Biomedical subjects
Publications and source records attributed to D S Latchman.
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Simulated ischaemia causes both necrotic and apoptotic death of primary cultures of neonatal rat cardiac myocytes. Simulated ischaemia is associated with increased expression of urocortin mRNA and with the release of urocortin peptide into the medium. Exogenous urocortin is more potent than corticotropin releasing hormone (CRH) in protecting cardiac myocytes from necrotic and apoptotic death induced by ischaemia, and the cardioprotective effects of ischaemia-preconditioned media are abrogated by antagonists to the CRH family of peptides. Simulated ischaemia increases cardiac myocyte expression of CCAAT enhancer binding (C/EBP) transcription factors, and of the p65 subunit of NFkappaB, and reporter activity of a construct incorporating a fragment of the urocortin promoter containing a C/EBP consensus site is also enhanced by simulated ischaemia. The data suggest that ischaemia, acting partly through increased expression of C/EBP transactivators, increases expression of urocortin mRNA, which is rapidly translated to the mature form. The mature peptide is rapidly released, and exerts autocrine/paracrine protective effects through the cardiac CRH-R2 receptor which preferentially binds urocortin.
NGFI-A is an immediate early gene (IEG) that is transcriptionally induced by nerve growth factor (NGF) in PC12 cells and has been implicated in a number of cellular responses. Studies have shown that elements within the first 106 base pairs of the NGFI-A promoter contribute to its induction by NGF in PC12 cells. One element, within the serum response element (SRE) bridge region, bears strong homology to a motif previously identified in promoters regulated by the Brn-3a POU domain transcription factor. We report here that Brn-3a activates the NGFI-A promoter in neurons (both primary and cell lines). Analysis revealed that this response requires sequences between positions -49 and -106. Whilst DNA-protein interaction studies failed to identify a site bound directly by Brn-3a, the data presented here suggest that Brn-3a may cooperate in the regulation of NGFI-A gene expression in neurons, possibly during the developmental switch between neurotrophin dependency that occurs during neurogenesis.
The BRCA-1 tumour supressor gene was identified on the basis of mutations which occur in familial breast cancer indicating that its inactivation can cause this disease. Although BRCA-1 does not appear to be mutated in sporadic breast cancer, its expression has been shown to be reduced in tumour material from such cases. We show here that mammary tumours which have reduced levels of BRCA-1 expression show enhanced expression of the Brn-3b POU family transcription factor at both the mRNA and protein levels. This elevated expression of Brn-3b is not found in normal mammary cells, benign tumours or in malignant tumour samples which do not exhibit reduced levels of BRCA-1. In contrast, no correlation was noted between BRCA-1 and expression of the related factor Brn-3a. Moreover, Brn-3b but not Brn-3a can strongly repress the BRCA-1 promoter approximately 20-fold in mammary tumour cells. To our knowledge, this is the first report of a transcription factor which regulates BRCA-1 expression. Thus, Brn-3b may play an important role in regulating expression of BRCA-1 in mammary tumours with enhanced expression of Brn-3b resulting in reduced BRCA-1 expression and thereby being potentially important in tumour development.
The Brn-3a POU family transcription factor has previously been shown to activate the human papilloma virus type 16 (HPV-16) promoter driving the expression of the E6- and E7-transforming proteins. Moreover, Brn-3a is overexpressed approximately 300-fold in cervical biopsies from women with cervical intra-epithelial neoplasia type 3 (CIN3) compared with normal cervical material. To test the role of Brn-3a in cervical neoplasia we have manipulated its expression in cervical carcinoma-derived cell lines with or without endogenous HPV genes. In HPV-expressing cells, reduction in Brn-3a expression specifically reduces HPV gene expression, growth rate, saturation density and anchorage-independent growth, whereas these effects are not observed when Brn-3a expression is reduced in cervical cells lacking HPV genomes. Together with our previous observations, these findings indicate a critical role for Brn-3a in regulating HPV gene expression and thereby in controlling the growth/transformation of cervical cells.
Apoptosis is a distinct form of cell death, induced, for example, by ischaemia/reperfusion injury, that results in characteristic alterations in cell morphology and fate. In tissue sections, the most commonly used technique to detect apoptosis is terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining which labels the ends of DNA strand breaks characteristic of the apoptotic process. However, without the employment of additional staining, TUNEL is only a qualitative procedure that gives no information about the proportion of negative cells nor the cell type undergoing apoptosis. We have utilised propidium iodide (PI) as a counterstain to visualise TUNEL negative nuclei together with anti-desmin antibody in order to assess quantitatively apoptosis in specific cell types. The procedure has been evaluated in tissue sections from isolated perfused rat hearts subjected to ischaemia and reperfusion. Hearts were cross-sectioned into four 2.5 mm thick slices which were fixed in 4% formaldehyde and embedded in paraffin. Serial sections (5 microns) were cut, dewaxed and pretreated by incubation with trypsin at 37 degrees C for 30 min. After the employment of the TUNEL assay, sections were labelled with anti-desmin antibody, counterstained with PI and finally examined by confocal fluorescent microscopy. Apoptosis was not seen in sections from hearts subjected to ischaemia alone nor in control hearts. After 35 min of ischaemia the percentages of TUNEL positive cells were very low both in myocytes (0.1%) and in non-myocytes (0.3%). In ischaemic-reperfused hearts, the number of TUNEL positive cells was only significantly higher in vascular cells (44+/-5%) and cardiac myocytes (6+/-2%). This simple method therefore allows quantification of apoptosis in myocytic and non-myocytic cells in tissue sections. Use of alternative immunohistochemical markers would permit adaptation of the method to the quantitative assessment of apoptosis in other tissues.
