STAT1 deficiency in the heart protects against myocardial infarction by enhancing autophagy.

Previous studies have shown that the transcription factor signal transducer and activator of transcription 1 (STAT1) activation is increased in primary cardiac myocytes exposed to simulated ischaemia/reperfusion injury. This promotes apoptotic cell death by enhancing the expression of pro-apoptotic proteins. Autophagy has been demonstrated to play a cardioprotective role in the heart following myocardial infarction (MI). We therefore investigated the role of STAT1 in the intact heart subjected to MI and examined the contribution of autophagy in modulating the protective effect of STAT1 after MI injury. STAT1-deficient hearts had significantly smaller infarcts than wild-type hearts and this correlated with increased levels of autophagy shown by light chain 3 (LC3)-I/LC3-II conversion, and up-regulation of Atg12 and Beclin 1. Moreover, pre-treatment with the autophagy inhibitor 3-methyladenine reversed the cardioprotection observed in the STAT1-deficient hearts. These results reveal a new function of STAT1 in the control of autophagy and indicate a cross-talk between the cardioprotective versus the damaging effects of STAT1 in the intact heart exposed to MI injury.

EWS/ETS proteins promote expression and regulate function of the homeodomain transcription factor BRN3A.

Ewing's sarcoma family tumors (ESFTs or EFTs) express neuronal markers, which indicates they may originate from cells at least partly committed to neuronal lineage. However, recent publications suggest EFT originates in mesenchymal stem cells, and EWS/ETS fusion proteins characteristic of EFT activate neuronal marker expression to confer a neural phenotype on EFT. Here we show that the neuronal marker BRN3A/POU4F1 is expressed abundantly at the protein level in primary EFT but not in rhabdomyosarcoma and neuroblastoma, and EFT cells exhibit high activity of the BRN3A proximal autoregulatory region. EWS/FLI-1 siRNA reduces BRN3A expression and promoter activity and EWS/ETS proteins are bound to the BRN3A locus, suggesting a direct function for EWS/ETS proteins in control of BRN3A expression. Differentiation-associated and autoregulatory activities of BRN3A are respectively impaired and altered in EFT cells, and EWS/FLI-1 siRNA can restore some BRN3A function. A potentially novel function for BRN3A in EFT cells is identified. These results extend the hypothesis that EWS/ETS proteins induce expression of neuronal markers such as BRN3A in EFT by showing that the function of those same markers may be restricted or controlled in an EWS/ETS-dependent manner.

The cellular transcription factor Brn-3a and the smoking-related substance nicotine interact to regulate the activity of the HPV URR in the cervix.

The cellular transcription factor Brn-3a differentially regulates different human papilloma virus (HPV)-16 variants that are associated with different risks of progression to cervical carcinoma in infected humans. The upstream regulatory regions (URRs) of high- and intermediate-risk HPV-16 variants are activated by the cellular transcription factor Brn-3a, whereas the URR of a low-risk HPV-16 variant is not. In this study, we show in transfection assays that Brn-3a and the smoking-related substance nicotine produce stronger responsiveness of the URR of the low- and high-risk variants than with either factor alone, but not the intermediate-risk variant. We determined that this synergistic activity of Brn-3a/nicotine is due to two nucleotide differences in the URR, crucial for oncogenic E6/E7 transactivation. Mutant constructs in which the nucleotide residues were substituted alter Brn-3a/nicotine responsiveness. Importantly, women smokers with high levels of Brn-3a infected with low- or high-risk HPV-16 variants have augmented E6 levels, and were more frequently diagnosed with higher grades of cervical intraepithelial neoplasia (CIN) and cancer, as compared with non-smokers who were infected with similar variants and expressed similar levels of Brn-3a. Therefore, this study defines the specific interplay between the cellular transactivator Brn-3a, the environmental smoking-related substance nicotine and specific HPV variants in cervical carcinogenesis, and thus helps to explain why some women are susceptible to rapid CIN progression and cancer and others are not.

Characterisation of a novel NR4A2 mutation in Parkinson's disease brain.

