Alicia R. Hovland

An N-terminal Inhibitory Function, IF, Suppresses Transcription by the A-isoform but Not the B-isoform of Human Progesterone Receptors

The B-isoform of human progesterone receptors (PR) contains three activation functions (AF3, AF1, and AF2), two of which (AF1 and AF2) are shared with the A-isoform. AF3 is in the B-upstream segment (BUS), the far N-terminal 164 amino acids of B-receptors; AF1 is in the 392-amino acid N-terminal region common to both receptors; and AF2 is in the C-terminal hormone binding domain. B-receptors are usually stronger transactivators than A-receptors due to transcriptional synergism between AF3 and one of the two downstream AFs. We now show that the N terminus of PR common to both isoforms contains an inhibitory function (IF) located in a 292-amino acid segment lying upstream of AF1. IF represses the activity of A-receptors but is not inhibitory in the context of B-receptors due to constraints imparted by BUS. As a result, IF inhibits AF1 or AF2 but not AF3, regardless of the position of IF relative to BUS. IF is functionally independent and strongly represses transcription when it is fused upstream of estrogen receptors. These data demonstrate the existence of a novel, transferable inhibitory function, mapping to the PR N terminus, which begins to assign specific roles to this large undefined region.

Role of Phosphorylation on DNA Binding and Transcriptional Functions of Human Progesterone Receptors

To study the function of human progesterone receptor (hPR) phosphorylation, we have tested four sets of serine to alanine substitution mutants: 10 serine clusters, located in regions common to both hPR isoforms (the M-series mutants) were mutated in A-receptors and B-receptors; 6 serine clusters located in the B-upstream segment (BUS; the B-series mutants) were mutated individually and collectively and cloned into B-receptors and into BUS-DBD-NLS, a constitutive transactivator, in which the AF3 function of BUS is fused to the DNA binding domain (DBD) and nuclear localization signal (NLS) of hPR. Transcription by most of the M-series mutants resembles that of wild-type A- or B-receptors. Mutation of 3 sites, Ser190 at the N terminus of A-receptors, a cluster of serines just upstream of the DBD, or Ser676 in the hinge region, inhibits transcription by 20-50% depending on cell or promoter context. These sites lie outside the AF1 activation function. M-series mutants are substrates for a hormone-dependent phosphorylation step, and they all bind well to DNA. Progressive mutation of the B-series clusters leads to the gradual dephosphorylation of BUS, but only the 6-site mutant, involving 10 serine residues, is completely dephosphorylated. These data suggest that in BUS alternate serines are phosphorylated or dephosphorylated at any time. However, even when BUS is completely dephosphorylated, both BUS-DBD-NLS and full-length B-receptors remain strong transactivators. Mutant B-receptors also do not acquire the dominant negative properties of A-receptors, and they retain the ability to activate transcription in synergy with 8-Br-cAMP and antiprogestins. We conclude that phosphorylation has subtle effects on the complex transcriptional repertoire that distinguishes the two hPR isoforms and does not influence transactivation mediated by AF1 or AF3, but subserves other functions.

Multiple antioxidants in the prevention and treatment of neurodegenerative disease: analysis of biologic rationale.

Parkinsons disease and Alzheimers disease are major progressive neurologic disorders, the risk of which increases with advancing age (65 years and over). In familial cases, however, early onset of disease (35-65 years) is observed. In spite of extensive basic and chemical research on Parkinsons disease and Alzheimers disease, no preventive or long-term effective treatment strategies are available. The analysis of existing literature suggests that oxidative stress is a major intermediary risk factor for the action of diverse groups of neurotoxins that are involved in these neurodegenerative diseases. In this review, it is proposed that the epigenetic components (mitochondria, other organelles, membranes, protein modification) rather than nuclear genes of neurons are the primary targets for the action of neurotoxins, including free radicals. In addition, a scientific rationale for using multiple antioxidants in clinical trials for the prevention of Parkinsons disease and Alzheimers disease among high-risk populations, and as an adjunct to standard therapy in the treatment of these diseases is presented.

