Biomedical subjects

Donald B DeFranco

Publications and source records attributed to Donald B DeFranco.

25 records · Page 2Linked to original sources

PubMed2003

The Group 3 LIM domain protein paxillin potentiates androgen receptor transactivation in prostate cancer cell lines.

Paxillin, a member of the group 3 subfamily of LIM domain proteins, is localized within focal adhesions and participates in a number of signal transduction pathways mobilized upon activation of cell surface receptors. In recent years, a number of group 3 LIM domain proteins have been found to also localize within the nucleus and exert direct effects on transcription. We show here that paxillin is present within nuclei and can target the nuclear matrix of CV-1 cells, cultured prostate cancer cell lines, and human prostate tissue. The increased targeting of androgen receptor to the nuclear matrix upon overexpression of paxillin may be brought about by direct interactions between paxillin and the receptor, which were detected in vitro. Paxillin functions as a coactivator for androgen receptor and glucocorticoid receptor, but not estrogen receptor alpha, similar to its close relative Hic-5/ARA55. Both paxillin and Hic-5/ARA55 use their COOH-terminal LIM domain to interact with steroid receptors. However, paxillin is distinguished from Hic-5/ARA55 by both the location of its receptor coactivation domain (i.e., COOH-terminal LIM domain) and by the dominant-negative activity of its NH(2)-terminal domain. Thus, highly related group 3 LIM domain proteins may use distinct mechanisms to modulate steroid hormone receptor transactivation.

Cytoskeletal Proteins↗

PubMed2003

Cell death triggered by polyglutamine-expanded huntingtin in a neuronal cell line is associated with degradation of CREB-binding protein.

Huntington's Disease belongs to the CAG repeat family of neurodegenerative diseases and is characterized by the presence of an expanded polyglutamine (polyQ) repeat in the huntingtin (htt) gene product. PolyQ-expanded htt accumulates within large aggregates that are found in various subcellular compartments, but are more often localized within the nucleus. It has been suggested that the sequestration of proteins essential to cell viability may be one mechanism that accounts for toxicity generated by polyQ-expanded proteins. Nuclear inclusions containing polyQ-expanded htt recruit the transcriptional cofactor, CREB-binding protein (CBP). PolyQ toxicity appears to involve alterations of gene transcription and reduced neuronal cell viability. In the HT22 hippocampal cell line, we find that toxicity within individual cells induced by polyQ-expanded htt, as revealed by a TUNEL assay, is associated with the localization of the mutant htt within either nuclear or perinuclear aggregates. However, in addition to CBP recruitment, we show here that CBP ubiquitylation and degradation can be selectively enhanced by polyQ-expanded htt. Thus, selected substrates may be directed to the ubiquitin/proteasome-dependent protein degradation pathway in response to polyQ-expanded htt within the nucleus.

Animals↗

PubMed2002

Hypothermic reperfusion after cardiac arrest augments brain-derived neurotrophic factor activation.

Induction of mild hypothermia improves neurologic outcome after global cerebral ischemia. This study measured levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in hippocampal tissue of rats after resuscitation from 8 minutes of normothermic, asphyxial cardiac arrest. After resuscitation, rats were maintained either at normal temperature (37 degrees C) or cooled to mild hypothermia (33 degrees C, beginning 60 minutes after resuscitation). After 12 or 24 hours, neurotrophin levels in hippocampus were measured by immunoblotting. Ischemia and reperfusion increased hippocampal levels of BDNF. Induction of hypothermia during reperfusion potentiated the increase in BDNF after 24 hours, but not after 12 hours. Levels of NGF were not increased by postresuscitation hypothermia. Hypothermia also increased tissue levels and tyrosine phosphorylation of TrkB, the receptor for BDNF. Increased BDNF levels were correlated with activation of the extracellularly regulated kinase (ERK), a downstream element in the signal transduction cascade induced by BDNF. In contrast to the many deleterious processes during ischemia and reperfusion that are inhibited by induced hypothermia, increasing BDNF levels is a potentially restorative process that is augmented. Increased activation of BDNF signaling is a possible mechanism by which mild hypothermia is able to reduce the neuronal damage typically occurring after cardiac arrest.

Animals↗

PubMed2002

Proteasomal inhibition enhances glucocorticoid receptor transactivation and alters its subnuclear trafficking.

