Cell stress genes and neuronal protection.
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Animals↗
Biomedical subjects
D S Latchman
Publications and source records attributed to D S Latchman.
Retinoic acid inhibits both the basal activity and phorbol ester-mediated activation of the HIV long terminal repeat promoter.
OBJECTIVE: To establish whether the steroid hormone retinoic acid (RA) was able to modulate the activity of the HIV-1 promoter. DESIGN: The effect of RA and nuclear factor (NF)-kappa B on HIV-1 promoter activity was investigated using HIV-1 long terminal repeat (LTR)-based reporter constructs. METHODS: The activity of wild type and mutant LTR sequences was compared in a variety of stably and transiently transfected human cell lines. RESULTS: It was shown that RA treatment inhibits both the basal activity of the HIV-1 LTR and its stimulation by phorbol ester treatment. This inhibition is observed in both HeLa cells (in the presence of exogenously supplied RA receptors) and the naturally RA-responsive U937 monocyte cell line. RA can also inhibit the stimulation of the HIV-1 LTR by overexpression of NF-kappa B subunits, while linkage of the NF-kappa B sites in the HIV promoter to a heterologous promoter results in its inhibition by RA. CONCLUSIONS: The data presented clearly demonstrate a negative effect of RA on HIV-1 LTR activity that may contribute to its effect on viral replication.
Base Sequence↗
The DNA target site for the Brn-3 POU family transcription factors can confer responsiveness to cyclic AMP and removal of serum in neuronal cells.
The POU factors Brn-3a and Brn-3b are closely related transcription factors which are expressed in neuronal cells. The levels of the transcripts encoding these factors are regulated in opposite directions in neuronal cells by specific cellular signalling pathways with dibutyryl cyclic AMP treatment and serum removal enhancing the level of Brn-3a and reducing the level of Brn-3b expression. This opposite expression pattern is paralleled by the ability of Brn-3a to specifically transactivate a target promoter bearing its DNA binding site whereas this promoter is repressed by Brn-3b. As predicted from these observations this target promoter is strongly activated by serum removal or addition of dibutyryl cyclic AMP. Therefore changes in Brn-3a and b expression can have a functional effect on promoter activity indicating that Brn-3a and Brn-3b can regulate gene expression via a specific binding site in response to the activation of specific cellular signalling pathways. The reasons for the differences in activity between these two related factors and their role in regulating gene activity in the nervous system are discussed.
Base Sequence↗
A common pathway mediates retinoic acid and PMA-dependent programmed cell death (apoptosis) of neuronal cells.
The extent of programmed cell death (apoptosis) which occurs upon transfer of a neuronal cell line (ND7) to serum-free medium can be greatly increased by addition of retinoic acid (RA) to the medium. Here we show that the degree of apoptosis can also be enhanced by agents which activate protein kinase C (PKC) and that such agents synergize with RA in inducing apoptosis. In contrast chronic down regulation of PKC dramatically reduces the ability of RA to induce apoptosis whilst ND7 cell lines selected for resistance to RA-induced apoptosis are also resistant to apoptosis induced by PKC activation. This indicates that a common death pathway mediates the induction of apoptosis by PKC activators and RA. The potential nature of this pathway and the role of PKC in neuronal cell apoptosis is discussed.
Animals↗
The Oct-2 transcription factor represses tyrosine hydroxylase expression via a heptamer TAATGARAT-like motif in the gene promoter.
The tyrosine hydroxylase (TH) gene promoter contains adjacent octamer and heptamer motifs which act as target sites for octamer binding transcription factors. Mutation of the heptamer motif but not the octamer motif enhances TH promoter activity in neuronal cells expressing Oct-2 but not in non-expressing fibroblasts. Similarly addition of the heptamer motif to a minimal TH promoter represses gene expression in neuronal cells but not in fibroblasts. These effects can be reproduced by the artificial expression of neuronal isoforms of Oct-2 in fibroblasts which results in the repression of transfected TH promoters containing an intact heptamer motif but not those in which this motif has been mutated or deleted. The TH promoter thus represents the first example of a cellular promoter which is repressed by Oct-2. The significance of this effect is discussed in terms of the cell type specificity of the TH promoter and its induction by different physiological stimuli.
Alternative Splicing↗
Inhibition of herpes simplex virus infection by ectopic expression of neuronal splice variants of the Oct-2 transcription factor.
