Hormones influence countless biological processes across the lifespan, and during developmental s... more Hormones influence countless biological processes across the lifespan, and during developmental sensitive periods hormones have the potential to cause permanent tissue-specific alterations in anatomy and physiology. There are numerous critical periods in development wherein different targets are affected. This review outlines the proceedings of the Hormonal Programming in Development session at the US-South American Workshop in Neuroendocrinology in August 2011. Here we discuss how gonadal hormones impact various biological processes within the brain and gonads during early development and describe the changes that take place in the aging female ovary. At the cellular level, hormonal targets in the brain include neurons, glia, or vasculature. On a genomic/epigenomic level, transcription factor signaling and epigenetic changes alter the expression of hormone receptor genes across development and following ischemic brain insult. In addition, organizational hormone exposure alters epigenetic processes in specific brain nuclei and may be a mediator of sexual differentiation of the neonatal brain. During development of the ovary, exposure to excess gonadal hormones leads to polycystic ovarian syndrome (PCOS). Exposure to excess androgens during fetal development also has a profound effect on the development of the male reproductive system. In addition, increased sympathetic nerve activity and stress during early life have been linked to PCOS symptomology in adulthood. Finally, we
Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 ... more Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and f85% of kringle 5. In culture, cancer cell surface globular B-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface B-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized B-actin similarly, with a K d of f140 nmol/L. The binding is inhibited by E-aminocaproic acid (EACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from B-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys 61 or Lys 68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys 61 and Lys 68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 Mmol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface B-actin, and the truncated kringle 5 in AS4.5 results in its release from B-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites.
... E-mail: [email protected] Immune Modulation by Estrogens Role in CNS HIV-1 Infection Melinda E. ... more ... E-mail: [email protected] Immune Modulation by Estrogens Role in CNS HIV-1 Infection Melinda E. Wilson,2 Filomena O. Dimayuga,1 Janelle L. Reed,1 Thomas E. Curry,3 Carol F. Anderson,4 Avindra Nath,4 and Annadora J. Bruce-Keller1 ...
Http Dx Doi Org 10 1089 Aid 2009 0065, Nov 29, 2009
Many human immunodeficiency virus (HIV) proteins including Tat are produced by HIV-infected astro... more Many human immunodeficiency virus (HIV) proteins including Tat are produced by HIV-infected astrocytes and secreted into the brain resulting in extensive neuronal damage that contributes to the pathogenesis of HIV dementia. The neuroprotective hormone 17beta-estradiol (E2) is known to negatively regulate the HIV transcriptional promoter in human fetal astrocytes (SVGA cell line) in a Tat-dependent manner. In the present study we extended our investigation in HIV-infected SVGA cells and found a reduction in HIV p24 levels following E2 treatment in comparison to control. Although many E2-mediated events occur through estrogen receptor alpha (ERalpha), we found low levels of ERalpha mRNA and failed to detect ERalpha protein in SVGA cells. Paradoxically, when ERalpha was overexpressed the E2-mediated decrease in Tat transactivation of the promotor was prevented. To determine whether ERalpha expression is altered in the human brain following HIV infection, postmortum hippocampal tissue was obtained from cognitively normal HIV- and HIV+ patients, patients diagnosed with either mild cognitive/motor disorder (MCMD) or HIV-associated dementia (HAD). Immunohistochemistry and quantitative real-time PCR (qRT-PCR) for ERalpha and glial fibrillary acidic protein (GFAP) showed that ERalpha mRNA levels were not significantly different between groups, while GFAP increased in the hippocampus in the HIV+ compared to the HIV- group and was decreased in the MCMD and HAD subgroups compared to HIV+ controls. Notably the ratio of ERalpha-positive reactive astrocytes to total reactive astrocytes increased and significantly correlated with the severity of cognitive impairment following HIV infection. The data suggest that E2 would have the most dramatic effect in reducing HIV transcription early in the disease process when the subpopulation of astrocytes expressing ERalpha is low.
In recent years our appreciation that estradiol is truly a pleiotropic hormone has grown dramatic... more In recent years our appreciation that estradiol is truly a pleiotropic hormone has grown dramatically. We will review the findings that suggest that estrogens may exert important non-reproductive actions on the brain. These studies provide important insights into the clinical effects of estrogen replacement therapy on age- and disease-related processes in the brain. We will also discuss the multiple cellular and molecular mechanisms that may underlie estradiol's neurotrophic and neuroprotective effects.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
We have shown that physiological levels of estradiol exert profound protective effects on the cer... more We have shown that physiological levels of estradiol exert profound protective effects on the cerebral cortex in ischemia induced by permanent middle cerebral artery occlusion. The major goal of this study was to begin to elucidate potential mechanisms of estradiol action in injury. Bcl-2 is a protooncogene that promotes cell survival in a variety of tissues including the brain. Because estradiol is known to promote cell survival via Bcl-2 in non-neural tissues, we tested the hypothesis that estradiol decreases cell death by influencing bcl-2 expression in ischemic brain injury. Furthermore, because estradiol may protect the brain through estrogen receptormediated mechanisms, we examined expression of both receptor subtypes ER␣ and ER in the normal and injured brain. We analyzed gene expression by RT-PCR in microdissected regions of the cerebral cortex obtained from injured and sham female rats treated with estradiol or oil. We found that estradiol prevented the injury-induced downregulation of bcl-2 expres-sion. This effect was specific to bcl-2, as expression of other members of the bcl-2 family (bax, bcl-x L , bcl-x S , and bad) was unaffected by estradiol treatment. We also found that estrogen receptors were differentially modulated in injury, with ER expression paralleling bcl-2 expression. Finally, we provide the first evidence of functional ER protein that is capable of binding ligand within the region of the cortex where estradiolmediated neuroprotection was observed in cerebral ischemia. These findings indicate that estradiol modulates the expression of bcl-2 in ischemic injury. Furthermore, our data suggest that estrogen receptors may be involved in hormone-mediated neuroprotection.
