Inflammation-associated upregulation of the sulfated steroid transporter Slc10a6 in mouse liver and macrophage cell lines.

AIM: Slc10a6, an incompletely characterized member of the SLC10A bile acid transporter family, was one of the most highly induced RNA transcripts identified in a screen for inflammation-responsive genes in mouse liver. This study aimed to elucidate a role for Slc10a6 in hepatic inflammation. METHODS: Mice were treated with lipopolysaccharide (LPS; 2 mg/kg) or interleukin (IL)-1ß (5 mg/kg) for various time points. Cells were treated with LPS (1 µg/mL) at various time points, with cell signaling inhibitors, nuclear receptor ligands and Slc10a6 substrates. All mRNA levels were determined by quantitative polymerase chain reaction. RESULTS: Slc10a6 mRNA levels were upregulated in mouse liver at 2 h (7-fold), 4 h (100-fold) and 16 h (50-fold) after LPS treatment, and 35-fold by the cytokine IL-1ß (4 h). Both absence of the nuclear receptor Fxr and pretreating mice with the synthetic retinoid X receptor-α ligand LG268 attenuated the LPS upregulation of Slc10a6 mRNA by 60-75%. In vitro, Slc10a6 mRNA was induced 30-fold by LPS in mouse RAW264.7 macrophages in a time-dependent manner (maximum at 8 h). The Slc10a6 substrate dehydroepiandrosterone sulfate (DHEAS) enhanced LPS induction of CCL5 mRNA, a pro-inflammatory chemokine, by 50% in RAW264.7 cells. This effect was abrogated in the presence of anti-inflammatory nuclear receptor ligands 9-cis-retinoic acid and dexamethasone. CONCLUSION: Dramatic upregulation of Slc10a6 mRNA by LPS combined with enhanced LPS stimulation of CCL5 expression by the Slc10a6 substrate DHEAS in macrophages suggests that Slc10a6 function contributes to the hepatic inflammatory response.

Neural distribution of vasotocin receptor mRNA in two species of songbird.

The neurohypophyseal hormones vasopressin and oxytocin are produced and released within the mammalian brain, where they act via multiple receptor subtypes. The neural distributions of these receptors, for example, V1a and oxytocin receptors, have been well described in many mammals. In birds, the distribution of binding sites for the homologous neuropeptides, vasotocin (VT) and mesotocin, has been studied in several species by using synthetic radioligands designed to bind to mammalian receptors. Such binding studies, however, may not reveal the specific distributions of each receptor subtype. To identify and map the receptors likely to bind VT and mesotocin, we generated partial cDNA sequences for four VT receptor subtypes, VT1, VT2 (V1b), VT3 (oxytocin-like), and VT4 (V1a), in white-throated sparrow (Zonotrichia albicollis) and zebra finch (Taeniopygia guttata). These genes shared high sequence identity with the homologous avian and mammalian neurohypophyseal peptide receptors, and we found evidence for VT1, VT3, and VT4 receptor mRNA expression throughout the brains of both species. As has been described in rodents, there was striking interspecific and intraspecific variation in the densities and distribution of these receptors. For example, whereas the VT1 receptor mRNA was more widespread in zebra finch brain, the VT3 (oxytocin-like) receptor mRNA was more prevalent in the sparrow brain. Although VT2 (V1b) receptor mRNA was abundant in the pituitary, it was not found in the brain. Because of their association with brain regions implicated in social behavior, the VT1, VT3, and VT4 receptors are all likely candidates for mediating the behavioral effects of VT.

Transcriptional enhancement by GATA1-occupied DNA segments is strongly associated with evolutionary constraint on the binding site motif.

Tissue development and function are exquisitely dependent on proper regulation of gene expression, but it remains controversial whether the genomic signals controlling this process are subject to strong selective constraint. While some studies show that highly constrained noncoding regions act to enhance transcription, other studies show that DNA segments with biochemical signatures of regulatory regions, such as occupancy by a transcription factor, are seemingly unconstrained across mammalian evolution. To test the possible correlation of selective constraint with enhancer activity, we used chromatin immunoprecipitation as an approach unbiased by either evolutionary constraint or prior knowledge of regulatory activity to identify DNA segments within a 66-Mb region of mouse chromosome 7 that are occupied by the erythroid transcription factor GATA1. DNA segments bound by GATA1 were identified by hybridization to high-density tiling arrays, validated by quantitative PCR, and tested for gene regulatory activity in erythroid cells. Whereas almost all of the occupied segments contain canonical WGATAR binding site motifs for GATA1, in only 45% of the cases is the motif deeply preserved (found at the orthologous position in placental mammals or more distant species). However, GATA1-bound segments with high enhancer activity tend to be the ones with an evolutionarily preserved WGATAR motif, and this relationship was confirmed by a loss-of-function assay. Thus, GATA1 binding sites that regulate gene expression during erythroid maturation are under strong selective constraint, while nonconstrained binding may have only a limited or indirect role in regulation.