FK506, transforming growth factor-beta1 and mesangial matrix synthesis: parallels and differences compared with cyclosporine A

Cyclosporine A (CsA)-induced glomerulosclerosis is a well-described side effect of CsA treatment. Current evidence indicates that FK506 causes similar morphologic changes. Recently, we demonstrated that CsA up-regulates the expression of transforming growth factor-beta1 (TGF-beta1), its receptors type I (TbetaR-I) and type II (TbetaR-II), as well as related matrix protein synthesis in mesangial cells (MCs). Here, we assessed the effect of FK506 on the expression of TGF-beta1, TbetaR-I, TbetaR-II, fibronectin (FN) and plasminogen activator inhibitor type-1 (PAI-1) in MCs. Resting MCs were incubated with/without FK506. Time- and concentration-dependent expression was measured at the mRNA and protein level. Compared to untreated controls, FK506 stimulated TGF-beta1 mRNA (maximum at 8 h, 100 ng/mL: 2.13+/-0.15-fold, P<0.005) and protein expression (maximum at 96 h, 100 ng/mL: 1.96+/-0.29-fold, P<0.005). In contrast, TbetaR-I and TbetaR-II protein expression remained unchanged. Concerning matrix protein synthesis, FK506 slightly increased FN production (96 h, 100 ng/mL: 1.38+/-0.28-fold, P<0.05), but not PAI-1 production. These results indicate that, comparable to CsA, FK506 induced glomerulosclerosis is also due to a direct effect on mesangial matrix production, which is at least in part mediated via up-regulation of TGF-beta1 expression. The fact that, unlike CsA, FK506 does not increase the expression of TbetaR-I, TbetaR-II, and PAI-1, deserves further investigation.     

Novel immunosuppressive agent, FK506. In vitro effects on the cloned T cell activation

The present study shows the in vitro effects of a novel immunosuppressive agent, FK506, in comparison with cyclosporin A (CsA). FK506 inhibited concanavalin A response and allo-mixed lymphocyte reaction of murine splenic lymphocytes in a dose-dependent manner, and at 40- to 200-fold lower concentrations than CsA. Allo-cytolytic T lymphocyte induction from murine thymocytes was also inhibited by FK506, whereas the ability of cytolytic T lymphocyte to lyse targets was not affected by the agent. Immunosuppressive effects of FK506 were further characterized by using antigen specific-proliferative T lymphocyte clones, BC.21 and KO.6. FK506 inhibited the proliferation of T cell clones stimulated with specific antigens in a dose-dependent manner, and at about 100-fold lower concentrations than CsA. However, cloned T cells, once activated, were scarcely affected by the agent; interleukin-2 (IL-2) driven proliferation of cloned T cells was not inhibited. On the other hand, it was found that FK506 inhibited both IL-2 secretion and IL-2 receptor expression of BC.21 after stimulation with the specific antigen. FK506 also inhibited the proliferation of BC.21 stimulated with phorbol 12-myristate 13-acetate plus calcium ionophore, indicating that it directly affected the signaling pathway downward from the perturbation of the Ti/T3 complex. Finally, it was suggested that FK506 and CsA synergistically inhibited the antigen-driven proliferation of cloned T cells. These results indicate that the novel immunosuppressive agent, FK506, affects T cell activation with mechanisms similar to those of CsA but at considerably lower concentrations.     

Two distinct action mechanisms of immunophilin-ligand complexes for the blockade of T-cell activation

The immunosuppressive effects of cyclosporin A (CsA) and FK506 are mediated through binding to immunophilins. Here we show that FK506–FKBP complex suppresses the activation of JNK and p38 pathways at a level upstream of mitogen-activated protein kinase (MAPK) kinase kinase (MAPKK-K) besides the calcineurin–NFAT pathway. A238L, a viral gene product that binds to immunophilin, also blocks activation of both pathways. In contrast, direct inhibitors of calcineurin, Cabin 1 and FR901725, suppress the activation of NFAT but not the JNK or p38 pathway. We further demonstrate that co-expression of a constitutively active NFAT and a constitutively active MEKK1 renders the interleukin-2 promoter in Jurkat T lymphocytes resistant to CsA and FK506, whereas Jurkat cells expressing a constitutively active NFAT alone are still sensitive to CsA or FK506. Therefore, CsA and FK506 exert their immunosuppressive effects through targeting both the calcineurin-dependent NFAT pathway and calcineurin-independent activation pathway for JNK and p38.     