In vitro experiments from our laboratory and others have suggested that herpes simplex virus thym... more In vitro experiments from our laboratory and others have suggested that herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy depends on gap junctional intercellular communication (GJIC) to produce a strong bystander effect. Furthermore, we have shown that cells transduced with HSV-TK can be protected from GCV-mediated toxicity by GJIC with bystander cells. We wished to determine whether GJIC affected either the bystander or protective effect of the cytosine deaminase (CD)/5-flucytosine (5-FC) gene therapy approach, in which CD converts 5-FC to 5-fluorouracil (5-FU). To test this, we designed a coculture system using communication-competent WB rat hepatocytes and a noncommunicating subclone (aB1), which were transduced with CD and with antibiotic resistance genes so that we could independently determine the survival of the CD-containing or bystander cells. We found that, compared to the HSV-TK/GCV strategy, bystander killing resulting from treatment with CD/5...
Many clinical studies suggest that estrogen enhances memory and cognition and protects against ne... more Many clinical studies suggest that estrogen enhances memory and cognition and protects against neurodegenerative diseases and injury associated with stroke or stress. These results are strongly supported by experiments performed in animal models using both in vivo and in vitro methods. We present here data from our lab that establishes that physiological levels of estradiol exert profound protective actions against ischemic injury. Further we will present evidence that these effects may be mediated through estrogen receptors that may influence the bcl-2 family of genes.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999
We have shown that physiological levels of estradiol exert profound protective effects on the cer... more We have shown that physiological levels of estradiol exert profound protective effects on the cerebral cortex in ischemia induced by permanent middle cerebral artery occlusion. The major goal of this study was to begin to elucidate potential mechanisms of estradiol action in injury. Bcl-2 is a proto-oncogene that promotes cell survival in a variety of tissues including the brain. Because estradiol is known to promote cell survival via Bcl-2 in non-neural tissues, we tested the hypothesis that estradiol decreases cell death by influencing bcl-2 expression in ischemic brain injury. Furthermore, because estradiol may protect the brain through estrogen receptor-mediated mechanisms, we examined expression of both receptor subtypes ERalpha and ERbeta in the normal and injured brain. We analyzed gene expression by RT-PCR in microdissected regions of the cerebral cortex obtained from injured and sham female rats treated with estradiol or oil. We found that estradiol prevented the injury-ind...
As part of highly active antiretroviral therapy, protease inhibitor treatment has significantly i... more As part of highly active antiretroviral therapy, protease inhibitor treatment has significantly increased the lifespan of human immunodeficiency virus (HIV)-infected individuals. Many patients, however, develop negative side effects, including premature atherosclerosis. We have previously demonstrated that in male low density lipoprotein receptor (LDL-R) null mice, HIV protease inhibitors induce atherosclerotic lesions and cholesterol accumulation in macrophages in the absence of changes in plasma lipid levels. We determined that these increases were due to an up-regulation of the scavenger receptor, CD36. In the present study, we examined the effects of HIV protease inhibitors in female LDL-R null mice. Female mice given ritonavir and amprenavir (23 and 10 microg/mouse/day, respectively) developed fewer atherosclerotic lesions than males. Furthermore, peritoneal macrophages isolated from ritonavir-treated females had reduced levels of cholesterol accumulation as compared with males...
American journal of physiology. Cell physiology, 2006
HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infecte... more HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that alpha-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using alpha-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of alpha-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, alpha-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase ...
Marek's disease virus (MDV) is an acutely oncogenic avian herpesvirus. The tightly cell-a... more Marek's disease virus (MDV) is an acutely oncogenic avian herpesvirus. The tightly cell-associated in vitro growth characteristics of MDV present unique problems when attempting to purify, analyze, and manipulate MDV genomes. To facilitate molecular characterization of MDV, contour-clamped homogeneous electric fields electrophoresis (CHEF) was used to purify infectious MDV genomes. CHEF techniques were optimized for evaluation of total genome size and alterations in structure which occur during in vitro attenuation of oncogenic MDV. Our results indicated that genomes of attenuated serotype 1 MDV strain JM may contain deletions totaling 15 kbp while high-passage serotype 2 nononcogenic MDV strains SB-1 and 281MI/1 were 5 and 3 kbp larger, respectively, than their low-passage counterparts. Using cell-free CHEF-purified MDV genomes as hybridization probes, we identified a 200-bp deletion in attenuated genomes of the very virulent MDV strain MD11. At present, it is unclear if this 200-bp is related to mutations which lead to loss of oncogenicity or pathogenicity in MD11. This study is the first report which describes procedures for purification of infectious herpesvirus genomes from pulsed-field gels. Our results demonstrate that pulsed-field-purified viral DNA will facilitate molecular characterization of MDV and other cell-associated herpesviruses.
