counterpart, 22B carcinoma cells. 11B cells demonstrated motility in response to soluble TSP with a maximal effect observed at 1 microM TSP. Checkerboard analysis indicated that motility was directional with a significant chemokinetic component. Monoclonal antibody C6.7, specific for the distal COOH terminus of TSP, inhibited chemotaxis by 60%. Studies with TSP fragments demonstrated that the M(r) 140,000 COOH-terminal domain (140K) supported chemotaxis to the same extent as intact TSP. The NH2-terminal heparin-binding domain was ineffective in stimulating chemotaxis. Substrate-bound TSP also elicited 11B cell motility with a maximal response at 100 nM TSP. Directionality of this response was confirmed by checkerboard analysis. Interestingly, as in chemotaxis, haptotaxis was mediated exclusively by 140K as demonstrated by TSP fragment studies and inhibition with monoclonal antibody C6.7. Therefore, 140K appeared to mediate both chemotaxis and haptotaxis. Compared with 11B cells, 22B carcinoma cells are nonmetastatic and synthesize and secrete low levels of TSP. Immunoprecipitation and Northern blot analysis confirmed that 11B cells expressed much higher levels of TSP than 22B cells. Although 22B cells are able to attach to TSP, they did not exhibit either chemotaxis or haptotaxis in response to TSP or TSP fragments. Similarly, an antisense TSP cell line responded poorly in chemotaxis assays to TSP and 140K. These data suggest that the ability of metastatic cells in vivo to establish secondary sites of proliferation may be related to their ability to migrate in response to extracellular matrix proteins such as TSP incorporated into basement membranes or interstitial matrices.

Carcinoma, Squamous Cell↗
PubMed1992

Thrombospondin 1 and thrombospondin 2 are expressed as both homo- and heterotrimers.

There exist two distinct thrombospondin molecules (designated TSP1 and TSP2) which are encoded by separate genes. TSP1 is a trimeric cell surface and extracellular matrix molecule. Sequence comparison reveals that the 2 cysteines involved in interchain disulfide linkage and trimer assembly in TSP1 are conserved in TSP2 (Laherty, C. D., O'Rourke, K., Wolf, F. W., Katz, R., Seldin, M. F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 3274-3281). Swiss 3T3 fibroblasts express both TSP1 and TSP2, and, therefore, an important question is whether TSP in such cells is expressed as homotrimers or as heterotrimers. We find that Swiss 3T3 cells and epithelial cells transfected with TSP expression vectors express both homo- and heterotrimeric forms of TSP. In addition, homotrimeric TSP2 has a lower affinity for heparin than homotrimeric TSP1. Thus, the heparin affinity of TSP can be modulated by the expression of TSP as homo- or heterotrimers.

3T3 Cells↗
PubMed1992

The Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B.

A20 is an inducible zinc finger protein that confers resistance to tumor necrosis factor alpha cytotoxicity. A survey of various cell lines revealed that A20 was constitutively expressed in Epstein-Barr virus (EBV)-immortalized B-cells. Transfection experiments demonstrated that the EBV latent membrane protein LMP1 induced A20 expression. LMP1 is a transforming protein of EBV that has dramatic effects on cell growth, activation, and survival. An integral membrane phosphoprotein, LMP1 bears no homology to other recognized membrane signaling molecules, and its signal transduction pathway is not known. However, studies using the A20 promoter demonstrated that LMP1 transcriptionally activates the A20 gene through cis-acting kappa B sites. In addition, electrophoretic mobility shift assays confirmed LMP1-inducible binding of an NF-kappa B-like factor to kappa B sequences within the A20 promoter. This is the first report implicating NF-kappa B in signaling by LMP1, a fundamentally important viral transforming protein.

Antigens, Viral↗
PubMed1992

Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements.

A20 was first identified as a tumor necrosis factor (TNF) primary response transcript encoding a 790-amino acid protein with a unique zinc finger motif. Recently, A20 was shown to protect cells from TNF-induced cytotoxicity in a variety of cell lines. Nuclear run-on studies previously established that TNF activates A20 at the transcriptional level. To further characterize the mechanism by which TNF activates the A20 gene, we have cloned the A20 5'-flanking sequences and identified TNF-responsive elements within the promoter. The transcription initiation site was mapped by both primer extension and S1 nuclease protection experiments to a position 4.2 kilobases (kb) upstream of the initiator methionine; the first and second exon were separated by a 3.9-kb intron. Sequences upstream of the transcription start site were 76% GC-rich and contained six Sp1 binding sites and a TATA-like sequence at -29 but lacked a consensus CCAAT site. Transfection of Jurkat T-cells with an array of A20 promoter CAT constructs showed that two kappa B elements residing at -54 and -66 were required for induction by TNF. Supporting this notion, DNA electrophoretic mobility shift assays using nuclear extracts from unstimulated and TNF-stimulated Jurkat cells demonstrated kappa B-specific binding of a TNF-activated factor to an end-labeled probe containing the two A20 kappa B sequences. Finally, evidence obtained from cotransfection experiments showed that A20 negatively regulated its own expression.

