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S M Hollenberg

Publications and source records attributed to S M Hollenberg.

At least 55 records · Page 3Linked to original sources
PubMed1993

Use of a conditional MyoD transcription factor in studies of MyoD trans-activation and muscle determination.

DNA sequences encoding the hormone-binding domains of several steroid hormone receptors were fused in frame to the MyoD gene. When the gene for this chimeric protein was expressed in NIH 3T3 or 10T1/2 fibroblasts, these cells displayed hormone-dependent induction of myogenesis. Our experiments focused on cell lines expressing estrogen receptor-MyoD chimeras. Induction of these lines in the presence of estradiol and an inhibitor of protein synthesis, cycloheximide, resulted in the activation of the endogenous myogenin gene but did not activate the muscle-specific creatine kinase or cardiac alpha-actin gene. This result suggests that MyoD is not a "direct" activator of these downstream myogenic genes but must first activate myogenin as an intermediary. Once muscle is induced by estrogen receptor-MyoD the muscle phenotype is very stable and does not need the continued presence of estradiol for its maintenance.

3T3 Cells↗
PubMed1993

Mammalian Ras interacts directly with the serine/threonine kinase Raf.

We have identified proteins that interact with H-Ras using a two hybrid system screen of a mouse cDNA library. Approximately 50% of the clones identified encoded portions of the c-Raf and A-Raf serine/threonine kinases. Overlaps among these clones define a conserved 81 residue region of the N-terminus of Raf as the Ras interaction region. We show that Raf interacts with wild-type and activated Ras, but not with an effector domain mutant of Ras or with a dominant-interfering Ras mutant. Using purified bacterially expressed fusion proteins, we show, furthermore, that Ras and the N-terminal region of Raf associate directly in vitro and that this interaction is dependent on GTP bound to Ras.

Amino Acid Sequence↗
PubMed1993

Tachyphylaxis to the vasopressor effects of endothelin in rat aortic rings.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide released by endothelial cells, binds with high affinity to surface receptors and is highly resistant to dissociation. We observed tachyphylaxis to the pressor effects of a second application of ET-1 in rat aortic rings and investigated the mechanism of this effect. Developed tension increased progressively with doses of ET-1 ranging from 1 to 500 nM (P < 0.001), and tensions with rechallenge were correspondingly decreased (P < 0.001). In response to 500 nM ET-1, tension increased 1,599 +/- 72 (SE) mg/mg ring wt. Rechallenge with 500 nM ET-1 led to contraction of only 33 +/- 40 mg/mg ring wt. Tachyphylaxis was seen up to 6 h after initial challenge. Pretreatment with nicardipine, lidoflazine, nitroglycerin, and sphingosine did not affect tachyphylaxis. Pretreatment with 500 microM dansylcadaverine (an inhibitor of endothelin internalization) markedly inhibited ET-1-induced contraction and also inhibited tachyphylaxis to ET-1. Further studies with radiolabeled ET-1 suggested that subsequent ET-1 binding is markedly decreased after an initial ET-1 challenge. Dansylcadaverine inhibited ET-1 internalization and also inhibited the decreased binding seen with ET-1 rechallenge. Rat aortic rings demonstrate tachyphylaxis to the pressor effect of a second dose of ET-1. The mechanism appears to be related to binding and subsequent internalization of endothelin-receptor complexes. This effect suggests a possible mechanism for sustained decreases in systemic vascular resistance.

Animals↗
PubMed1993

Nitric oxide synthase inhibition reverses arteriolar hyporesponsiveness to catecholamines in septic rats.