T cell receptor-mediated signaling induces GRP78 expression in T cells: the implications in maintaining T cell viability

The 78-kDa glucose-regulated protein (GRP78) is an important molecular chaperone in the endoplasmic reticulum (ER) induced by various stresses. This study showed that stimulation with anti-CD3 mAb, PMA plus ionomycin, or an antigen increased the levels of GRP78 mRNA in primary T cells, which was inhibited by Ca²⁺ chelators EGTA and BAPTA-AM and by an inhibitor of calcineurin FK506. In addition, the specific knockdown of GRP78 protein expression induced apoptosis in mouse EL-4 T cell line associated with CHOP induction and caspase-3 activation. Furthermore, overexpression of GRP78 inhibited PMA/ionomycin-induced cell death in EL-4 cells. Collectively, GRP78 expression is induced by TCR activation via a Ca²⁺-dependent pathway and may play a critical role in maintaining T cell viability in the steady and TCR-activated states. These results suggest a novel regulatory mechanism and an essential function of GRP78 in T cells.     

FK506 and Cyclosporin A Enhance IL-6 Production in Monocytes: A single-Cell Assay

The effect of FK506 and cyclosporin A (CsA) on the production of interleukin 6 (IL-6) in adherent monocytes was studied at a single-cell level by the avidinbiotin- peroxidase complex methods. The percentage of IL-6-producing monocytes increased when stimulated with lipopolysaccharide (LPS) at concentrations between 10 ng/ml and 10 mug/ml, in a dose dependent manner. Both FK506 and CsA enhanced the percentage of IL-6- producing monocytes stimulated with 100 pg/ml-1 mug/ml of LPS up to values near those obtained with 10 mug/ml of LPS. The enhancement by FK506 and CsA was not seen when monocytes were stimulated with a high concentration of LPS (10 mug/ml). When monocytes were stimulated with a low concentration of LPS (10 ng/ml), FK506 and CsA enhanced IL-6 production in a dose dependent manner, at a drug concentration of 0.12 nM-1.2 muM (0.1-1 000 ng/ml) for FK506 and 0.83 nM-8.3 muM (1-10 000 ng/ml) for CsA. The optimal effect of FK506 was achieved at a concentration 7-fold lower than that of CsA. In contrast, production of turnout necrosis factor-alpha (TNFalpha and interleukin 1beta (IL-1beta) was slightly suppressed by FK506 and CsA at the concentrations tested. Moreover, pretreatment of monocytes with FK506 and CsA had a significant enhancing effect on LPS-induced IL-6 production, while treatment with FK506 or CsA after LPS stimulation had no effects on IL-6 production, suggesting that the enhancing effect of each drug is exerted before LPS stimulation or at an early stage of the post-receptor pathway after LPS stimulation. These experiments demonstrate that FK506 and CsA can selectively enhance IL-6 production in monocytes under certain conditions in vitro and, possibly, also in vivo.     

Modulation of the electrophoretic mobility of the linker for activation of T cells (LAT) by the calcineurin inhibitors CsA and FK506: LAT is a potential substrate for PKC and calcineurin signaling pathways

The linker for activation of T cells (LAT) is essential for T cell activation. Cyclosporin A (CsA) and FK506, inhibitors of T cell proliferation, have been very useful for preventing autoimmune and inflammatory disease and graft rejection. However, both compounds are associated with side effects. We show that TCR ligation in the presence of FK506 or CsA induced rapid modifications in LAT that modulate the electrophoretic mobility of the molecule in SDS-PAGE. Calcineurin, a target for CsA and FK506, dephosphorylated LAT in vitro and restored its electrophoretic mobility. Stimulating T cells with the protein kinase C (PKC) activator PMA induced a shift in the mobility of LAT, whereas inhibitors of PKC blocked the effect of PMA. Thus, manipulating calcineurin or PKC activation alters the electrophoretic mobility of LAT. These results shed light on the molecular actions of CsA and FK506 in T cells and implicate LAT in mediating the drugs' actions.     

Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.     

A comparative study on immunosuppressive effects of cyclosporin A and FK 506 on peripheral blood lymphocytes in dogs

Immunosuppressive effects of cyclosporin A (CsA) and FK 506 (FK) on peripheral blood lymphocytes were studied in dogs in respect to mixed lymphocyte reaction, proliferative responses to recombinant interleukin-2 (rIL-2), phytohemagglutinin (PHA) and concanavalin-A (Con-A); phenotypes of OKIa1, CD3, CD8 and surface IgM; cytotoxic activity against xenogeneic tumor cells. CsA (2.0 or 5.0 mg/kg, intravenously) or FK (0.16 mg/kg, intramuscularly) was given to mongrel dogs every morning for serial 21 days. The blood concentrations of CsA, measured as trough levels by fluorescence polarization method, ranged from 37 to 350 ng/ml in dogs administered at 2.0 mg/kg and from 170 to 894 ng/ml in dogs administered at 5.0 mg/kg during treatment, respectively. In dogs treated with FK at a dose of 0.16 mg/kg, the drug concentrations in the plasma during treatment ranged from 0.16 to 1.8 ng/ml. Mixed lymphocyte reaction and proliferative responses to rIL-2, PHA and Con-A, which were declined by CsA, were not affected by FK. In contrast, the proportion of OKIa1+ cells was not affected by CsA, whereas FK decreased the proportion of OKIa1+ cells progressively during the course of treatment. Cytotoxic activity was suppressed by both CsA and FK. These results possibly indicate that CsA and FK exert their immunosuppressive effects via different mechanisms.     

Cyclosporin A, but not FK506, increases arachidonic acid release and inhibits proliferation of pituitary corticotrope tumor cells

The selective immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) are used in the prevention of allogenic transplant rejection and in the therapy of chronic autoimmune inflammatory pathologies. Chronic treatment with CsA leads to secondary functional and trophic alterations of multiple organs and cell systems among which endocrine ones, through insofar uncharacterized mechanisms. With the recent use of FK506 there have been reports of an improved therapeutic efficacy and a reduction of side-effects, as compared to CsA. An intriguing hypothesis is that toxic damage could be due to a systemic CsA activation of arachidonic acid (AA) metabolism, through pathways as yet only partially characterized. The side-effects of both drugs have been poorly studied on cells from tissues other than blood or kidney. We have thus proceeded to study their action on AA release in corticotropic AtT-20/D16-16 cells. The results obtained are as follows: 1) during incubation times > or =12 h, basal AA release is increased by CsA, but not FK506; the acute effect (10 min) of melittin, a PLA2 activator, is significantly potentiated starting from a 30 min pretreatment with CsA but not FK506; manoalide, a PLA2 inhibitor, antagonizes the melittin potentiation of AA release by CsA whereas the inhibition of the melittin stimulus by glucocorticoids is antagonized both by CsA and FK506. 2) during longer (>2 d) incubation times, cell growth is inhibited by CsA but not FK506. These results indicate a role for CsA, not apparent for FK506, in the activation of PLA2 and in the inhibition of cell growth. They also suggest that CsA does not have a direct (i.e. not mediated by the immune system) therapeutic effect in inflammatory processes.     

Calcineurin enhances MEF2 DNA binding activity in calcium-dependent survival of cerebellar granule neurons.