OBJECTIVE: We performed a mutation screen of NR4A2 (also known as NURR1) in 409 Parkinson's disease (PD) patients. We identified a novel single base substitution in the 5'UTR of the NR4A2 (also known as NURR1) gene (c.-309C>T). RESULTS: We have performed expression studies in neuronal cell lines showing that the c.-309C>T mutation reduces NR4A2 mRNA expression in vitro. We have confirmed this finding in vivo by performing allele specific real-time PCR from brain tissue harbouring the 309C>T mutation and show a 3.48+/-1.62 fold reduction in mRNA expression of the mutant allele compared to wild-type. In addition we have undertaken genome wide expression analysis of the mutant NR4A2 brain and shown underexpressed genes were significantly enriched for gene ontology categories in nervous system development and synaptic transmission and overexpressed genes were enriched for unfolded protein response and morphogenesis. Lastly we have shown that the c.-309C>T mutation abrogates the protective effect of wild-type NR4A2 against apoptopic stress. CONCLUSIONS: Our findings indicate the c.-309C>T mutation reduces NR4A2 expression resulting in the downregulation of genes involved in the development and maintenance of the nervous system and synaptic transmission. These downregulated pathways contained genes known to be transactivated by NR4A2 and were not disrupted in idiopathic PD brain suggesting causality of the mutation.

In vivo inhibition of antiphospholipid antibody-induced pathogenicity utilizing the antigenic target peptide domain I of beta2-glycoprotein I: proof of concept.

SUMMARY OBJECTIVES: In the antiphospholipid syndrome (APS), the immunodominant epitope for the majority of circulating pathogenic antiphospholipid antibodies (aPLs) is the N-terminal domain I (DI) of beta(2)-glycoprotein I. We have previously shown that recombinant DI inhibits the binding of aPLs in fluid phase to immobilized native antigen, and that this inhibition is greater with the DI(D8S/D9G) mutant and absent with the DI(R39S) mutant. Hence, we hypothesized that DI and DI(D8S/D9G) would inhibit aPL-induced pathogenicity in vivo. METHODS: C57BL/6 mice (n = 5, each group) were injected with purified IgG derived from APS patients (IgG-APS, 500 microg) or IgG from normal healthy serum (IgG-NHS) and either recombinant DI, DI(R39S), DI(D8S/D9G), or an irrelevant control peptide (at 10-40 microg). Outcome variables measured were femoral vein thrombus dynamics in treated and control groups following standardized vessel injury, expression of vascular cell adhesion molecule-1 (VCAM-1) on the aortic endothelial surface, and tissue factor (TF) activity in murine macrophages. RESULTS: IgG-APS significantly increased thrombus size as compared with IgG-NHS. The IgG-APS thrombus enhancement effect was abolished in mice pretreated with recombinant DI (P

Brn-3a/POU4F1 interacts with and differentially affects p73-mediated transcription.

The Brn-3a/POU4F1 POU transcription factor is critical for the survival and differentiation of specific sensory neurons during development or upon injury; by regulating expression of target genes, either directly or indirectly upon interaction with other proteins. In this study, we demonstrated the physical interaction of Brn-3a with different p73 isoforms and showed co-localization in sensory neurons arising from the neural crest. The biological effects of p73/ Brn-3a interaction depend on the particular p73 isoform, because co-expression of Brn-3a with TAp73 enhanced cell cycle arrest, whereas Brn-3a and DeltaNp73 cooperated to increase protection from apoptosis. Brn-3a antagonized TAp73 transactivation of pro-apoptotic Bax, but co-operated to increase transcription of the cell cycle regulator p21 CIP1/Waf1. The region 425-494 amino acids within the TAp73 C terminus were critical for Brn-3a to repress Bax transactivation, but not for cooperation on the p21 CIP1/Waf1 promoter. Our results suggest that co-factors binding to the p73 C terminus facilitate maximal activation on the Bax but not p21 CIP1/Waf1 promoter and that Brn-3a modulates this interaction. Thus, the physical interaction of Brn-3a with specific p73 isoforms will be critical for determining cell fate during neuronal development or in injured neurons expressing both factors.