Prostaglandins as Putative Neurotoxins in Alzheimer's Disease

Abstract Chronic inflammatory reactions in the brain appear to be one of the primary etiological factors in the pathogenesis of Alzheimers disease (AD). This is supported by the fact that the secretory products of inflammatory reactions, which include cytokines, complement proteins, adhesion molecules, and free radicals, are neurotoxic. We have recently reported that prostaglandins (PGs), which are also released during inflammatory reactions, cause rapid degenerative changes in differentiated murine neuroblastoma cells (NB) in culture. PGA1 is more effective than PGE1. Similar observations were made in a primary culture of fetal rat hippocampal cells. Epidemiological and clinical studies on AD also support the involvement of PGs in neuronal degeneration. Thus, we propose a hypothesis that PGs are one of the major extracellular signals that initiate neuronal degeneration, which is mediated by intracellular signals such as the β-amyloid peptide (Aβ) and ubiquitin, since the levels of these proteins are increased by PG treatment. We further suggest that adenosine 3′, 5′-cyclic monophosphate (cAMP) is one of the factors that regulate the levels of both Aβ and ubiquitin in NB cells. Increases in the level of Ap in NB cells following an elevation of intracellular cAMP levels appear to be due to an increase in the rate of processing of the amyloid precursor protein (APP) rather than an increase in the expression of APP. The mechanisms underlying Aβ-induced neuronal degeneration have been under intense investigation, and several mechanisms of action have been proposed. We postulate that PG-induced elevation of Aβ may lead to an increased binding of Aβ to the 20S proteasome, resulting in a reduction of 20S proteasome-mediated degradation of ubiquitin-conjugated proteins. This is predicted to lead to an increase in an accumulation of abnormal proteins, which ultimately contribute to neuronal degeneration and death. Based on our hypothesis and on studies published by others, we propose that a combination of nonsteroidal anti-inflammatory drugs, which inhibit the synthesis of PGs, and antioxidant vitamins, which quench free radicals and both of which have been recently reported to be of some value in AD treatment when used individually, may be much more effective in the prevention and treatment of AD than the individual agents alone.

Effects of altered cyclophilin A expression on growth and differentiation of human and mouse neuronal cells.

Abstract1. Cyclophilin A (CyP-A), a soluble cytoplasmic immunophilin, is known for its involvement in T cell differentiation and proliferation. Although CyP-A has a pivotal role in the immune response, it is most highly concentratedin brain, where its functions are largely unknown.2. We reported previously that a murine neuroblastoma (NB-P2) cellline can partially differentiate into neurons when treated with cyclosporin A (CyS-A), implicating a role for CyP-A in neuronal differentiation (Hovland et al. [1999]. Neurochem. Int. 3:229–235).3. The role of CyP-A in regulating neuronal growth and differentiation is not well defined. To investigate this, we first tested the utility of retroviral-mediated gene transfer and expression in human embryonic brain (HEB) and NB-P2 cells. Second, we examined the effects of retroviral-mediated overexpression or antisense-mediated reduction of CyP-A in HEB and NB-P2 cells.4. Our data show that retroviral vectors are efficient for stable gene transfer and expression in both cell lines. Moreover, neither overexpression nor reduction of CyP-A expression in NB-P2 cells altered the growth rate or induced differentiation. More importantly, the up- or down-regulation of CyP-A expressiondid not affect the magnitude of cAMP-induced NB-P2 differentiation. However, overexpression of CyP-A increased the growth rate of HEB cells.5. In summary, the utility of retroviral vectors for stable gene expression in human embryonic brain and murine neuroblastoma cells was shown. Furthermore,a novel role for CyP-A in augmenting the proliferation of human embryonic braincells was demonstrated in vitro.

Identifying genes involved in regulating differentiation of neuroblastoma cells

The genes regulating the induction of differentiation in neurons are not definitively known. Some neuronal tumors retain the ability to differentiate into mature, functional neurons in response to pharmacological agents, despite the presence of genetic anomalies. We hypothesized that some of the genes whose expression is altered between undifferentiated and differentiated states may be those responsible for inducing differentiation. To investigate this, we used a mouse neuroblastoma (NB) cell line, NBP2, in which ≥90% of the cells in the culture terminally differentiate upon elevation of intracellular adenosine 3′,5′‐cyclic monophosphate (cAMP) levels. Gene expression was analyzed using cDNA array blots containing 588 known genes. mRNA from cultures of undifferentiated and differentiated NB cells was used to make cDNA probes for blot hybridization. We identified several genes that are predominantly expressed in either undifferentiated or differentiated NB cells. In addition, numerous genes are moderately up‐ or down‐regulated during differentiation of NB cells. We identified the N‐myc protooncogene, cyclin B1, and protease nexin 1 as genes that are expressed in undifferentiated NB cells and whose levels are significantly down‐regulated upon differentiation. In contrast, the c‐fes and c‐fos protooncogenes and the RAG‐1 gene activator are genes whose expression is significantly up‐regulated during differentiation of NB cells. These findings were confirmed by RT‐PCR analysis. The transcript size and expression level of N‐myc, cyclin B1, protease nexin 1, c‐fes, and c‐fos were verified by Northern blotting. These genes may represent key mediators involved in the regulation of NB cell differentiation. J. Neurosci. Res. 64:302–310, 2001.

Differentiation Genes: Are They Primary Targets for Human Carcinogenesis?

