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G M Ringold

Publications and source records attributed to G M Ringold.

At least 37 records · Page 2Linked to original sources
PubMed1989

Two amino acids within the knuckle of the first zinc finger specify DNA response element activation by the glucocorticoid receptor.

The specificity of target gene activation by steroid receptors is encoded within a small, cysteine-rich domain that is believed to form two zinc-coordinated fingers. Here we show that the ability of glucocorticoid and estrogen receptors to discriminate between their closely related response elements resides in the two amino acids located between the two cysteines in the C-terminal half of the first finger. Unexpectedly, chimeric glucocorticoid receptors harboring portions of the interfinger and/or second finger of the estrogen receptor have the ability to activate transcription from either a GRE- or ERE-containing promoter. We surmise that whereas the "knuckle" region of the first finger may be the primary determinant of sequence recognition, the remainder of the DNA binding domain normally confers structural information required for preventing promiscuous HRE recognition.

Amino Acid Sequence↗
PubMed1989

Mutational analysis of the mouse glucocorticoid receptor.

We have cloned the mouse glucocorticoid receptor (GR) from both wild-type and glucocorticoid-resistant variants of the mouse lymphoma cell lines WEHI-7 and S49. Mapping of the mutations present in the variant receptors, together with deletion analysis of wild-type receptor, reveals that the receptor has three clearly defined domains. The COOH-terminal domain contains the hormone-binding site. Within this domain is a small region which is important for the suppression of receptor activity in the absence of hormone. The large NH2-terminal domain is essential for full receptor activity and contains within it a highly acidic region that potentiates receptor activity. The presence of this acidic region reduces nonspecific DNA binding and may therefore be crucial in the discrimination between specific and nonspecific DNA-binding sites by the receptor. A small centrally located domain contains all the information necessary to bind specifically to DNA and to activate transcription. Although this region is absolutely conserved in the GR of different species, many mutations introduced in vitro give rise to functional receptor. In addition, part of this region in the GR can be substituted for by the corresponding sequence in the estrogen receptor to give a GR with the DNA-binding and transcriptional specificity of an estrogen receptor. Lastly, we have succeeded in obtaining stable high-level expression of wild-type and mutant GR in transfected Chinese hamster ovary cells.

Amino Acid Sequence↗
PubMed1989

Evidence for two mechanisms by which tumor necrosis factor kills cells.

Tumor necrosis factor (TNF) can inhibit the differentiation of preadipocytes to adipocytes and will revert differentiated adipocytes to the preadipocyte state. TNF is not toxic to either adipocytes or preadipocytes when used alone but is highly toxic to these cells when used in conjunction with cycloheximide, yielding virtually 100% killing within 4-6 h of treatment. A cell line (TA1 R-6) was isolated which is resistant to the combined toxic effects of TNF and cycloheximide. This cell line is stable and, unlike the parental cell line, does not morphologically differentiate to adipocytes or express adipocyte-specific mRNAs. It has a more transformed appearance and growth pattern and, while resistant to the toxic effects of TNF and cycloheximide in a 6-h assay, has become sensitive to cytotoxicity induced by TNF used alone in a 3-day assay. The adipocyte differentiation-inducing agents, dexamethasone and indomethacin, block the cytotoxicity induced by TNF alone in the TA1 R-6 line but do not block the rapid cytotoxicity of TNF and cycloheximide in the parental line. These results provide both genetic and pharmacologic evidence that there are at least two distinct or overlapping pathways by which TNF mediates its effects.

Adipose Tissue↗
PubMed1989

Cationic liposome-mediated transfection.

A lipid named DOTMA has been created that forms unilamellar liposomes which complex with DNA and RNA for the transfection of mammalian cells, including suspension cells and hybridomas.

DNA↗
PubMed1989

Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta.

