Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype.

The multidrug-resistant (MDR) phenotype is characterized in vitro by the resistance displayed by cell lines to a broad spectrum of natural product cytotoxic agents. This high level of cross-resistance is due to the increased expression of a membrane glycoprotein termed P-glycoprotein. Encoded in humans by the mdr1 gene, P-glycoprotein functions as an energy-dependent efflux pump of these cytotoxic agents. In this report, we demonstrate that the newly characterized immunosuppressant FK506 and its structural analogue, rapamycin, are capable of functioning as MDR reversal agents. FK506 and rapamycin increase both intracellular, cytotoxic drug (daunomycin) accumulation, and the cytotoxicity of chemotherapeutic agents in multidrug-resistant cells. The increase in cytotoxic drug accumulation is observed at concentrations of FK506 and rapamycin 1,000-fold greater than the concentrations required for FK506 and rapamycin to inhibit T-lymphocyte activation and similar to those shown to be effective for other MDR reversal agents such as cyclosporine A (CsA) and verapamil. The effect of FK506 or rapamycin on both intracellular accumulation and cytotoxicity of daunomycin is additive. This is supported by the ability of FK506 and rapamycin to directly compete the binding of the photoaffinity analogue 125I-iodoaryl azidoprazosin to the P-glycoprotein. The data demonstrate that FK506 and rapamycin represent a new class of structurally distinct molecules that can function as MDR reversal agents and suggest a previously unidentified, potential clinical role for these compounds.     

FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae.

FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T-cell activation by interfering with signal transduction. In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-trans isomerase). Cyclosporin A acts similarly on a different proline rotamase, cyclophilin. Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo. We have isolated the gene encoding the FKBP proline rotamase (FPR1) from Saccharomyces cerevisiae. The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin. Disruption of FPR1 and CPR1 (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes. Overexpression or disruption of FPR1 confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast. However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506.     

Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux.

The immunophilin FK506 binding protein 12 (FKBP12) is associated with and modulates the ryanodine receptor calcium release channel of skeletal muscle. Ryanodine receptor has amino acid homology and functional similarity with another intracellular Ca2+ release channel, the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we show that highly purified preparations of IP3R contain FKBP12. The complex of these two proteins is disrupted by the immunosuppressants FK506 and rapamycin, both of which are known to bind FKBP12 with high affinity. Disrupting the IP3R-FKBP12 interaction increases Ca2+ flux through IP3R, an effect that is reversed by added FKBP12. FKBP12 appears to be physiologically linked to IP3R, regulating its Ca2+ conductance.     

Role of FK506 binding protein 5 (FKBP5) in osteoclast differentiation

Objectives

We previously disclosed the enhanced expression of FK506 binding protein 5 (FKBP5) messenger RNA (mRNA) in bone marrow (BM) CD34⁺ cells in rheumatoid arthritis (RA), in which systemic osteoporosis takes place. Since BM CD34⁺ cells are precursors of osteoclasts, it is possible that FKBP5 overexpression might lead to osteoporosis by affecting osteoclastogenesis. We therefore explore the influences of FKBP5 in osteoclast differentiation.

Methods

Stable transfectants of RAW264.7 overexpressing murine FKBP5 gene were established. Osteoclast differentiation was induced by receptor activator of nuclear factor kappa B (NF-κB) ligand and was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay.

Results

FKBP5 transfectants of RAW264.7 generated higher numbers of TRAP-positive multinucleated cells with increased activity of pit formation on calcium phosphate-coated culture slides than mock transfectants. The enhancement of osteoclast differentiation of FKBP5 transfectants was only partially inhibited by N-acetyl l-cysteine. Finally, glucocorticoid enhanced FKBP5 mRNA expression as well as osteoclast differentiation of RAW264.7 cells in a dose-dependent manner.

Conclusions

These results indicate that FKBP5 promotes osteoclast differentiation by a mechanism distinct from NF-κB activation. Moreover, the data suggest that FKBP5 might play a role in bone destruction and development of osteoporosis in RA as well as in glucocorticoid-induced osteoporosis.     

