Showing papers by "Bruce M. Spiegelman published in 1990"

A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo

Abstract: The murine gene for adipocyte P2 encodes an adipocyte-specific member of the family of intracellular lipid binding proteins. The region upstream from the start of transcription of this gene has been found to contain binding sites for the transcription factors c-jun/c-fos and C/EBP (CCAAT/enhancer binding protein) and several short sequence elements found in other adipocyte gene promoters, termed fat-specific elements. To identify DNA sequences that were responsible for the high level of transcription of the gene for adipocyte P2 in vivo, we made a series of transgenic mice containing 168 base pairs (bp), 247 bp, 1.7 kilobases (kb), and 5.4 kb of 5' flanking sequence linked to the bacterial gene chloramphenicol acetyltransferase. Although plasmids containing only 168 bp of 5' sequence including the C/EBP and AP-1 (activation protein 1) binding sites were expressed well in cultured adipocytes, high levels of chloramphenicol acetyltransferase activity in the adipose tissue of transgenic mice were not observed until the 5' flanking region was extended to kb -54. An enhancer mapping between kb -4.9 and kb -5.4 upstream from the start of transcription was identified by transfection of further deletions into cultured adipocytes. This enhancer, when linked to a bp -63 promoter fragment from the gene for adipocyte P2, directed very high level chloramphenicol acetyltransferase expression specifically to adipose tissue in transgenic mice. These results identify a functional adipose-specific enhancer and indicate that it is the major determinant of tissue specificity of the gene for adipocyte P2. These results also demonstrate that the proximal-promoter binding sites for AP-1 and C/EBP are not sufficient or necessary to give adipose-tissue-specific expression in vivo, though they may play an important role in the response of this promoter to glucocorticoids.

Protooncogene c-fos as a transcription factor.

Abstract: Publisher Summary This chapter discusses the recent advances in the understanding of cellular gene regulation by c-fos—the cellular homolog of the transforming gene (v-fos) of the murine sarcoma viruses FBJ and FBR. The v-fos and c-fos genes are both capable of transforming fibroblasts, although the c-fos gene requires structural alterations. One of the ways of elevating c-fos levels is to transfect cells with an activated c-fos gene. The level of modification for c-fos appears to be dependent on the inducing agent used. If c-fos and c-jun are found in a single complex, then transcription factor AP-1 purified by sequence-specific DNA affinity chromatography can be expected to contain c-fos. The c-fos plays a role in the trans-activation of the TRE as antisense RNA to c-fos completely prevents trans-activation via the TRE when cells are stimulated by a number of agents. The role of c-fos in modulating the DNA-binding activity of c-jun is investigated in the chapter using proteins synthesized in rabbit reticulocyte translation systems from purified mRNA.

DNA-binding activity of Jun is increased through its interaction with Fos.

Abstract: Transcription factor AP-1 mediates induction of a set of genes in response to the phorbol ester tumor promoter TPA Recently, AP-1 preparations from HeLa cells were shown to contain a product of the c-JUN protooncogene (Jun/AP-1) which forms a tight complex with the Fos protein In this paper, we examine the role of the Fos protein in the DNA-binding activity of the AP-1 complex We show that the DNA-binding activity of bacterially expressed trpE-Jun fusion proteins is increased many-fold upon their interaction with Fos (or a Fos-related antigen) expressed from a baculovirus vector The site of Fos interaction is within the DNA-binding domain of Jun/AP-1, and anti-Fos antibodies interfere with the binding of affinity purified AP-1 to DNA These results suggest that, by associating with Jun/AP-1, Fos is responsible for the formation of a multimeric protein complex that has greater affinity for the target sequence than does Jun/AP-1 alone

Reduced adipsin expression in murine obesity: effect of age and treatment with the sympathomimetic-thermogenic drug mixture ephedrine and caffeine.