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BACKGROUND: Cell migration is a major contributor to injury-induced neointimal hyperplasia and depends on alteration of the proteolytic balance within the arterial wall toward matrix breakdown. This is partly mediated by the matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). METHODS AND RESULTS: An increase in expression of biologically active and immunoreactive TIMP-1 was seen in vitro after infection of rat smooth muscle cells (SMCs) with Av1.TIMP1 (an adenoviral vector containing the human TIMP1 cDNA). Infection of rat SMCs with Av1.TIMP1 reduced migration in vitro by 27% compared with control virus-infected cells (37.6+/-4.34 versus 51+/-5.01 cells per high-power field, P<0.05). The adenoviral vector was delivered to the injured rat carotid artery, and 4 days later, immunoreactive protein was identified and migration of SMCs reduced by 60% (5.2+/-0. 5 versus 12.8+/-1.5 cells per section, P<0.05, n=5). Neointimal area 14 days after injury showed a 30% reduction in the animals receiving the Av1.TIMP1 virus compared with controls (0.09+/-0.01 versus 0. 14+/-0.01 mm2, P=0.02, n=14). CONCLUSIONS: The response to arterial balloon injury involves MMP-dependent SMC migration and can be attenuated in vivo by the transmural expression of TIMP-1 by adenoviral gene transfer.
The Brn-3a POU family transcription factor has been shown to strongly activate expression of the Bcl-2 proto-oncogene and thereby protect neuronal cells from programmed cell death (apoptosis). This activation of the Bcl-2 promoter by Brn-3a is strongly inhibited by the p53 anti-oncogene protein. This inhibitory effect of p53 on Brn-3a-mediated transactivation is observed with nonoverlapping gene fragments containing either the Bcl-2 p1 or p2 promoters but is not observed with other Brn-3a-activated promoters such as in the gene encoding alpha-internexin or with an isolated Brn-3a binding site from the Bcl-2 promoter linked to a heterologous promoter. In contrast, p53 mutants, which are incapable of binding to DNA, do not affect Brn-3a-mediated activation of the Bcl-2 p1 and p2 promoters. Moreover, Brn-3a and p53 have been shown to bind to adjacent sites in the p2 promoter and to directly interact with one another, both in vitro and in vivo, with this interaction being mediated by the POU domain of Brn-3a and the DNA binding domain of p53. The significance of these effects is discussed in terms of the antagonistic effects of Bcl-2 and p53 on the rate of apoptosis and the overexpression of Brn-3a in specific tumor cell types.
The Bcl-2 protein has an anti-apoptotic effect in neuronal and other cell types. We show for the first time that the Bcl-2 promoter is activated by the neuronal survival factor nerve growth factor (NGF) and that this effect is dependent on a region of the promoter from -1472 to -1414. This activation requires the Rap-1 G protein and the MEK-1 and p42/p44 MAPK enzymes but is independent of other NGF-activated signalling pathways involving protein kinase A or protein kinase C.
The ability of the CBP (CREB binding protein) coactivator to stimulate transcription has previously been shown to be stimulated by treatment of neuronal cells with nerve growth factor (NGF). This effect is dependent upon activation of the p42/p44 MAPK (mitogen activated protein kinase) pathway. Here we show that both CBP and the related p300 protein directly associate with the p42/p44 MAPK enzymes both prior to and following their activation by NGF and that CBP is phosphorylated following NGF treatment. These results indicate that phosphorylation of CBP itself by the p42/p44 MAPK pathway is likely to be critical for its role in NGF-mediated stimulation of gene expression.
Overexpression of the gene encoding the 70-kDa heat shock protein (hsp70) has previously been shown to protect neuronal cells against subsequent thermal or ischemic stress. It has no protective effect, however, against stimuli that induce apoptosis, although a mild heat shock (sufficient to induce hsp synthesis) does have a protective effect against apoptosis. We have prepared disabled herpes simplex virus-based vectors that are able to produce high level expression of individual hsps in infected neuronal cells without damaging effects. We have used these vectors to show that hsp27 and hsp56 (which have never previously been overexpressed in neuronal cells) as well as hsp70 can protect dorsal root ganglion neurons from thermal or ischemic stress. In contrast, only hsp27 can protect dorsal root ganglion neurons from apoptosis induced by nerve growth factor withdrawal, and hsp27 also protects the ND7 neuronal cell line from retinoic acid-induced apoptosis. However, hsp70 showed no protective effect against apoptosis in contrast to its anti-apoptotic effect in non-neuronal cell types. These results thus identify hsp27 as a novel neuroprotective factor and show that it can mediate this effect when delivered via a high efficiency viral vector.
