Dual control of C-reactive protein gene expression by interleukin-1 and interleukin-6.

Human C-reactive protein (CRP) is the major acute phase reactant during acute inflammation. The human CRP promoter is expressed in an inducible and cell-specific manner when linked to the bacterial CAT gene and transfected into human hepatoma cell cultures. In this paper we analyze the effect of several recombinant cytokines or CRP promoter inducibility in human Hep3B cells. When cytokines are tested singly the major inducer of CRP-CAT fusions is interleukin-6 (IL-6). Maximal CAT gene expression, however, is only achieved when both interleukin-1 beta (IL-1 beta) and IL-6 are present. The response to the two cytokines is cooperative. Cooperativity is maintained when the CRP promoter is linked to a different coding region, that of the bacterial neomycin phosphotransferase II gene. With a series of 5' and 3' deletions we show the existence of two distinct and independent regions responsive to IL-6 and located upstream to the TATA box. The IL-1 effect is exerted at the level of downstream sequences that are probably important for optimal mRNA translatability or nuclear-cytoplasmic transport. Inducibility is not influenced by the activation of protein kinases C or A and does not require new protein synthesis.     

Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages.

Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene.     

Suppression of interleukin-6-induced C-reactive protein expression by FXR agonists

C-reactive protein (CRP), a human acute-phase protein, is a risk factor for future cardiovascular events and exerts direct pro-inflammatory and pro-atherogenic properties. The farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. In this study, we report that two synthetic FXR agonists, WAY-362450 and GW4064, suppressed interleukin-6-induced CRP expression in human Hep3B hepatoma cells. Knockdown of FXR by short interfering RNA attenuated the inhibitory effect of the FXR agonists and also increased the ability of interleukin-6 to induce CRP production. Furthermore, treatment of wild type C57BL/6 mice with the FXR agonist, WAY-362450, attenuated lipopolysaccharide-induced serum amyloid P component and serum amyloid A3 mRNA levels in the liver, whereas no effect was observed in FXR knockout mice. These data provide new evidence for direct anti-inflammatory properties of FXR.     

The human haptoglobin gene promoter: interleukin-6-responsive elements interact with a DNA-binding protein induced by interleukin-6

[This corrects the article on p. 1145 in vol. 8, PMID: 2787245.].     

High altitude increases circulating interleukin-6, interleukin-1 receptor antagonist and C-reactive protein

Hypoxic pulmonary vasoconstriction is associated with but may not be sufficient for the development of high-altitude pulmonary oedema (HAPO). Hypoxia is known to induce an inflammatory response in immune cells and endothelial cells. It has been speculated that hypoxia-induced inflammatory cytokines at high altitude may contribute to the development of HAPO by causing capillary leakage in the lung. We were interested if such an inflammatory response, possibly involved in a later development of HAPO, is detectable at high altitude in individuals without HAPO. We examined the plasma levels of interleukin 6 (IL-6), interleukin 1 receptor antagonist (IL-1ra) and C-reactive protein (CRP) in two independent studies: study A, Jungfraujoch, Switzerland, three overnight stays at 3458 m, n=12; study B: Capanna Regina Margherita, Italy, 3 overnight stays at 3647 m and one overnight stay at 4559 m, n=10. In both studies, probands showed symptoms of acute mountain sickness but no signs of HAPO. At the Jungfraujoch, IL-6 increased from 0.1+/-0.03 pg/ml to 2. 0+/-0.5 pg/ml (day 2, P=0.03), IL-1ra from 101+/-21 to 284+/-73 pg/ml (day 2, P=0.01), and CRP from 1.0+/-0.4 to 5.8+/-1.5 micrograms/ml (day 4, P=0.01). At the Capanna Margherita, IL-6 increased from 0. 5+/-0.2 pg/ml to 2.0+/-0.8 pg/ml (P=0.02), IL-1ra from 118+/-25 to 213+/-28 pg/ml (P=0.02), and CRP from 0.4+/-0.03 to 3.5+/-1.1 micrograms/ml (P=0.03). IL-8 was below the detection limit of the ELISA (<25 pg/ml) in both studies. The increase of IL-6 and IL-1ra in response to high altitude was delayed and preceded the increase of CRP. We conclude that: (1) circulating IL-6, IL-1ra and CRP are upregulated in response to hypobaric hypoxic conditions at high altitude, and (2) the moderate systemic increase of these inflammatory markers may reflect considerable local inflammation. The existence and the kinetics of high altitude-induced cytokines found in this study support the hypothesis that inflammation is involved in the development of HAPO.     

Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.

Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.     

Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression.

The Polycomb group and trithorax group genes of Drosophila are required for maintaining the differential expression state of developmental regulators, such as the homeotic genes, in a stable and heritable manner throughout development. The Polycomb group genes have been suggested to act by regulating higher order chromatin and packaging repressed chromosomal domains in a heterochromatin-like structure. We have mapped, at high resolution, the distribution of Polycomb protein on the bithorax complex of Drosophila tissue culture cells, using an improved formaldehyde cross-linking and immunoprecipitation technique. Polycomb protein is not distributed homogeneously on the regulatory regions of the repressed Ultrabithorax and abdominal-A genes, but is highly enriched at discrete sequence elements, many of which coincide with previously mapped Polycomb group response elements (PREs). Our results further suggest that Polycomb protein spreads locally over a few kilobases of DNA surrounding PREs, perhaps to stabilize silencing complexes. GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus-binding sites. Two modes of binding can be distinguished: a high level binding to elements in the regulatory domain of the expressed Abdominal-B gene, and a low level of binding to Polycomb-bound PREs in the inactive domains of the bithorax complex. We propose that GAGA factor binds constitutively to regulatory elements in the bithorax complex, which function both as PREs and as trithorax group response elements.