Communicated by Fotis C. Kafatos, December 6, 1988 (received for review October 6, 1988) ABSTRACT rev (trs/art) is an essential human immunodeficiency virus type 1 (HIV-1) regulatory protein. rev increases the levels of the gag-and env-producing mRNAs via a cis-acting element in the env region of HIV-1, named revresponsive element. Our results show that rev increases the stability of the unspliced viral mRNA, while it does not affect the stability of the multiply spliced viral mRNAs that do not contain the rev-responsive element. The study of mutated proviral constructs producing mRNA that cannot be spliced revealed that the effect of rev on stability is independent of splicing. Our experiments also indicate that rev promotes the transport of the viral mRNA containing the rev-responsive element from the nucleus to the cytoplasm. The proposed functions of rev are consistent with its nuclear localization as shown by immunofluorescence. The selective effects of rev on the levels of the viral mRNA suggest a model for feedback regulation by rev leading to a steady state of viral expression.
Summary Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24 and miR-629. Moreover, we show that once this circuit is activated, it maintains suppression of HNF4a and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side-effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.
The orphan nuclear hormone receptor SHP interacts with a number of other nuclear hormone receptors and inhibits their transcriptional activity. Several mechanisms have been suggested to account for this inhibition. Here we show that SHP inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4 (HNF-4) and the retinoid X receptor (RXR) by at least two mechanisms. SHP interacts with the same HNF-4 surface recognized by transcriptional coactivators and competes with them for binding in vivo. The minimal SHP sequences previously found to be required for interaction with other receptors are sufficient for interaction with HNF-4, although deletion results indicate that additional C-terminal sequences are necessary for full binding and coactivator competition. These additional sequences include those associated with direct transcriptional repressor activity of SHP. SHP also competes with coactivators for binding to ligand-activated RXR, and based on the ligand-dependent interaction with other nuclear receptors, it is likely that coactivator competition is a general feature of SHP-mediated repression. The minimal receptor interaction domain of SHP is sufficient for full interaction with RXR, as previously described. This domain is also sufficient for full coactivator competition. Functionally, however, full inhibition of RXR transactivation requires the presence of the C-terminal repressor domain, with only weak inhibition associated with this receptor interaction domain. Overall, these results suggest that SHP represses nuclear hormone receptor-mediated transactivation via two separate steps: first by competition with coactivators and then by direct effects of its transcriptional repressor function.Nuclear hormone receptors are transcription factors whose activities are regulated by direct binding of small lipophilic molecules such as steroids, thyroid hormone, retinoids, and vitamin D 3 (24). These receptors have been studied extensively due to their important roles in the control of development and cellular homeostasis. The nuclear hormone receptor superfamily also includes numerous orphan receptors, which do not have identified ligands (10, 23). The structural similarities shared by superfamily members reside in two distinct functional domains. The first and most highly conserved is the DNA binding domain, also called the C domain, which targets receptors to specific DNA sequences known as hormone response elements. The second is the ligand binding domain (LBD), which is also called the E domain. It is located in the C-terminal region and is involved in several functions in addition to ligand binding, including dimerization and ligand-dependent transcriptional activation.Crystallographic studies (e.g., references 30, 43, and 46) have revealed that ligand binding provokes a conformational change in the receptors that allows them to bind a diverse group of proteins termed coactivators (reviewed in references 14, 37, and 47). These coactivators bind to a common surface of the receptors formed by sever...
The hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor superfamily and participates in the regulation of several genes involved in diverse metabolic pathways and developmental processes. To date, the functional domains of this nuclear receptor have not been identified, and it is not known whether its transcriptional activity is regulated by a ligand or other signals. In this report, we show that HNF-4 contains two transactivation domains, designated AF-1 and AF-2, which activate transcription in a cell type-independent manner. AF-1 consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic ␣-helical structure. In contrast, the AF-2 transactivator is complex, spanning the 128 -366 region of HNF-4, and it cannot be further dissected without impairing activity. The 360 -366 region of HNF-4 contains a motif that is highly conserved among transcriptionally active nuclear receptors, and it is essential for AF-2 activity, but it is not necessary for dimerization and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the 361-465 region bind efficiently to DNA as homo-and heterodimers and behave as dominant negative mutants. Remarkably, the full transactivation potential of AF-2 is inhibited by the region spanning residues 371-465 (region F). The inhibitory effect of region F on the HNF-4 AF-2 activity is a unique feature among members of the nuclear receptor superfamily, and we propose that it defines a distinct regulatory mechanism of transcriptional activation by HNF-4.The nuclear receptor superfamily comprises a large set of ligand-regulated transcription factors. This superfamily includes receptors for steroid hormones, retinoids, thyroid hormone, and vitamin D 3 , as well as a large number of structurally and functionally related transcription regulatory proteins whose natural ligands are not yet known, the so-called orphan receptors (reviewed in Refs. 1 and 2). Nuclear receptors exhibit a modular structure with six distinct regions (referred to as regions A-F), which correspond to functional domains. The N-terminal region A/B is highly variable among nuclear receptors and contains a ligand-independent transactivation function AF-1 (2). Region C contains a highly conserved DNA binding domain (DBD) 1 composed of two zinc-coordinated modules and is responsible for specific binding to cognate response elements (Refs. 1 and 2 and references therein). The exact functions of regions D and F are not clear, although they appear to be well conserved for each receptor across species. Region D is postulated to function as a flexible hinge between the DBD and the ligand-binding domain (LBD), allowing rotational differences between these domains when dimeric receptors bind to direct, inverted, or palindromic repeats (2). Interestingly, the D regions of the thyroid hormone (TR) and retinoic acid receptors (RARs) interact with ...
The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trslart) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels ofgag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.
Fat mass, adipocyte size and metabolic responsiveness, and preadipocyte differentiation decrease between middle and old age. We show that expression of CCAAT/enhancer binding protein (C/EBP)-alpha, a key regulator of adipogenesis and fat cell function, declined substantially with aging in differentiating preadipocytes cultured under identical conditions from rats of various ages. Overexpression of C/EBP alpha in preadipocytes cultured from old rats restored capacity to differentiate into fat cells, indicating that downstream differentiation-dependent genes maintain responsiveness to regulators of adipogenesis. C/EBP alpha-expression also decreased with age in fat tissue from three different depots and in isolated fat cells. The overall level of C/EBP beta, which modulates C/EBP alpha-expression, did not change with age, but the truncated, dominant-negative C/EBP beta-liver inhibitory protein (LIP) isoform increased in cultured preadipocytes and isolated fat cells. Overexpression of C/EBP beta-LIP in preadipocytes from young rats impaired adipogenesis. C/EBP delta, which acts with full-length C/EBP beta to enhance adipogenesis, decreased with age. Thus processes intrinsic to adipose cells involving changes in C/EBP family members contribute to impaired adipogenesis and altered fat tissue function with aging. These effects are potentially reversible.
The N-termnal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pue-chase experiments showed that the depletion of intracellular apoB-17follows an apparent first-order kinetics with tl2 = 51 min.Under conditions of continuous labeling, >60% of the total synthesized apoB-17 was secreted in a soluble form, =98% lipid-poor and -2% lipid-bound. Inclusion of 1.2 mM oleate resulted in 5-and 2.5-fold increases in the amount of labeled apoB-17 in the p < 1.063 g/ml and 1.063 < p < 1.21 g/ml fractions, respectively, which was coordinated with increased secretion of radiolabeled core lipids, triacylglycerols, and cholesteryl esters. Thus under conditions in which lipid pools are enriched a greater fraction of apoB-17 may be secreted on lipoprotein-like particles. The lipid-poor apoB-17 present in p > 1.21 g/ml readily associates with exogenously added dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles to form discoidal particles. Discs formed with DMPC/ apoB-17, 7:1 (wt/wt), are 239 ± 43 A in diameter and 61 ± 4 A thick and contain -2 molecules of apoB-17 and 2250 molecules of DMPC per disc. Based on volume calculations we conclude that apoB-17 forms an annulus about one bilayer high and 10 A thick surrounding the DMPC disc. Circular dichroic spectra of apoB-17 on DMPC discs showed apoB-17 to contain 39% a-helix, 36% 13-sheet, 9% (3-turn, and 16% random coil.To be consistent with this model >70% of apoB-17 on DMPC discs must bind to lipid. These data suggest that the N-terminal 17% of apoB-100 can bind lipid and may contribute to some extent to the stabilization of triglyceride-rich lipoproteins.
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