Genetic assoaation studies for complex diseases, like Alzheimer's disease (AD). offer several adv... more Genetic assoaation studies for complex diseases, like Alzheimer's disease (AD). offer several advantages; they are simpler to organize at a relatively low cost and are more powerful to detect small effects than family-based linkage studies. However. using a case-control design to rtudy association always raises the concern that inappropriate control or population stratification could generate false positive results. Thus, case-control studies muAt be interpreted with caution. The associatton of the APOE* allele with AD has been well established. However, as with other complex genetic disorders the underlying cause of AD is not noted in one gene. The observation that the APOE* allele is neither necessary nor sufficient for the development of AD emphasizes on the involvement of other genetic and/or environmental factora, which etther alone or in conjunction with APOE*4, increase an individual's risk of developing AD. In addttion to APOE, several other genes have been implicated in the risk of late-onset AD, but the results of these association studies are equtvocal. The controversial re%dts of some of these association studies underscores the need that association studies be carried out on relatively large and genetically homogenous case-control cohorts. This nlao underscore\ the m~portancr that AD is a complex, heterogeneous and multifactorial disorder where several genes. each with a small effect, are involved in affecting the risk of AD. To Eearch fol additional risk genes for AD, It is important to focus on those genes whose products have been implicated in the pathogene\is of AD. Determination of relationship between these, so called "susceptibility genes" as well as between these and the APOE gene will help to unravel the complex genetic and physiologic mechanism\ leading to AD. We have evaluated the role of genetic variation in the alantichymotrypsin (ACT) gene and the risk of AD in a large case-control cohort. Our result? indicate that the ACT gene harbors several potentially important variable sites which are associated with either an increased or decreased risk of AD, but their contribution is relatively small.
Experimental and epidemiological data suggest that estrogen can be protective in both brain injur... more Experimental and epidemiological data suggest that estrogen can be protective in both brain injury and infection. While estrogens can act directly on neurons to promote neuronal survival, estrogen also has antiinflammatory properties that may contribute to overall neuroprotection. Accordingly, estrogens may have particular relevance in chronic neuroimmune disorders such as HIV dementia. As AIDS is now a leading cause of death among women in their reproductive years, understanding the role that female sex hormones might play in the physiology of HIV-1 infection is especially critical. Indeed, there is accumulating evidence that many manifestations of HIV differ in women. For instance, it is now well established that women present with a lower viral titer at the time of seroconversion, have lower HIV viral loads compared to men at similar stages of disease, and may have altered disease progression during pregnancy. Conversely, while epidemiological studies suggest that women may be more vulnerable to certain late-stage AIDS-related illnesses including HIV dementia, there is accumulating data that strongly suggest an estrogen-deficient state is associated with long-term HIV infection in some women. Evaluated as a whole, existing evidence indicates that estrogen can directly protect neurons from damage, can modulate brain inflammation, and could act to maintain low titers of the HIV-1 virus. Accordingly, it can be hypothesized that maintenance of serum estradiol levels could decrease the incidence of HIV dementia and other AIDS-related neurological syndromes in HIV-1 positive women. In this article, we both summarize current understanding and present new data related to the potential mechanisms whereby estrogen could modulate the mechanics and the consequences of HIV-1 infection in the brain and thereby thwart the development of HIV dementia.
Communication mechanisms [extra-, intra-, and gap junctional inter-cellular communication (GJIC)]... more Communication mechanisms [extra-, intra-, and gap junctional inter-cellular communication (GJIC)] control, from the fertilized egg, through embryogenesis to maturity and aging, whether a cell proliferates, differentiates, dies by apoptosis, or if differentiated, adaptively responds to endogenous and exogenous signals. From the egg to the 100 trillion cells in the human body, health is maintained when these communication processes between stem, progenitor and terminally differentiated cells are integrated. Each cell choice involves 'epigenetic' mechanisms to alter the expression of genes at the transcriptional, translational or post-translational levels. Disruption of the communication mechanisms can be either adaptive or maladaptive. Modulation of extra-cellular communication, either by genetic imbalances of growth factors, hormones or neurotransmitters or by environmental, exogenous chemicals can trigger signal transducing intra-cellular mechanisms. These intra-cellular signals can modulate gene expression at the transcriptional, translational or post-translational levels while also modulating GJIC. Untimely or chronic disruption of GJIC during embryonic or fetal development could lead to embryonic lethality or teratogenesis. By modulation of GJIC, homeostatic control of cell growth, differentiation or apoptosis could lead to specific diseases, such as neurological, cardiovascular, reproductive or endocrinological dysfunction. Chemical modulation or oncogene down-regulation of GJIC in initiated tissues has been shown to lead to tumor promotion. Genetic syndromes carrying a mutated gap junction gene, together with some transgenic and knock-out gap junction gene mice, provide evidence for the importance of this organelle found only in metazoans. Implications for 'thresholds' to toxicants and for risk assessment are evident.
Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens. A considerable amount of rese... more Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens. A considerable amount of research has been devoted to predicting the genotoxic, tumor-initiating potential of PAHs based on chemical structure. However, information on the correlation of structure with the non-genetoxic, epigenetic events of tumor promotion is sparse. PAHs containing a bay or bay-like region were shown to be potent inhibitors of gap-junctional intercellular communication (GJIC), an epigenetic event involved in the removal of an initiated cell from growth suppression. We tested the epigenetic toxicity of PAHs containing bay-like regions by comparing the effects of methylated vs. chlorinated isomers of anthracene on the temporal activation of mitogen-activated protein kinase (MAPK) and the regulation of GJIC. Specifically, we used anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9,10-dimethylanthracene, 1-chloroanthracene, 2-chloroanthracene, and 9-chloroanthracene. We determined the effect of these compounds on GJIC and on the activation of extracellular receptor kinase , a MAPK, in F344 rat liver epithelial cells. Results showed that bay or bay-like regions, formed by either chlorine or a methyl group, reversibly inhibited GJIC at the same doses, time, and time of recovery, whereas the linear-planar isomers had no effect on GJIC. Similarly, the GJIC-inhibitory isomers also induced the phosphorylation of ERK 1 and ERK 2, while the non-inhibitory isomers had no effect on the activation of these MAPKs. MAPK activation occurred 10 -20 min after the inhibition of GJIC, which indicates that MAPK is not involved in the initial regulation of GJIC; instead altered GJIC may be affecting MAPK activation. The present study revealed that there are structural determinants of PAHs, which clearly affect epigenetic events known to be involved in the non-genetoxic steps of tumor promotion. These events are the release of a cell from growth suppression involving the reduction of GJIC, followed by the activation of intracellular mitogenic events.
The Journal of Steroid Biochemistry and Molecular Biology, 1998
FSH-beta mRNA is dramatically regulated in the infantile female rat anterior pituitary. Elevated ... more FSH-beta mRNA is dramatically regulated in the infantile female rat anterior pituitary. Elevated plasma levels of FSH correspond with increased FSH-beta mRNA levels which peak on PND 12. The source of this regulation does not appear to be GnRH, since the administration of a potent GnRH antagonist does not suppress FSH-beta mRNA levels. Consequently, we have examined the effects of the gonadal steroid hormones, estrogen and androgen, on the maintenance of gonadotropin secretion and gene expression both in vivo and in vitro. Androgen and estrogen action was blocked in vivo with the specific receptor antagonists, flutamide (150 microg) and tamoxifen (200 microg). Administration of antagonists during two different three day time-periods of infantile life [postnatal day (PND) 8-11 and PND 11-14] resulted in differing effects on both FSH and LH secretion as well as on FSH-beta and LH-beta mRNA levels. Flutamide and tamoxifen treatment both suppressed FSH secretion at either age examined (p < 0.01). LH secretion was suppressed by both treatments but only at the younger of the two ages (p < 0.01). In contrast to its effects on FSH secretion, tamoxifen suppressed FSH-beta mRNA levels in the later group only. LH-beta mRNA levels were suppressed by tamoxifen, but only in the younger age group (p < 0.05). The direct effects of steroid hormones on infantile pituitary gonadotrophs were examined in vitro by incubating cells with dihydrotestosterone propionate (DHTP; 10(-8) M) or 17beta-estradiol (E; 10(-8) M). Both DHT and E treatment stimulated FSH secretion when measured 48 h later (p < 0.01). There were no effects on LH secretion. FSH-beta mRNA levels were also stimulated by DHT at 48 h (p < 0.01). Estradiol treatment transiently increased FSH-beta mRNA levels at 2 and 6 h following treatment (p < 0.01) but not at 48 h. LH-beta levels were suppressed by DHT treatment (p < 0.05), and E transiently elevated LH-beta mRNA levels at 2 h (p < 0.05). Taken together these studies indicate that gonadotrophs from infantile female rats are capable of responding directly to steroid hormones, and may play a role in the selective stimulation of FSH secretion and expression in vivo.
During aging, there is an increase in neurodegenerative diseases and a decrease in cognitive perf... more During aging, there is an increase in neurodegenerative diseases and a decrease in cognitive performance. Postmenopausal women are more vulnerable as their estrogen levels decline, but most hormone replacement therapies do not prevent cognitive decline. One potential reason is that the timing of hormone replacement is critical and changes in estrogen receptor expression may over-ride hormonal intervention. In rodents, ERβ mRNA decreases in the cortex with age. One mechanism by which ERβ mRNA could be regulated is by epigenetic modification of ERβ promoter. Here we show an increase in methylation of ERβ promoter corresponding to decreases in ERβ mRNA in aging female cortex.