Proliferation-associated Brn-3b transcription factor can activate cyclin D1 expression in neuroblastoma and breast cancer cells.

Brn-3b transcription factor enhances proliferation of neuroblastoma (NB) and breast cancer cell lines in vitro and increases the rate and size of in vivo tumour growth, whereas reducing Brn-3b slows growth, both in vitro and in vivo. Brn-3b is elevated in >65% of breast cancer biopsies, and here we demonstrate that Brn-3b is also elevated in NB tumours. We show a significant correlation between Brn-3b and cyclin D1 (CD1) in breast cancers and NB tumours and cell lines. Brn-3b directly transactivates the CD1 promoter in co-transfection experiments, whereas electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrate that Brn-3b protein binds to an octamer sequence located in the proximal CD1 promoter. Site-directed mutagenesis of this sequence resulted in loss of transactivation of the CD1 promoter by Brn-3b. Thus, Brn-3b may act to alter growth properties of breast cancer and NB cells by enhancing CD1 expression in these cells.

Beta-subunits of voltage-gated sodium channels in human prostate cancer: quantitative in vitro and in vivo analyses of mRNA expression.

We previously identified high levels of Na(v)1.7 voltage-gated sodium channel alpha-subunit (VGSCalpha) mRNA and protein in human prostate cancer cells and tissues. Here, we investigated auxillary beta-subunit (VGSCbetas) expression. In vitro, the combined expression of all four VGSCbetas was significantly (approximately 4.5-fold) higher in strongly compared to weakly metastatic cells. This was mainly due to increased beta1-expression, which was under androgenic control. In vivo, beta1-beta4 mRNAs were detectable and their expression in CaP vs non-CaP tissues generally reflected the in vitro levels in relation to metastatic potential. The possible role(s) of VGSCbetas (VGSCalpha-associated and VGSCalpha-independent) in prostate cancer are discussed.

The heat shock proteins and plastic surgery.

Heat shock proteins are diverse and essential components of cell physiology. Their expression is elevated in the cell undergoing stress, where they protect the cell from death by necrosis or apoptosis and accelerate recovery. Significant advances have been made in studies relevant to plastic surgery regarding these proteins and their manipulation. This review introduces the heat shock proteins and appraises these studies in skin, ultraviolet light exposure, neoplasia, wound healing, ageing, burns, and reconstructive surgery.

The Urocortins: mechanisms of cardioprotection and therapeutic potential.

The study of the Urocortin family of peptides is becoming increasingly important clinically, as new discoveries have revealed that their roles in the body are extremely diverse. They range from being involved in the aetiology of affective disorders, boosting the immune system, to cardioprotection during ischaemia and reperfusion injury. Therefore, it is important to understand how these peptides become activated, how they activate different cell types and finally their intracellular signalling pathways. Such studies may enable scientists to develop clinical therapies based on their mechanism of action, which may be used to alleviate a diverse range of pathologies.

PINK1 protein in normal human brain and Parkinson's disease.

