Interleukin-1 Receptor Antagonist Gene Expression Research Articles

Influence of melatonin on IL-1Ra gene and IL-1 expression in rats with liver ischemia reperfusion injury.

The aim of the present study was to explore the influence of melatonin (MT) on rats with liver ischemia reperfusion injury (IRI) and its mechanism. A total of 66 male Sprague-Dawley rats were randomly divided into 3 groups: i) Normal control group, ii) ischemia reperfusion group (IR group) and iii) melatonin treatment group (MT group). Rats in the MT group were administered an intraperitoneal injection of MT (10 mg/kg, 1 ml) at 70 and 35 min before ischemia, early reperfusion, and 1 and 2 h after reperfusion, respectively. Blood was removed at the normal time-point (prior to any processes), 35 min before ischemia, 2, 4, 8 and 24 h after reperfusion. Subsequently the rats were sacrificed. The pathological changes of liver tissues, interleukin-1 receptor antagonist (IL-1Ra) gene and IL-1 expression levels were detected. There were no evident differences between the immediate reperfusion and 2 h IR group and MT group. The liver structure injury of the 4, 8 and 24 h MT groups were improved to various differences compared to the corresponding IR group; liver IL-1β of the MT group at 35 min after ischemia, and 2, 4, 8 and 24 h after reperfusion was evidently lower than that of the IR group (P<0.05); IL-1Ra mRNA expression in the 2 h MT group was higher compared to the 2 h IR group by 4.85-fold; and IL-1Ra mRNA expression in the 4 h MT group was higher compared to the 4 h IR group by 9.34-fold. Differences between two groups at other time-points were <2-fold. In conclusion, MT can upregulate IL-1Ra gene expression by reducing generation of IL-1 thus reducing IRI.

Lentivirus transduced interleukin-1 receptor antagonist gene expression in murine bone marrow-derived mesenchymal stem cells in vitro.

Genetically modified mesenchymal stem cells have been used in attempts to increase the expression of interleukin-1 receptor antagonist (IL-1Ra); however, the attempts thus far have been unsuccessful. The aim of the present study was to investigate whether the lentivirus transduced IL-1Ra gene was able to be stably expressed in murine bone marrow-derived mesenchymal stem cells (mBMSCs) in vitro. In the present study, third generation lentiviral (Lv) vectors transducing the IL-1Ra/green fluorescent protein (copGFP) gene were constructed and transfected into mBMSCs to establish the Lv.IL-1Ra.copGFP/mBMSCs, which were evaluated using fluorescence microscopy, flow cytometry, cell viability analysis using a cell counting kit-8 kit, Trypan blue staining and an MTT growth kinetics assay. The expression of IL-1Ra was analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that the Lv.IL-1Ra/copGFP vector was successfully constructed. The mBMSCs exhibited a short proliferation life, however they had good growth kinetics at an early stage and improved viability following efficient transduction of the IL-1Ra gene. IL-1Ra was overexpressed following transfection of mBMSCs. In conclusion, lentiviral vector transduced mBMSCs were able to efficiently express exogenous Il-1Ra under certain conditions and had a marked capacity for proliferation.

SB203580 enhances interleukin-1 receptor antagonist gene expression in IFN-γ-stimulated BV2 microglial cells through a composite nuclear factor-κB/PU.1 binding site

Interleukin-1 receptor antagonist (IL-1ra) is a naturally occurring antagonist of IL-1α and IL-1β binding to the IL-1 receptors and alleviates various inflammatory reactions. Therefore, the upregulation of IL-1ra expression is important for preventing and/or treating inflammatory diseases including many neurodegenerative diseases. This study found that SB203580, which is generally known as a p38 MAP kinase inhibitor and an anti-inflammatory agent, increased the level of IL-1ra expression in IFN-γ-stimulated BV2 microglial cells. This effect is believed to occur through the inhibition of protein kinase B (PKB), independently of the p38 MAP kinase pathways. Further mechanistic studies using an IL-1ra promoter revealed that a composite NF-κB/PU.1 binding site plays an important role in this SB203580-mediated upregulation of IL-1ra. Considering that IFN-γ is a major stimulator of the innate and adaptive immune responses, the upregulation of anti-inflammatory IL-1ra expression by SB203580 in the IFN-γ-stimulated microglia might provide a new therapeutic modality for various inflammatory diseases of the central nervous system.

