FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition.

The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.

Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.

Identification of a gene induced by glucocorticoids in murine T-cells: a potential G protein-coupled receptor.

Using cDNA cloning techniques we previously identified a set of genes induced by glucocorticoids and cAMP in murine T-lymphocytes. We report here the sequence of one of these cDNA clones (clone 4.2), renamed here as glucocorticoid-induced receptor (GIR), which encodes a potential new member of the family of receptors that couple to G-proteins. Several different forms of cDNA for this gene were isolated and shown to correspond to multiple mRNA species in lymphoid cells using an RNase protection assay. The cDNA clone corresponding to the most abundant form of GIR mRNA encodes a precursor protein of 423 amino acids, with a putative signal peptide of 17 amino acids. A hydropathy plot reveals the presence of seven hydrophobic regions, with significant similarities to other G-protein-coupled receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Genes newly identified as regulated by glucocorticoids in murine thymocytes.

Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.

Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function.

Murine lymphoma cell lines such as WEHI-7 exhibit a cytolytic response to both cAMP and glucocorticoids. We have exploited this behavior to ask if cyclic AMP-dependent protein kinase plays a role in regulating glucocorticoid receptor function. We have found that cAMP-resistant cell lines containing a defective cAMP-dependent protein kinase activity give rise to spontaneous steroid-resistant variants at a high frequency (approximately 10(-7)) relative to wild type cells (less than 10(-10)). Unlike previous results with wild type cells, nearly complete loss of glucocorticoid receptor function was observed in a single selection using unmutagenized cAMPr derivatives of WEHI-7. Thus, the initial selection of the cAMPr phenotype serves as a permissive step toward the acquisition of glucocorticoid resistance in WEHI-7. In addition, cAMP was found to increase the levels of steroid binding in these cell lines, and the dose response was dependent upon the phenotype of the cyclic AMP-dependent protein kinase. The results demonstrate an important role for cAMP in regulating glucocorticoid receptor activity and strongly suggest that this novel two-step selection scheme leads to the isolation of new forms of glucocorticoid resistance.

High-resolution anion exchange chromatography of the glucocorticoid receptor from WEHI-7 cells.

A simple refinement of the current methods of DEAE chromatography of steroid receptors has been developed which takes advantage of the characteristics of Fast Flow DEAE Sepharose (Pharmacia). The approach provides a convenient and inexpensive means to carry out high-resolution chromatography of the glucocorticoid receptor. Using this method, at least five separate species of receptor have been detected within the single so-called "low-salt" peak normally seen using the current methods of receptor anion exchange chromatography.

Detection of poly (U) hybridization using azido modified poly (A) coated piezoelectric crystals.

A technique has been developed which uses AT-cut, 9 MHz piezoelectric crystals for the immobilization of single stranded nucleic acids and the subsequent hybridization reactions. This hybridization technique is based on detecting small changes in mass on the surface of a piezoelectric crystal which occur upon immobilizing azido containing probe nucleic acid on the surface and hybridizing complementary target nucleic acid to previously immobilized probe. The immobilized probe could be used for repeated hybridization tests. The hybridization assays were performed in solution and the frequency measurements were performed in the dry state.

Development of a chromatographic method for the isolation and detection of hygromycin B in biological fluids.

An affinity chromatography method was developed for the purification of hygromycin B from biological fluids. Lysozyme and alpha-lactalbumin were immobilized on an N-hydroxysuccinimide activated agarose support. Hygromycin B solubilized in water was bound by the proteins and subsequently eluted using 10 mM sodium citrate buffer, pH 4.0. Hygromycin B was purified from swine plasma, bovine serum and bovine milk samples using a combination of ion-exchange chromatography for initial clean-up of spiked biological samples followed by affinity chromatography. Thin layer chromatographic analysis of the isolated hygromycin B revealed one band with the same R(F) value as the hygromycin B standard.

Isolation of new types of dexamethasone-resistant variants from a cAMP-resistant lymphoma.

We have developed a sequential selection procedure for the isolation of novel steroid-resistant variants of the murine thymoma WEHI-7. The first step involves the isolation of cell lines with an altered cAMP-dependent protein kinase (cAPK) activity by selection for resistance to dibutyryl cAMP (dbcAMP). The second step involves the selection for resistance to dexamethasone (dex) which results in the isolation of variants with decreased receptor function and a cAMPrdexr phenotype. The initial selection, to cAMPr, serves as a permissive step since isolation of spontaneous glucocorticoid resistance from wild-type WEHI-7 does not occur at a measurable frequency. The results demonstrate a potential role for cAPK in regulating the functional levels of glucocorticoid receptor and suggest that mutations in other cellular functions that affect receptor activity could lead to steroid resistance in lymphoid cells.

The efficacy of reducing agents or antioxidants in blocking the formation of dense cells and irreversibly sickled cells in vitro.

We show that N-acetylcysteine (NAC) has the ability to cause statistically significant diminishment in the in vitro formation of irreversibly sickled cells (ISCs) at concentrations greater than 250 micromol/L. Other antioxidants, approved for human use (cysteamine, succimer, dimercaprol), were not efficacious. NAC had the ability to cause statistically significant conversion of ISCs formed in vivo back to the biconcave shape. NAC was also shown to reduce the formation of dense cells and increase the available thiols in beta-actin. We showed that diminishing reduced glutathione (GSH), by treatment with 1-chloro-2,4-dinitrobenzene, resulted in increased dense cells. We conclude the NAC blocks dense cell formation and ISC formation by targeting channels involved in cellular dehydration and beta-actin, respectively. The efficacy of NAC is probably due to its combined antioxidant activity and ability to increase intracellular GSH.