Stephen P. Squinto

Morphine activation of c-fos expression in rat brain.

The post-receptor mechanism of opiate action has been studied by examining the activation by morphine of the proto-oncogene c-fos and its encoded nucleoprotein pp55c-fos (FOS) in rat caudate-putamen, which is rich in the mu-type opiate receptor. Following an acute morphine treatment, c-fos mRNA levels in rat caudate-putamen were increased to maximum (420% of control level) at 45 minutes and returned to control levels at 90 minutes. This induction was completely abolished by naloxone, a morphine antagonist. Fos protein, detected by immunocytochemistry, was also increased 3 hours after morphine injection, in the caudate-putamen, but not in the olfactory tubercle, which does not have the mu-type opiate receptor. Upon activation of opiate receptors by morphine, the c-fos gene is activated and Fos protein may act as a signal transducer uniquely involved in the mechanism of opiate addiction at the level of gene regulation.

Platelet-activating factor and polyunsaturated fatty acids in cerebral ischemia or convulsions: Intracellular PAF-binding sites and activation of a fos/jun/AP-1 transcriptional signaling system

Platelet-activating factor (PAF) is a lipid mediator formed in the early response of the central nervous system to ischemia or convulsions. Free polyunsaturated fatty acids and arachidonic and docosahexaenoic acids are accumulated along with PAF. Antagonists of PAF have been found to improve cerebral blood flow and partially block the rise in free fatty acids, an effect that may arise by way of inhibition of PAF receptors or stimulation of the reacylation of free fatty acids released upon insult. Three intracellular PAF-binding sites have been identified in rat cerebral cortex. These very high-affinity binding sites are inhibited by PAF antagonists, with certain antagonists exhibiting specificity for a particular binding site. This specificity indicates heterogeneity in these binding sites. Ischemia or stimulation also leads to protooncogene transcriptional activation. Here, we discuss studies with cells in culture showing that PAF promotes transcriptional activation of immediate-early genes. PAF activates the transcription of the immediate-early genesfos andjun, whose gene products are regulators of the transcription of other genes. Transcription offos is also activated by convulsion or ischemia in the central nervous system. The activation of these genes by PAF can be inhibited by PAF antagonists, and is apparently accomplished by way of an AP-1 transcription regulatory sequence in the promoter region of the target genes. Studies with deletion mutants show that PAF can also exert its activating properties by way of cyclic adenosine-3′,5′-monophosphate-(cAMP) and Ca2+-responsive elements, and suggest that PAF is involved in an interconnected network of cell signaling that may coordinate short-term and long-term responses of cells to stimulus and injury.

Platelet-activating factor activates HIV promoter in transfected SH-SY5Y neuroblastoma cells and MOLT-4 T lymphocytes

Transfected gene constructs comprising the long terminal repeat (LTR) sequence of the human immunodeficiency virus (HIV) genome spliced to an assayable reporter gene have made possible the evaluation of a lipid mediator, platelet-activating factor (PAF), as a potential HIV transcriptional regulatory molecule. We assessed the activation of the HIV LTR promoter sequence linked to the chloramphenicol acetyltransferase (CAT) reporter gene (HIV-CAT) by PAF in both a human neural (SH-SY5Y neuroblastoma) and a human leukocytic (MOLT-4 T-lymphocyte) cell line. PAF activated expression of the HIV-CAT construct in both the SH-SY5Y and MOLT-4 T-cell lines. PAF-induced CAT activity was approximately six to seven times higher in the SH-SY5Y cells than in the MOLT-4 cells. Preincubation of cells with the specific PAF antagonist BN 52021 completely inhibited CAT expression in both cell lines. The biologically inactive PAF precursor lyso-PAF did not activate CAT expression. Assays for CAT mRNA demonstrated an increase after PAF treatment, an effect that was completely inhibited by BN 52021, and which was not elicited by lyso-PAF. These results show that PAF represents a potential cellular mediator evoking the expression of the HIV genome.

Prolonged Activation of c-fos and Optimal Activation of Pro-opiomelanocortin mRNA after Repeated Morphine Exposure in SH-SY5Y Cells

The time course of change in the mRNA concentrations of the proto-oncogene c-fos and pro-opiomelanocortin after two different methods of morphine treatment was examined in SH-SY5Y human neuroblastoma cells. In a repeated treatment design, SH-SY5Y cells exposed to morphine sulfate (MS) for 12 h or more were periodically given fresh morphine (10 or 1 muM). In a single-dose design, 10 muM MS was added to the flasks at designated times without changing the medium. Slot-blotting hybridization analysis of total cellular RNA using a [(32)P]-fos cDNA probe revealed that repeated morphine treatment caused both an early transient induction of c-fos and a later prolonged increase in c-fos. Single treatment with morphine caused only a transient and rapid induction of c-fos. Slot-blotting hybridization with a [(32)P]-POMC cRNA probe revealed that POMC mRNA was significantly activated at 6 h and remained significantly elevated up to 7 days in the cells with repeated morphine treatment. In the single-dose experiments, however, the POMC mRNA was not significantly elevated at 2 days or less. It was significantly activated at 6 days, but at a much lower level than that seen in the repeated-dose design. These results indicate that repeated exposure to morphine induces a prolonged activation of c-fos mRNA which may be functionally related to the significant activation of POMC mRNA in SH-SY5Y cells.