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Biomedical subjects

G Litwack

Publications and source records attributed to G Litwack.

At least 55 records · Page 3Linked to original sources
PubMed1995

Modulator inhibits nuclear translocation of the glucocorticoid receptor and inhibits glucocorticoid-induced apoptosis in the human leukemic cell line CEM C-7.

Modulator is an endogenous low-molecular-weight regulator of both glucocorticoid and mineralocorticoid receptors as well as protein kinase C. Structural analysis of modulator purified to apparent homogeneity suggests that it is a novel ether aminophosphoglyceride. In this report, we show that modulator inhibits cytosolic human glucocorticoid receptor (GR) complex activation as measured by DNA-cellulose binding. In addition, modulator blocks glucocorticoid-induced nuclear translocation of the GR in intact human leukemic (CEM C-7) cells, as illustrated by immunocytochemical localization. Furthermore, we demonstrate that modulator, by blocking the activation and subsequent translocation of GR, inhibits glucocorticoid-mediated apoptosis, characterized by chromatin condensation, internucleosomal DNA fragmentation, and cell death in glucocorticoid-sensitive CEM C-7 cells. Modulator inhibits glucocorticoid-induced c-myc gene repression and glucocorticoid receptor gene up-regulation. These data suggest that modulator functions to regulate the GR in intact cells as well as in cytosolic preparations. In addition, the inhibition of glucocorticoid-induced programmed cell death by modulator sheds light on the cellular function of modulator as well as on the mechanism by which apoptosis occurs in CEM C-7 cells.

Amino Acid Sequence↗
PubMed1995

Contributions of distal and proximal promoter elements to glucocorticoid regulation of osteocalcin gene transcription.

Previous studies identified several glucocorticoid response elements (GREs) in the 5'-promoter region of the rat osteocalcin (OC) gene by purified receptor binding. The present study addresses functionality of the GRE sequences in the proximal promoter at nucleotide (nt) -16 to -1 downstream of the TATA element together with the GRE half-element in the OC box at nt -86 to -81. This was done by assaying glucocorticoid responsiveness [at 10(-6) M dexamethasone (DEX)], and in combination with 10(-8) M 1,25-dihydroxyvitamin D3, of a series of deleted and mutated OC promoter reporter constructs (OCCAT) in osteoblast-like cells, the ROS 17/2.8 rat osteosarcoma line. Promoter deletion analysis revealed an additional GRE in the distal promoter at nt -697 to -683 that functions to suppress OC transcription. In the absence of this upstream negative GRE (nGRE), the -531 OCCAT construct exhibited enhanced promoter activity in response to DEX (1.8-fold DEX/Control), but further deletion (-348 and -108 OCCAT constructs) restored DEX suppression to OC promoter activity (0.6- and 0.8-fold DEX/Control, respectively). Mutations introduced in both the proximal GRE (nt -16 to -1) and the half-GRE in the OC box, or in the proximal GRE alone, nearly abrogated DEX responsiveness of OC promoter activity. Both distal and proximal GREs specifically bound glucocorticoid receptor present in ROS 17/2.8 nuclear extracts as shown by competition with wild type and mutated oligonucleotides and antibody inhibition of binding. Furthermore, both GREs, independently, conferred DEX-responsive transcriptional repression to the heterologous thymidine kinase basal promoter. We also report that glucocorticoid suppression of 1,25-dihydroxyvitamin D3-stimulated transcription occurs independently of distal or proximal GREs. Taken together, these results demonstrate that in vivo responsiveness of OC to DEX involves the integrative activities of several functional promoter elements.

Animals↗
PubMed1995

Synthetic peptides derived from the steroid binding domain block modulator and molybdate action toward the rat glucocorticoid receptor.

Modulators are endogenous low-mol-wt inhibitors of glucocorticoid receptor activation, and are mimicked by exogenous sodium molybdate. Activation involves the dissociation of the 90-kDa heat-shock protein from the receptor. The heat-shock protein is thought to bind to a conserved 20-amino-acid region in the steroid-binding domain of the receptor (595-614 of the rat protein). Synthetic peptides corresponding to this amino acid sequence prevented the modulators and sodium molybdate from inhibiting receptor activation. These results imply that the region 595-614 of the rat glucocorticoid receptor is also a modulator/molybdate binding site.