Induction of nitric oxide synthase by cytokines has been hypothesized as a mechanism of the hyporesponsiveness to catecholamines that occurs in clinical septic shock. We measured responses of resistance arterioles in rat cremaster muscle to topically suffused norepinephrine in vivo with the use of image-shearing videomicroscopy. Rats made septic by cecal ligation and puncture were compared with controls that underwent sham ligation. The norepinephrine concentration-response curve was shifted to the right in septic rats [50% effective concentration (EC50) 9.1 +/- 5.4 vs. 0.10 +/- 0.02 microM, P < 0.05]. Contractions at doses of 10(-9), 10(-8), and 10(-7) M norepinephrine were 26, 41, and 38%, respectively, of sham controls. Superfusion of the muscle with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine at 100 microM restored the arteriolar responsiveness of the septic rats (EC50 0.14 +/- 0.07 vs. 6.8 +/- 3.1 microM, P < 0.05). This effect was reversed with superfusion of excess (1 mM) L-arginine. These experiments demonstrate impaired vasoconstriction in response to norepinephrine in resistance arterioles of septic rats in vivo. NG-monomethyl-L-arginine reversed this hyporesponsiveness, implying that nitric oxide synthase may mediate the decreased catecholamine responsiveness associated with sepsis.

Amino Acid Oxidoreductases↗
PubMed1992

Effect of septic serum on vascular smooth muscle: in vitro studies using rat aortic rings.

BACKGROUND AND METHODS: Septic shock in humans is characterized by hypotension, low systemic vascular resistance, and high cardiac output. We hypothesized that circulating vasodilatory substances are, in part, responsible for this low systemic vascular resistance. To investigate this possibility, we exposed isolated rat aortic rings to sera from patients with septic shock and to sera from dogs made septic by intraperitoneal implantation of infected clots. Isolated rings from rat thoracic aortas were mounted on hooks in chambers filled with modified Krebs' buffer and bubbled with 95% oxygen/5% CO2. After documentation of functional endothelium, the rings were precontracted with norepinephrine. Serum was then added and ring tension measured continuously over the next 20 mins. RESULTS: Sera from normal humans increased ring tension by 6% (60 +/- 39 [SEM] mg tension/mg ring). Sera from nine patients with septic shock decreased tension by 30% (350 +/- 42 mg tension/mg ring; p less than .001). In response to sera from control dogs, tension increased by 16% (122 +/- 37 mg tension/mg ring). In contrast, septic dog sera caused tension to decrease by 36% (278 +/- 38 mg tension/mg ring;p less than .001). Vasodilation was unaffected when the septic patient and dog sera were dialyzed to remove molecules less than 10,000 molecular weight. Chemical deendothelialization with sodium deoxycholate also did not affect the vasodilatory response to septic patient or dog sera. CONCLUSIONS: Septic sera can relax rat aortic smooth muscle. Dialyzing to exclude the effects of small molecules and removing the endothelium did not eliminate this vasodilatory response. These data suggest the presence in septic serum of circulating substances capable of relaxing vascular smooth muscle that may play an important role in the pathogenesis of the cardiovascular abnormalities in septic shock.

Animals↗
PubMed1991

Heat shock protein 90 as a critical factor in maintaining glucocorticosteroid receptor in a nonfunctional state.

Previous work demonstrated that the ligand binding domain (LBD) was required to determine the formation of the cytosolic, untransformed, inactive, 8 S, heterooligomeric form of the human glucocorticosteroid receptor (hGR) which includes the 90-kDa heat shock protein (hsp90) (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguère, V., Cadepond, F.M., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). Truncations of hGR deleting all or almost all of the LBD give GR derivatives in the non-hsp90-interacting 4 S form able to stimulate transcription in a hormone-independent manner. To identify the LBD subregion(s) involved in 8 S formation, we analyzed the sedimentation behavior of hGR mutants with various LBD internal deletions and/or truncations transiently expressed in cells that contain hsp90 but very low levels of endogenous GR, and we correlated the results with their transcriptional activity. LBD has been divided into three subregions: two of them, LBD1 (between amino acids 551 and 626) and LBD2 (between amino acids 627 and 696), include amino acid sequences highly conserved in the steroid receptor superfamily, and LBD3 consists of the carboxyl-terminal part of the molecule (amino acids 697-777). Each of these subregions can be deleted without impeding 8 S heterooligomer formation, and the corresponding receptors do not have transcriptional activity in the absence as well as in the presence of hormone. When linked to hGR mutants truncated after amino acids 532 or 550, each subregion does separately promote 8 S heterooligomeric complex formation and repress the intrinsic constitutive transcriptional activity of the truncated receptors. These 8 S complexes contain hsp90. In a control experiment, the linkage of 1,017 amino acids of beta-galactosidase to the carboxyl-terminal of 1-532 hGR gave a hybrid receptor still constitutively transcriptionally active which did not bind hsp90. These results provide evidence that there is a strong correlation between the association with hsp90 and the loss of GR functional properties and that hsp90 may play a critical role in maintaining the receptor in a nonfunctional state.