Herpes simplex virus (HSV) is capable of lytic replication in most cells, such replication in epithelial cells resulting in the mucocutaneous lesions observed following in vivo infection. In addition however, the virus also establishes asymptomatic latent infections in sensory neurons which serve as a reservoir for further cycles of peripheral lytic infections. These latent infections are dependent upon the inhibition of viral immediate-early (IE) gene expression via the octamer-related TAATGARAT motif in the IE promoters resulting in the failure of the viral lytic cycle. Here we show that the ectopic expression of neuronal isoforms of the octamer/TAATGARAT-binding transcription factor Oct-2 in permissive BHK cells represses IE gene expression following HSV infection and inhibits the viral lytic cycle whereas the B lymphocyte isoform of Oct-2 does not have this effect. These results suggest that the neuronal isoforms of Oct-2 play a critical role in rendering neuronal cells non-permissive for the viral lytic cycle thereby allowing the establishment of latent infection. Moreover, this is the first time that the ectopic expression of a cellular transcription factor has been shown to inhibit infection with any virus, raising the possibility of therapeutically inhibiting lytic viral infections by inducing such ectopic expression.
Animals↗
Functional interaction between different isoforms of the Oct-2 transcription factor expressed in neuronal cells.
The predominant neuronal isoforms of the Oct-2 transcription factor, Oct 2.4 and Oct 2.5, repress the herpes simplex virus immediate-early promoter both in neuronal cells and in fibroblasts normally lacking Oct-2. In contrast, the predominant B lymphocyte form Oct 2.1, which is present at a lower level in neuronal cells, activates the immediate-early promoter in fibroblasts but represses it in neuronal cells. We show here that both Oct 2.4 and Oct 2.5 can functionally interact with Oct 2.1 and convert it from an activator into a repressor. Hence, the cell type-specific activity of Oct 2.1 results from the presence of Oct 2.4 and 2.5 in neuronal cells and their absence in other cell types. The significance of this effect is discussed in terms of the role of Oct-2 in the regulation of viral and cellular gene expression in neuronal cells.
Animals↗
Cell cycle arrest and morphological differentiation can occur in the absence of apoptosis in a neuronal cell line.
Apoptotic cell death plays a critical role in the development of the nervous system. Although the apoptotic death of mature non-dividing neurons has been extensively studied, the mechanisms mediating the extensive cell death in areas of the developing brain where proliferating neuroblasts are differentiating into mature neurons have not been analyzed. We have previously shown that the cell cycle arrest of a proliferating neuronal cell line by transfer from medium containing 10% foetal calf serum (FCS) to serum-free medium results in the morphological differentiation of some cells and the death of others by apoptosis. Here we show that the effect of 10% FCS can be mimicked by medium containing either similar concentrations of newborn or adult bovine serum or 1% FCS all of which maintain cellular proliferation and inhibit differentiation and apoptosis. In contrast, the presence of 0.5% FCS in the medium effectively prevents apoptosis but does not allow cellular proliferation or inhibit morphological differentiation. Hence cell cycle arrest and differentiation can occur in the absence of apoptosis in cells of neuronal origin and the factors in serum responsible for modulating these processes are likely to be distinct.
Animals↗
Stable high level expression of a transfected human HSP70 gene protects a heart-derived muscle cell line against thermal stress.
Heat shock protein 70 (HSP70) has been shown to play a fundamental role in the induction of thermotolerance in many biological systems. Elevated synthesis of HSP70 in response to diverse stresses such as heat, anoxia, ischaemia, ethanol and heavy metals has been correlated with protection against subsequent more severe stress and cross-tolerance to differing stresses. In this respect, exposure of the mammalian heart to sublethal heat treatment or ischaemia has been shown to protect against subsequent myocardial ischaemia with concomitant elevation of HSP70. However, direct demonstration that HSP70 can alone confer thermotolerance has until recently been restricted to transfection of fibroblasts with an HSP70 gene, although preliminary data from others suggests that transfection of H9c2 myocytes with an HSP70 gene can confer tolerance to substrate-free hypoxia. The purpose of this study was, therefore, to test directly whether a myocyte cell line which retains certain features of cardiac cells (as opposed to non-myocyte cells) can be protected against lethal thermal stress by transfection with a single HSP70 gene. Rat heart-derived H9c2 cells were transfected either with a plasmid from which high level expression of a human HSP70 gene is driven by a strong, heterologous (human) beta-actin promoter (APr-HS70), or with an analogous control plasmid containing no HSP70 gene (pAPr-1 neo). Following selection with the neomycin analogue G418, several clones were isolated which either expressed no HSP70 (control: pAPr-1 neo-derived) or stably expressed high constitutive levels of an inducible isoform of HSP70 (HSP70i) (APr-HS70-derived) as determined by Western blotting with a specific monoclonal antibody to HSP70i.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Antagonistic effects of retinoic acid and thyroid hormone on the expression of the tissue-specific splicing protein SmN in a clonal cell line derived from rat heart.