Amino Acid Sequence↗
PubMed1995

Purification of the glucocorticoid receptor-mineralocorticoid receptor modulator-2 from rabbit liver.

Modulators-1 and -2 are endogenous low-mol-wt regulators of glucocorticoid and mineralocorticoid receptors and protein kinase C. Structural analysis of apparently purified modulators suggested that these molecules were novel ether aminophosphoglycerides. Subsequent X-ray crystallography and NMR spectroscopy indicated that the ultra-large scale modulator preparations were contaminated with glutamate and aspartate, although these amino acids lacked modulator activity. In this article, we describe the purification of modulator-2 from rabbit liver cytosol and the separation of this phosphoglyceride from these amino acids. This purification was similar to the ultra-large scale version (Bodine, P.V. and Litwack, G. [1990] J. Biol. Chem. 265, 9544-9554), but involved the chromatography of trypsinized rabbit liver cytosol on the 7-L bed volume Sephadex G-15 gel-filtration column. As before, two peaks of modulator activity (modulator-1 and -2), as well as a DNA-binding inhibitor (peak-3), eluted from the gel-filtration column. The resulting modulator-2 pool was incubated with glutamate decarboxylase and treated batch-wise with Dowex-50W cation-exchange resin and Chelex-100 resin. This enzyme/resin-treated modulator-2 preparation was then chromatographed on a Dowex-1 anion-exchange column. Finally, modulator-2 was purified by preparative silica TLC. This last purification step resulted in the separation of modulator-2 from glutamate, aspartate, and gamma-aminobutyrate. In summary, rabbit liver cytosol appears to be a reasonable source of modulator-2. In addition, treatment of the preparation with glutamate decarboxylase seems to facilitate the subsequent separation of modulator-2 from the contaminating amino acids.

Adrenalectomy↗
PubMed1994

FKBP46, a novel Sf9 insect cell nuclear immunophilin that forms a protein-kinase complex.

Recently, we identified a 59-kDa nuclear phosphoprotein that is associated with a recombinant mouse FKBP-52 (Alnemri, E. S., Fernandes-Alnemri, T., Nelki, D. S., Dudley, K., DuBois, G. C., and Litwack, G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6839-6843). Here we describe the cloning, overexpression, and characterization of this protein from Spodoptera frugiperda insect cells (Sf9 cells). The cloned cDNA codes for an acidic protein of 412 amino acids with distinct structural domains. Starting with the N terminus, the first 218 amino acids contain two highly acidic domains separated by a short basic domain. Following the second large acidic domain is another basic domain of 87 amino acids with significant sequence and structural homology to HMG1 and HMG2 DNA binding proteins. The two basic domains contain several nuclear targeting signals. The last 108 C-terminal amino acids contain a binding domain for immunosuppressive drugs FK506 and rapamycin, which makes this protein a new member of the immunophilin family. We provide evidence that the new immunophilin (FKBP46) is a DNA binding protein that can bind immunosuppressive drug FK506 and possesses peptidylprolyl isomerase activity. FKBP46 is localized in the nucleus and is associated with a nuclear kinase that specifically phosphorylates it in the presence of Mg2+ and ATP. Upon subsequent sequence analysis of the mouse FKBP52 cDNA used in our previous study, it was observed that a spermatid nuclear transition protein 2 (TP2) sequence is fused in frame with the C terminus of the recombinant FKBP52 probably as a result of a cloning artifact. We demonstrate that the FKBP46 does not form a complex with the FKBP52 but rather with the highly basic nuclear protein TP2. Our data suggest that interaction of FKBP46 with TP2 is mediated by the N-terminal acidic domains of FKBP46. This implies that the acidic domains of FKBP46 are involved in protein-protein interaction between nuclear FKBP46 and other basic chromatin proteins.

Amino Acid Isomerases↗
PubMed1994

CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme.