Parkinson's disease is a common incurable neurodegenerative disease whose molecular aetiology remains unclear. The identification of Mendelian genes causing rare familial forms of Parkinson's disease has revealed novel proteins and pathways that are likely to be relevant in the pathogenesis of sporadic Parkinson's disease. Recently, mutations in a novel gene, PINK1, encoding a 581 amino acid protein with both mitochondrial targeting and serine/threonine kinase domains, were identified as a cause of autosomal recessive parkinsonism. This provided important evidence for the role of the mitochondrial dysfunction and kinase pathways in neurodegeneration. In this study, we report the first characterization of the PINK1 protein in normal human and sporadic Parkinson's brains, in addition to Parkinson's cases with heterozygous PINK1 mutations. The possible role of the PINK1 protein was also assessed in a number of neurodegenerative diseases characterized by proteinaceous inclusions. For these studies, rabbit polyclonal antibodies were raised against two peptide sequences within the N-terminal hydrophilic loops of PINK1 protein. Using immunohistochemistry and western blotting we were able to demonstrate that PINK1 is a ubiquitous protein expressed throughout the human brain and it is found in all cell types showing a punctate cytoplasmic staining pattern consistent with mitochondrial localization. Fractionation studies of human and rat brain confirm that PINK1 is localized to the mitochondrial membranes. In addition, we show that PINK1 is detected in a proportion of Lewy bodies in cases of sporadic Parkinson's disease and Parkinson's disease associated with heterozygous mutations in the PINK1 gene, which are clinically and pathologically indistinguishable from the sporadic cases. PINK1 was absent in cortical Lewy bodies, in neurofibrillary tangles in Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, and in the glial and neuronal alpha-synuclein positive inclusions in multiple system atrophy. These studies provide for the first time in vivo morphological and biochemical evidence to support a mitochondrial localization of PINK1 and underpin the significance of mitochondrial dysfunction in the pathogenesis of nigral cell degeneration in Parkinson's disease.

Differential regulation of different human papilloma virus variants by the POU family transcription factor Brn-3a.

The Brn-3a POU family transcription factor is overexpressed in human cervical carcinoma biopsies and is able to activate expression of the human papilloma virus type 16 (HPV-16) upstream regulatory region (URR), which drives the expression of the E6 and E7 oncoproteins. Inhibition of Brn-3a expression in human cervical cancer cells inhibits HPV gene expression and reduces cellular growth and anchorage independence in vitro as well as the ability to form tumours in vivo. Here, we show that Brn-3a differentially regulates different HPV-16 variants that have previously been shown to be associated with different risks of progression to cervical carcinoma. In human cervical material, Brn-3a levels correlate directly with HPV E6 levels in individuals infected with a high risk variant of HPV-16, whereas this is not the case for a low-risk variant. Moreover, the URRs of high- and intermediate-risk variants are activated by Brn-3a in transfection assays, whereas the URR of a low-risk variant is not. The change of one or two bases in a low-risk variant URR to their equivalent in a higher-risk URR can render the URR responsive to Brn-3a and vice versa. These results help explain why the specific interplay between viral and cellular factors necessary for the progression to cervical carcinoma only occurs in a minority of those infected with HPV-16.

Brn-3a neuronal transcription factor functional expression in human prostate cancer.

Neuroendocrine differentiation has been associated with prostate cancer (CaP). Brn-3a (short isoform) and Brn-3c, transcriptional controllers of neuronal differentiation, were readily detectable in human CaP both in vitro and in vivo. Brn-3a expression, but not Brn-3c, was significantly upregulated in >50% of tumours. Furthermore, overexpression of this transcription factor in vitro (i) potentiated CaP cell growth and (ii) regulated the expression of a neuronal gene, the Nav1.7 sodium channel, concomitantly upregulated in human CaP, in an isoform-specific manner. It is concluded that targeting Brn-3a could be a useful strategy for controlling the expression of multiple genes that promote CaP.

Opposing actions of STAT-1 and STAT-3.

The signal transducers and activators of transcription (STATs) are a family of transcription factors, which were originally identified on the basis of their ability to transduce a signal from a cellular receptor into the nucleus and modulate the transcription of specific genes. Interestingly, recent studies have demonstrated that STAT-1 plays a key role in promoting apoptosis in a variety of cell types, whereas STAT-3 has an anti-apoptotic effect. Moreover, whilst STAT-3 promotes cellular proliferation and is activated in a variety of tumour cells, STAT-1 appears to have an anti-proliferative effect. Although the initially characterised signal transduction events mediated by STAT-1 and STAT-3 involve the DNA binding and transcriptional activation domains of the factor, some of their other effects appear not to require DNA binding. Therefore, STAT-1 and STAT-3 can mediate the regulation of gene transcription both by direct DNA binding and via a co-activator mechanism and despite their very similar structures, have antagonistic effects on cellular proliferation and apoptosis.

Representational difference analysis of cDNA identifies novel genes expressed following preconditioning of the heart.

