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D A Fruman

Publications and source records attributed to D A Fruman.

30 records · Page 2Linked to original sources
PubMed1995

FK506 binding protein 12 mediates sensitivity to both FK506 and rapamycin in murine mast cells.

The immunosuppressive drugs FK506 and rapamycin bind to a family of intracellular proteins termed FK506-binding proteins (FKBP). FK506 and rapamycin inhibit lymphocyte-activation pathways by forming complexes with an FKBP; subsequently, the drug/FKBP complexes interact with target molecules involved in signal transduction. A key target of FK506/FKBP12 complexes is calcineurin, a calcium- and calmodulin-dependent serine/threonine phosphatase. In mammalian cells, rapamycin treatment is associated with inhibition of the activity of several cellular serine/threonine kinases, including p70 S6 kinase. These kinases may function in signaling pathways involving TOR gene producs, which have been shown to interact with rapamycin/FKBP12 complexes in vitro. To determine if FKBP12 mediates the effects of both FK506 and rapamycin in mammalian cells, we overexpressed FKBP12 in a murine mast cell line. Increased expression of FKBP12 resulted in increased sensitivity to FK506 and rapamycin, as measured by inhibition of calcineurin activity and p70 S6 kinase activity, respectively. In contrast, overexpression of FKBP25 had no effect on sensitivity to either drug. Two distinct point mutations in FKBP12, one altering a hydrophobic residue within the drug-binding pocket and the other changing a charged surface residue of FKBP12, abrogated its ability to mediate sensitivity to FK506 and rapamycin. These results establish that FKBP12 can mediate sensitivity to both FK506 and rapamycin in mammalian cells.

Animals↗
PubMed1995

Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells.

The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.

Amino Acid Sequence↗
PubMed1994

Inhibition of calcineurin phosphatase activity in adult bone marrow transplant patients treated with cyclosporine A.

In vitro studies have demonstrated that cyclosporine A (CsA) acts by inhibiting the phosphatase activity of calcineurin, an important mediator of T-cell activation. The relationship of CsA administration in vivo, calcineurin activity, and graft-versus-host disease (GVHD) has yet to be studied. The calcineurin activities of mononuclear cells isolated from 62 bone marrow transplant recipients and 12 normal volunteers were determined and analyzed with respect to administration of CsA, presence or absence of CsA in plasma, and presence or absence of GVHD. Of 62 patients, 33 were taking CsA and 29 were not. Early posttransplant (< 100 days), the calcineurin activity of patients on CsA was significantly lower than that of patients not on CsA (P = .0004) and than that of normal volunteers (P < .0001). Similarly, late posttransplant (> 100 days), the calcineurin activity of patients taking CsA was inhibited compared with normal volunteers (P < .05). The calcineurin activity of patients with acute GVHD who were taking CsA was lower than that of patients on CsA without acute GVHD matched for time posttransplant (P = .02). Calcineurin activity in patients on CsA with chronic GVHD was similar to those without chronic GVHD on drug. In conclusion, calcineurin activity is significantly suppressed by in vivo administration of CsA. The lower calcineurin activity of patients on CsA with acute GVHD suggests that CsA-resistant GVHD is not the result of inadequate suppression of calcineurin activity. These data suggest that if inhibition of calcineurin is the only physiologic target of CsA administration, simply increasing doses of CsA or treatment with other inhibitors of calcineurin, such as FK506, would not be expected to ameliorate GVHD.

Acute Disease↗
PubMed1994

Disruption of T cell development and repertoire selection by calcineurin inhibition in vivo.

The microbial products FK506 and CsA are potent immunosuppressive agents that prevent early transcriptional events in TcR-mediated activation. Their mode of action is dependent upon the inhibition of calcineurin, a serine/threonine phosphatase positioned within the calcium-dependent signaling pathway. TcR-mediated activation of thymocytes constitutes an important prerequisite for their development and selection to mature T cells. Disruption of the cross-talk between thymic APC and thymocytes results in the loss of normal T cell ontogeny. To study the role of calcineurin in T cell maturation and repertoire selection in vivo, mice were treated with either FK506 or CsA. Administration of either drug inhibited the progression of CD4+CD8+ positive thymocytes to mature single positive T cells. Furthermore, both drugs disrupted the process of negative thymic selection, causing an increased frequency of self-reactive cells among the few positively selected T cells. These effects correlated directly with the degree of inhibition of in vivo calcineurin enzyme activity. Blocking calcineurin activity appears to disrupt positive thymic selection and to prevent the deletion of self-reactive thymocytes.