We have cloned a novel apoptotic gene from human Jurkat T-lymphocytes. The new gene encodes a 32-kDa putative cysteine protease (CPP32) with significant homology to Caenorhabditis elegans cell death protein Ced-3, mammalian interleukin-1 beta-converting enzyme (ICE), and the product of the mouse nedd2 gene. The CPP32 transcript is highly expressed and most abundant in cell lines of lymphocytic origin. Overexpression of CPP32 or ICE in Sf9 insect cells resulted in apoptosis. In addition, coexpression of recombinant p20 and p11 derived from the parental full-length CPP32 sequence resulted in apoptosis in Sf9 cells. Our data suggest that similar to ICE, CPP32 is made of two subunits, p20 and p11, which form the active CPP32 complex. The apoptotic activity of CPP32 and its high expression in lymphocytes suggest that CPP32 is an important mediator of apoptosis in the immune system.

Amino Acid Sequence↗
PubMed1994

A new synthetic ether aminophosphoglyceride exhibits partial modulator activity towards the glucocorticoid receptor.

Modulator is an endogenous low-molecular weight regulator of both glucocorticoid and mineralocorticoid receptors, as well as protein kinase C. Analogs of the putative modulator structure have been synthesized. These compounds include 1-O-(3'-carboxypropyl) or (5'-carboxypentyl)-L-glycero-3-phospho-L-serine or L-threonine, and the D-glycerol stereoisomers. These compounds were tested for in vitro modulator activity using the glucocorticoid-receptor complex activation inhibition and steroid-binding stabilization assays. One of the ether phosphoglycerides, 1-O-(5'-carboxypentyl)-L-glycero-3-phospho-L-threonine (H-GPT-1), partially inhibited steroid-receptor complex activation in a dose-dependent manner. However, none of the other compounds exhibited any modulator activity towards the glucocorticoid-receptor complex. Like modulator, H-GPT-1 did not inhibit activated glucocorticoid-receptor complex binding to DNA-cellulose. Surprisingly, in contrast to modulator, H-GPT-1 partially inhibited unoccupied receptor steroid-binding in a dose-dependent manner. These results suggest that although modulator is not exactly mimicked by this compound, H-GPT-1 is the first synthetic organic molecule to exhibit some modulator activity towards the glucocorticoid receptor.

Adrenalectomy↗
PubMed1994

Mineralocorticoid and glucocorticoid receptor steroid binding and localization in colonic cells.

In rat colon epithelium glucocorticoids and mineralocorticoids regulate Na transport by binding to distinct receptors and stimulating different pathways. The distribution and intracellular localization of mineralocorticoid (MR) and glucocorticoid (GR) receptors in colonic Na-absorbing surface cells and Cl-secreting crypt cells is unknown. Surface and crypt cells were sequentially isolated from rat distal colon by EDTA chelation and mechanical dissociation. Cell viability was confirmed by trypan blue exclusion and low rates of 2',7'-bis(2-carboxyethyl)-5(6)-carboxylfluorescein leak. Histologic examination, alkaline phosphatase activity, and rates of [3H]leucine incorporation confirmed separation of surface from crypt cells. Scatchard analysis of [3H]aldosterone and [3H]triamcinolone acetonide binding demonstrated that the number of MR decreased from 7,228 +/- 1,067 in surface to 2,299 +/- 434 receptors/cell in crypt cells, whereas the number of GR increased from 20,857 +/- 4,241 in surface to 58,598 +/- 8,207 receptors/cell in crypt cells. The dissociation constants were 2.8 +/- 0.4 nM for the MR and 12 +/- 3 nM for the GR. Indirect immunofluorescence using the specific anti-MR antibody hMRsN and the anti-GR antibody BuGR-2 demonstrated that both unliganded receptors were cytoplasmic and translocated to the nucleus after hormone binding. These data indicate that both surface and crypt cells are potentially responsive to mineralocorticoids and glucocorticoids and that both the MR and GR require hormone for nuclear translocation.

Aldosterone↗
PubMed1993

Assembly of glucocorticoid receptor and c-JUN homodimer on the promoter of mouse mammary tumor virus-long terminal repeat is influenced by order of addition.

