Induction of fibronectin gene expression by inhibitors of protein phosphatase type 2B in normal and transformed fibroblasts. (41/2304)

Two intracellular signal pathways mediated by cAMP and protein kinase C (PKC) were involved in the regulation of FN gene expression (Lee et al., Exp. Mol. Med. 30: 240, 1998). In this study, a possible involvement of protein phosphatase-dependent pathways in the regulation of FN gene expression was investigated by using protein phosphatase type 2B (PP2B) inhibitors, cyclosporin A and ascomycin. Both cyclosporin A and ascomycin increased the levels of FN mRNA in WI-38 human lung fibroblasts and the SV40-transformed WI-38 cells but not in MC3T3-E1 osteoblasts. The expression of FN appears to increase from six hours up to 48 hours after treatment suggesting that it is not an immediate effect. In addition, this effect required a new protein synthesis. Neither cyclosporin A nor ascomycin affects the phorbol myristate acetate (PMA)-induced stimulation of FN gene expression and the same result occurred in vice versa suggesting the mechanism of PMA and cyclosporin A/ascomycin in the regulation of FN gene expression may share a common downstream pathway. Taken together, this study suggests that PP2B is involved in the regulation of FN gene expression in normal and transformed fibroblasts but not in osteoblasts.  (+info)

Tacrolimus for prevention of graft-versus-host disease after mismatched unrelated donor cord blood transplantation. (42/2304)

Ten children with hematologic malignancies or a storage disease underwent transplantation using cord blood cells from an unrelated donor mismatched for 1 (n = 7) or 2 (n = 3) HLA antigens. The median total nucleated cell dose was 4.0 (range, 2.2-7.1) x 10(7)/kg. GVHD prophylaxis consisted of tacrolimus dose-adjusted to maintain a whole blood level of 5-15 ng/ml with or without methotrexate 5 mg/m2 i.v. on days 1, 3, 6 and 11. Corticosteroids were not administered prophylactically. Median follow-up is 12 months (range, 5-28 months). One patient had autologous recovery and subsequently relapsed 153 days post transplant. For the remainder of the patients, the median time to an ANC >0.5 x 10(9)/l was 21 days (range, 19-38 days), and the median time to platelets >20 x 10(9)/l was 39 days (range, 21-97 days). The actuarial risk of grade 2 GVHD was 77% (95% CI, 49-100%), and no patient had grades 3-4 GVHD. Two patients developed chronic GVHD. The survival rate is 90% (95% CI, 81-100%). The combination of tacrolimus and minidose methotrexate is active for the prevention of severe but not moderate acute GVHD after mismatched unrelated donor cord blood transplantation.  (+info)

Calcineurin is required for skeletal muscle hypertrophy. (43/2304)

Molecular signaling pathways linking increases in skeletal muscle usage to alterations in muscle size have not been identified. In the present study, we tested the hypothesis that calcineurin, a calcium-regulated phosphatase recently implicated in the signaling of some forms of cardiomyopathic growth, is required to induce skeletal muscle hypertrophy and muscle fiber type conversions associated with functional overload in vivo. Administration of the specific calcineurin inhibitors cyclosporin (CsA) or FK506 to mice, for which the fast plantaris muscle was overloaded for 1-4 weeks, prevented the rapid doubling of mass and individual fiber size and the 4-20-fold increase in the number of slow fibers that characterize this condition. CsA treatment influenced the expression of muscle myofibrillar protein genes in a way reflective of fiber phenotype transformations but only in the long term of the overload condition, suggesting that the control of this growth response by calcineurin is not limited to the transcriptional activation of these muscle-specific genes. Clinically, these results provide insight to the post-surgical muscle wasting and weakness observed in recovering transplant recipients administered therapeutic dosages of these immunosuppressants.  (+info)

Approach to withdrawal from tacrolimus in a fully allogeneic murine skin graft model. (44/2304)