Regulation of ryanodine receptors by FK506 binding proteins

Ryanodine receptors (RyRs) are the major sarcoplasmic reticulum calcium-release channels required for excitation-contraction coupling in skeletal and cardiac muscle. Mutations in RyRs have been linked to several human diseases. Mutations in the cardiac isoform of RyR2 are associated with catecholaminergic polymorphic ventricular arrhythmias (CPVT), and arrhythmogenic right ventricular dysplasia type 2 (ARVD2), whereas mutations in the skeletal muscle isoform (RyR1) are linked to malignant hyperthermia (MH) and central core disease (CCD). RyRs are modulated by several other proteins, including the FK506 binding proteins (FKBPs), FKBP12 and FKBP12.6. These immunophilins appear to stabilize a closed state of the channel and are important for cooperative interactions among the subunits of RyRs. This review discusses the regulation of RyRs by FKBPs and the possibility that defective modulation of RyR2 by FKBP12.6 could play a role in heart failure, CPVT, and ARVD2. Also discussed are the consequences of FKBP12 depletion to skeletal muscle and the possibility of FKBP12 involvement in certain forms of MH or CCD.     

Neointimal expression of rapamycin receptor FK506-binding protein FKBP12: postinjury animal and human in-stent restenosis tissue characteristics

Despite excellent clinical results for sirolimus (rapamycin)-eluting stents, the exact mechanisms of antirestenotic activity and affected cellular targets are incompletely understood. Therefore, we determined the presence and tem- porospatial expression pattern of FKBP12, the primary intracellular receptor of rapamycin, in rat carotid arteries after balloon injury, as well as in human in-stent restenosis and primary stable coronary atheroma. FKBP12 expression was assessed by immunohistochemistry. Rat carotid arteries revealed maximal expression in 57.7 +/- 4.0% of neointimal cells at day 7. A large proportion of these FKBP12+ cells showed luminally confined co-expression with dendritic cell markers. Despite a considerably thicker neointima at day 28, presence of FKBP12 decreased (8.5 +/- 1.9%, p = 0.02) with a scattered pattern in luminal and deep neointima. Likewise, human in-stent restenosis atherectomy specimens (time after stent implantation 2-12 months) revealed a comparable extent of cellular rapamycin receptor expression (9.3 +/- 1.0%) that significantly differed from that found in primary stable atheroma (1.3 +/- 0.4%, p < 0.001). In conclusion, the rapamycin receptor is predominantly present during early neointima formation, while mature neointimal atheromas show a relatively low expression without confinement to luminal areas. Co-expression of FKBP12 and dendritic cell markers suggests that dendritic cells may be another important target for early and long-term rapamycin effects.     

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.     

A novel FK506-like binding protein interacts with the glucocorticoid receptor and regulates steroid receptor signaling

FKBP-like (FKBPL) protein is a novel immunophilin-like protein that plays a role in the cellular stress response. Its three tetratricopeptide repeat motifs are homologous to the heat shock protein 90 interaction sites of other immunophilins that have roles in steroid hormone receptor signaling. In this study, using biomolecular complementation and coimmunoprecipitation techniques, we show that FKBPL also colocalizes and interacts with the components of the heat shock protein 90-glucocorticoid receptor (GR) complex and demonstrate that the PPIase domain of FKBPL is important for the interaction between this complex and the dynein motor protein, dynamitin. Treatment of DU145 cells with the GR ligand, dexamethasone, induced a rapid and coordinated translocation of both GR and FKBPL to the nucleus; this response was perturbed when FKBPL was knocked down with a targeted small interfering RNA. Furthermore, overexpression of FKBPL increased GR protein levels and transactivation of a luciferase reporter gene in response to dexamethasone in DU145 cells. However, these responses were cell line dependent. In summary, these data suggest that FKBPL can be classed as a new member of the FKBP protein family with a role in steroid receptor complexes and signaling.     