Interactions between the glucocorticoid receptor (GR) and c-Jun/c-Jun homodimer (JUN) on the promoter DNA of mouse mammary tumor virus-long terminal repeat (MMTV-LTR) are reported here using the electrophoretic mobility shift assay (EMSA). Both GR and JUN are capable of independently binding to their respective response elements, including glucocorticoid response element (GRE) and phorbol ester response element (TRE), on MMTV-LTR promoter. The protein-DNA complex, assembled by pre-incubating JUN and DNA before the addition of GR, migrates slower (supershift) on gel electrophoresis than do the complexes formed by the other orders of addition. The formation of the supershifted complex is GR and JUN dose-dependent. The supershift is not detected with the cleaved fragments of MMTV-LTR promoter that separate GRE from TRE, indicating that the integrity of the promoter and possibly the spacing between GRE and TRE are important. The interaction of GR and JUN on the MMTV-LTR promoter appears to be more complex than simple protein-protein interaction.

Adrenalectomy↗
PubMed1993

Identification of multiple glucocorticoid receptor binding sites in the rat osteocalcin gene promoter.

The biosynthesis of osteocalcin (OC), a bone-specific, noncollagenous protein, is stringently regulated during differentiation of the osteoblast phenotype. Glucocorticoids, and also 1,25(OH)2D3, mediate the developmental regulation of OC gene transcription. In this study, we established that the -1097 to +23 promoter (pOCZCat) of the rat OC gene confers glucocorticoid responsiveness to both basal and vitamin D-induced OC expression. The presence of multiple glucocorticoid receptor (GR) binding sites in the proximal rat OC gene promoter was determined by the combined use of DNase I footprinting, dimethyl sulfate fingerprinting, and gel mobility shift analysis with glucocorticoid receptor protein. One glucocorticoid receptor binding element (GRE) resides immediately downstream of the TATA box (-16 to -1). In vivo activity was established by cotransfection of ROS 17/2.8 osteosarcoma cells with an OC-CAT construct in the presence of cloned GRE sequences (wild type or mutant) as competitors. A putative second, less protected GR binding site is located further upstream in the OC gene basal promoter within the region overlapping the TATA box. This is in direct contrast to the organization of GREs in the human OC proximal promoter wherein GR binding at the upstream GRE overlapping the TATA is stronger than at the downstream GRE. In addition, we detected sequence-specific binding of GR protein to another basal promoter element, the OC box (-99 to -76), which contains a central CCAAT motif. The presence of multiple GR binding sites in the rat OC gene proximal promoter indicates that regulation of basal and vitamin D-enhanced transcription by glucocorticoids may involve the integrated activities of multiple, independent GREs.

Animals↗
PubMed1993

Specific regulation of male rat liver cytosolic estrogen receptor by the modulator of the glucocorticoid receptor.

Modulator is a novel low-molecular-weight organic compound that regulates activities of glucocorticoid and mineralocorticoid receptors as well as protein kinase C. In this study we show that male rat liver cytosolic estrogen receptor activation is inhibited by modulator in a dose-dependent manner. Fifty percent inhibition is obtained with 1 unit/ml modulator purified from bovine liver which is within the physiological concentration for modulator. However, sheep uterine cytosolic estrogen and androgen receptors are insensitive to regulation by modulator. Exogenous sodium molybdate treatment inhibits activation of all of these receptors of liver or uterus origin in an identical manner, further differentiating the effects of modulator and the molybdate anion.

Adrenalectomy↗
PubMed1993

Overexpression, characterization, and purification of a recombinant mouse immunophilin FKBP-52 and identification of an associated phosphoprotein.