Myocyte enhancer factor 2 (MEF2) has been shown recently to be necessary for mediating activity-dependent neuronal survival. In this study, we show that calcium signals regulate MEF2 activity through a serine/threonine phosphatase calcineurin. In cultured primary cerebellar granule neurons, the electrophoretic mobility of MEF2A protein was sensitive to the level of extracellular potassium chloride (KCl) and depolarizing concentrations of KCl led to hypophosphorylation of the protein. The specific inhibitors of calcineurin cyclosporin A (CsA) and FK506 could overcome KCl-dependent MEF2A hypophosphorylation. The effects of CsA and FK506 were KCl specific as they had little effect on MEF2A phosphorylation when granule neurons were cultured in the presence of full media. Hyperphosphorylation of MEF2A led to the loss of its DNA binding activity as determined by DNA mobility shift assay. Consistent with this, CsA/FK506 also inhibited MEF2-dependent reporter gene expression. These findings demonstrate that regulation of MEF2A by calcium signals requires the action of protein phosphatase calcineurin. By maintaining MEF2A in a hypophosphorylated state, calcineurin enhances the DNA binding activity of MEF2A and therefore maximizes its transactivation capability. The identification of MEF2 as a novel target of calcineurin may provide in part a biochemical explanation for the therapeutic and toxic effects of immunosuppressants CsA and FK506.     

A proposed molecular model for the interaction of calcineurin with the cyclosporin A-cyclophilin A complex.

Cyclosporin A (CsA) and FK506 are potent natural product immunosuppressants that induce their biological effects by forming an initial complex with cytosolic proteins termed immunophilins. These drug immunophilin complexes then bind to and inhibit the serine/threonine protein phosphatase calcineurin (CN). Two classes of immunophilin have been identified with cyclophilins (CyP's) being proteins specifically binding CsA and FKBPs specifically binding FK506. Solution and crystal structures of various CsA-CyP and FK506-FKBP complexes have been determined and show no apparent structural similarity between the two classes of drug protein complexes. These findings raise the question as to how, given their structural differences, these two complexes can both inhibit CN. While the crystal structure of the FK506-FKBP12-CN complex has been reported, no structure for a CsA-CyP CN complex has been determined. Here are reported studies that use various modelling strategies to construct a model for the interaction of the cyclosporin A- cyclophilin A complex with calcineurin. The first stage of constructing this model consisted of using conformational comparison of CsA and FK506, GRID and GROUP analysis and restrained molecular dynamics to dock CsA into the FK506 binding site of the FK506-FKBP12-CN structure. An initial model for the CsA-CyPA-CN complex was then constructed by superimposing the structure of the CsA-CyPA complex onto the docked CsA molecule. This model was then optimised with molecular dynamics simulations run on sterically clashing regions. The validity of the model for the CsA-CyPA-CN complex was then examined with respect to the effect of chemical modifications to CsA and amino acid substitutions within CyPA on the ability of the drug-immunophilin complex to inhibit calcineurin.     

Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A.

The immunosuppressive complexes cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 inhibit calcineurin, a heterodimeric Ca(2+)-calmodulin-dependent protein phosphatase that regulates signal transduction. We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK506-sensitive Saccharomyces cerevisiae strain. Three mutations that confer dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1. One mutation that confers dominant FK506 resistance alters a single residue (W430C) in the calcineurin A catalytic subunit CMP2. In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have reduced affinity for cyclophilin A-CsA. When introduced into the CMP1 subunit, the FK506 resistance mutation (W388C) blocks binding by FKBP12-FK506, but not by cyclophilin A-CsA. Co-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and to FK506 but not to CsA plus FK506. Double mutant calcineurin A subunits (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506. These studies identify cyclophilin A-CsA and FKBP12-FK506 binding targets as distinct, highly conserved regions of calcineurin A that overlap the binding domain for the calcineurin B regulatory subunit.     

Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506

Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membrane-based steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.     

Reversible inhibition of calcineurin by the polyphenolic aldehyde gossypol

The reversible inhibition of calcineurin (CaN), which is the only Ca(2+)/calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of (33)P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC(50) value of 15 microm. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC(50) of 9 and 6 microm, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18.CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.