Animals↗
PubMed1994

Immunophilins in protein folding and immunosuppression.

Lymphocyte activation requires the transmission of signals from molecules at the plasma membrane to nuclear signals that regulate gene expression. In recent years, several immunosuppressive compounds have been used as probes to identify important and potentially novel molecules involved in lymphocyte signal transduction processes. The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin have been studied in particular detail. Two distinct classes of immunosuppressant binding proteins have been identified, and collectively termed immunophilins. The cyclophilin family of immunophilins binds CsA, whereas the FK506-binding protein (FKBP) family binds FK506 and rapamycin. This review will discuss both the endogenous functions of immunophilins as well as their roles in mediating immunosuppression.

Amino Acid Isomerases↗
PubMed1994

T cell receptor (beta chain) transgenic mice have selective deficits in gamma delta T cell subpopulations.

TCR-beta (T cell receptor-beta chain) transgenic mice have altered lymphocyte development. TCR-beta transgenic mice are hyporesponsive to alloantigens in vivo and are deficient in gamma delta T cells. In order to begin a study of the relationship between a deficiency of alloreactive gamma delta cells and the defective function of in vivo alloantigen recognition, we analysed the gamma delta T cell development in TCR-beta mice. The presence of the TCR-V beta 8.2 chain transgene is associated with inhibition of gamma chain gene rearrangement. In order to determine how the presence of the TCR-beta transgene affects gamma delta T cell development, gamma delta T cells were studied in the skin, intestine and spleen. TCR-beta mice have dramatically reduced numbers of gamma delta T cells in the spleen and moderately reduced numbers of gamma delta T cells among intestinal intraepithelial lymphocytes. In contrast, these mice have normal numbers of gamma delta dendritic epidermal cells (DEC). These selective deficits could be due to the developmental regulation of transgene transcription during fetal life. We examined transcription of the TCR-beta transgene in the fetal thymus and found that the TCR-beta transgene is first transcribed at high levels on day 16 of fetal life, after DEC have already migrated from the thymus to the epidermis. Furthermore, mRNA from the transgene was detected in DEC, ruling out the formal possibility that DEC bear a gamma delta receptor only because they are incapable of expressing the transgene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A role for calcineurin in degranulation of murine cytotoxic T lymphocytes.

The immunosuppressive drugs cyclosporin A (CsA) and FK506 bind to distinct families of intracellular proteins, cyclophilins, and FK506 binding proteins (FKBP) respectively, termed immunophilins. Immuno-suppressant-immunophilin complexes bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. CsA is known to inhibit degranulation in CTL as assessed by N benzyloxylcarbonyl-L-lysine thiobenzyl ester-esterase release assays. We have investigated whether calcineurin phosphatase activity is involved in this degranulation. Both CsA and FK506 are shown to inhibit N benzyloxylcarbonyl-L-lysine thiobenzyl esteresterase release in murine CTL clones induced either by cognate target or by PMA and the calcium ionophore A23187. Inhibition is concentration dependent and is observed at drug concentrations that specifically inhibit cellular calcineurin. The FK506-binding immunophilin FKBP12, as well as calcineurin, are shown to be present in these cells by immunoblotting analysis. Rapamycin, a macrolide antibiotic thought to compete with FK506 for binding to common FKBP receptor sites, antagonizes the effects of FK506 on both degranulation and calcineurin activity. Neither the degranulation nor the effect of the immunosuppressants is affected by the protein synthesis inhibitor cycloheximide. These observations suggest a role for calcineurin in CTL degranulation. Thus, in addition to its previously described role in lymphokine gene activation, calcineurin also appears to be involved in T cell activation processes which do not require protein synthesis.

Animals↗
PubMed1992

Effects of cyclosporin A and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12.