Interleukin-1 Receptor Antagonist Induction as an Additional Mechanism for Liver Receptor Homolog-1 to Negatively Regulate the Hepatic Acute Phase Response

The liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor believed to play a key role in bile acid metabolism, cholesterol homeostasis, and intestinal cell crypt renewal. LRH-1 has recently been reported to negatively regulate the hepatic acute phase response by antagonizing, at least in part, the CCAAT/enhancer-binding protein signaling pathway. Here we have shown, using adenovirus-mediated LRH-1 overexpression and gene-silencing experiments, that the interleukin-1 receptor antagonist (IL-1RA) gene is a novel LRH-1 target gene in hepatic cells. Promoter mapping and chromatin immunoprecipitation experiments revealed that LRH-1 regulates IL-1RA gene expression under inflammatory conditions at the transcriptional level via the binding to an LRH-1 response element. Interestingly, IL-1RA induction by an intraperitoneal injection of lipopolysaccharide is significantly lower in LRH-1 heterozygous compared with wild-type mice, demonstrating the contribution of LRH-1 in IL-1RA gene regulation. Finally, RNA interference experiments indicate that LRH-1 blocks the hepatic acute phase response by, at least in part, inducing IL-1RA expression. Taken together, these results lead to the identification of IL-1RA as a novel LRH-1 target gene and demonstrate the existence of multiple mechanisms contributing to the overall anti-inflammatory properties of LRH-1 in hepatic cells.

Association of interleukin-1 and interleukin-1 receptor antagonist gene expression and preterm birth

Association of interleukin-1 and interleukin-1 receptor antagonist gene expression and preterm birth

Specificity from Toll Receptors

The Toll-like receptors 2 and 4 (TLR2 and TLR4) function in detection of bacterial infection, responding to lipopolysaccharide (LPS) from Gram-negative bacteria or peptidoglycan (PGN) from Gram-positive bacteria, respectively. Although the two receptors generate similar signaling outputs, evidence is accumulating that responses to the two are notably distinct. In fact, Carl et al. show that even when using the same signaling elements, the TLR2 and TLR4 signals act distinctly to control expression of a single gene target. The authors monitored activation of the gene encoding the secretory interleukin-1 receptor antagonist, sIL-1Ra. Although activation of either TLR2 or TLR4 activated expression of sIL-1Ra mRNA, the two signals acted synergistically. In both cases, promoter deletion analysis showed that the transcription factor PU.1 was required for a full response, and pharmacological inhibition of the p38 stress-activated protein kinase showed that the kinase was also required for full transcriptional activation. Nevertheless, these common elements had distinct roles depending on whether cultured murine macrophages were treated with LPS or PGN. The promoter regions required for the responses to TLR2 or TLR4 were different and even the p38-dependent response mapped to distinct regions of the promoter, with the response to TLR2-activated p38 requiring PU.1, whereas the p38-mediated signal from TLR4 did not. The authors conclude that TLR2 and TL4 must activate different DNA binding proteins (to account for the distinct promoter regions required) and that, even when using very similar pathways (through p38 and PU.1), signals from the two receptors result in distinct gene regulatory responses. V. S. Carl, K. Brown-Steinke, M. J. H. Nicklin, M. F. Smith Jr., Toll-like receptor 2 and 4 (TLR2 and TLR4) agonists differentially regulate secretory interleukin-1 receptor antagonist gene expression in macrophages. J. Biol.Chem. 277 , 17448-17456 (2002). [Abstract] [Full Text]

Toll-like Receptor 2 and 4 (TLR2 and TLR4) Agonists Differentially Regulate Secretory Interleukin-1 Receptor Antagonist Gene Expression in Macrophages

Treatment of macrophages with lipopolysaccharide (LPS) from Gram-negative bacteria or peptidoglycan (PGN) from Gram-positive bacteria activates multiple intracellular signaling pathways and a large, diverse group of nuclear transcription factors. The signaling receptors for PGN and LPS are now known to be the Toll-like receptors 2 and 4 (TLR2 and -4, respectively). While a large body of literature indicates that the members of the TLR family activate nearly identical cytoplasmic signaling programs, several recent reports have suggested that the functional outcomes of signaling via TLR2 or TLR4 are not equivalent. In the current studies, we compared the responses of the secretory IL-1 receptor antagonist (sIL-1Ra) gene to both LPS and PGN. Both LPS and PGN induced IL-1Ra gene expression; however, the combination of both stimuli synergistically increased sIL-1Ra mRNA expression and promoter activity, suggesting that the signals induced by PGN and LPS are not equivalent. While both LPS and PGN utilized the PU.1-binding sites in the proximal sIL-1Ra promoter region to generate a full response, additional distinct promoter elements were utilized by LPS or PGN. Activation of p38 stress-activated protein kinase was required for LPS- or PGN-induced IL-1Ra gene expression, but the p38-responsive promoter elements localized to distinct regions of the sIL-1Ra gene. Additionally, while the LPS-induced, p38-dependent response was dependent upon PU.1 binding, the PGN-induced, p38 response was not. Collectively, these data indicated that while some of the intracellular signaling events by TLR2 and TLR4 agonists are similar, there are clearly distinct differences in the responses elicited by these two bacterial products.