With few exceptions, transplant patients must take immunosuppressants throughout their lives. In this study, we used anti-T-cell receptor (TCR/CD3) monoclonal antibodies (mAbs) to induce immunological tolerance to alloantigens after withdrawal from tacrolimus in a fully allogeneic murine skin graft model. Skin grafts from AKR donor mice were maintained in C57BL/6 recipients by administering tacrolimus for one month. Anti-T-cell receptor (TCR) alphabeta mAb was administered to recipient mice on the day of withdrawal from tacrolimus administration. Seven days after mAb administration, the recipient mice were treated with various combinations of the following treatments: low-dose whole body irradiation, AKR bone marrow transfer (BMT), and anti-CD3 mAb administration. The control recipient mice did not receive treatment with either mAb, nor any other treatment. All the control recipient mice showed rejection of AKR skin grafts 42 days after tacrolimus withdrawal (mean skin graft survival: 77 days). Mice treated with a combination of anti-TCR alphabeta antibody, low-dose irradiation and AKR BMT showed stable chimerism in their peripheral blood lymphocytes and significantly prolonged skin graft survival (mean skin graft survival: >151.2+/-15.3 days). Mice given the combination of anti-TCR alphabeta mAb, anti-CD3 mAb, low-dose irradiation, and AKR BMT exhibited more stable chimerism but had earlier skin graft rejection (mean skin graft survival: 116.7+/-17.6 days) than the mice that did not receive anti-CD3 mAb. These results suggest that anti-TCR alphabeta mAb, but not anti-CD3 mAb, in combination with low-dose irradiation and BMT, is useful for long-lasting allograft survival after withdrawal from tacrolimus in mice with fully allogeneic skin grafts.  (+info)

ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation. (45/2304)

BACKGROUND: Early loss of neurites followed by delayed damage of neuronal somata is a feature of several neurodegenerative diseases. Death by apoptosis would ensure the rapid removal of injured neurons, whereas conditions that prevent apoptosis may facilitate the persistence of damaged cells and favor inflammation and disease progression. MATERIALS AND METHODS: Cultures of cerebellar granule cells (CGC) were treated with microtubule disrupting agents. These compounds induced an early degeneration of neurites followed by apoptotic destruction of neuronal somata. The fate of injured neurons was followed after co-exposure to caspase inhibitors or agents that decrease intracellular ATP (deoxyglucose, S-nitrosoglutathione, 1-methyl-4-phenylpyridinium). We examined the implications of energy loss for caspase activation, exposure of phagocytosis markers, and long-term persistence of damaged cells. RESULTS: In CGC exposed to colchicine or nocodazole, axodendritic degeneration preceded caspase activation and apoptosis. ATP-depleting agents or protein synthesis inhibition prevented caspase activation, translocation of the phagocytosis marker, phosphatidylserine, and apoptotic death. However, they did not affect the primary neurite loss. Repletion of ATP by enhanced glycolysis restored all apoptotic features. Peptide inhibitors of caspases also prevented the apoptotic changes in the cell bodies, although the axodendritic net was lost. Under this condition cell demise still occurred 48 hr later in a caspase-independent manner and involved plasma membrane lysis at the latest stage. CONCLUSIONS: Inhibition of the apoptotic machinery by drugs, energy deprivation, or endogenous mediators may result in the persistence and subsequent lysis of injured neurons. In vivo, this may favor the onset of inflammatory processes and perpetuate neurodegeneration.  (+info)

Secretion of FK506/FK520 and rapamycin by Streptomyces inhibits the growth of competing Saccharomyces cerevisiae and Cryptococcus neoformans. (46/2304)

FK506 and rapamycin are immunosuppressants that inhibit signalling cascades required for T-cell activation, yet both are natural products of Streptomyces that live in the soil. FK506 and rapamycin also have potent antimicrobial activity against yeast and pathogenic fungi, suggesting a natural role in inhibiting growth of competing micro-organisms. The immunosuppressive and antimicrobial activities of FK506 and rapamycin are mediated by binding to the FKBP12 prolyl isomerase and the resulting FKBP12/FK506 and FKBP12/rapamycin complexes inhibit conserved protein targets, either the phosphatase calcineurin or the TOR (target of rapamycin) kinases, respectively. Streptomyces sp., 'Streptomyces hygroscopicus subsp. ascomyceticus' and Streptomyces hygroscopicus, which produce FK506, FK520 (also known as ascomycin, a C21 ethyl derivative of FK506) and rapamycin, respectively, produced toxins that inhibited the growth of competing cells of the yeast Saccharomyces cerevisiae and the pathogenic fungus Cryptococcus neoformans. Yeast and fungal mutants lacking FKBP12 or expressing dominant drug-resistant calcineurin or TOR mutants were resistant to FK506 and rapamycin, and to the toxins produced by Streptomyces. Streptomyces strains with mutations in the FK506 or rapamycin biosynthetic enzymes were impaired in toxin production. Finally, the toxins secreted by 'S. hygroscopicus subsp. ascomyceticus' and S. hygroscopicus promoted formation of FKBP12/calcineurin and FKBP12/TOR complexes in a two-hybrid assay and mutations that rendered calcineurin or TOR drug-resistant prevented interaction. These observations support the hypothesis that Streptomyces evolved to secrete FK506, FK520 and rapamycin as toxins to inhibit the growth of competing yeast and fungi.  (+info)