Hormones influence countless biological processes across the lifespan, and during developmental s... more Hormones influence countless biological processes across the lifespan, and during developmental sensitive periods hormones have the potential to cause permanent tissue-specific alterations in anatomy and physiology. There are numerous critical periods in development wherein different targets are affected. This review outlines the proceedings of the Hormonal Programming in Development session at the US-South American Workshop in Neuroendocrinology in August 2011. Here we discuss how gonadal hormones impact various biological processes within the brain and gonads during early development and describe the changes that take place in the aging female ovary. At the cellular level, hormonal targets in the brain include neurons, glia, or vasculature. On a genomic/epigenomic level, transcription factor signaling and epigenetic changes alter the expression of hormone receptor genes across development and following ischemic brain insult. In addition, organizational hormone exposure alters epigenetic processes in specific brain nuclei and may be a mediator of sexual differentiation of the neonatal brain. During development of the ovary, exposure to excess gonadal hormones leads to polycystic ovarian syndrome (PCOS). Exposure to excess androgens during fetal development also has a profound effect on the development of the male reproductive system. In addition, increased sympathetic nerve activity and stress during early life have been linked to PCOS symptomology in adulthood. Finally, we
Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 ... more Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and f85% of kringle 5. In culture, cancer cell surface globular B-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface B-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized B-actin similarly, with a K d of f140 nmol/L. The binding is inhibited by E-aminocaproic acid (EACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from B-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys 61 or Lys 68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys 61 and Lys 68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 Mmol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface B-actin, and the truncated kringle 5 in AS4.5 results in its release from B-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites.
... E-mail: [email protected] Immune Modulation by Estrogens Role in CNS HIV-1 Infection Melinda E. ... more ... E-mail: [email protected] Immune Modulation by Estrogens Role in CNS HIV-1 Infection Melinda E. Wilson,2 Filomena O. Dimayuga,1 Janelle L. Reed,1 Thomas E. Curry,3 Carol F. Anderson,4 Avindra Nath,4 and Annadora J. Bruce-Keller1 ...
Http Dx Doi Org 10 1089 Aid 2009 0065, Nov 29, 2009
Many human immunodeficiency virus (HIV) proteins including Tat are produced by HIV-infected astro... more Many human immunodeficiency virus (HIV) proteins including Tat are produced by HIV-infected astrocytes and secreted into the brain resulting in extensive neuronal damage that contributes to the pathogenesis of HIV dementia. The neuroprotective hormone 17beta-estradiol (E2) is known to negatively regulate the HIV transcriptional promoter in human fetal astrocytes (SVGA cell line) in a Tat-dependent manner. In the present study we extended our investigation in HIV-infected SVGA cells and found a reduction in HIV p24 levels following E2 treatment in comparison to control. Although many E2-mediated events occur through estrogen receptor alpha (ERalpha), we found low levels of ERalpha mRNA and failed to detect ERalpha protein in SVGA cells. Paradoxically, when ERalpha was overexpressed the E2-mediated decrease in Tat transactivation of the promotor was prevented. To determine whether ERalpha expression is altered in the human brain following HIV infection, postmortum hippocampal tissue was obtained from cognitively normal HIV- and HIV+ patients, patients diagnosed with either mild cognitive/motor disorder (MCMD) or HIV-associated dementia (HAD). Immunohistochemistry and quantitative real-time PCR (qRT-PCR) for ERalpha and glial fibrillary acidic protein (GFAP) showed that ERalpha mRNA levels were not significantly different between groups, while GFAP increased in the hippocampus in the HIV+ compared to the HIV- group and was decreased in the MCMD and HAD subgroups compared to HIV+ controls. Notably the ratio of ERalpha-positive reactive astrocytes to total reactive astrocytes increased and significantly correlated with the severity of cognitive impairment following HIV infection. The data suggest that E2 would have the most dramatic effect in reducing HIV transcription early in the disease process when the subpopulation of astrocytes expressing ERalpha is low.
In recent years our appreciation that estradiol is truly a pleiotropic hormone has grown dramatic... more In recent years our appreciation that estradiol is truly a pleiotropic hormone has grown dramatically. We will review the findings that suggest that estrogens may exert important non-reproductive actions on the brain. These studies provide important insights into the clinical effects of estrogen replacement therapy on age- and disease-related processes in the brain. We will also discuss the multiple cellular and molecular mechanisms that may underlie estradiol's neurotrophic and neuroprotective effects.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
We have shown that physiological levels of estradiol exert profound protective effects on the cer... more We have shown that physiological levels of estradiol exert profound protective effects on the cerebral cortex in ischemia induced by permanent middle cerebral artery occlusion. The major goal of this study was to begin to elucidate potential mechanisms of estradiol action in injury. Bcl-2 is a protooncogene that promotes cell survival in a variety of tissues including the brain. Because estradiol is known to promote cell survival via Bcl-2 in non-neural tissues, we tested the hypothesis that estradiol decreases cell death by influencing bcl-2 expression in ischemic brain injury. Furthermore, because estradiol may protect the brain through estrogen receptormediated mechanisms, we examined expression of both receptor subtypes ER␣ and ER in the normal and injured brain. We analyzed gene expression by RT-PCR in microdissected regions of the cerebral cortex obtained from injured and sham female rats treated with estradiol or oil. We found that estradiol prevented the injury-induced downregulation of bcl-2 expres-sion. This effect was specific to bcl-2, as expression of other members of the bcl-2 family (bax, bcl-x L , bcl-x S , and bad) was unaffected by estradiol treatment. We also found that estrogen receptors were differentially modulated in injury, with ER expression paralleling bcl-2 expression. Finally, we provide the first evidence of functional ER protein that is capable of binding ligand within the region of the cortex where estradiolmediated neuroprotection was observed in cerebral ischemia. These findings indicate that estradiol modulates the expression of bcl-2 in ischemic injury. Furthermore, our data suggest that estrogen receptors may be involved in hormone-mediated neuroprotection.