Cultured TA1 adipocytes treated with tumor necrosis factor alpha (TNF) lose intracytoplasmic lipid and, over a period of days, come to resemble their predifferentiated progenitors (preadipocytes). To examine the extent to which this phenotypic reversion represents a return to a less differentiated cell, we examined three major characteristics that distinguish preadipocytes from adipocytes: (a) pattern of gene expression; (b) hormonal requirement for accelerated adipogenesis; and (c) pattern of protein synthesis. We found that within hours of TNF addition to adipocytes, mRNAs for genes whose expression is augmented during adipogenesis decreased to predifferentiated levels; in addition, like preadipocytes, TNF-treated adipocytes required exposure to hormones to accelerate adipogenesis. Further, the pattern of protein synthesis seen on polyacrylamide gels reverted to that seen before differentiation. Transforming growth factor-beta (TGF-beta) also caused a rapid decrease in expression of adipose genes when added to fully differentiated cells, an effect that was achieved by treatment with either TGF-beta 1 or TGF-beta 2. These effects were seen in the absence of a demonstrable proliferative response to either TNF or TGF-beta. Thus characteristics that define the "terminally" differentiated state in adipocytes are subject to modulation by environmental influences.

Adipose Tissue↗
PubMed1989

Differential effects of fibroblast growth factor and tumor promoters on the initiation and maintenance of adipocyte differentiation.

Fibroblast growth factor (FGF) has been shown to inhibit the differentiation of myogenic and adipogenic cell lines without inducing a proliferative response. We have previously shown that agents capable of activating protein kinase C (PKC), such as FGF and the phorbol ester tetradecanoyl phorbol-13-acetate (TPA), inhibit the differentiation of the adipogenic cell line TA1, as measured by the rapid loss of adipocyte-specific RNAs. We report here that the treatment of fully differentiated TA1 adipocytes with FGF at 10 ng/ml induces the reversal of adipocyte differentiation, even in cells where PKC activity has been down-regulated by TPA pretreatment. In contrast, TPA or lower concentrations of FGF (1 ng/ml), both effective inducers of c-fos RNA in adipocytes, fail to reverse adipocyte differentiation. The adipocytes, however, will extinguish differentiation-specific functions in response to TPA by the addition of a calcium ionophore. Therefore, we propose that there are two FGF-sensitive pathways in TA1 cells: one responsible for the initiation of differentiation (TPA sensitive) and another required for maintenance of the adipose phenotype (TPA insensitive). These results suggest that activation of two distinct signaling pathways--one PKC and calcium dependent, the other FGF activated but PKC independent--are capable of inhibiting the biochemical events responsible for the maintenance of adipocyte differentiation.

Adipose Tissue↗
PubMed1988

The molecular cloning and characterization of murine ferritin heavy chain, a tumor necrosis factor-inducible gene.

Ferritin is a ubiquitous and highly conserved protein which plays a major role in iron homeostasis. We have identified and sequenced a full-length cDNA for murine ferritin heavy chain. The isolated cDNA is 819 nucleotides in length. It includes 546 nucleotides which encode a protein of 182 amino acids, a 5' noncoding sequence of 120 nucleotides, and a 3'-noncoding region of 153 nucleotides. The sequence displays a high degree of homology to human ferritin H, and includes a portion of the iron-responsive element conserved in chick, frog, and human ferritin. Tumor necrosis factor (TNF), a cytokine which mediates elements of the stress response, induces expression of ferritin H mRNA. Both mouse TA1 adipocytes and human muscle cells increase expression of ferritin H mRNA 4-6-fold after 48 h exposure to TNF. This increase occurs both prior and subsequent to differentiation of adipocytes and muscle cells, and is accompanied by an increase in the synthesis of the ferritin H subunit. These findings suggest a novel role for TNF in iron metabolism.

Adipose Tissue↗
PubMed1988

A growth factor-repressible gene associated with protein kinase C-mediated inhibition of adipocyte differentiation.