Abstract: Adipsin gene expression is greatly diminished in certain forms of genetic and acquired obesity. In the present study we evaluate the time course for the development of adipsin deficiency in obesity and its regulation by the sympathomimeticthermogenic drug mixture ephedrine and caffeine. Previously, it was unknown whether adipsin deficiency occurred before or after the development of massive obesity. In the first series of experiments in which mice were treated with monosodium glutamate (MSG) for the first week of life, we demonstrate that adipsin deficiency occurs early in the development of MSG-induced obesity as evidenced by decreased circulating adipsin concentrations by 1 week of age and deficient adipsin mRNA levels in white adipose tissue (WAT) by 2 weeks. In db/db mice, diminished circulating adipsin was noted at 2 weeks of age. In both models, decreased adipsin gene expression precedes the development of marked obesity. Little is known about the factors which regulate adipsin gene expression in ob...

A direct role for c-fos in AP-1-dependent gene transcription.

Abstract: Transcription factor activator protein 1 (AP-1) is a protein fraction that contains c-fos, c-jun, and several other related proteins. Although this protein fraction can stimulate transcription in vitro, the relative contributions of c-fos and c-jun to the transcriptional effect of AP-1 are not clear. In order to approach this question, we have overexpressed both proteins using a baculovirus-mediated expression system and defined their DNA-binding and transcriptional enhancement activities in vitro. Gel mobility-shift and DNase 1 footprinting assays showed that c-jun protein specifically binds to DNA through an AP-1 binding site. Under the same conditions, no detectable binding of c-fos protein was observed. However, when the DNA binding assays were performed in the presence of both c-jun and c-fos, a marked increase in the affinity of c-jun for the AP-1 site was observed. An AP-1-dependent transcription assay was used to test the capability of both proteins to stimulate correctly initiated RNA synthesis in vitro. Under our conditions, c-jun protein was capable of stimulating specific RNA transcription in an AP-1 site-dependent manner. In contrast, c-fos protein showed no detectable transcriptional activation by itself. However, a transcription assay carried out in the presence of both c-fos and c-jun proteins showed that the c-fos/c-jun complex was more active as a transcriptional regulator than c-jun protein alone. These experimental results indicate that c-fos and c-jun proteins are required to reconstitute full AP-1-dependent transcriptional activation and directly demonstrate that c-fos is a regulator of gene expression.

Reduced adipsin mRNA and circulating adipsin protein are modulated by adrenal steroids in obese Zucker rats

Abstract: We investigated expression of the adipose-specific serine protease adipsin in genetically obese Zucker rats and whether adrenalectomy modifies expression. Adipsin mRNA levels were determined by slot blot and Northern blot analysis of total RNA samples extracted from epididymal adipose tissue and isolated retroperitoneal adipocytes of obese (fa/fa) and homozygous lean (Fa/Fa) Zucker rats. Both 30-day-old and 4-mo-old animals were analyzed in experiment 1. In experiment 2, 10-wk-old obese and lean rats were either bilaterally adrenalectomized or sham operated, and adipsin mRNA levels were determined on tissue and cell samples 2 wk postsurgery. In both experiments, serum adipsin protein was determined by Western blot analysis and plasma insulin by radioimmunoassay. The data show that both adipsin mRNA and adipsin protein are reduced in obese compared with lean rats and that adrenalectomy restores these values toward normal in obese rats. The data thus suggest that adrenal steroids are involved in modulating adipsin expression in obese Zucker rats and that insulin may be an intermediary factor in such modulation.

Angiogenic butyric acid analogs and their prodrugs

Abstract: Butyric acid and its analogs of formula (1) or the physiological salts thereof, wherein each X2, X3 and X4 is independently H, OH, OR, SH, SR, NH2, NHR, NR2, or halo, wherein each R is independently lower alkyl(1-4C); or one or two of X3 and X4 taken together form a pi-bond, or wherein one of X2 and X3 taken together form a pi-bond; with the proviso that at least four of said X must be either H or a participant in a pi-bond, or a prodrug which generates said compound of formula (1) or its salt, are capable of stimulating angiogenesis in vivo. These compounds, and their pharmaceutical compositions, are therefore useful in wound healing and other therapeutic applications where stimulation of vascularization is beneficial. Antibodies to these materials have also been prepared and are useful in diagnosis and therapy.