In vitro experiments from our laboratory and others have suggested that herpes simplex virus thym... more In vitro experiments from our laboratory and others have suggested that herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy depends on gap junctional intercellular communication (GJIC) to produce a strong bystander effect. Furthermore, we have shown that cells transduced with HSV-TK can be protected from GCV-mediated toxicity by GJIC with bystander cells. We wished to determine whether GJIC affected either the bystander or protective effect of the cytosine deaminase (CD)/5-flucytosine (5-FC) gene therapy approach, in which CD converts 5-FC to 5-fluorouracil (5-FU). To test this, we designed a coculture system using communication-competent WB rat hepatocytes and a noncommunicating subclone (aB1), which were transduced with CD and with antibiotic resistance genes so that we could independently determine the survival of the CD-containing or bystander cells. We found that, compared to the HSV-TK/GCV strategy, bystander killing resulting from treatment with CD/5...
Many clinical studies suggest that estrogen enhances memory and cognition and protects against ne... more Many clinical studies suggest that estrogen enhances memory and cognition and protects against neurodegenerative diseases and injury associated with stroke or stress. These results are strongly supported by experiments performed in animal models using both in vivo and in vitro methods. We present here data from our lab that establishes that physiological levels of estradiol exert profound protective actions against ischemic injury. Further we will present evidence that these effects may be mediated through estrogen receptors that may influence the bcl-2 family of genes.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999
We have shown that physiological levels of estradiol exert profound protective effects on the cer... more We have shown that physiological levels of estradiol exert profound protective effects on the cerebral cortex in ischemia induced by permanent middle cerebral artery occlusion. The major goal of this study was to begin to elucidate potential mechanisms of estradiol action in injury. Bcl-2 is a proto-oncogene that promotes cell survival in a variety of tissues including the brain. Because estradiol is known to promote cell survival via Bcl-2 in non-neural tissues, we tested the hypothesis that estradiol decreases cell death by influencing bcl-2 expression in ischemic brain injury. Furthermore, because estradiol may protect the brain through estrogen receptor-mediated mechanisms, we examined expression of both receptor subtypes ERalpha and ERbeta in the normal and injured brain. We analyzed gene expression by RT-PCR in microdissected regions of the cerebral cortex obtained from injured and sham female rats treated with estradiol or oil. We found that estradiol prevented the injury-ind...
As part of highly active antiretroviral therapy, protease inhibitor treatment has significantly i... more As part of highly active antiretroviral therapy, protease inhibitor treatment has significantly increased the lifespan of human immunodeficiency virus (HIV)-infected individuals. Many patients, however, develop negative side effects, including premature atherosclerosis. We have previously demonstrated that in male low density lipoprotein receptor (LDL-R) null mice, HIV protease inhibitors induce atherosclerotic lesions and cholesterol accumulation in macrophages in the absence of changes in plasma lipid levels. We determined that these increases were due to an up-regulation of the scavenger receptor, CD36. In the present study, we examined the effects of HIV protease inhibitors in female LDL-R null mice. Female mice given ritonavir and amprenavir (23 and 10 microg/mouse/day, respectively) developed fewer atherosclerotic lesions than males. Furthermore, peritoneal macrophages isolated from ritonavir-treated females had reduced levels of cholesterol accumulation as compared with males...
American journal of physiology. Cell physiology, 2006
HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infecte... more HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that alpha-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using alpha-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of alpha-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, alpha-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase ...
Marek's disease virus (MDV) is an acutely oncogenic avian herpesvirus. The tightly cell-a... more Marek's disease virus (MDV) is an acutely oncogenic avian herpesvirus. The tightly cell-associated in vitro growth characteristics of MDV present unique problems when attempting to purify, analyze, and manipulate MDV genomes. To facilitate molecular characterization of MDV, contour-clamped homogeneous electric fields electrophoresis (CHEF) was used to purify infectious MDV genomes. CHEF techniques were optimized for evaluation of total genome size and alterations in structure which occur during in vitro attenuation of oncogenic MDV. Our results indicated that genomes of attenuated serotype 1 MDV strain JM may contain deletions totaling 15 kbp while high-passage serotype 2 nononcogenic MDV strains SB-1 and 281MI/1 were 5 and 3 kbp larger, respectively, than their low-passage counterparts. Using cell-free CHEF-purified MDV genomes as hybridization probes, we identified a 200-bp deletion in attenuated genomes of the very virulent MDV strain MD11. At present, it is unclear if this 200-bp is related to mutations which lead to loss of oncogenicity or pathogenicity in MD11. This study is the first report which describes procedures for purification of infectious herpesvirus genomes from pulsed-field gels. Our results demonstrate that pulsed-field-purified viral DNA will facilitate molecular characterization of MDV and other cell-associated herpesviruses.