To gain insight into the structure and function of the immunophilin FKBP-52, a mouse FKBP-52 was overexpressed in Spodoptera frugiperda insect cells (Sf9 cells) with the baculovirus expression system. The purification and characterization of the recombinant FKBP-52 (rFKBP-52) was facilitated by incorporating a histidine 6-mer domain at its N terminus. The rFKBP-52 was highly purified on a N(i)2+ affinity resin with an estimated recovery of 10 mg of pure protein from 1 liter of Sf9 cell culture. Subcellular fractionation revealed that the rFKBP-52 is expressed predominantly in the nuclei of infected Sf9 cells maximally at 48 hr after infection, consistent with the nuclear localization of FKBP-52 in mammalian cells. The rFKBP-52 can be assembled in vitro with the glucocorticoid receptor complex, establishing its functionality and confirming that it is a component of the unactivated glucocorticoid receptor complex. The rFKBP-52 possesses an ATP/GTP binding activity that is stimulated by divalent cations. Furthermore, incubation of purified rFKBP-52 with [gamma-32P]ATP and MgCl2 resulted in the phosphorylation of a 59-kDa nuclear protein. Amino acid sequence analysis of this protein revealed that it is a phosphoprotein or kinase that is associated with the rFKBP-52.

Amino Acid Sequence↗
PubMed1993

The steroid binding domain influences intracellular solubility of the baculovirus overexpressed glucocorticoid and mineralocorticoid receptors.

The expression and formation of the oligomeric steroid binding form of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) were further examined in the baculovirus expression system. Analysis of the steroid binding ability and accumulation of the GR and MR in recombinant baculovirus-infected insect cells revealed that only 0.35% of the total expressed receptors are actually assembled into cytoplasmic oligomeric receptor complexes. The majority of the overexpressed GR and MR, which amounts to 50 pg/cell, appear to self-aggregate and form insoluble aggregates which fractionate with the nucleus. We believe that this large amount of receptors far exceeds the limited amounts of hsp90, hsp70, and other cellular factors which are required to assemble the heteromeric receptor complex. Attempts to assemble the GR and the MR in vivo by coexpression of the receptors with hsp90 or hsp70 failed to cause any increase in the formation of the steroid binding receptor complex and also in preventing the aggregation of the receptors. On the other hand, in vitro incubation of monomeric GR or MR partially purified from the insoluble receptor aggregates with reticulocyte lysate resulted in complete reconstitution of the oligomeric receptor complex with a concomitant restoration of full steroid binding ability. These data suggest that interaction of hsp90 with the GR or the MR is complex and highly regulated and requires the participation of other cellular factors which are limited in insect cells but can be supplied in vitro by reticulocyte lysate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Demonstration of nuclear translocation of the mineralocorticoid receptor (MR) using an anti-MR antibody and confocal laser scanning microscopy.

Using a synthetic peptide that corresponds to a unique region of the N-terminal domain of the human mineralocorticoid receptor (MR), amino acids 87-96, we have generated a polyclonal antibody, human (h) MRsN. This sequence shares no homology with the corresponding sequences of the glucocorticoid receptor or other steroid/thyroid hormone receptor superfamily members. Antibody hMRsN cross-reacts with MR from human, rat, and mouse cells and recognizes denatured MR from either crude preparations or partially purified rat kidney cytosol, rat colon, or recombinant hMR overexpressed in baculovirus-infected Sf9 cells. Immunoprecipitation of the native MR from either partially purified or crude preparations of rat kidney cytosol with hMRsN, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain, demonstrated a major protein band with a mol wt of 116 kilodaltons. In addition, using confocal laser scanning microscopy and digital image analysis, the immunocytochemical localization of the recombinant hMR over-expressed in Sf9 cells 24 h post-transfection was determined. In the absence of ligand, the MR was detected solely in the cytoplasm, after a 30-min exposure to 100 nM aldosterone the MR was perinuclear, and after 60 min, the MR was predominantly nuclear. To ascertain that this phenomenon was not unique to insect cells, aldosterone induced MR nuclear translocation in mouse macrophage cells was also demonstrated immunocytochemically, clearly indicating a role for nuclear translocation in MR function.

Amino Acid Sequence↗
PubMed1992

Overexpressed full-length human BCL2 extends the survival of baculovirus-infected Sf9 insect cells.