Preconditioning of the myocardium rapidly induces a number of transcription factors, which are likely to be responsible for a cascade of transcriptional changes underlying the development of delayed adaptation. Identifying these changes provides insight into the molecular pathways elicited by sub-lethal ischaemia and the mechanism leading to delayed adaptation. Genes up-regulated in rabbit myocardium in vivo by ischaemic preconditioning following reperfusion for 2 h, 4 h and 6 h post-treatment were identified by representational difference analysis of cDNA (cDNA. RDA). The area of the left ventricle rendered ischaemic by preconditioning or the equivalent area of sham-treated animals was isolated and cDNA.RDA performed. Three novel genes and six genes with known function where identified, including the TGFbeta receptor interacting protein 1, the alpha isoform of the A subunit of PP2 and the cap binding protein NCBP1. To determine whether expression of these genes correlated with preconditioning per se, expression was measured in myocardium after both ischaemic as well as heat shock induced preconditioning following 2 h, 4 h, and 6 h reperfusion. These genes were induced in rabbit myocardium in vivo by both ischaemia and heat shock, consistent with a fundamental role in the development of delayed adaptation. The well described role of PP2 in modulating the mitogen-activated protein kinase pathway and promoting cell survival is consistent with our previous work, which identified the reperfusion injury salvage kinase pathway in mediating the protective effects of ischaemic preconditioning. Expression of Trip1 and Ncbp1 also implicates TGFbeta signalling pathways and RNA processing and transport in delayed adaptation to stress in the myocardium.

HSP27 and cell survival in neurones.

Although the majority of studies on the protective effect of individual hsps have concentrated on the major inducible heat shock protein Hsp70, a variety of evidence suggests that the small heat shock protein Hsp27 may have a more potent protective effect in the nervous system. Thus, for example, in cultured neurones over-expression of Hsp70 can protect against subsequent exposure to thermal or ischaemic stress but not against exposure to some other stressful stimuli, whereas over-expression of Hsp27 protects against a variety of stresses. Similarly, although transgenic animals over-expressing Hsp70 are protected against cardiac ischaemia, more equivocal results have been obtained in terms of their protection against cerebral ischaemia and other stresses to the nervous system. In contrast, transgenic animals over-expressing Hsp27 have recently been shown to show neuroprotection as well as being protected against cardiac ischaemia. Recent findings have also implicated Hsp27 and related proteins in human disease. Thus, it has been demonstrated that mutation of either Hsp27 or the related protein hsp22 can be observed in specific families with hereditary motor neuropathy caused by premature axonal loss, possibly due to neuronal death and subsequent degeneration. Moreover, the mutations are associated with a reduced ability to promote neuronal survival compared to the wild type protein. Hence, Hsp27 appears to be a potent protective factor for neuronal cells whose mutation results in neuronal cell death and disease, whilst enhanced expression of the wild type protein may be a therapeutic option for human diseases involving excessive neuronal cell death.

Raised levels of interleukin 6 in systemic lupus erythematosus correlate with anaemia.

OBJECTIVES: Raised levels of the cytokines interleukin (IL) 6 and IL10 have been reported in patients with systemic lupus erythematosus (SLE). OBJECTIVE: To determine if levels of IL6 and IL10 correlate with organ/system-specific disease activity in SLE, using the British Isles Lupus Assessment Group (BILAG) Disease Activity Index. METHODS: Levels of IL6 and IL10 in serum samples from 171 patients with SLE and 50 normal controls were determined by enzyme linked immunosorbent assay (ELISA). Levels of cytokines in individual patients with SLE were compared with the presence or absence of active disease in eight organ/systems using the BILAG index. RESULTS: Levels of IL6 were significantly higher (p = 0.005) in patients with active compared with inactive haematological disease, as scored by the BILAG index. Further analysis showed that this association was dependent on an inverse correlation (p = 0.002, r = -0.26) between IL6 levels and haemoglobin levels in patients with SLE. In contrast, IL10 levels did not correlate with individual organ/system disease activity. CONCLUSIONS: Raised levels of IL6 in SLE may influence the development of anaemia in this disease. These findings are in agreement with an increasing number of studies, which support physiological links between IL6 and anaemia. Importantly, with the exception of the haematological system, our studies do not provide evidence of any individual organ/system which would respond to therapeutic manipulation of either IL6 or IL10 levels.

