Identification of multiple, oxygen-stable HIF1 alpha isoforms, and augmented expression of adrenomedullin in rheumatoid arthritis.

OBJECTIVES: To identify and quantitate hypoxia inducible factor 1 alpha (HIF1 α) isoforms in circulating peripheral blood mononuclear cells (PBMCs), and to assess their effects on target gene expression in rheumatoid arthritis (RA) patients. METHODS: PBMCs from healthy controls and from RA patients were analysed ex-vivo for expression of HIF isoforms, and target genes were assessed by RT-PCR. RESULTS: Transcripts of multiple HIF1α isoforms exist in circulating PBMCs. Expression of all these isoforms is dramatically, and maximally, augmented by foreign surface recognition. However, HIF1α protein stabilisation requires additional cell activation with phorbol ester. No difference in the expression or regulation of the HIF1α isoforms was seen between patients with active RA and healthy controls. However, analysis of a panel of HIF1α target genes revealed increased basal expression of the adrenomedullin gene in RA PBMCs, with resulting loss of further induction upon cell activation. CONCLUSIONS: Even in normoxia PBMCs express stable HIF1α protein on cell activation. Whilst multiple HIF1α isoforms exist in PBMCs no differences in expression were seen in RA compared with healthy controls. RA causes constitutive adrenomedullin expression in PBMCs that is not explicable by altered HIF expression, or stabilisation. Adrenomedullin has a variety of potential biological roles in RA, including regulation of angiogenesis, and aberrant gene regulation may be relevant in RA pathogenesis.

Cell cycle phase regulates glucocorticoid receptor function.

The glucocorticoid receptor (GR) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. In contrast to many other nuclear receptors, GR is thought to be exclusively cytoplasmic in quiescent cells, and only translocate to the nucleus on ligand binding. We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1). Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early G(1). This suggests that the heterogeneity in GR distribution is reflective of cell cycle phase. The impact of cell cycle-driven GR trafficking on a panel of glucocorticoid actions was profiled. In G2/M-enriched cells there was marked prolongation of glucocorticoid-induced ERK activation. This was accompanied by DNA template-specific, ligand-independent GR transactivation. Using chimeric and domain-deleted receptors we demonstrate that this transactivation effect is mediated by the AF1 transactivation domain. AF-1 harbours multiple phosphorylation sites, which are consensus sequences for kinases including CDKs, whose activity changes during the cell cycle. In G2/M there was clear ligand independent induction of GR phosphorylation on residues 203 and 211, both of which are phosphorylated after ligand activation. Ligand-independent transactivation required induction of phospho-S211GR but not S203GR, thereby directly linking cell cycle driven GR modification with altered GR function. Cell cycle phase therefore regulates GR localisation and post-translational modification which selectively impacts GR activity. This suggests that cell cycle phase is an important determinant in the cellular response to Gc, and that mitotic index contributes to tissue Gc sensitivity.

Glucocorticoid receptor over-expression promotes human small cell lung cancer apoptosis in vivo and thereby slows tumor growth.

Small cell lung cancer (SCLC) is an aggressive tumor, associated with ectopic ACTH syndrome. We have shown that SCLC cells are glucocorticoid receptor (GR) deficient, and that restoration of GR expression confers glucocorticoid sensitivity and induces apoptosis in vitro. To determine the effects of GR expression in vivo, we characterized a mouse SCLC xenograft model that secretes ACTH precursor peptides, and so drives high circulating corticosterone concentrations (analogous to the ectopic ACTH syndrome). Infection of SCLC xenografts with GR-expressing adenovirus significantly slowed tumor growth compared with control virus infection. Time to fourfold initial tumor volume increased from a median of 9 days to 16 days (P=0.05; n=7 per group). Post-mortem analysis of GR-expressing tumors revealed a threefold increase in apoptotic (TUNEL positive) cells (P<0.01). Infection with the GR-expressing adenovirus caused a significant reduction in Bcl-2 and Bcl-xL transcripts. Furthermore, in both the GR-expressing adenovirus-infected cells and tumors, a significant number of uninfected cells underwent apoptosis, supporting a bystander cell killing effect. Therefore, GR expression is pro-apoptotic for human SCLCs in vivo, as well as in vitro, suggesting that loss of GR confers a survival advantage to SCLCs.

Absence of Chlamydia pneumoniae in brain of vascular dementia patients.

We recently detected cytomegalovirus (CMV) in brains of 83% of vascular dementia (VaD) patients and 34% of age-matched normal people. Since CMV and also Chlamydia pneumoniae (Cpn) have been found in some studies to be associated with coronary artery disease (which shares several risk factors with VaD), we sought Cpn DNA in VaD brain DNA. We examined brain specimens from 19 VaD patients, 16 elderly normal people and four Alzheimer's disease (AD) patients for the presence of a sequence in the Cpn gene for rRNA, using polymerase chain reaction (PCR) and taking stringent precautions against contamination. We did not detect Cpn DNA in any of the brain specimens, the sensitivity of detection being 10 copies or fewer bacterial DNA sequences per tube or, in terms of infectious units (IFU), 0.025 IFU. Our results do not support a role for Cpn in the aetiology of VaD, either in the 83% of patients in whose brains we detected CMV, or in the remaining 17% without CMV in brain.