The conversion of determined adipoblasts to fully differentiated adipocytes requires appropriate environmental conditions. A strict dependence on cell confluence and a facilitation by glucocorticoid hormones have previously been described. We have found that agents that are capable of activating protein kinase C, such as basic fibroblast growth factor and phorbol esters, inhibit the differentiation of the adipogenic cell line TA1 without stimulating cell growth. Here we describe the sequence and characterization of a cDNA (clone 5) that detects an RNA, the expression of which is enhanced by glucocorticoids and increasing cell density. In contrast, activators of protein kinase C including basic fibroblast growth factor, phorbol esters, and synthetic diacylglycerols inhibit clone 5 gene expression. It appears that clone 5 expression is closely linked to environmental and hormonal factors that promote the differentiation of adipogenic cells.

Adipose Tissue↗
PubMed1988

Induction of adenosine 3',5'-monophosphate-dependent protein kinase subunits during adipogenesis in vitro.

Fatty acid metabolism in adipocytes is known to be regulated by the intracellular transducer cAMP. This study was undertaken to determine the temporal and hormonal regulation of cAMP-dependent protein kinase during the differentiation of preadipocyte mesenchymal cells to adipocytes. For this we have used a stable cell line (TA1) in which the undifferentiated preadipocyte acquires adipocyte functions and morphology after growth to confluence. We observed that synthesis of type I and II cAMP-dependent protein kinases was induced during the adipogenic conversion of growth-arrested TA1 cells. In preconfluent cells, neither mRNAs encoding regulatory subunits (RI, RII beta) and catalytic subunit (C alpha) nor the peptides themselves were detectable. Within several days of growth arrest at high cell density, mRNAs for RI, RII beta, and C alpha were detectable in total RNA extracted from cell populations. The subunits themselves were detectable in some, but not all, of the cells by indirect immunofluorescence. Immunoblotting of cytosolic extracts indicated that RI and the beta-isoform of RII (mol wt = 52,000) were expressed in these cells. Analysis of subunit presence or absence in single cells by immunofluorescence also indicated that kinase subunit expression preceded the accumulation of lipid droplets within the cells. Further, the subunits were predominantly associated with a reticular cytoplasmic structure (Golgi apparatus?) abutting the nucleus. Conversion of TA1 cells to adipocytes can be accelerated by indomethacin (125 microM) or dexamethasone (1 microM) treatment, compounds that also enhanced the accumulation of RII beta and C alpha mRNAs. Within 2-3 days of addition of indomethacin to confluent cultures, RII beta message content is increased about 20-fold, and protein content is increased about 5-fold relative to those in untreated cultures. C alpha mRNA content is increased about 5-fold relative to that in untreated cells. The response to dexamethasone requires 6-7 days, and changes in RII beta message levels were the most pronounced. We also observed the induction of mRNAs for the functionally relevant mRNA lipoprotein lipase in indomethacin-treated cells. In addition to this apparent transcriptional regulation of kinase subunit expression, we provide evidence for regulation at the posttranscriptional level. Within a differentiated culture, there exist stem cells that can be selected, will repopulate the dish, and will again differentiate into adipocytes upon growth arrest at high cell density. In preconfluent populations of these stems cells, unlike the preconfluent TA1 cells originally plated, both RII beta and C alpha messages were present.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Sequences downstream of the glucocorticoid regulatory element mediate cycloheximide inhibition of steroid induced expression from the rat alpha 1-acid glycoprotein promoter: evidence for a labile transcription factor.

The glucocorticoid induction of alpha 1-acid glycoprotein (AGP) RNA in rat hepatoma cells is diminished by inhibiting protein synthesis. We now show that the AGP 5'-flanking region contains a DNA sequence (position -121 to -107), exhibiting a high degree of homology to the glucocorticoid regulatory element (GRE) consensus sequence ACAXXXTGTTCT, which serves to specifically bind purified rat glucocorticoid receptor in vitro. A 15 base pair oligonucleotide representing the AGP GRE confers glucocorticoid responsiveness on a heterologous promoter; such regulation is not diminished by concurrent inhibition of protein synthesis. However, inclusion of the AGP sequences immediately downstream of the AGP GRE (position -106 to -42) renders the hormonal induction sensitive to inhibition of protein synthesis. Furthermore, inclusion of these downstream sequences results in a more pronounced induction mediated by the AGP GRE. In vitro DNase-1 treatment using nuclear extracts prepared from HTC hepatoma cells generate footprints that indicate the presence of DNA-protein interactions spanning the region from -110 to -68 of the AGP gene. We propose that one or more labile factors acting within this domain, immediately downstream of the GRE, is required for efficient transcription at the AGP promoter.

