Structure, hormonal regulation, and identification of the interleukin-6- and dexamethasone-responsive element of the rat haptoglobin gene.

Hepatic expression of the haptoglobin (Hp) gene in mammalian species is stimulated severalfold during an acute-phase reaction. To identify the molecular mechanism responsible for this regulation, the single-copy rat Hp gene has been isolated. The genomic sequences showed a high degree of homology with the primate Hp gene. Activity of the rat Hp gene was increased in cultured liver cells by interleukin-1 (IL-1), IL-6, and glucocorticoids. The genomic Hp gene sequence spanning from -6500 to +6500, when transiently introduced into human hepatoma (HepG2) cells, directed IL-6- and dexamethasone-stimulated expression of rat Hp mRNA and protein. No response to IL-1 was detected, suggesting that the corresponding regulatory element(s) might lie outside of the tested gene sequences. An IL-6- and dexamethasone-responsive element has been localized to the promoter proximal region -146 to -55. Although the nucleotide sequences of this rat Hp gene region showed substantial divergence from that of the human gene, analysis of sequential 5' and 3' deletion constructs indicated an arrangement of functional IL-6 response elements in the rat Hp promoter sequence comparable to that of the human homolog. The magnitude of IL-6 regulation through the rat Hp gene promoter was severalfold lower than that of the human Hp gene. The reduced activity could be ascribed to a single-base difference in an otherwise conserved sequence corresponding to an active element in the human gene. The IL-6 response of the rat Hp element was improved severalfold by substituting that base with the human nucleotide.     

Divergent homeobox gene hex regulates promoter of the Na(+)-dependent bile acid cotransporter

The divergent homeobox gene Hex is expressed in both developing and mature liver. A putative Hex binding site was identified in the promoter region of the liver-specific Na(+)-bile acid cotransporter gene (ntcp), and we hypothesized that Hex regulates the ntcp promoter through this site. Successive 5'-deletions of the ntcp promoter in a luciferase reporter construct transfected into Hep G2 cells confirmed a Hex response element (HRE) within the ntcp promoter (nt -733/-714). Moreover, p-CMHex transactivated a heterologous promoter construct containing HRE multimers (p4xHRELUC), whereas a 5-bp mutation of the core HRE eliminated transactivation. A dominant negative form of Hex (p-Hex-DN) suppressed basal luciferase activity of p-4xHRELUC and inhibited activation of this construct by p-CMHex. Interestingly, p-CMHex transactivated the HRE in Hep G2 cells but not in fibroblast-derived COS cells, suggesting the possibility that Hex protein requires an additional liver cell-specific factor(s) for full activity. Electrophoretic mobility shift assays confirmed that liver and Hep G2 cells contain a specific nuclear protein that binds the native HRE. We have demonstrated that the liver-specific ntcp gene promoter is the first known target of Hex and is a useful tool for evaluating function of the Hex protein.     

Structural and functional homology between the 29 kD rat liver nucleoprotein and the high mobility group 1 protein

A 29 kD soluble rat liver nucleoprotein (p29) has increased binding affinity for the hormone responsive element (RE) of the rat haptoglobin (Hp) gene during the acute-phase reaction. In this work the possibility of its structural and functional homology to the high mobility group 1 (HMG1) nonhistone protein constituent of chromatin was examined. The results of two-dimensional gel electrophoresis, Southwestern and Western immunoblot analyses, showed that p29 and HMG1 are homologous protein species. On the basis of in vitro and in vivo phosphorylation/dephosphorylation experiments, we discuss the modulatory role of phosphate groups in view of the structure and function of p29.     

Expression of the rat liver haptoglobin gene is mediated by isoforms of C/EBPalpha, -beta and -delta proteins

CCAAT/enhancer binding proteins (C/EBP) alpha, -beta and -delta play an important role in mediating I interleukin-6 (IL-6) dependent expression of acute-phase protein (APP) genes in liver during acute-phase (AP) response. Based on the presence of type IL-6 responsive element (IL-6 RE) in the rat haptoglobin (Hp) gene promoter we assumed that some C/EBPalpha, -beta and/or -delta isoforms could mediate the expression of this gene during turpentine-induced AP response. By Western immunoblot and Northern blot assays, we found that turpentine treatment of rats led to a coordinate induction of C/EBPbeta and -delta as well as repression of C/EBPalpha isoforms pool levels in rat liver nuclear extracts (NEs) which was preceded by an adequate alteration of their mRNAs expression in liver. Consequently, results of DNA affinity chromatography revealed that affinity of certain C/EBPalpha isoforms to bind the type I IL-6 RE within the rat Hp gene promoter decreased whereas affinities of certain C/EBPbeta isoforms and C/EBPdelta were increased and induced, respectively. Our data suggest that turpentine-induced alterations of C/EBPalpha, -beta and -delta pool levels and DNA-binding activities can be regarded as an integral part of activation of the Hp gene expression in the course of AP response.     

Progesterone and regulation of the follicle-stimulating hormone (FSH-beta) gene.

Progesterone (P) biphasically modulates follicle-stimulating hormone (FSH) secretion in the rat both in vivo and in vitro with the duration of estrogen priming determining the biphasic nature of the P action, probably through estrogen up-regulation of the anterior pituitary progesterone receptor (PR) levels. P has been also shown to regulate anterior pituitary levels of FSH-beta mRNA in the rat. Although the mechanism of this action has not been determined, steroids may regulate gene expression through the binding of liganded receptors to gene sequences known as hormone response elements (HRE); however, it is not known whether HRE's exist on the rat FSH-beta gene. We have localized a series of progesterone response elements (PRE)-like sequences on the rat FSH-beta gene and have begun testing the hypothesis that P modulates the expression of the rat FSH-beta gene through the direct binding of the P/PR complex to these PRE-like sequences. Electromobility shift assays indicate that these PRE-like sequences bind PR with high affinity and specificity. In addition, when a 361-base pair sequence, which contains the three PRE-like sequences localized in the upstream region of the gene, was cloned into a luciferase expression vector driven by a heterologous promoter and transiently transfected into anterior pituitary cell cultures, progestin stimulation elicited increased luciferase expression. These results indicated that the 361-base pair sequence conferred P-responsiveness to a heterologous promoter. The data further suggest that FSH synthesis in the rat is modulated by direct binding of PR to PRE-like sequences.     

O-GlcNAc inhibits interaction between Sp1 and sterol regulatory element binding protein 2

O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine on the serine and threonine residues of nucleocytoplasmic proteins, is a novel protein modification that is ubiquitous among eukaryotes and implicated in cell regulation. Recent evidence indicates that O-GlcNAc regulates protein-protein interactions. Here we provide evidence that O-GlcNAc interrupts a known interaction between Sp1 and sterol regulatory element binding protein 2 (SREBP2), thereby inhibiting expression of the gene encoding acetyl-CoA synthetase 1, which is involved in lipid synthesis. This study suggests a novel mechanism in which lipid biosynthesis may be regulated by O-GlcNAc.