The ubiquitin-proteasome pathway regulates the turnover of many transcription factors, including steroid hormone receptors such as the estrogen receptor and progesterone receptor. For these receptors, proteasome inhibition interferes with steroid-mediated transcription. We show here that proteasome inhibition with MG132 results in increased accumulation of the glucocorticoid receptor (GR), confirming that it is likewise a substrate for the ubiquitin-proteasome degradative pathway. Using the mouse mammary tumor virus (MMTV) promoter integrated into tissue culture cells, we found that proteasome inhibition synergistically increases GR-mediated transactivation. This increased activation was observed in a number of cell lines and on various MMTV templates, either as transiently transfected reporters or stably integrated into chromatin. These observations suggest that the increase in GR-mediated transcription due to proteasome inhibition may occur downstream of the initial chromatin remodeling step. In support of this concept, the increase in transcription did not correlate with an increase in chromatin remodeling, as measured by restriction enzyme hypersensitivity, or transcription factor loading, as exemplified by nuclear factor 1. To investigate the relationship between GR turnover, transcription, and subnuclear trafficking, we examined the effect of proteasome inhibition on the mobility of the GR within the nucleus and association of the GR with the nuclear matrix. Blocking GR turnover reduced the mobility of the GR within the nucleus, and this correlated with increased association of the receptor with the nuclear matrix. As a result of proteasome inhibition, GR mobility within the nucleus was reduced while its association with the nuclear matrix was increased. Thus, while altered nuclear mobility of steroid receptors may be a common feature of proteasome inhibition, GR is unique in its enhanced transactivation activity that results when proteasome function is compromised. Proteasomes may therefore impact steroid receptor action at multiple levels and exert distinct effects on individual receptor types.

Breast Neoplasms↗

PubMed2002

Glucocorticoid receptors in hippocampal neurons that do not engage proteasomes escape from hormone-dependent down-regulation but maintain transactivation activity.

The glucocorticoid receptor (GR) protein is subjected to hormone-dependent down-regulation in most cells and tissues. This reduction in receptor levels that accompanies chronic hormone exposure serves to limit hormone responsiveness and operates at transcriptional, posttranscriptional, and posttranslational levels. The ability of glucocorticoid hormones to trigger GR down-regulation may be not universal, particularly in mature and developing neurons in which conflicting results regarding hormone control of GR protein have been reported. We find that endogenous GR is not down-regulated in the HT22 mouse hippocampal cell line and in primary hippocampal neurons derived from embryonic rats. Because GR has the capacity to be ubiquitylated in HT22 cells, receptor down-regulation must be limited by defects in either targeting of polyubiquitylated receptor to the proteasome or processing of the targeted receptor by the proteasome. Despite the lack of GR down-regulation in the HT22 cells, glucocorticoid-induced transcription from transiently transfected templates is attenuated upon prolonged hormone treatment. This termination of GR transactivation is not due to inefficient nuclear import or nuclear retention of the receptor. Furthermore, GR efficiently exports from HT22 cell nuclei in hormone-withdrawn cells, indicating that the receptor has access to both nuclear and cytoplasmic degradation pathways. Our results suggest that appropriate maturation of proteasomal degradative or targeting activities may be required, particularly in hippocampal neurons, for hormone-dependent down-regulation of GR.

Active Transport, Cell Nucleus↗

PubMed2002

Navigating steroid hormone receptors through the nuclear compartment.

Steroid hormone receptors exert much of their effects on cellular physiology through regulating the rate of transcription from unique target genes. Much has been learned about the actions of steroid hormone receptors at regulated promoters through model in vitro studies, but it has always been a challenge to extrapolate these mechanistic insights to molecular events that occur in live cells. However, novel insights have recently been gained regarding the nature of receptor encounters with the transcriptional machinery from elegant experimental approaches that used advances gained in biochemical, molecular biological, cell biological, and biophysical disciplines. Although these is no doubt that steroid hormone receptors represent some of the most mobile proteins within the nucleus, they still maintain their ability to orchestrate a highly ordered recruitment of cofactors and coregulators at specific sites and remain accessible to alternative processing pathways that limit their action. As highlighted in this review, there may be interrelationships between seemingly distinct pathways of receptor trafficking and processing within the nucleus that impact receptor action at regulated promoters.

Animals↗

PubMed2001

Prolonged nuclear retention of activated extracellular signal-regulated protein kinase promotes cell death generated by oxidative toxicity or proteasome inhibition in a neuronal cell line.

In the HT22 mouse hippocampal cell line and primary immature embryonic rat cortical neurons, glutamate-induced oxidative toxicity is associated with a delayed but chronic activation of extracellular signal-regulated kinase-1/2 (ERK-1/2). ERK-1/2 is also activated in HT22 cells that undergo caspase-dependent cell death upon inhibition of proteasome-dependent protein degradation brought about by MG132 treatment. As in glutamate-treated HT22 cells and primary neurons, inhibition of MEK-1, an upstream activator of ERK-1/2 protects against MG132-induced toxicity. Furthermore, activated ERK-1/2 is retained within the nucleus in glutamate- and MG132-treated HT22 cells. Although previous studies suggested that ERK-1/2 activation was downstream of many cell death-inducing signals in HT22 cells, we show here that cycloheximide, the Z-vad caspase inhibitor, and a nonlethal heat shock protect against glutamate- and MG132-induced toxicity without diminishing ERK-1/2 activation. In these cases, ERK-1/2, although chronically activated, is not retained within the nucleus but accumulates within the cytoplasm. Thus, persistent nuclear retention of activated ERK-1/2 may be a critical factor in eliciting proapoptotic effects in neuronal cells subjected to oxidative stress or proteasome inhibition.

Animals↗