Interaction of FK506-binding protein 13 with brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1): effects of FK506

BIG1 and BIG2 are brefeldin A-inhibited guanine nucleotide-exchange proteins that activate ADP-ribosylation factors (ARFs), critical components of vesicular trafficking pathways. These proteins can exist in macromolecular complexes and move between Golgi membranes and cytosol. In the BIG1 molecule, a centrally located Sec7 domain is responsible for ARF activation, but functions of other regions are largely unknown. Yeast two-hybrid screens of a human placenta cDNA library with BIG1 cDNA constructs revealed specific interaction of the N-terminal region (amino acids 1-331) with FK506-binding protein 13 (FKBP13). The association was confirmed by immunoprecipitation of both endogenous BIG1 and FKBP13 from Jurkat T cells with antibodies against either one. Binding of BIG1, BIG2, and ARF to cell membranes in vitro was increased by guanosine 5'-[gamma-thio]triphosphate, and further increases were induced by FK506. Incubation of Jurkat T cells with FK506 increased binding of BIG1, BIG2, and ARF to Golgi and other membranes in a time- and concentration-dependent manner, without effects on clathrin or gamma-adaptin binding. Binding of BIG1, BIG2, and ARF to membranes was also increased by L-732,531, an agonist structurally related to FK506, but was not increased by a related antagonist, L-685,818, nor by cyclosporin A or rapamycin. These findings are consistent with a role for FKBP13 and FK506 in vesicular trafficking, influencing ARF activity through their guanine nucleotide-exchange proteins.     

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.     

Asymmetrical blockade of the Ca2+ release channel (ryanodine receptor) by 12-kDa FK506 binding protein.

A soluble 12-kDa FK506 binding protein (FKBP12), the cellular receptor of the immunosuppressive drug FK506, is tightly associated with the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum [Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P. & Marks, A. (1992) J. Biol. Chem. 267, 9474-9477]. We have assessed the role of excess free FKBP12 in the function of single Ca2+ release channels incorporated into planar lipid bilayers. The addition of human recombinant FKBP12 (hFKBP12) to the cytoplasmic face of the Ca2+ release channel blocked the flow of cytoplasmic to luminal current (outward current) in a concentration-dependent manner but had no significant effect on the flow of luminal to cytoplasmic current (inward current). The luminal to cytoplasmic flow of current was modulated by Ca2+, Mg2+, ATP, caffeine, and ryanodine in the presence and absence of FKBP12. An immunosuppressive drug, L-683,590, an analog of FK506, did not block or reverse the asymmetrical hFKBP12 blockade of single Ca2+ release channels in planar lipid bilayers. FKBP12 may play a role in regulation of the flow of ions into the lumen of the sarcoplasmic reticulum through the Ca2+ release channel.     

Short-term recognition memory impairment is associated with decreased expression of FK506 binding protein 51 in the aged mouse brain

Evidence suggests that increased glucocorticoid receptor (GR) signaling may contribute to cognitive decline with age. We hypothesized that alterations in GR signaling pathway molecules, FK506 binding protein (FKBP) 51 and FKBP52, were associated with memory impairment in aged mice. We used the single-trial object recognition test to measure short-term memory in 18 aged mice compared to 22 young mice, and employed quantitative immunohistochemistry to assess cellular expression of those three proteins in the frontal cortex, hippocampal CA1, and dentate gyrus. Values of the discrimination ratio (DR, a measure of novelty preference) in aged mice were significantly lower than those in young mice (mean 0.54 vs. 0.67, p = 0.003, t test). Aged mice with DR below 0.54 were considered impaired (n = 9). In the three neuroanatomic regions studied, the immunoreactivity normalized to the area measured (IRn) for GR was significantly increased in aged mice regardless of their task performance compared to young mice (p < 0.005), as was the FKBP52 IRn (p < 0.007, U test). In the frontal cortex and CA1, the FKBP51 IRn was significantly lower in impaired aged mice than in unimpaired aged mice (p < 0.01 and <0.05, respectively) and in young mice (p < 0.05 and <0.01, respectively, Dunn’s post hoc test). In aged mice, the frontal cortex FKBP51 IRn correlated directly with DR (r _s = 0.68, p = 0.002, Spearman rank correlation). These observations suggest that recognition memory impairment in aged mice is associated with decreased FKBP51 expression that may promote GR-mediated glucocorticoid signaling to a greater extent than in unimpaired aged mice.     