In spite of extensive research in molecular carcinogenesis, genes that can be considered primary targets in human carcinogenesis remain to be identified. Mutated oncogenes or cellular growth regulatory genes, when incorporated into normal human epithelial cells, failed to Immortalize or transform these cells. Therefore, they may be secondary events In human carcinogenesis. Based on some experimental studies we have proposed that downregulation of a differentiation gene may be the Primary event in human carcinogenesis. Such a gene could be referred to as a tumor-initiating gene. Downregulation of a differentiation gene can be accomplished by a mutation in the differentiation gene, by activation of differentiation suppressor genes, and by inactivation of tumor suppressor genes. Downregulation of a differentiation gene can lead to immortalization of normal cells. Mutations in cellular proto-oncogenes, growth regulatory genes, and tumor suppressor genes in immortalized cells can lead to transformation. Such genes could be called tumor-promoting genes. This hypothesis can be documented by experiments published on differentiation of neuroblastoma (NB) cells in culture. The fact that terminal differentiation can be induced In NB cells by adenosine 3′,5′-cyclic monophosphate (cAMP) suggests that the differentiation gene in these cells Is not mutated, and thus can be activated by an appropriate agent. The fact that cAMP-resistant cells exist In NB cell populations suggests that a differentiation gene is mutated in these cancer cells, or that differentiation regulatory genes have become unresponsive to cAMP. In addition to cAMP, several other differentiating agents have been identified. Our proposed hypothesis of carcinogenesis can also be applied to other human tumors such as melanoma, pheochromocytoma, medulloblastoma, glioma, sarcoma, and colon cancer.

Adenosine 3', 5'- cyclic monophosphate increases processing of amyloid precursor protein (APP) to β-amyloid in neuroblastoma cells without changing APP levels or expression of APP mRNA

Abstractβ-Amyloid (Aβ), a 39–43 residue peptide generated by splicing of the amyloid precursor protein (APP), is one of the major components of senile plaques which are the hallmark of Alzheimers disease (AD); and therefore, a role of Aβ in neuronal degeneration has been proposed. The factors which regulate the levels of Aβ have not been fully identified. Since an elevation of the intracellular levels of adenosine, 3′, 5′-cyclic monophosphate (cAMP) in neuroblastoma cells (NB) induces terminal differentiation, and since these differentiated NB cells undergo spontaneous degeneration, the role of cAMP in the regulation of Aβ levels in these cells have been investigated. In order to determine the specificity of the effect of cAMP on nerve cells, rat glioma cells (C-6) were investigated in a similar manner. Results showed that an elevation of the levels of cAMP in NB cells enhances the intensity of Aβ immunostaining without changing the levels of APP or APP mRNA. This suggests that the rate of processing of APP to Aβ increases following an elevation of cAMP level in NB cells. Data also revealed that an elevation of cAMP level in glioma cells did not alter the intensity of staining with APP or Aβ.

RELATIVE SENSITIVITY OF UNDIFFERENTIATED AND CYCLIC ADENOSINE 3′,5′-MONOPHOSPHATE–INDUCED DIFFERENTIATED NEUROBLASTOMA CELLS TO CYCLOSPORIN A: POTENTIAL ROLE OF β-AMYLOID AND UBIQUITIN IN NEUROTOXICITY

SummaryCyclosporin A is routinely used in transplant therapy following allogeneic or xenogeneic tissue transplantation to prevent rejection. This immunosuppressive drug is also neurotoxic; however, its mechanisms of action for neurotoxicity are poorly understood. Undifferentiated and cyclic adenosine 3′, 5′-monophosphate (cAMP)-induced differentiated neuroblastoma (NB) cells were used as an experimental model to study the toxicity of cyclosporin A. Results showed that cyclosporin A promoted the outgrowth of neurites and inhibited the growth of undifferentiated NB cells. When cyclosporin A was added simultaneously with RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, or with prostaglandin E1, a stimulator of adenylate cyclase, it markedly enhanced the growth inhibitory and differentiation effects of these cAMP-stimulating agents. In addition, cyclosporin A added to cAMP-induced differentiated NB cells caused dose-dependent degeneration of these cells as evidenced by the vacuolization of cytoplasm and the fragmentation of nuclear and cytoplasmic materials; however, neurites remained intact. Cyclosporin A alone did not alter the intensity of cell immunostaining for ubiquitin or β-amyloid peptide (amino acids 1–14) (Aβ1–14); however, it enhanced the intensity of staining for both ubiquitin and Aß in cells that were treated with cAMP-stimulating agents. The intensity of staining of amyloid precursor protein (amino acids 44–63) (APP44–63) did not change in any treated group, suggesting that the increase in Aß staining is due to increased processing of APP to Aß. We propose that one of the mechanisms of cyclosporin A-induced neurotoxicity involves increased levels of Aß and ubiquitin.