Animals↗
PubMed1987

Glucocorticoid-regulated expression from the 5'-flanking region of the rat alpha 1-acid glycoprotein gene. Requirement for ongoing protein synthesis.

The induction of alpha 1-acid glycoprotein (AGP) mRNA by glucocorticoids in rat hepatoma cells requires ongoing protein synthesis. Here we show that the 5'-flanking region of the AGP gene confers glucocorticoid responsiveness on the expression of heterologous coding sequences. Moreover, the induction of beta-globin mRNA directed by the AGP promoter is inhibited by concurrent treatment with cycloheximide, thereby suggesting that the requirement for protein synthesis is mediated through 5'-flanking sequences. Analysis of the effects of varying durations of cycloheximide treatment indicates that both the endogenous AGP gene and the transfected AGP-beta-globin chimeric gene are induced normally by dexamethasone during the first 1-2h of concurrent treatment. In addition, pretreatment with cycloheximide yields a decrease in the subsequent induction of both beta-globin and AGP RNAs. These data support the notion that a pre-existing and labile protein, perhaps in concert with the glucocorticoid receptor, acts through the 5'-flanking region of the AGP gene to stimulate transcription from the AGP promoter.

Animals↗
PubMed1987

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.

Animals↗
PubMed1987

Requirements for triggering of adipocyte differentiation by glucocorticoids and indomethacin.

The stable adipogenic cell line TA1, spontaneously differentiates into mature adipocytes after several days at confluence. Glucocorticoids (e.g. dexamethasone) accelerate the onset of differentiation by precociously activating the transcription of genes that are expressed in the mature adipocyte but not in the preadipocyte. Thus the hormone may induce a critical regulatory factor required for activating the entire set of differentiation-dependent genes. We have found that the nonsteroidal antiinflammatory drug indomethacin also stimulates differentiation of TA1 cells but even more rapidly and completely than does dexamethasone. Contrary to previous suggestions we find that this activity of indomethacin's cannot be ascribed to inhibition of cyclo-oxygenase, the critical enzyme in prostaglandin biosynthesis. Finally, indomethacin's ability to stimulate TA1 cell differentiation synchronously and rapidly has allowed us to document that cell confluence is required for efficient differentiation and that the drug needs only to trigger rather than maintain the differentiation process.

Adipose Tissue↗
PubMed1987

Domains of the glucocorticoid receptor involved in specific and nonspecific deoxyribonucleic acid binding, hormone activation, and transcriptional enhancement.

We have analyzed the domain structure of the mouse glucocorticoid receptor by expression of in vitro mutated receptor in COS-7 cells. The receptor consists of a core domain rich in Cys, Lys, and Arg amino acids which can bind specific DNA sequences (glucocorticoid response elements) and activate transcription. The activity of this centrally located domain is modulated by the activity of the other two domains. The N-terminal domain of the receptor plays a role in decreasing nonspecific DNA binding and may therefore improve the ability of the protein to discriminate between specific and nonspecific DNA binding sites. This activity maps to a small, highly acidic region of the N-terminal domain. The C-terminal domain of the receptor contains the glucocorticoid binding site and in addition represses the transcriptional activity of the receptor in the absence of hormone. Hormone binding relieves the repression allowing transcription activation. The C-terminal domain contains a short sequence conserved among steroid receptors; its deletion yields a receptor that activates transcription in the absence of hormone.

Amino Acid Sequence↗
PubMed1986

Analysis of glucocorticoid unresponsive cell variants using a mouse glucocorticoid receptor complementary DNA clone.