FK506 in the maturation of dendritic cells

BACKGROUND AND OBJECTIVES: FK506 (tacrolimus) is a potent immunosuppressive agent that inhibits interleukin-2 (IL-2) and interferon-g production by CD4+ cells. The effect of this agent on dendritic cells (DCs), the highly professional antigen-presenting cells for T-cells, has not been completely defined. We investigated the effect of FK506 on DC differentiation from monocytes, and on the shift from immature to mature immunophenotypes. DESIGN AND METHODS: DCs were generated in vitro from monocytes of healthy donors. Cells were exposed to lipopolysaccharide (LPS) and two doses of FK506, with variations in time of exposure and sequence of FK506 and LPS addition. Immunophenotype analysis in immature and mature DCs under FK506 treatment was performed by flow cytometry at the end of cell culture. The Student's t-test was used for statistical analyses. RESULTS: FK506 did not affect dendritic cell generation or viability. There were no changes in cell surface markers with addition of FK506 at physiologic concentrations (10 ng/mL). We found a decrease in CD1a median fluorescence intensity (MFI) and an increase in percentage of CD86-positive cells with lengthy exposure (6 days) to FK506 at 5000 ng/mL. In the sequential study, 5000 ng/mL FK506 before LPS addition resulted in a significant decrease in CD1a MFI and in the percentage of cells co-expressing CD83 and CD86. INTERPRETATION AND CONCLUSIONS: Our results indicate that lengthy exposure to 5000 ng/mL FK506 modified the expression of some DC-cell surface markers, maintaining DCs in a low maturity stage.     

Effects of FK506 on exocrine pancreas in rats.

The efficacy of FK506 on exocrine pancreas was studied in rats. Male Sprague-Dawley rats (230-250 g) received an i.m. daily injection of FK506 (0.1, 0.5, or 5.0 mg/kg), cyclosporine (CS; 25 mg/kg), or saline for 2 weeks. Isolated dispersed pancreatic acini were prepared from rats, and enzyme content of the cells and secretory response to cholecystokinin (CCK) were determined. Amylase and trypsin contents were increased in a dose-related manner by FK506 (p less than 0.01) and by CS at 25 mg/kg (p less than 0.01). The release of amylase in response to CCK was reduced by FK506 in a dose-related manner (p less than 0.01) and by CS at 25 mg/kg (p less than 0.01). Histologic examination showed that treatment of rats with FK506 at 0.1 mg/kg did not affect morphology of the acinar cells. FK506 at 0.5 mg/kg induced a minimal number of small vacuoles in cytoplasm of acinar cells and FK506 at 5.0 mg/kg, and CS at 25 mg/kg induced numerous cytoplasmic vacuoles and pyknotic nuclei. Increased enzyme storage and suppressed responsiveness of amylase release may have an association with the histologic changes. Therefore, the results of this study suggest that FK506, even when used in a low dose, may have adverse effects on the exocrine pancreas. Understanding of the mechanism of action of FK506 on pancreas will provide essential basic information that will allow transplant practitioners to more fully explore the benefits of this drug.     

FKB1 encodes a nonessential FK 506-binding protein in Saccharomyces cerevisiae and contains regions suggesting homology to the cyclophilins.

