Gudrun Toresson

GPS2 Is Required for Cholesterol Efflux by Triggering Histone Demethylation, LXR Recruitment, and Coregulator Assembly at the ABCG1 Locus

Transcriptional coregulators, rather than ligand signals, are suspected to confer context and pathway specificity to nuclear receptor signaling, but the identity of such specifying coregulators and the underlying molecular mechanisms remain largely enigmatic. Here we address this issue in metabolic oxysterol receptor LXR pathways and describe the selective requirement of GPS2 for ABCG1 cholesterol transporter gene transcription and cholesterol efflux from macrophages. We implicate GPS2 in facilitating LXR recruitment to an ABCG1-specific promoter/enhancer unit upon ligand activation and identify functional links to histone H3K9 demethylation. We further describe fundamental differences between ABCG1 and ABCA1 with regard to GPS2 in relation to other coregulators, which are likely to apply to additional LXR-regulated genes. Our work identifies a coregulator-dependent epigenetic mechanism governing the access of a nuclear receptor to communicating regulatory regions in the genome. The pathway and coregulator selectivity of this mechanism implies pharmacological possibilities for the development of selective LXR agonists.

A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP

SHP (short heterodimer partner, NROB2) is an atypical orphan member of the mammalian nuclear receptor family that consists only of a putative ligand‐binding domain and thus cannot bind DNA. Instead, SHP acts as a transcriptional coregulator by inhibiting the activity of various nuclear receptors (downstream targets) via occupation of the coactivator‐binding surface and active repression. However, repression mechanisms have remained elusive and may involve coinhibitory factors (upstream targets) distinct from known nuclear receptor corepressors. Here, we describe the isolation of mouse E1A‐like inhibitor of differentiation 1 (EID1) as a candidate coinhibitor for SHP. We characterize the interactions between SHP and EID1 and identify two repression‐defective SHP mutations that have lost the ability to bind EID1. We suggest histone acetyltransferases and histones as targets for EID1 action and propose that SHP inhibition of transcription involves EID1 antagonism of CBP/p300‐dependent coactivator functions.

Structure of the retinoid X receptor α–liver X receptor β (RXRα–LXRβ) heterodimer on DNA

Nuclear receptors (NRs) are conditional transcription factors with common multidomain organization that bind diverse DNA elements. How DNA sequences influence NR conformation is poorly understood. Here we report the crystal structure of the human retinoid X receptor α–liver X receptor β (RXRα–LXRβ) heterodimer on its cognate element, an AGGTCA direct repeat spaced by 4 nt. The complex has an extended X-shaped arrangement, with DNA- and ligand-binding domains crossed, in contrast to the parallel domain arrangement of other NRs that bind an AGGTCA direct repeat spaced by 1 nt. The LXRβ core binds DNA via canonical contacts and auxiliary DNA contacts that enhance affinity for the response element. Comparisons of RXRα–LXRβs in the crystal asymmetric unit and with previous NR structures reveal flexibility in NR organization and suggest a role for RXRα in adaptation of heterodimeric complexes to DNA.

Detection of N‐Terminally Extended Substance P but Not of Substance P in Human Cerebrospinal Fluid: Quantitation with HPLC‐Radioimmunoassay

Abstract: A reversed‐phase HPLC system was used to concentrate and separate components of substance P‐like immunoreactivity (SP‐LI) from human CSF. When CSF was injected and fractions collected, no SP‐LI could be detected by radioimmunoassay (RIA) at the retention time of SP or SP‐sulfoxide. Instead, SP‐LI was detected in later eluting fractions. This SP‐LI reacted with two different antisera raised against the C‐terminal part of SP, but not with an antiserum against the N‐terminal part. A compound with similar properties was also found to be present in neutral extracts of rat dorsal spinal cord. When the late‐eluting compound from human CSF was treated with trypsin and rechromatographed on HPLC, an immunoreactive component eluting at the position of SP could be detected with both the C‐ and N‐terminally directed SP antisera. These results suggest that an N‐terminally extended form of SP is present in human CSF. Trypsinization also gave two other compounds with affinity for the N‐ but not the C‐terminally directed antisera. This may indicate that N‐terminal fragments of SP extended at the N‐terminus or SP molecules extended at both the N‐ and the C‐terminus (i.e., preprotachykinins) also are present in human CSF. In 32 CSF samples from depressed patients, SP‐LI was determined with a C‐terminally directed antiserum with and without prior HPLC separation. SP itself could not be detected, but the late‐eluting form of SP‐LI could be quanti‐tated in all samples by combined HPLC‐RIA. In most samples, there was a relatively good agreement between the SP‐LI levels measured with and without HPLC.

Pancreatic exocrine insufficiency in LXRβ-/- mice is associated with a reduction in aquaporin-1 expression

Liver X receptors (LXRs) α and β are nuclear oxysterol receptors with a key role in cholesterol, triglyceride, and glucose metabolism. In LXRβ−/− mice on a normal diet, there is a reduction in size of perigonadal fat pad and, on high-fat diet there is resistance to obesity. In the present study, we investigated the reason for the resistance of LXRβ−/− mice to weight gain. In LXRβ−/− mice we found pancreatic exocrine insufficiency with reduced serum levels of amylase and lipase, reduced proteolytic activity in feces, chronic inflammatory infiltration, and, in the ductal epithelium, an increased apoptosis without compensatory proliferation. Electron microscopy revealed ductal dilatation with intraductal laminar structures characteristic of cystic fibrosis. To investigate the relationship between LXRβ and pancreatic secretion, we studied the expression of LXRβ and the water channel, aquaporin-1 (AQP1), in the ductal epithelium of the pancreas. In WT mice, ductal epithelial cells expressed LXRβ in the nuclei and AQP1 on the plasma membrane. In LXRβ−/− mice neither LXRβ nor AQP1 was detectable. Moreover, in WT mice the LXR agonist (T2320) increased AQP1 gene expression. These data demonstrate that in LXRβ−/− mice dietary resistance to weight gain is caused by pancreatic insufficiency and that LXRβ regulates pancreatic exocrine secretion through the control of AQP1 expression. Pancreatic exocrine insufficiency is the main cause of malabsorption syndrome responsible for weight loss in adults and growth failure in children. Several genes are known to be involved in the pathogenesis and susceptibility to pancreatic insufficiency. LXRβ should be included in that list.

