Linda A. Kobierski

Cyclic AMP‐Dependent Activation of the Proenkephalin Gene Requires Phosphorylation of CREB at Serine‐133 and a Src‐Related Kinase

Abstract: The transcription factor CREB [cyclic AMP response element (CRE)‐binding protein] is activated by several kinase pathways on phosphorylation of serine‐133. Phosphorylation of CREB at serine‐133 is required for the induction of target gene expression. The proenkephalin gene is a target of cyclic AMP‐dependent agonists like forskolin, and its expression is driven by the enhancer element CRE‐2. It has been shown that CREB binds CRE‐2 in extracts from striatum and hypothalamus. However, these studies did not show a functional requirement for CREB serine‐133 phosphorylation in CRE‐2 function. We demonstrate that CREB binds CRE‐2 in primary astrocyte cultures and that transcriptional activation of CRE‐2 requires CREB phosphorylation at serine‐133. In addition, it has recently been shown that, at least in some contexts, CREB phosphorylation is not sufficient to activate target gene expression and that another intracellular signal seems to be required. Therefore, we also sought to determine if another signaling event, in addition to CREB phosphorylation, might be involved in cyclic AMP‐mediated induction of the proenkephalin gene. We have found that the inhibition of src‐related nonreceptor tyrosine kinases blocks forskolin‐induced proenkephalin gene expression without having any effect on serine‐133‐phosphorylated CREB levels and that constitutively activated src kinase can activate the proenkephalin promoter.

NFAT4 is expressed in primary astrocytes and activated by glutamate

Calcium signaling is a critical component of astrocyte activation but little information is available regarding the identity and function of transcriptional targets of calcium signaling in these cells. As a first step in elucidating the mechanisms that astrocytes use to regulate transcription in response to raised intracellular calcium concentrations, we have investigated expression and activation of the calcium activated transcription factors of the NFAT family. We report here that NFAT4 is expressed in U373 astrocytoma cells and in primary cultures of astrocytes. Treatment of U373 cells or primary astrocytes with the calcium ionophore A23187, or the neurotransmitter glutamate, leads to NFAT nuclear translocation and increased DNA binding to a NFAT consensus site through a cyclosporin A‐sensitive process. These data suggest that NFAT4 functions as a calcium‐responsive transcription factor in astrocytes. Published 2003 Wiley‐Liss, Inc.

Systemic lipopolysaccharide and interleukin-1β activate the interleukin 6: STAT intracellular signaling pathway in neurons of mouse trigeminal ganglion

We have studied the activation of signal transducers and activators of transcription (STATs) in trigeminal ganglion after intraperitoneal injection of lipopolysaccharide (LPS) and Interleukin-1beta (IL-1beta). Using electrophoretic mobility shift assays we have shown that STAT1 and STAT3 are activated within 1 to 2 h. of injection of either LPS or IL-1beta. Eight hours after LPS injection the DNA binding activity of these complexes is still elevated while induction by IL-1beta returns to baseline levels within 4 h. By immunohistochemistry, using an antibody specific for the tyrosine phosphorylated, activated form of STAT-1, we show that this induction occurs in sensory neurons. IL-6 may be important in this cascade since induction of STATs by IL-1beta is blocked in Interleukin-6 knock out mice.

PhosphoCREB and CREM/ICER: positive and negative regulation of proenkephalin gene expression in the paraventricular nucleus of the hypothalamus.

In the hypothalamic paraventricular nucleus (PVN), the proenkephalin gene may be upregulated by lipopolysaccharide (LPS) and downregulated by the GABA-Aagonist muscimol. Candidate transcription factors regulating the proenkephalin gene in opposite directions are cAMP-response-element-binding protein (CREB) (when phosphorylated, a positive regulator) and cAMP-responsive modulatory inducible cAMP early repressor (CREM/ICER) (a negative regulator). Our results demonstrate that CREM α,β,γ transcripts and ICER are induced in the PVN by LPS and remain elevated for periods of up to 12 h. PhosphoCREB is elevated after LPS administration, peaking at 8 h, but remaining elevated over control levels at 12 h. PhosphoCREB induction by LPS is also seen in primary hypothalamic cultures. Cotransfection of ICER with ENK-CAT12 into primary hypothalamic cultures produced a decrease in chloramphenicol acetyl transferase (CAT) levels following cAMP or LPS stimulation. PhosphoCREB is down-regulated and CREM/ICER is upregulated in the PVN by muscimol, suggesting that the regulation of these transcription factors may underlie the inhibitory effect of muscimol on target genes in the PVN.

A water-soluble synthetic spicamycin derivative (San-Gly) decreases mechanical allodynia in a rodent model of neuropathic pain

The goal of this study was to examine the effects of synthetic spicamycin derivative, SAN-Gly, on mechanical allodynia in a spared nerve injury animal model of neuropathic pain. Adult male rats underwent surgical ligation and cutting of the common peroneal and tibial nerves, which produced a mechanical allodynia within 2-4 days. One week after the surgery, SAN-Gly was administered via intravenous injection. Mechanical allodynia was measured using von-Frey hairs. Spicamycin produced a significant reduction in mechanical allodynia for up to 6 weeks. This study demonstrates that SAN-Gly may be of potential use in treating patients with neuropathic pain.

A single intravenous injection of KRN5500 (antibiotic spicamycin) produces long-term decreases in multiple sensory hypersensitivities in neuropathic pain.

Neuropathic pain is a significant clinical problem. Currently, there are no drugs that produce complete amelioration of this type of pain. We have previously shown that KRN5500, a derivative of the antibiotic spicamycin, produces a prolonged (7-day), and significant reduction in neuropathic pain, but not nociceptive pain. Herein, we provide further evidence for the efficacy of this drug in inhibiting pain after IV injection in a spared nerve injury model of neuropathic pain. A single IV dose of the drug produces an increase in pain thresholds to punctuate mechanical stimuli and to cold stimuli over a period of 7 days, whereas IV injection of the vehicle is without any effect. No change in pain threshold was observed in the contralateral foot. In addition, a significant antiallodynic effect to mechanical stimuli was observed at 1, 2, 4, and 6 wk. The drug may be a potential candidate for cancer-related neuropathic pain as well as a marker for discovery of effective analgesics for neuropathic pain.

Isolating Developmentally Important Genes from the Lobster Nervous System

We are examining the roles that specific gene expression play in development and behavior in the nervous system of the American lobster, Homarus americarus. In particular, we have isolated genomic DNA sequences that encompass several putative lobster genes— one possibly encoding a receptor for the steroid molting hormone, 20-hydroxyecdysone, and at least one homologous to the family of mammalian genes encoding the biopterin-dependent amine hydroxylases (tryptophan hydroxylase, tyrosine hydroxylase, and phenylalanine hydroxylase). Tryptophan hydroxylase is the rate-limiting enzyme for the synthesis of serotonin, a developmentally regulated lobster neurohormone. The control of expression of these genes in individual lobster neurons can be assayed by in situ hybridization and could provide insights into the linkage between behavior, development, and gene expression in this organism.