Blotting, Western↗
PubMed1991

The effect of tumor necrosis factor on vascular smooth muscle. In vitro studies using rat aortic rings.

The mechanisms of vasodilation in septic shock have not been well elucidated. We used isolated rat aortic rings as an in vitro model of vascular reactivity to assess the acute, direct effects on vascular smooth muscle relaxation of four cytokines: interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 6 (IL-6), and tumor necrosis factor (TNF). The rings were precontracted with catecholamines and then test cytokines were added. The changes in tension with IL-1 at 1,000 U/ml, IL-2 at 1,000 U/ml, and IL-6 at 500 U/ml differed from controls by -67 +/- 59, -5 +/- 42, and 8 +/- 56 mg/mg of tissue, respectively (for each, n = 10, p = NS). The change in tension with TNF at 1,000 U/ml differed from controls by -176 +/- 42 mg/mg of tissue (n = 20), p less than 0.001). Chemical removal of the endothelium with deoxycholate diminished TNF-induced vasodilation to -62 +/- 14 mg/mg of tissue (p less than 0.05). The relaxation with TNF occurred in a concentration-dependent fashion and was unaffected by indomethacin. This study demonstrates that TNF has an acute, concentration-dependent, cyclooxygenase-independent, vasodilatory effect on vascular smooth muscle. The effect of TNF was partially, but not fully, dependent on the presence of an intact endothelium, implying that TNF acts on both the endothelium and the smooth muscle. These findings suggest that TNF may play an important role in the vasodilation characteristic of septic shock.

Animals↗
PubMed1989

Identification of two regions of the human glucocorticoid receptor hormone binding domain that block activation.

The steroid-binding domain of the human glucocorticoid receptor has a dominant, negative influence over the activity of the remainder of the molecule. We have determined sequences within this domain that are responsible for this inhibition by creating truncations and internal deletions which remove part or all of the ligand-binding domain. Two apparently unrelated sequences within this region are shown to block the ability to stimulate transcription when fused to the carboxyl terminus of a functional, hormone-independent receptor derivative. These results support the possibility of two distinct protein-binding sites within the steroid-binding domain.

Amino Acid Sequence↗
PubMed1989

Fusion of adenovirus E1A to the glucocorticoid receptor by high-resolution deletion cloning creates a hormonally inducible viral transactivator.

The 289-amino-acid E1A protein of adenovirus type 2 stimulates transcription from early viral and certain cellular promoters. Its mechanism is not known, and there exist no temperature-sensitive mutants of E1A that could help to elucidate the details of E1A transcriptional activation. To create for E1A such a conditional phenotype, we fused portions of E1A to the human glucocorticoid receptor (GR) to make transactivation by E1A dependent on the presence of dexamethasone. Nested subsets of the E1A coding region, centered around the 46-amino-acid transactivating domain, were substituted for the DNA-binding domain of the GR. One of the resulting chimeric proteins (GR/E1A-99), which included the entire E1A transactivating domain, stimulated expression from a viral early promoter (E3) exclusively in the presence of hormone. GR/E1A-99 did not transactivate a GR-responsive promoter. It therefore exhibited the promoter specificity of E1A while possessing the hormone inducibility of the GR. Two smaller chimeras that contained only portions of the E1A transactivating domain failed to transactivate E3. These three chimeras were constructed by a novel strategy, high-resolution deletion cloning. In this procedure, series of unidirectional deletions were made with exonuclease III on each side of the E1A coding region at a resolution of 1 to 2 nucleotides. The large number of in-frame fragments present in the collection of deleted clones facilitated the construction of the GR/E1A chimeras and can be used to create many additional fusions.