Elevated expression of the Brn-3a and Brn-3b transcription factors in systemic lupus erythematosus correlates with antibodies to Brn-3 and overexpression of Hsp90.

OBJECTIVE: Important developmental and antiapoptotic roles have been described for the Brn-3 family of transcription factors in mammalian cells. Following a report of pathogenic autoantibody-inducing T cell reactivity to the Brn-3 transcription factors in murine lupus, we undertook this study to investigate serum levels of antibodies to Brn-3 and levels of expression of Brn-3 in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). METHODS: Serum and PBMC samples were obtained from 87 SLE patients and 30 normal control subjects. Serum antibodies to the Brn-3a and Brn-3b transcription factors were measured by enzyme-linked immunosorbent assay. Levels of Brn-3a and Brn-3b messenger RNA (mRNA) in PBMCs were measured by reverse transcription and real-time quantitative polymerase chain reaction. RESULTS: Elevated serum levels of antibodies to Brn-3a and Brn-3b were found in 43% and 32%, respectively, of SLE patients. This elevation paralleled enhanced expression of Brn-3a and Brn-3b in PBMCs of 44% and 31%, respectively, of SLE patients. Furthermore, we observed a significant correlation (P = 0.002) between elevated levels of anti-Brn-3b antibodies and elevated levels of Brn-3b mRNA in individual patients. A preliminary analysis of possible target genes for Brn-3a and Brn-3b revealed a significant correlation (P = 0.01) between the level of Brn-3a mRNA and the level of Hsp90 protein (90-kd heat-shock protein, which is overexpressed in SLE) in PBMCs of SLE patients. In addition, we observed that overexpression of Brn-3a and Brn-3b in cultured cells enhanced expression of Hsp90 protein and transcription of Hsp90 promoter-reporter constructs. Finally, we observed an association between elevated levels of Brn-3a mRNA and active SLE (P = 0.002). CONCLUSION: Expression of both Brn-3a and Brn-3b was found to be enhanced in SLE, and this correlated with enhanced levels of autoantibodies to these proteins and with the previously reported overexpression of Hsp90, which was shown to be a novel gene regulated by Brn-3a and Brn-3b. The overexpression of Brn-3a correlated with active disease, suggesting that it may play a role in the disease process via its targeting by the immune system and its ability to induce the expression of specific genes.

Cardioprotection mediated by urocortin is dependent on PKCepsilon activation.

Urocortin (Ucn) is an endogenous cardioprotective agent that protects against the damaging effects of ischemia and reperfusion injury in vitro and in vivo. We have found that the mechanism of action of Ucn involves both acute activation of specific target molecules, and using Affymetrix (Santa Clara, CA) gene chip technology, altered gene expression of different end effector molecules. Here, from our gene chip data, we show that after a 24 h exposure to Ucn, there was a specific increase in mRNA and protein levels of the protein kinase C epsilon (PKCepsilon) isozyme in primary rat cardiomyocytes compared with untreated cells and in the Langendorff perfused ex vivo heart. Furthermore, a short 10 min exposure of these cells to Ucn caused a specific translocation/activation of PKCepsilon in vitro and in the Langendorff perfused ex vivo heart. The importance of the PKCepsilon isozyme in cardioprotection and its relationship to cardioprotection produced by Ucn was assessed using PKCepsilon-specific inhibitor peptides. The inhibitor peptide, when introduced into cardiomyocytes, caused an increase in apoptotic cell death compared with control peptide after ischemia and reperfusion. When the inhibitor peptide was present with Ucn, the cardioprotective effect of Ucn was lost. This loss of cardioprotection by Ucn was also seen in whole hearts from PKCepsilon knockout mice. These findings indicate that the cardioprotective effect of Ucn is dependent upon PKCepsilon.