Treatment of depression with E-10-hydroxynortriptyline — a pilot study on biochemical effects and pharmacokinetics

The major metabolite of nortriptyline, i.e. E-10-hydroxynortriptyline (E-10-OH-NT), was given as a racemate in increasing doses from 75 to 225 mg/day to five patients with major depressive episode. Plasma concentrations of both the (−)- and (+)-enantiomers were linearly related to the doses. The mean ratio between them was 3.6±0.53, indicating stereospecific kinetics during maintenance treatment. Lumbar punctures were performed in four of the patients before and after 3 weeks of E-10-OH-NT treatment. There was a 18% mean decrease (P<0.01) in the noradrenaline metabolite HMPG in cerebrospinal fluid (CSF), supporting previous in vitro data showing that E-10-OH-NT inhibits noradrenaline uptake in vivo. During treatment, the median depression score measured by the Montgomery-Åsberg Depression Rating Scale declined from 32 to 14 (P<0.05). As the study was open, the clinical outcome is not conclusive but does not contradict the hypothesis that E-10-OH-NT has antidepressant properties. If present at all, side effects were mild and did not interfere with the treatment.

N-terminally extended tachykinins in human cerebrospinal fluid

With HPLC-RIA methods we were unable to demonstrate SP and NKA in measurable amounts in human CSF. Instead N-terminally extended forms of these peptides were found to be present and could be quantitated. The finding that these forms of tachykinins can be released from nervous tissue might suggest that their levels in CSF can be used as markers of the activity in central SP and NKA neurons.

N-terminally extended substance P is released together with substance P from rat spinal cord

The release of different forms of substance P-like immunoreactivity (SP-LI) from superfused slices of rat spinal cord was studied. The released SP-LI was characterized by reverse-phase high-performance liquid chromatography and radioimmunoassay with two antisera directed to the C- and N-terminal parts of SP, respectively. The SP-LI detected in the superfusates with the C-terminally directed antiserum was found to consist of (undeca) SP, SP-sulfoxide and a late eluting component which was not detectable with the N-terminally directed antiserum. This component was also found in neutral extracts of the spinal cord. Upon trypsin digestion, it produced SP-LI detectable with both C- and N-terminally directed antiserum which also coeluted with SP. From these results we conclude that this form of SP-LI most likely corresponds to an N-terminally extended form of SP. An increase of the potassium concentration in the superfusion fluid from 5 to 50 mM evoked an increased overflow of both SP and the N-terminally extended SP. The present results indicate that N-terminally extended SP is released by a calcium-dependent mechanism together with SP from terminals in the spinal cord in response to potassium stimulation.

Neuropeptide K is present in human cerebrospinal fluid.

Neurokinin A-like immunoreactivity (NKA-LI) in human cerebrospinal fluid (CSF) was determined by radioimmuno assay (RIA) combined with high performance liquid chromatography (HPLC). The major immunoreactive component did not coelute with NKA, but coeluted with neuropeptide K (NPK), which contains the NKA sequence in its C-terminus. Trypsin treatment of this component from human CSF and of synthetic NPK, produced a substance which coeluted with NKA in the HPLC system. When the NKA-LI was oxidized with hydrogen peroxide and rechromatographed, the immunoreactivity coeluted with NPK sulfoxide. The results indicate that the main part of the NKA-LI in CSF is identical with NPK. The mean concentration of NPK measured in CSF from 6 healthy subjects by HPLC-RIA was 23 +/- 11 (SD) pmol/L.

Quantitation of N-terminally extended tachykinins in cerebrospinal fluid from healthy subjects

N-terminally extended substance P (SP) and neuropeptide K (NPK), an N-terminally extended form of neurokinin A (NKA), were determined in cerebrospinal fluid (CSF) from healthy human subjects by combined high performance liquid chromatography and radioimmunoassay. The concentrations of the peptides were similar in fresh CSF and in CSF which had been kept frozen for up to 5 months. SP and NKA were not present in measurable amounts in neither fresh CSF nor in CSF that had been frozen. On the other hand, when synthetic SP and NKA were added to approx. 2 pM concentration to fresh CSF samples, both peptides were recovered to 85 and 98%, respectively. There were no significant concentration gradients of the peptides in the first 18 ml (three consecutive 6 ml fractions) of CSF (n = 10). In contrast, we confirmed previous findings, that there are gradients of the amine metabolites 5-HIAA (P < 0.01) and HVA (P < 0.001) (n = 5). The concentrations of extended SP (expressed in SP equivalents) and NPK in the first 6 ml of CSF were 1.5 +/- 0.7 pM and 14.2 +/- 6.4 pM (mean +/- S.D., n = 10), respectively. The present results thus show that the levels of N-terminally extended SP and NKA are stable in frozen CSF samples for up to 5 months. The virtual lack of SP and NKA in CSF does not seem to be due to losses during sample preparation or storage.