Angiogenic monobutyrin and its analogs

Abstract: Methods are disclosed to stimulate angiogenesis in a warm-blooded animal, which method comprises administering to a subject in need of such treatment, an amount of an angiogenic glyceride of formulae (1) or (2) wherein X is O, NH, S, or CH2, and R1 is alkyl (1-10C) or acyl (2-10C) which is saturated or unsaturated and which is unsubstituted or substituted with one or more substituents which do not interfere with angiogenic activity, said substituents selected from the group consisting of OH, OR, SH, SR, NH?2?, NHR, NR2 and halo wherein each R is independently lower alkyl (1-4C); and each of R?2 and R3? is independently H, PO?3?-2, or is alkyl or acyl as defined above, or in formula 1, R?2 and R3? taken together are an alkylene moiety or OR?2 and OR3? form an epoxide. The compounds of formulae (1) or (2) or mixtures thereof can be used in combination with various peptide growth factors to enhance the angiogenesis effect.

A Direct Role for c-fos in AP-1-dependent Gene

Abstract: Transcription factor activator protein 1 (AP-1) is a protein fraction that contains c-fos, c-jun, and several other related proteins. Although this protein fraction can stimulate transcription in vitro, the relative contributions of c-fos and c-jun to the transcriptional effect of AP-1 are not clear. In order to approach this question, we have overexpressed both proteins using a baculovirus-mediated expression system and defined their DNA-binding and transcriptional enhancement activities in vitro. Gel mobility-shift and DNase 1 footprinting assays showed that c-jun protein specifically binds to DNA through an AP-1 binding site. Under the same conditions, no detectable binding of cfos protein was observed. However, when the DNA binding assays were performed in the presence of both c-jun and c-fos, a marked increase in the affinity of cjun for the AP-1 site was observed. An AP-1-dependent transcription assay was used to test the capability of both proteins to stimulate correctly initiated RNA synthesis in vitro. Under our conditions, c-jun protein was capable of stimulating specific RNA transcription in an AP-1 site-dependent manner. In contrast, c-fos protein showed no detectable transcriptional activation by itself. However, a transcription assay carried out in the presence of both c-fos and c-jun proteins showed that the c-fos/cjun complex was more active as a transcriptional regulator than c-jun protein alone. These experimental results indicate that c-fos and c-jun proteins are required to reconstitute full AP-1 -dependent transcriptional activation and directly demonstrate that c-fos is a regulator of gene expression.

Monobutyrine angiogenique et ses analogues

Abstract: On decrit des procedes pour stimuler l'angiogenese chez les animaux a sang chaud, qui consistent a administrer au sujet necessitant un tel traitement une certaine quantite d'un glyceride angiogenique de la formule (1) ou (2) dans lesquelles X est O, NH, S, ou CH2, et R1 est alkyle (1-10C) ou acyle (2-10C), sature ou non sature, et qui comporte une substitution ou non d'un ou plusieurs element(s) de substitution qui n'interfere(nt) pas avec l'activite angiogenique, lesdits elements de substitution etant choisis dans le groupe comprenant OH, OR, SH, SR, NH?2?, NHR, NR2 et halo, ou R est independamment alkyle (1-4C) inferieur; et chacun des R?2 et R3? est independamment H, PO?3?-2 ou un alkyle ou acyl tel que defini plus haut; ou dans la formule 1, R2 et R3 forment ensemble une moitie alkylene ou OR?2 et OR3? forment un epoxyde. Les composes de la formule 1 ou 2 ou les melanges de ceux-ci peuvent etre utilises en combinaison avec divers facteurs de croissance de peptides pour augmenter l'effet d'angiogenese.