Secretory Interleukin-1 Receptor Antagonist Gene Expression Requires both a PU.1 and a Novel Composite NF-κB/PU.1/ GA-binding Protein Binding Site

The human secretory interleukin-1 receptor antagonist (secretory IL-1Ra) gene is controlled through three lipopolysaccharide (LPS)-responsive promoter elements, one of which was identified as an NF-kappaB binding site. Sequence analysis of the secretory IL-1Ra promoter identified a potential PU.1 binding site located between positions -80 and -90 on the complementary strand overlapping the NF-kappaB site. Gel shift analysis using this potential binding site with nuclear extracts from RAW 264.7 macrophages demonstrated the formation of three complexes, one LPS-inducible and two constitutive. The inducible factor was identified as NF-kappaB, and the constitutive factors were identified as PU.1 and GA-binding protein. Site-directed mutagenesis of the -93 to -79 promoter region demonstrated that mutation of either the NF-kappaB 5'-half site or the PU.1/GA-binding protein half-site alone did not significantly decrease LPS responsiveness. However, a mutation that disrupted the binding of all three factors resulted in a 50% decrease in LPS responsiveness. A second PU.1 binding site centered at -230 was identified by gel shift and supershift assays. Mutation of the core GGAA region resulted in a 50% decrease in LPS-responsive promoter activity. Mutation of both the distal and proximal LPS response elements led to an almost complete loss of responsiveness. These data therefore suggest that the regulation of IL-1Ra gene expression is a complex event involving the interactions of three different transcription factors with a single cis-acting element and that the two PU.1 binding sites are the major response elements for LPS-induced IL-1Ra gene expression.

Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms.

Lipopolysaccharide (LPS) treatment of monocytic cells has been shown to activate the Raf-1/mitogen-activated protein kinase (MAPK) signaling pathway and to increase secretory interleukin-1 receptor antagonist (sIL-1Ra) gene expression. The significance of the activation of the Raf-1/MAPK signaling pathway to LPS regulation of sIL-1Ra gene expression, however, has not been determined. This study addresses the role of the Raf-1/MAPK signaling pathway in regulation of sIL-1Ra gene expression by LPS. Cotransfection of the murine macrophage cell line RAW 264.7 with a 294-bp sIL-1Ra promoter/luciferase construct (pRA-294-luc) and a constitutively active Raf-1 kinase expression vector (pRSV-Raf-BXB) resulted in induction of sIL-1Ra promoter activity, indicating that Raf-1, like LPS, can regulate sIL-1Ra promoter activity. An in vitro MAPK analysis indicated that both LPS treatment and pRSV-Raf-BXB transfection of RAW 264.7 cells increases p42 MAPK activity. An in vitro Raf-1 kinase assay, however, failed to detect LPS-induced Raf-1 kinase activity in RAW 264.7 cells, suggesting that in RAW 264.7 cells, Raf-1 kinase is not an activating component of the LPS signaling pathway regulating MAPK activity or sIL-1Ra promoter activity. This observation was supported by results from transfection studies which demonstrated that expression of a dominant-inhibitory Raf-1 mutant in RAW 264.7 cells does not inhibit LPS-induced MAPK activity or sIL-1Ra promoter activity, indicating that LPS-induced sIL-1Ra promoter activation occurs independent of the Raf-1/MAPK signaling pathway. In additional studies, cotransfection of RAW 264.7 cells with pRA-294-luc and increasing amounts of pRSV-Raf-BXB caused a dose-dependent inhibition of LPS-induced sIL-1Ra promoter activity, indicating that the role of the Raf-1 pathway in the regulation of sIL-1Ra promoter activity by LPS is as an antagonizer. Interestingly, LPS treatment of RAW 264.7 cells, cotransfected with pRA-294-luc and pRSV-Raf-BXB, also inhibited pRSV-Raf-BXB-induced sIL-1Ra promoter activity, suggesting that inductions of sIL-1Ra promoter activity by LPS and Raf-1 actually occur by mutually antagonistic mechanisms. In support of this conclusion, sIL-1Ra promoter mapping studies indicated that LPS and Raf-1 responses localized to different regions of the sIL-1Ra promoter. Further studies demonstrated that mutual antagonism between the LPS and Raf-1 kinase pathways is not promoter specific, as the same phenomenon is observed in assays using a c-fos enhancer/thymidine kinase promoter/luciferase construct (pc-fos-TK81-luc). Additionally, mutual antagonism with regard to sIL-1Ra promoter activity also was observed between the LPS and MEK kinase pathways, indicating that mutual antagonism can occur in more than one MAPK activation pathway.

Interleukin-1β and interleukin-1 receptor antagonist gene expression in bone: Correlation with bone loss in healthy postmenopausal women

Interleukin-1β and interleukin-1 receptor antagonist gene expression in bone: Correlation with bone loss in healthy postmenopausal women