Genetic assoaation studies for complex diseases, like Alzheimer's disease (AD). offer several adv... more Genetic assoaation studies for complex diseases, like Alzheimer's disease (AD). offer several advantages; they are simpler to organize at a relatively low cost and are more powerful to detect small effects than family-based linkage studies. However. using a case-control design to rtudy association always raises the concern that inappropriate control or population stratification could generate false positive results. Thus, case-control studies muAt be interpreted with caution. The associatton of the APOE* allele with AD has been well established. However, as with other complex genetic disorders the underlying cause of AD is not noted in one gene. The observation that the APOE* allele is neither necessary nor sufficient for the development of AD emphasizes on the involvement of other genetic and/or environmental factora, which etther alone or in conjunction with APOE*4, increase an individual's risk of developing AD. In addttion to APOE, several other genes have been implicated in the risk of late-onset AD, but the results of these association studies are equtvocal. The controversial re%dts of some of these association studies underscores the need that association studies be carried out on relatively large and genetically homogenous case-control cohorts. This nlao underscore\ the m~portancr that AD is a complex, heterogeneous and multifactorial disorder where several genes. each with a small effect, are involved in affecting the risk of AD. To Eearch fol additional risk genes for AD, It is important to focus on those genes whose products have been implicated in the pathogene\is of AD. Determination of relationship between these, so called "susceptibility genes" as well as between these and the APOE gene will help to unravel the complex genetic and physiologic mechanism\ leading to AD. We have evaluated the role of genetic variation in the alantichymotrypsin (ACT) gene and the risk of AD in a large case-control cohort. Our result? indicate that the ACT gene harbors several potentially important variable sites which are associated with either an increased or decreased risk of AD, but their contribution is relatively small.
Experimental and epidemiological data suggest that estrogen can be protective in both brain injur... more Experimental and epidemiological data suggest that estrogen can be protective in both brain injury and infection. While estrogens can act directly on neurons to promote neuronal survival, estrogen also has antiinflammatory properties that may contribute to overall neuroprotection. Accordingly, estrogens may have particular relevance in chronic neuroimmune disorders such as HIV dementia. As AIDS is now a leading cause of death among women in their reproductive years, understanding the role that female sex hormones might play in the physiology of HIV-1 infection is especially critical. Indeed, there is accumulating evidence that many manifestations of HIV differ in women. For instance, it is now well established that women present with a lower viral titer at the time of seroconversion, have lower HIV viral loads compared to men at similar stages of disease, and may have altered disease progression during pregnancy. Conversely, while epidemiological studies suggest that women may be more vulnerable to certain late-stage AIDS-related illnesses including HIV dementia, there is accumulating data that strongly suggest an estrogen-deficient state is associated with long-term HIV infection in some women. Evaluated as a whole, existing evidence indicates that estrogen can directly protect neurons from damage, can modulate brain inflammation, and could act to maintain low titers of the HIV-1 virus. Accordingly, it can be hypothesized that maintenance of serum estradiol levels could decrease the incidence of HIV dementia and other AIDS-related neurological syndromes in HIV-1 positive women. In this article, we both summarize current understanding and present new data related to the potential mechanisms whereby estrogen could modulate the mechanics and the consequences of HIV-1 infection in the brain and thereby thwart the development of HIV dementia.
Communication mechanisms [extra-, intra-, and gap junctional inter-cellular communication (GJIC)]... more Communication mechanisms [extra-, intra-, and gap junctional inter-cellular communication (GJIC)] control, from the fertilized egg, through embryogenesis to maturity and aging, whether a cell proliferates, differentiates, dies by apoptosis, or if differentiated, adaptively responds to endogenous and exogenous signals. From the egg to the 100 trillion cells in the human body, health is maintained when these communication processes between stem, progenitor and terminally differentiated cells are integrated. Each cell choice involves 'epigenetic' mechanisms to alter the expression of genes at the transcriptional, translational or post-translational levels. Disruption of the communication mechanisms can be either adaptive or maladaptive. Modulation of extra-cellular communication, either by genetic imbalances of growth factors, hormones or neurotransmitters or by environmental, exogenous chemicals can trigger signal transducing intra-cellular mechanisms. These intra-cellular signals can modulate gene expression at the transcriptional, translational or post-translational levels while also modulating GJIC. Untimely or chronic disruption of GJIC during embryonic or fetal development could lead to embryonic lethality or teratogenesis. By modulation of GJIC, homeostatic control of cell growth, differentiation or apoptosis could lead to specific diseases, such as neurological, cardiovascular, reproductive or endocrinological dysfunction. Chemical modulation or oncogene down-regulation of GJIC in initiated tissues has been shown to lead to tumor promotion. Genetic syndromes carrying a mutated gap junction gene, together with some transgenic and knock-out gap junction gene mice, provide evidence for the importance of this organelle found only in metazoans. Implications for 'thresholds' to toxicants and for risk assessment are evident.
Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens. A considerable amount of rese... more Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens. A considerable amount of research has been devoted to predicting the genotoxic, tumor-initiating potential of PAHs based on chemical structure. However, information on the correlation of structure with the non-genetoxic, epigenetic events of tumor promotion is sparse. PAHs containing a bay or bay-like region were shown to be potent inhibitors of gap-junctional intercellular communication (GJIC), an epigenetic event involved in the removal of an initiated cell from growth suppression. We tested the epigenetic toxicity of PAHs containing bay-like regions by comparing the effects of methylated vs. chlorinated isomers of anthracene on the temporal activation of mitogen-activated protein kinase (MAPK) and the regulation of GJIC. Specifically, we used anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9,10-dimethylanthracene, 1-chloroanthracene, 2-chloroanthracene, and 9-chloroanthracene. We determined the effect of these compounds on GJIC and on the activation of extracellular receptor kinase , a MAPK, in F344 rat liver epithelial cells. Results showed that bay or bay-like regions, formed by either chlorine or a methyl group, reversibly inhibited GJIC at the same doses, time, and time of recovery, whereas the linear-planar isomers had no effect on GJIC. Similarly, the GJIC-inhibitory isomers also induced the phosphorylation of ERK 1 and ERK 2, while the non-inhibitory isomers had no effect on the activation of these MAPKs. MAPK activation occurred 10 -20 min after the inhibition of GJIC, which indicates that MAPK is not involved in the initial regulation of GJIC; instead altered GJIC may be affecting MAPK activation. The present study revealed that there are structural determinants of PAHs, which clearly affect epigenetic events known to be involved in the non-genetoxic steps of tumor promotion. These events are the release of a cell from growth suppression involving the reduction of GJIC, followed by the activation of intracellular mitogenic events.
The Journal of Steroid Biochemistry and Molecular Biology, 1998
FSH-beta mRNA is dramatically regulated in the infantile female rat anterior pituitary. Elevated ... more FSH-beta mRNA is dramatically regulated in the infantile female rat anterior pituitary. Elevated plasma levels of FSH correspond with increased FSH-beta mRNA levels which peak on PND 12. The source of this regulation does not appear to be GnRH, since the administration of a potent GnRH antagonist does not suppress FSH-beta mRNA levels. Consequently, we have examined the effects of the gonadal steroid hormones, estrogen and androgen, on the maintenance of gonadotropin secretion and gene expression both in vivo and in vitro. Androgen and estrogen action was blocked in vivo with the specific receptor antagonists, flutamide (150 microg) and tamoxifen (200 microg). Administration of antagonists during two different three day time-periods of infantile life [postnatal day (PND) 8-11 and PND 11-14] resulted in differing effects on both FSH and LH secretion as well as on FSH-beta and LH-beta mRNA levels. Flutamide and tamoxifen treatment both suppressed FSH secretion at either age examined (p < 0.01). LH secretion was suppressed by both treatments but only at the younger of the two ages (p < 0.01). In contrast to its effects on FSH secretion, tamoxifen suppressed FSH-beta mRNA levels in the later group only. LH-beta mRNA levels were suppressed by tamoxifen, but only in the younger age group (p < 0.05). The direct effects of steroid hormones on infantile pituitary gonadotrophs were examined in vitro by incubating cells with dihydrotestosterone propionate (DHTP; 10(-8) M) or 17beta-estradiol (E; 10(-8) M). Both DHT and E treatment stimulated FSH secretion when measured 48 h later (p < 0.01). There were no effects on LH secretion. FSH-beta mRNA levels were also stimulated by DHT at 48 h (p < 0.01). Estradiol treatment transiently increased FSH-beta mRNA levels at 2 and 6 h following treatment (p < 0.01) but not at 48 h. LH-beta levels were suppressed by DHT treatment (p < 0.05), and E transiently elevated LH-beta mRNA levels at 2 h (p < 0.05). Taken together these studies indicate that gonadotrophs from infantile female rats are capable of responding directly to steroid hormones, and may play a role in the selective stimulation of FSH secretion and expression in vivo.
During aging, there is an increase in neurodegenerative diseases and a decrease in cognitive perf... more During aging, there is an increase in neurodegenerative diseases and a decrease in cognitive performance. Postmenopausal women are more vulnerable as their estrogen levels decline, but most hormone replacement therapies do not prevent cognitive decline. One potential reason is that the timing of hormone replacement is critical and changes in estrogen receptor expression may over-ride hormonal intervention. In rodents, ERβ mRNA decreases in the cortex with age. One mechanism by which ERβ mRNA could be regulated is by epigenetic modification of ERβ promoter. Here we show an increase in methylation of ERβ promoter corresponding to decreases in ERβ mRNA in aging female cortex.
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