Amino Acid Sequence↗
PubMed1992

The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity.

Resistance against the cytotoxic actions of tumor necrosis factor alpha (TNF) is an active process requiring the synthesis of TNF-inducible proteins. The specific TNF-induced proteins so far identified (manganese superoxide dismutase and plasminogen activator inhibitor type 2) as having a role in resistance against TNF cytotoxicity are able to confer only partial protection to cells, suggesting that other genes are involved. A20 is a TNF-induced primary response gene which encodes a novel zinc finger protein. In this report we demonstrate that A20 protein is induced by TNF in a variety of cells. A survey of A20 expression in human breast carcinoma cell lines that are either sensitive or resistant to TNF cytotoxicity revealed increased expression of A20 message and protein in TNF-resistant cells. Constitutive expression of A20 after stable transfection of NIH 3T3 and WEHI 164 cells results in significant, but partial, resistance to TNF cytotoxicity. This work gives additional support to a role for TNF-induced immediate early response genes in protecting cells from TNF-induced death.

3T3 Cells↗
PubMed1992

Cloning of a novel tumor necrosis factor-alpha-inducible primary response gene that is differentially expressed in development and capillary tube-like formation in vitro.

TNF is a proinflammatory cytokine that has pleiotropic effects on cells and tissues, mediated in large part by alterations in target tissue gene expression. We have used the technique of differential hybridization to identify several primary response genes induced by TNF in human umbilical vein endothelial (HUVE) cells, a cell type that is profoundly activated by cytokine treatment. One of these cDNA, designated B94, detects a rapidly and transiently induced 4-kb transcript in TNF-treated HUVE cells, and this transcript is superinduced in the concomitant presence of cycloheximide. Other proinflammatory stimuli including IL-1 beta and LPS are also able to induce B94 mRNA expression. Nuclear run-on experiments demonstrate that TNF induction of B94 transcript occurs primarily at the level of transcriptional activation. Further, B94 is shown to be a single copy gene that is evolutionarily conserved. The gene is localized to the q32 region of chromosome 14, a region that is often rearranged in lymphoid neoplasms. B94 transcript expression is also found to be regulated during mouse development and in an in vitro model of endothelial capillary tube formation. Developmental regulation occurs most prominently in mouse embryonic liver and kidney, and a second smaller form of B94 transcript is detected in the placenta and testes. B94 and other TNF-responsive transcripts are also induced during capillary tube formation suggesting overlap between genes induced by TNF and those induced during angiogenesis. Sequence analysis of the B94 cDNA reveals an open reading frame encoding a 73-kDa polypeptide that has no homology to any known protein. Polyclonal antisera directed against the carboxyl-terminal portion of the B94 protein immunoprecipitates a protein of the predicted molecular mass both from COS cells transfected with a B94 expression vector and from TNF-treated HUVE cells.

Amino Acid Sequence↗
PubMed1992

Characterization of mouse thrombospondin 2 sequence and expression during cell growth and development.

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose expression has been associated with a variety of cellular processes including growth and embryogenesis. The recent discovery of the existence of a second mouse TSP gene necessitates careful examination of the discrete biochemical and functional properties associated with each molecule. In this report, the primary structures of human TSP, mouse TSP1 (mTSP1), mouse TSP2 (mTSP2), and chicken TSP are compared; and the expression of mTSP1 and mTSP2 during embryogenesis and growth factor-mediated cell proliferation is examined. The cloning and sequencing of the entire coding regions of mTSP1 and mTSP2 revealed considerable conservation of residues critical for TSP structure and function; these data suggest that TSP2 is capable of trimer formation and many of the same cell-surface and ligand interactions that mediate TSP function. Comparison of the various TSP sequences also allowed the assignment based on sequence homology of previously reported human TSP as TSP1 and chicken TSP as TSP2. mTSP2, like mTSP1, was shown to be a primary response gene when quiescent Swiss 3T3 cells were stimulated with serum, platelet-derived growth factor BB, basic fibroblast growth factor, or interleukin-1 beta. Interestingly, TSP1 and TSP2 exhibited markedly different tissue- and stage-specific patterns of mRNA expression during mouse embryogenesis, implying that the two TSP molecules possess discrete functional properties important for development. Additionally, the TSP genes (Thbs1 and Thbs2) were mapped to single loci on mouse chromosomes 2 and 17, respectively.

3T3 Cells↗
PubMed1992

Characterization of a novel tumor necrosis factor-alpha-induced endothelial primary response gene.