Full-length and truncated human BCL2 lacking the entire C-terminal hydrophobic domain have been overexpressed in Spodoptera frugiperda insect cells with the baculovirus expression system. Immunoblot analysis with BCL2-specific antibodies revealed that both full-length and truncated BCL2 are expressed as multiple immunoreactive species, suggesting posttranslational modifications. The expression of the full-length but not the truncated BCL2 extended the survival of baculovirus-infected cells by preventing virus-induced DNA cleavage. This result is consistent with the reported protective effect of BCL2 against apoptosis in mammalian lymphocytes and suggests a conserved function in evolution. Subcellular fractionation and indirect immunofluorescence studies in intact cells demonstrated that the recombinant full-length and truncated BCL2 proteins were expressed predominantly as nuclear membrane-associated proteins. These results imply that BCL2 must utilize hydrophobic domains other than the deleted domain for its association with the subcellular membranes. Metabolic labeling of insect cells expressing the full-length and the truncated form of BCL2 with 32P(i) demonstrated that BCL2 is a phosphoprotein.

Amino Acid Sequence↗
PubMed1992

Modulators of the glucocorticoid receptor also regulate mineralocorticoid receptor function.

Modulators are proposed to be novel ether aminophosphoglycerides that stabilize unoccupied and occupied glucocorticoid receptor steroid binding and inhibit glucocorticoid receptor complex activation. Two isoforms, modulator 1 and modulator 2, have been purified from rat liver cytosol [Bodine, P.V., & Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554]. Since the mineralocorticoid receptor is relatively resistant to activation, modulator's effect on rat distal colon mineralocorticoid receptor function was examined. Warming of unoccupied receptor decreased residual specific [3H]aldosterone binding by 86 +/- 2%. Both modulator isoforms completely prevented this destabilization with Km's of 2 +/- 1 microM modulator 1 and 24 +/- 5 microM modulator 2. Warming of occupied mineralocorticoid receptors decreased [3H]aldosterone binding by 56 +/- 3%. Modulator only partially stabilized occupied receptor binding with Km's of 10 +/- 2 microM modulator 1 and 68 +/- 8 microM modulator 2. Modulator inhibited receptor activation with Km's of 3 +/- 1 microM modulator 1 and 33 +/- 10 microM modulator 2. Double-reciprocal analysis showed linear kinetics, and mixing modulator isoforms together had additive effects on unoccupied and occupied receptor steroid binding stabilization and activation inhibition. Colon cytosol contained a low molecular weight, heat-stable factor(s) which inhibited receptor activation and stabilized occupied receptor steroid binding. Molybdate completely stabilized unoccupied mineralocorticoid receptor steroid binding and inhibited activation with half-maximal effects at 3-4 mM but only stabilized occupied receptor binding by approximately 40%. These data indicate that (i) apparent physiologic concentrations of modulator stabilize mineralocorticoid receptor steroid binding and inhibit receptor activation, (ii) an aldosterone-responsive tissue contains a modulator-like activity, and (iii) molybdate mimics the effects of modulator.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1992

Involvement of BCL-2 in glucocorticoid-induced apoptosis of human pre-B-leukemias.

To determine the role of BCL-2 in the glucocorticoid-induced apoptosis of lymphocytes, we analyzed the effect of glucocorticoid on two human pre-B-cell lines which express different levels of BCL-2. Glucocorticoid treatment of the 380 cell line which expresses high levels of BCL-2 resulted in inhibition of cellular proliferation without induction of apoptosis. On the other hand, glucocorticoid treatment of the 697 cell line which expresses lower levels of the BCL-2 resulted in both inhibition of cellular proliferation and apoptosis with characteristic internucleosomal DNA cleavage. The glucocorticoid-induced inhibition of cellular proliferation in both cell lines was also associated with repression of the c-myc mRNA expression. Taken together, our data suggest that BCL-2 blocks the glucocorticoid-induced apoptosis of the 380 pre-B-lymphocytes by extending their survival when the level of c-myc expression is repressed. Also by repressing the expression of c-myc, glucocorticoid causes apoptosis of the 697 pre-B-lymphocytes in the absence of high level of BCL-2 expression.

Cell Death↗