FK 506, a powerful immunosuppressant that blocks allograft rejection by preventing T-cell activation, binds to an 11-kDa protein called the FK 506-binding protein (FKBP). Like cyclophilin, a cytosolic protein that binds another immunosuppressant, cyclosporin A, FKBP possesses peptidylprolyl cis-trans isomerase activity. We have isolated a genomic clone encoding the yeast FKBP (FKB1). The gene encodes a protein of 114 amino acids having a calculated Mr of 12,158. Disruption of the gene shows that FKB1 is not essential for growth. A search of translated nucleic acid data bases revealed bacterial FKBP homologs in Neisseria meningiditis and Pseudomonas aeruginosa. Comparison of the conserved amino acids in FKBP homologs with the conserved amino acids in the cyclophilins has revealed a region of similarity that we speculate to be a homologous domain related to the functional similarities of the two proteins.     

FK506 binding protein 12/12.6 depletion increases endothelial nitric oxide synthase threonine 495 phosphorylation and blood pressure

Chronic treatment with the immunosuppressive drug rapamycin leads to hypertension; however, the mechanisms are unknown. Rapamycin binds FK506 binding protein 12 and its related isoform 12.6 (FKBP12/12.6) and displaces them from intracellular Ca2+ release channels (ryanodine receptors) eliciting a Ca2+ leak from the endoplasmic/sarcoplasmic reticulum. We tested whether this Ca2+ leak promotes conventional protein kinase C-mediated endothelial NO synthase phosphorylation at Thr495, which reduces production of the vasodilator NO. Rapamycin treatment of control mice for 7 days, as well as genetic deletion of FKBP12.6, increased systolic arterial pressure significantly compared with controls. Untreated aortas from FKBP12.6-/- mice and in vitro rapamycin-treated control aortas had similarly decreased endothelium-dependent relaxation responses and NO production and increased endothelial NO synthase Thr495 phosphorylation and protein kinase C activity. Inhibition of either conventional protein kinase C or ryanodine receptor restored endothelial NO synthase Thr495 phosphorylation and endothelial function to control levels. Rapamycin induced a small increase in basal intracellular Ca2+ levels in isolated endothelial cells, and rapamycin or FKBP12.6 gene deletion decreased acetylcholine-induced intracellular Ca2+ release, all of which were reversed by ryanodine. These data demonstrate that displacement of FKBP12/12.6 from ryanodine receptors induces an endothelial intracellular Ca2+ leak and increases conventional protein kinase C-mediated endothelial NO synthase Thr495 phosphorylation leading to decreased NO production and endothelial dysfunction. This molecular mechanism may, in part, explain rapamycin-induced hypertension.     

Heat capacity and entropy changes in processes involving proteins.

Six possible sources of the large heat capacity and entropy changes frequently observed for processes involving proteins are identified. Of these the conformational, hydrophobic, and vibrational effects seem likely to be of greatest importance. A method is proposed for estimating the magnitudes of the hydrophobic and vibrational contributions. Application of this method to several protein processes appears to achieve significant clarification of previously confusing and apparently contradictory data.     

Molecular cloning of a membrane-associated human FK506- and rapamycin-binding protein, FKBP-13.

The 12-kDa FK506-binding protein (FKBP-12) is a cytosolic receptor for the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and subcellular localization of a 13-kDa FKBP (FKBP-13), which has a 21-amino acid signal peptide and appears to be membrane-associated. Although no internal hydrophobic region, and thus no transmembrane domain, is apparent within the 120 amino acids of mature FKBP-13, a potential endoplasmic reticulum retention sequence (Arg-Thr-Glu-Leu) is found at its C terminus. FKBP-13 has 51% nucleotide sequence identity and 43% amino acid sequence identity to FKBP-12; the N-terminal sequences are divergent, but the 92-amino acid C-terminal sequence of FKBP-13 has 46 identical and 20 related residues when compared with FKBP-12. The conserved residues that comprise the drug binding site and rotamase active site of FKBP-12 are completely conserved in FKBP-13. Therefore, the three-dimensional structures of FKBP-12 and the FKBP-12/FK506 complex are likely to be excellent models of the corresponding FKBP-13 structure.