The inhibitory effects of cyclosporin A (CsA) and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA and the expression of their intracellular binding proteins were studied in interleukin 3 (IL-3)-dependent, mouse bone marrow-derived mast cells (BMMCs). In BMMCs sensitized with IgE anti-trinitrophenyl, CsA inhibited trinitrophenylated bovine serum albumin-induced increases in mRNA for IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 in a dose-related manner (IC50 values of 4, 65, and 130 nM, respectively). FK506 did not inhibit hapten-specific increases of mRNA for TNF-alpha or IL-6, and for IL-1 beta the IC50 was greater than 50-fold higher than that of CsA. Neither agent inhibited exocytosis of the endogenous secretory granule mediators beta-hexosaminidase and histamine at the IC50 values for inhibition of increases in cytokine mRNA. BMMCs expressed cyclophilin, and CsA inhibited the phosphatase activity of cellular calcineurin with an IC50 of approximately 8 nM. That CsA inhibited IL-1 beta mRNA accumulation in IgE-activated BMMCs with an IC50 similar to that for inhibition of calcineurin activity, whereas the IC50 values were approximately 20-fold higher for the inhibition of TNF-alpha and IL-6 mRNA, suggests that the induction of TNF-alpha and IL-6 is less dependent upon calcineurin activity than is the induction of IL-1 beta. BMMCs were deficient in the 12-kDa FK506-binding protein FKBP12, but not FKBP13, as assessed by RNA and protein blot analyses. FK506 did not inhibit calcineurin phosphatase activity in BMMCs, even at drug concentrations of 1000 nM. The resistance of BMMCs to inhibition of Fc epsilon receptor type I-mediated increases in cytokine mRNA by FK506 is most likely due to their deficiency of FKBP12 and the related inability to inhibit the activity of calcineurin.

Amino Acid Isomerases↗
PubMed1992

Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A.

The immunosuppressive agents cyclosporin A (CsA) and FK 506 bind to distinct families of intracellular proteins (immunophilins) termed cyclophilins and FK 506-binding proteins (FKBPs). Recently, it has been shown that, in vitro, the complexes of CsA-cyclophilin and FK 506-FKBP-12 bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. We have investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specifically inhibit cellular calcineurin at drug concentrations that inhibit interleukin 2 production in activated T cells. Rapamycin, which binds to FKBPs but exhibits different biological activities than FK 506, has no effect on calcineurin activity. Furthermore, excess concentrations of rapamycin prevent the effects of FK 506, apparently by displacing FK 506 from FKBPs. These results show that calcineurin is a target of drug-immunophilin complexes in vivo and establish a physiological role for calcineurin in T-cell activation.

Calcineurin↗
PubMed1992

Correlation of calcineurin phosphatase activity and programmed cell death in murine T cell hybridomas.

Ligation of T cell receptor/CD3 complexes induces programmed cell death, or apoptosis, in immature thymocytes and many T cell hybridomas. While it has been demonstrated that T cell receptor-mediated apoptosis requires an increase in intracellular calcium concentration, the specific calcium-dependent signalling events leading to cell death are poorly defined. We have previously shown that T cell receptor/CD3-mediated induction of apoptosis in a murine T cell hybridoma is inhibited by the immunosuppressive drugs cyclosporin A (CsA) and FK506. Recently, it has been determined that these agents inhibit the activity of calcineurin, a calcium- and calmodulin-dependent serine/threonine phosphatase. Using an assay which measures calcineurin activity in cell lysates, we find that calcineurin-dependent dephosphorylation of a phosphopeptide substrate is potently inhibited in hybridomas treated with CsA or FK506. Drug dose-response analyses indicate that the level of cellular calcineurin activity correlates closely with the ability of these cells to undergo apoptosis. Thus, calcineurin appears to be a critical mediator of T cell receptor/CD3 signalling leading to programmed cell death in T cell hybridomas.

Amino Acid Sequence↗
PubMed1991

Changes in inositol 1,4,5-trisphosphate mass in agonist-stimulated human neutrophils.

Changes in the endogenous synthesis of inositol 1,4,5-trisphosphate (IP3) mass have been quantitated in human peripheral neutrophils stimulated with FMLP, LTB4 and PAF using a recently described, highly specific radioreceptor assay. Each agonist induced a concentration-dependent synthesis of IP3 which was detectable within 10 seconds after stimulation. IP3 production was short-lived, returning to basal levels within 90 seconds. The maximal stimulated level of IP3 in response to FMLP and LTB4 was 30-50 50 pmoles/10(7) neutrophils. PAF was more effective (approximately 100 pmoles IP3/10(7) neutrophils). The response to FMLP was inhibited by pertussis toxin, but was unaffected by cholera toxin. Pretreatment with cytochalasin B did not enhance IP3 synthesis. These findings are generally consistent with previous studies employing [3H]myo-inositol-prelabeled cells, and provide one of the first measurements of IP3 synthesis by mass in agonist-stimulated human neutrophils.

Calcium Channels↗