Harri Ahola

The three-dimensional structure of the liver X receptor beta reveals a flexible ligand-binding pocket that can accommodate fundamentally different ligands.

The structures of the liver X receptor LXRβ (NR1H2) have been determined in complexes with two synthetic ligands, T0901317 and GW3965, to 2.1 and 2.4 Å, respectively. Together with its isoform LXRα (NR1H3) it regulates target genes involved in metabolism and transport of cholesterol and fatty acids. The two LXRβ structures reveal a flexible ligand-binding pocket that can adjust to accommodate fundamentally different ligands. The ligand-binding pocket is hydrophobic but with polar or charged residues at the two ends of the cavity. T0901317 takes advantage of this by binding to His-435 close to H12 while GW3965 orients itself with its charged group in the opposite direction. Both ligands induce a fixed “agonist conformation” of helix H12 (also called the AF-2 domain), resulting in a transcriptionally active receptor.

Interleukin-1 beta induces expression of cyclooxygenase-2 mRNA in human gingival fibroblasts.

The effect of interleukin-1β (IL-1β) on the expression of cyclooxygenase-1 and −2 (COX-1 and COX-2) mRNA and its relation to prostaglandin E2 (PGE2) biosynthesis in human gingival fibroblasts was studied. IL-1β increased levels of mRNA for COX-2 whereas the COX-1 mRNA level was unaffected. The increased COX-2 mRNA levels were accompanied by enhanced PGE2 formation. The phorbol, 12-myristate 13-acetate (PMA), known to stimulate protein kinase C (PKC), also induced expression of COX-2 mRNA. When gingival fibroblasts were treated simultaneously with IL-1β and PMA, the cytokine IL-1β synergistically increased levels of COX-2 mRNA, accompanied by a corresponding increase in PGE2 biosynthesis. The anti-inflammatory steroid, dexamethasone (DEX) abolished the enhanced expression of COX-2 mRNA as well as PGE2 formation induced by IL-1β, PMA or the combination of IL-1β and PMA. The study indicates that the IL-1β induced PGE2 formation is mediated by an enhanced gene expression of COX-2 in gingival fibroblasts suggesting that the enzyme COX-2 may play an important role in the regulation of prostanoid formation at inflammatory lesions in gingival tissue.

Induction of Cytosolic Phospholipase A2 mRNA Expression by Interleukin-1β and Tumor Necrosis Factor α in Human Gingival Fibroblasts

The involvement of phospholipase A2 (PLA2) enzymes, the 85 kDa cytosolic PLA2 (cPLA2) and the 14 kDa secretory PLA2 (sPLA2), on PGE2 production in human gingival fibroblasts was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the inflammatory mediators interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) induce the mRNA expression of cPLA2 in gingival fibroblasts. In addition, treatment of the cells with calcium ionophore, A23187, or the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), also induced cPLA2 mRNA expression, accompanied by enhanced PGE2 production. The anti-inflammatory steroid dexamethasone (DEX) reduced basal cPLA2 mRNA expression as well as blocked the induction of cPLA2 mRNA by IL-1β and TNFα in gingival fibroblasts. In contrast to cPLA2, the expression of sPLA2 mRNA was not detected either in untreated or in treated gingival fibroblasts. The study demonstrates that cPLA2 mRNA expression is upregulated by IL-1β and TNFα in human gingival fibroblasts suggesting an important role for the enzyme cPLA2 in the cytokine-induced PGE2 production. Furthermore, the enzyme cPLA2, rather than sPLA2, may be involved in the pathogenesis of periodontal disease by mediating PGE2 production in gingival fibroblasts.

Application of a Novel Method for the Comparison of DNA Binding Parameters of the Two Human Thyroid Hormone Receptor Subtypes hTRα1 and hTRβ1

DNA-binding characteristics of the two human thyroid hormone receptors alpha 1 and beta 1 (hTR alpha 1 and hTR beta 1) were studied by applying the recently developed solid-phase scintillation technique. Biotinylated double stranded oligonucleotides containing thyroid hormone response elements (TRE) were immobilized to streptavidin coated scintillating microtiter plates. The TRE:s consisted of variants of the consensus core sequence AGGTCA as monomers or as dimers in direct repeats. Equilibrium binding of radioactive labelled hTR alpha 1 and hTR beta 1 were studied. Metabolically 35S-labelled hTR (in vitro translated cDNA) as well as hTR expressed in the baculovirus-system and labelled with 125I-triiodothyronine (125I-T3) were used. In binding saturation experiments, the affinity for the TRE:s investigated did not differ greatly between hTR alpha 1 and hTR beta 1. No significant effects of T3 on the amplitude of DNA binding of either hTR alpha 1 or hTR beta 1 to the single site response elements could be demonstrated. Receptor binding to direct repeats was stimulated by the hormone in the case of the hTR beta 1. The hTR alpha 1 binding to direct repeats was not significantly altered by T3. The single site octameric variant of a TRE, TAAGGTCA, was observed to bind tighter to the hTR:s as compared to the hexameric variant AGGTCA. In the binding competition format, with one response element immobilized and other (un-biotinylated) added to the reaction mixture, there was a larger dynamic range for the affinity constants (IC50) as compared to the affinity constants (Kd) obtained in the binding saturation experiments. The present quantitative results confirm previous reports obtained with qualitative methods like gel shift assays. The method described here is applicable in basic research concerning characterisation of DNA binding of nuclear receptors. It also lends itself to automatization in high capacity formats.