Neuronal survival activity of s100betabeta is enhanced by calcineurin inhibitors and requires activation of NF-kappaB. (47/2304)

S100betabeta is a calcium binding, neurotrophic protein produced by nonneuronal cells in the nervous system. The pathway by which it enhances neuronal survival is unknown. Here we show that S100betabeta enhances survival of embryonic chick forebrain neurons in a dose-dependent manner. In the presence of suboptimal amounts of S100betabeta, neuronal survival is enhanced by the immunosuppressants FK506 and cyclosporin A at concentrations that inhibit calcineurin, which is present in these cells. Rapamycin, an immunosuppressant that does not inhibit calcineurin, did not enhance cell survival. Cypermethrin, a direct and highly specific calcineurin inhibitor, mimicked the immunophilin ligands in its neurotrophic effect. None of the drugs stimulated neuronal survival in the absence of S100betabeta. In the presence of suboptimal amounts of S100betabeta, FK506, cyclosporin A, and cypermethrin (but not rapamycin) also increased NF-kappaB activity, as measured by immunofluorescence of cells stained with antibody to the active subunit (p65) and by immunoblotting of nuclear extracts. Antioxidant and glucocorticoid inhibitors of NF-kappaB decreased both the amount of active NF-kappaB and the survival of neurons caused by S100betabeta alone or in the presence of augmenting drugs. We conclude that S100betabeta enhances the survival of chick embryo forebrain neurons through the activation of NF-kappaB.  (+info)

Mechanism of FK 506/520 action on rat renal proximal tubular Na+, K+-ATPase activity. (48/2304)

BACKGROUND: The neurotransmitter in renal sympathetic nerves, norepinephrine (NE), regulates the activity of proximal tubule (PT) Na+,K+-ATPase in a bidirectional manner via stimulation of alpha- and beta-adrenoceptors. The stimulatory alpha-adrenergic pathway is mediated by calcineurin, the target molecule for FK 506 and related compounds. We examined whether the FK 506 analogue FK 520, by interrupting the calcineurin-mediated alpha-adrenergic signaling pathway, enhance the inhibitory beta-adrenergic effect of NE on PT Na+,K+-ATPase activity. METHODS: The effects of three days of treatment with FK 520 were examined on rat renal PT Na+,K+-ATPase activity, measured as ouabain-sensitive ATP hydrolysis in single, microdissected PT segments. Renal function studies, including glomerular filtration rate (GFR) and urinary excretion of N-acetyl-3-D-glucoseaminidase (NAG), were examined using conventional clearance techniques after three days of treatment with FK 506. RESULTS: FK 520 treatment induced a pronounced and dose-dependent decrease in PT Na+,K+-ATPase activity. This effect was completely reversed by the competitive FK 520 antagonist, L 685 818, indicating that the effect was dependent on inhibition of calcineurin. To test whether the FK 520-induced decrease in Na+, K+-ATPase activity was mediated by enhanced beta-adrenoceptor signaling, the FK 520 effect was examined in rats pretreated with a beta-adrenoceptor antagonist (propranolol) or rats subjected to renal denervation. Both of these procedures prevented the FK 520-induced decrease in Na+,K+-ATPase activity. Thus, during FK 520 treatment, renal sympathetic nerves mediate an inhibitory effect on PT Na+,K+-ATPase activity via beta-adrenoceptors. Propranolol pretreatment also prevented FK 506-induced decreases in GFR and urinary excretion of NAG, an index of PT dysfunction. CONCLUSIONS: The results support the hypothesis that the net effect of the neurotransmitter NE on Na+,K+-ATPase activity is dependent on the balance between the alpha- and beta-adrenergic signaling pathways and suggest that agents that interfere with these pathways may, by altering the activity of tubular Na+,K+-ATPase, also alter the function of the renal tubular epithelial cell.  (+info)