We have previously demonstrated that interleukin-6 (IL-6) increases the levels of the heat shock protein 90 (Hsp-90) and activates the Hsp-90beta promoter via the IL-6-activated transcription factors NF-IL6 and signal transducer and activator of transcription-3 (STAT-3). Here, we show that interferon-gamma (IFN-gamma) treatment increases the levels of Hsp-70 and Hsp-90 and also enhances the activity of the Hsp-70 and Hsp-90beta promoters with these effects being dependent on activation of the STAT-1 transcription factor by IFN-gamma. These effects were not seen in a STAT-1-deficient cell line, indicating that IFN-gamma modulates Hsp induction via a STAT-1-dependent pathway. The effect of IFN-gamma/STAT-1 was mediated via a short region of the Hsp-70/Hsp-90 promoters, which also mediates the effects of NF-IL6 and STAT-3 and can bind STAT-1. This region also contains a binding site for the stress-activated transcription factor HSF-1. We show that STAT-1 and HSF-1 interact with one another via a protein-protein interaction and produce a strong activation of transcription, which is in contrast to our previous finding that STAT-3 and HSF-1 antagonize one another. To our knowledge this is the first report of HSF-1 interacting directly via a protein-protein interaction with another transcription factor. Such protein-protein interactions and the binding of a number of different stress and cytokine-activated transcription factors to a short region of the Hsp-90 and Hsp-70 gene promoters are likely to play a very important role in Hsp gene activation by non-stressful stimuli and the integration of these responses with the stress response of these genes.
The POU (Pit-Oct-Unc) family of transcription factors was originally defined on the basis of a common DNA binding domain in the mammalian factors Pit-1, Oct-1, and Oct-2 as well as the nematode protein Unc-86. Subsequently, a number of other POU family factors have been identified in both vertebrates and invertebrates. Many of these original and subsequently isolated members of the family have been shown to play critical roles in the development and functioning of the nervous system. To exemplify this, studies are described involving the functional characterisation of the Oct-2 factor, one of the original POU factors, and of the Brn-3 factors, which were isolated subsequently and are the mammalian factors most closely related to Unc-86.
Over expression of heat shock proteins (hsps) by transfection of plasmid constructs in vitro and in transgenic animals in vivo can protect primary cardiac cells from subsequent exposure to severe thermal or hypoxic stress. Here we show that such protection can also be achieved by over-expressing the hsps using herpes simplex virus (HSV) vectors capable of efficient gene delivery in vivo. Moreover, the convenience and high efficiency of this system has allowed us to show, for the first time, that over-expression of hsp27 or hsp70 can protect cardiac cells against three different apoptosis-inducing stimuli as well as against thermal or hypoxic stress whereas hsp56 has no protective effect. The potential therapeutic use of inducing the over-expression of specific hsps in cardiac cells in vivo using pharmacological or gene therapy procedures is discussed.
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Elevated levels of the cytokine interleukin-10 (IL-10) have been reported in patients with active systemic lupus erythematosus (SLE). Any role for IL-10 in the pathogenesis of SLE is likely to involve the activation of expression of specific genes within its target cells. We have previously reported elevated levels of the 90 000 MW heat-shock protein (hsp 90) and autoantibodies to hsp 90 in patients with SLE. Recent studies have shown that the cytokine IL-6 activates hsp 90 gene expression via specific transcription factors that include STAT-3 (signal transducer and activator of transcription 3). In view of the known role of STAT proteins in IL-10 signalling pathways, we have investigated the effect of IL-10 on hsp 90 gene expression. Here we report that IL-10 enhances the expression of hsp 90 in both a human hepatoma cell line (HepG2) stably expressing the human IL-10 receptor and peripheral blood mononuclear cells (PBMC). In reporter gene assays IL-10 is able to activate both the hsp 90alpha and hsp 90beta promoters directly. Furthermore, a short region of the hsp 90beta promoter which is activated in response to IL-10, contains a STAT-3 binding site. This element but not a mutant derivative unable to bind STAT-3, is able to confer a response to IL-10 on a heterologous promoter. These results may be understood in terms of the shared signalling mechanisms of IL-10 and IL-6 and provide evidence of a role for IL-10 in the overexpression of hsp 90 in SLE, with possible pathological consequences.
Cardiotrophin-1 (CT-1) is a member of the IL-6 family of cytokines which was originally discovered as a factor which can induce hypertrophy of cardiac myocytes both in vitro and in vivo. Subsequently, CT-1 has been shown to have a wide variety of different effects on cardiac and non cardiac cells including the ability to stimulate the survival of both cardiac and neuronal cells. Interestingly, whilst activation of the p42/p44 MAP kinase pathway is necessary for the survival promoting effects of CT-1 in cardiac cells, it is not required for its hypertrophic effect which is likely to involve activation of the Jak/STAT-3 pathway. CT-1 may therefore be of use as a novel cardio-protective agent, particularly if its hypertrophic effect can be specifically inhibited.