Adenovirus Early Proteins↗
PubMed1988

Transcriptional inhibition by a glucocorticoid receptor-beta-galactosidase fusion protein.

Binary developmental decisions and homeostatic regulation by steroids require negative transcriptional regulation to balance steroid-mediated stimulatory effects. Human glucocorticoid receptor mutants were used to identify regions important for trans-repression of the gene encoding the alpha subunit of chorionic gonadotropin. While the amino terminus is not critical, the DNA binding and ligand binding domains are required for efficient repression. However, the function of the carboxyl terminus can be substituted by a polypeptide from the human mineralocorticoid receptor or beta-galactosidase gene. The function of these fusion repressors supports the model that the human glucocorticoid receptor negatively regulates transcription via a steric hindrance mechanism. These results suggest a potentially general strategy for creation of sequence-specific transcriptional repressors.

Cell Line↗
PubMed1988

Multiple and cooperative trans-activation domains of the human glucocorticoid receptor.

A 30 amino acid peptide (referred to as tau 2) that functions as an activation domain has been localized in the carboxyl terminus of the human glucocorticoid receptor. This sequence, when fused to yeast GAL4 as part of the ligand binding domain, generates a hormone-inducible activator. Tau 2 functions in a position-independent fashion and leads to a progressive gain of function when multimerized. A similar and independent activity has also been identified in the amino terminus of the receptor. These two sequences, although structurally unrelated, are both acidic in character and thus may have certain properties in common with yeast activator sequences.

Amino Acid Sequence↗
PubMed1988

Expression of synthetic thaumatin genes in yeast.

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.

Amino Acid Sequence↗
PubMed1988

A region in the steroid binding domain determines formation of the non-DNA-binding, 9 S glucocorticoid receptor complex.

This work was initiated to determine if a specific region of the glucocorticoid receptor determines the formation of the inactive (i.e. non-DNA-binding) 9 S form of the receptor recovered in cytosol preparations. It is known that the murine glucocorticoid receptor of the nti phenotype, which consists of only the carboxyl-terminal 40-kDa peptide containing the DNA-binding and steroid-binding domains separated by a short linker region, is recovered in hypotonic lysates as a 9 S heteromeric complex (Gehring, U., and Arndt, H. (1985) FEBS Lett. 179, 138-142). To further localize the domain required for formation of the 9 S complex, we have determined the sedimentation coefficients of receptors produced in COS-7 cells transfected with several mutants of the human glucocorticoid receptor gene. Deletion of the DNA-binding domain results in a 9 S complex that is somewhat less stable than the wild type receptor during sucrose gradient centrifugation. Deletion of the linker region yields a molybdate-stabilized 9 S complex, but deletion of the entire steroid-binding domain or internal deletion of the amino-terminal two-thirds of this domain yields receptors that are constitutive transcriptional activators and are present in cytosol only in the 4 S form. Taken together, these observations demonstrate that the steroid-binding domain contains the features required for formation of the 9 S heteromeric complex, and they are consistent with the proposal that the steroid-binding domain normally represses receptor function.

Amino Acid Sequence↗
PubMed1987

Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor.

Using a combination of a transient expression assay and in vitro mutagenesis, we showed previously that the human glucocorticoid receptor (hGR) is composed of a series of discrete functional domains. Here we report the effects of selective deletion of each of these domains on hGR ability to activate transcription of the MTV-CAT fusion gene. Deletion of the immunogenic domain or the entire amino-terminal half of the protein reduces but does not abolish the ability of the hGR to induce transcriptional activation. Somewhat surprisingly, deletion of the steroid-binding domain engenders a constitutively active receptor, revealing that this domain normally represses receptor function. However, the central, cysteine-rich region contains all the information required for both DNA binding and trans-activation. Taken together, these data delineate a core domain in the hGR spanning 88 amino acids that determines both DNA-binding and transcriptional activation functions. This physical linkage distinguishes the glucocorticoid receptor from other described eukaryotic regulatory proteins, where these two functions have been shown to be separable.

Animals↗