We have used a glucocorticoid receptor cDNA isolated from a mouse lymphoma cell line to characterize receptor mRNA and genomic sequences present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Wild type rat and mouse cell lines contain two receptor mRNAs, 5 and 7 kilobase pairs (kb) in length, which differ in the length of their 3'-untranslated regions. Levels of receptor mRNA present in mutant HTC, WEHI7, and S49 cells of the r- (receptorless) phenotype are decreased compared to wild type cells. This decreased level of receptor mRNA parallels the decreased level of total immunoreactive receptor protein found in these cells. S49 nt- (nuclear transfer minus) cells contain receptor mRNA levels which parallel their hormone binding and immunoreactive receptor levels. Cells of the r- and nt- phenotype contain no detectable deletions or rearrangements of the receptor gene. We conclude that r- cells have lesions which affect the expression of receptor mRNA. Surprisingly, HTC cells of the r- phenotype differ from WEHI7 and S49 r- cells in that HTC r- cells contain a lower level of receptor DNA than does their parental wild type cell line. Although these cells may contain multiple lesions, it appears that loss of receptor genomic sequences is responsible, in part, for the phenotype of the HTC r- cells. The S49 nti (nuclear transfer increase) cells produce glucocorticoid receptors of molecular weights 40,000 and 94,000. These cells produce, in addition to the wild type 5- and 7-kb receptor mRNAs, two other receptor messages 5.5 and 3.5 kb in length. RNA blot analysis using various portions of our receptor cDNA indicates that these are 5' truncated messages and suggests that the 40-kDa nti receptor is truncated at its NH2-terminal end. These data also indicate that the hormone and DNA-binding regions of the receptor are located in the COOH-terminal half of the protein.

Alleles↗
PubMed1986

The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins.

We have isolated mouse glucocorticoid receptor (GR) cDNAs which, when expressed in transfected mammalian cells, produce a fully functional GR protein. Sequence analysis reveals an open reading frame of 2349 bp which could encode a protein of approximately 86,000 daltons. We have also isolated two receptor cDNAs from mouse S49 nuclear transfer-deficient (nt-) cells which encode mutant forms of the receptor protein. One cDNA encodes a protein that is unable to bind hormone and represents the endogenous hormone binding deficient receptor recently discovered in S49 cells. The lesion in this receptor is due to a single amino acid substitution (Glu-546 to Gly). The second cDNA from nt- cells produces a receptor protein that is able to bind hormone but has reduced nuclear binding. This cDNA, therefore, encodes for the S49 nt- receptor which has been shown to have reduced affinity for DNA. The lesion maps to a single amino acid substitution (Arg-484 to His) located in a highly Cys, Lys, Arg-rich region of the protein previously implicated in DNA binding. Our studies provide unambiguous identification of receptor domains and specific amino acids critical for the hormone and DNA binding properties of this transcriptional regulatory protein. Contained within the first 106 amino acids of the mouse GR is a stretch of nine glutamines with two prolines which are related to the family of transcribed repetitive elements, opa, found in Drosophila melanogaster. A truncated receptor lacking these 106 amino acids is functionally indistinguishable from the wild-type receptor.

Amino Acid Sequence↗
PubMed1986

Glucocorticoid control of developmentally regulated adipose genes.

We have analyzed the hormonal basis for the acceleration of differentiation by dexamethasone in the stable adipogenic cell line TA1. These cells, which were derived from 5-azacytidine-treated 10T1/2 mouse embryo fibroblasts, undergo differentiation in culture after reaching confluence. Using cDNA clones corresponding to mRNAs that are induced during adipogenesis, we find that dexamethasone elicits the precocious accumulation of differentiation-specific gene products. This effect appears to be mediated by the glucocorticoid receptor, yet unlike standard steroid inductions, most of the RNAs reach the same maximal levels in the absence of dexamethasone. Glucocorticoids thus may increase the expression of a regulatory factor required for activating the entire set of differentiation-dependent genes. We also describe a gene whose transcription is not only activated during adipogenesis, but is also specifically inducible by dexamethasone in the mature adipocyte. Moreover, the glucocorticoid responsiveness of this gene in differentiated cells appears to be dependent on its prior developmental activation.

Adipose Tissue↗