The response of endothelial cells to the cytokine tumor necrosis factor-alpha (TNF) is complex, involving the induction and suppression of multiple genes and gene products. Differential screening of a TNF-stimulated, cycloheximide-treated human umbilical vein endothelial cell library has resulted in the cloning of several novel cDNAs whose protein products are involved in the primary response of the endothelium to TNF. One of these cDNAs, designated B12, is further characterized here. B12 is encoded by a 3.5-kilobase transcript and is induced rapidly and transiently by TNF. Transcript expression is found to be developmentally regulated in a tissue-specific manner, with B12 message being differentially expressed in the heart and liver during mouse embryogenesis. The open reading frame of B12 predicts a 316-amino acid sequence rich in charged residues, particularly at the carboxyl terminus, and has neither significant homology to other known proteins nor to any extent sequence motifs. B12 is found to be a highly conserved single-copy gene which is located in the q22----q23 region of human chromosome 17. Polyclonal antibodies raised against a large portion of the B12 open reading frame immunoprecipitate a 36-kilodalton polypeptide from wheat germ lysates programmed to translate in vitro transcribed B12 mRNA. The B12 protein is further shown to be induced in human umbilical vein endothelial cells by TNF, and the protein is shown to be rapidly degraded.

Amino Acid Sequence↗
PubMed1992

Monocyte diapedesis through an in vitro vessel wall construct: inhibition with monoclonal antibodies to thrombospondin.

Human peripheral blood monocytes were examined for migration across an endothelial cell monolayer in an in vitro vessel wall construct. Few monocytes invaded in the absence of a chemotactic gradient, despite significant adhesion to the endothelial monolayer. However, the addition of zymosan-activated human plasma to the lower compartment, to create a chemotactic gradient across the vessel wall, resulted in significantly enhanced monocyte migration. Pretreatment of the monocytes with monoclonal antibodies to thrombospondin (TSP) dramatically inhibited monocyte diapedesis into the vessel wall. The same treatment inhibited monocyte adhesion to endothelial cells in two-dimensional monolayer cultures as well as in vessel wall constructs (no chemotactic gradient). Of interest, however, the monoclonal antibodies had no inhibitory effect on monocyte migration into collagen gels devoid of endothelial cells in response to the same chemotactic gradient, suggesting the importance of TSP in monocyte-endothelial cell interactions. Monoclonal antibodies to fibronectin and normal mouse immunoglobulin G did not inhibit migration in this model of a vessel wall. Furthermore, monoclonal antibodies to TSP showed no inhibition of human peripheral blood neutrophil migration. Previous studies have shown that monocytes synthesize TSP and express this moiety on their surface. The present data suggest that monocytes may utilize TSP to interact with endothelial cells lining the vessel wall during diapedesis.

Antibodies, Monoclonal↗
PubMed1992

Keratinocyte activation following T-lymphocyte binding.

T lymphocytes infiltrate the epidermis and follicular epithelium adhering to keratinocytes within hours following induction of cutaneous inflammation. To determine if the physical binding interaction between a T cell and keratinocyte induces transmission of activation pathways, CD3+ T cells (HUT 78) were allowed to directly bind to non-cytokine-treated cultured keratinocytes. When these T cells bound to keratinocytes, the keratinocytes were activated as evidenced by detection of tumor necrosis factor-alpha, interleukin-6, and intercellular adhesion molecule-1 mRNA. This induction was relatively mRNA specific, as several other mRNA were not found to be altered. This activation process appeared to be one-sided, as no change in HUT cell mRNA levels was detectable. The keratinocyte activation process was confined to cultures that had direct physical binding by HUT cells, because co-culturing the HUT cells immediately above the keratinocyte monolayer (but not in direct contact), resulted in no such mRNA alterations. This direct adhesion-mediated activation of keratinocytes by T lymphocytes may be important in the genesis of cutaneous inflammation by amplifying the original stimulus, as well as contributing to the trafficking pattern of inflammatory cells as they leave the general circulation and enter the skin.

Cell Adhesion Molecules↗
PubMed1992

Induction of thrombospondin 1 by retinoic acid is important during differentiation of neuroblastoma cells.

Neuroblastoma, a malignant neoplasm that arises in the adrenal medulla or sympathetic ganglion, is one of the most common solid tumors of childhood. Reports that neuroblastomas spontaneously mature to form benign ganglioneuromas have prompted investigations into the efficacy of using agents that induce neuronal differentiation in the treatment of this malignancy. Retinoic acid is one agent in particular that has been shown to induce growth inhibition and terminal differentiation of neuroblastoma cell lines in vitro. Using the human neuroblastoma cell line SMH-KCNR, we have investigated the role of the extracellular matrix protein thrombospondin in retinoic acid induced neuroblastoma differentiation. Treatment with retinoic acid results in a rapid induction (within 4 h) of thrombospondin (TSP) message which is independent of intervening protein synthesis and superinducible in the presence of cycloheximide. This suggests that TSP functions as a retinoic acid inducible immediate early response gene. A concomitant increase in both cell associated and soluble forms of TSP protein can be detected within 24 h of retinoic acid treatment. A functional role for TSP in SMH-KCNR differentiation was established in experiments which showed that exposure to anti-TSP monoclonal antibodies delay retinoic acid differentiation for 48 h. At the time the cells overcome the effects of TSP inhibition, laminin production becomes maximal. Treatment of the cells with a combination of anti-TSP and antilaminin antibodies results in complete inhibition of differentiation.

Cell Differentiation↗