The SmN protein is a tissue-specific splicing factor which is expressed only in adult heart and brain but not in other tissues. Although a role for SmN in modulating tissue specific alternative splicing decisions in the heart and brain has been suggested, its precise function and the processes regulating its expression remain unclear. We show here that SmN is expressed in the H9c2 clonal cell line derived from rat heart and that its expression in these cells is affected by a variety of members of the steroid/thyroid hormone family. In particular thyroid hormone and retinoic acid exhibit antagonistic effects on SmN expression with thyroid hormone treatment producing large increases in expression whilst retinoic acid treatment virtually abolishes expression. These findings are discussed in terms of the regulation of SmN expression and the potential role of this factor in the response of cardiac cells to members of the steroid/thyroid hormone family.
Animals↗
Mice lacking Snrpn expression show normal regulation of neuronal alternative splicing events.
The SmN protein is closely related to the constitutively expressed RNA splicing protein SmB but is expressed only in brain and heart tissue. Mice which lack expression of SmN die shortly after birth suggesting a critical role for this protein possibly in the regulation of neuronal-specific alternative splicing events. We show here however that the neuronal-specific alternative splicing of the RNAs encoding several different classes of protein proceeds normally in mice lacking SmN expression. The potential role of SmN and the reasons for the lethal effect observed in non-expressing mice are discussed.
Alternative Splicing↗
Herpes simplex virus vectors for gene therapy.
Herpes simplex virus (HSV) has a number of advantages as a vector for delivering specific genes to the nervous system. These include its large size, wide host range, and its ability to establish long-lived asymptomatic infections in neuronal cells in which a specific region of the viral genome continues to be expressed. Unfortunately, the large size of this virus and difficulty in manipulating it has led to its use as a vector lagging behind that of other, smaller viruses such as the retroviruses. In addition, the virus's ability to replicate lytically in the brain, under some circumstances, causing encephalitis, has led to fears about its potential safety for ultimate use in humans. This review will discuss a number of new approaches that are aimed at rendering simpler the insertion of foreign genes into the virus and making it as safe as possible. Ultimately, these advances offer real hope for the use of HSV vectors in gene therapy procedures.
Brain↗
Nerve growth factor induces expression of immediate-early genes NGFI-A (Egr-1) and NGFI-B (nur 77) in adult rat dorsal root ganglion neurons.
We have used primary cultures of adult rat dorsal root ganglia (DRG), enriched in sensory neurons, to investigate the induction of immediate-early genes by NGF and a variety of other growth factors. Using the polymerase chain reaction we have quantitatively amplified specific mRNA transcripts induced by NGF, in the presence and absence of the protein synthesis inhibitor cycloheximide. NGFIA (Egr-1) and NGFIB (nur 77) mRNAs were elevated in level within 60 min of NGF treatment and independently of de novo protein synthesis. This was consistent with the behaviour of immediate-early genes. These kinetics were seen at a range of NGF concentrations. NGFIA and NGFIB mRNAs were also found to be induced in DRG cultures by a variety of other growth factors. Different patterns of induction of NGFIA and NGFIB mRNA observed in DRG cultures suggested that transcript-specific pathways of signal transduction were operating within neurons, dependent upon the particular growth factor stimulus. Comparison of data reported from growth factor treatment of other cell types with data from DRG cultures also revealed patterns of NGFIA and NGFIB mRNA induction specific to DRG neurons.
Animals↗
Heat shock proteins hsp90 and hsp70 protect neuronal cells from thermal stress but not from programmed cell death.
Prior exposure to a mild thermal stress can protect neuronal cells from a subsequent more severe stress including high temperature, ischemia, glutamate toxicity, or stimuli inducing apoptosis. Although the protective effect of thermal stress correlates with the elevated expression of the heat shock proteins (hsps), the protective effect of individual hsps has never been directly demonstrated in neuronal cells. Here we show that the constitutive overexpression of either of the major hsps, hsp90 or hsp70, can protect neuronal cells from thermal stress but not from stimuli that induce apoptosis. The possible mechanisms by which thermal stress can protect neuronal cells from apoptosis are discussed.
Animals↗
Detection of autoantibodies to the 90 kDa heat shock protein in systemic lupus erythematosus and other autoimmune diseases.
Expression of the highly conserved 90 kDa heat shock protein (Hsp90) is elevated in the peripheral blood mononuclear cells of approximately 25% of patients with SLE. Conflicting data have been published about the frequency of antibodies to Hsp90 with the previous methodology using a complex Western blot system. We now describe an ELISA to measure autoantibodies to Hsp90 and Hsp70 in SLE patients, healthy controls and patients with a variety of autoimmune rheumatic diseases. IgG and IgM antibodies were elevated in 26 and 35% of SLE patients, respectively. These results show autoantibodies to Hsp90 (but not Hsp70) are elevated in a significant proportion of patients with SLE (P < 0.025) compared to healthy controls; and that those with raised antibody levels were more likely to have renal disease and a low C3 level (P < 0.02).
Arthritis, Rheumatoid↗
The opposite and antagonistic effects of the closely related POU family transcription factors Brn-3a and Brn-3b on the activity of a target promoter are dependent on differences in the POU domain.
The Brn-3a, Brn-3b, and Brn-3c POU family transcription factors are closely related to one another and are members of the group IV subfamily of POU factors. Here we show that despite this close relationship, the factors have different effects on the activity of a target promoter: Brn-3a and Brn-3c stimulate the promoter whereas Brn-3b represses it. Moreover, Brn-3b can antagonize the stimulatory effect of Brn-3a on promoter activity and can also inhibit promoter activation by the Oct-2.1 POU factor. The difference in the transactivation activities of Brn-3a and Brn-3b is dependent upon the C-terminal region containing the POU domain of the two proteins, since exchange of this domain between the two factors converts Brn-3a into a repressor and Brn-3b into an activator.
Amino Acid Sequence↗
Repression of a herpes simplex virus immediate-early promoter by the Oct-2 transcription factor is dependent on an inhibitory region at the N terminus of the protein.
The B-cell form of the Oct-2 transcription factor Oct 2.1 can activate the herpes simplex virus immediate-early gene 3 (IE3) promoter, whereas the neuronally expressed Oct 2.4 and 2.5 forms of the protein, which contain a different C terminus, can repress this promoter. Here we show that partial or full deletion of the C terminus of Oct 2.1 in the presence of an intact N terminus results in a protein which can strongly repress the IE3 promoter. In contrast, deletion of the entire N terminus or a short region within it leaving the C terminus intact results in a very strong activator. Deletion of both N and C termini leaving only the isolated POU domain generates only a very weak repressor. The N-terminal region defined in this way can repress a heterologous promoter when linked to the DNA-binding domain of the GAL4 factor, indicating that it can function as an independent inhibitory domain. These results indicate that a specific region within the N terminus common to Oct 2.1, 2.4, and 2.5 plays a critical role in the ability of neuronally expressed forms of Oct-2 to repress the IE3 promoter but can do so only when the C-terminal region of Oct 2.1 is altered or deleted.
Animals↗
Myocardial protection after whole body heat stress in the rabbit is dependent on metabolic substrate and is related to the amount of the inducible 70-kD heat stress protein.
The aims of this study were to examine the effects of whole body heat stress and subsequent stress protein induction on glycolytic metabolism, mitochondrial metabolism, and calcium handling within the heart. The effect of heat stress on glycolytic and mitochondrial pathways was examined by measuring contractile performance in the presence of glucose and pyruvate, respectively. Calcium handling was assessed using force-interval relationships. Right ventricular papillary muscles taken from heat-stressed and control rabbit hearts were superfused with Kreb's solution containing either glucose or pyruvate and rendered hypoxic for 30 min. After reoxygenation, the greatest recovery of contractile function occurred in the heat-stressed muscles with pyruvate as substrate; there was, however, no difference in the force-interval relationship between the groups. The degree of contractile recovery was related to the content of the inducible 70-kD but not the 65-kD, heat stress protein. This study suggests that heat stress enhances the ability of rabbit papillary muscle to use pyruvate, but not glucose, after reoxygenation, and that the differences seen in contractility may be secondary to induction of the 72-kD stress protein.
Animals↗