The CREB transcription factor regulates differentiation, survival, and synaptic plasticity. The complement of CREB targets responsible for these responses has not been identified, however. We ...developed a novel approach to identify CREB targets, termed serial analysis of chromatin occupancy (SACO), by combining chromatin immunoprecipitation (ChIP) with a modification of SAGE. Using a SACO library derived from rat PC12 cells, we identified ∼41,000 genomic signature tags (GSTs) that mapped to unique genomic loci. CREB binding was confirmed for all loci supported by multiple GSTs. Of the 6302 loci identified by multiple GSTs, 40% were within 2 kb of the transcriptional start of an annotated gene, 49% were within 1 kb of a CpG island, and 72% were within 1 kb of a putative cAMP-response element (CRE). A large fraction of the SACO loci delineated bidirectional promoters and novel antisense transcripts. This study represents the most comprehensive definition of transcription factor binding sites in a metazoan species.
Activation of the transcription factor cAMP response element-binding protein (CREB) by neurotrophins is believed to regulate the survival, differentiation, and maturation of neurons in the CNS and ...PNS. Although phosphorylation of Ser133 is critical for the expression of CREB-regulated genes, the identity of neurotrophin-regulated Ser133 kinases has remained controversial. We show here that neurotrophin-induced CREB phosphorylation in CNS neurons depends exclusively on the extracellular signal-regulated kinase 1/2-activated kinase mitogen- and stress-activated protein kinase 1 (MSK1). Small interfering RNA directed against ribosomal S6 kinase 1 (RSK1) and RSK2 reduced phosphorylation of a RSK substrate but did not effect CREB-dependent transcription. However, expression of a selective inhibitory MSK1 mutant markedly attenuated BDNF-stimulated CREB phosphorylation and CREB-mediated transcription. Moreover, the ability of neurotrophins to stimulate CREB phosphorylation was abolished in CNS neurons from MSK1 knock-out mice. Consistent with a role for MSK1 in Ser133 phosphorylation, neurotrophin-induced expression of CREB-regulated genes was attenuated in MSK-deficient neurons. These results indicate that MSK1 is the major neurotrophin-activated Ser133 kinase in CNS neurons.
Background Inflammatory responses can include recruitment of cells of hematopoietic origin to the tunica muscularis. These cells can secrete a variety of factors which can reset the gain of smooth ...muscle cells (SMC) and influence motor patterns. Histamine (H), a major mediator in inflammation, is released by mast cells and exerts diverse effects in SMC by binding to H receptors. The profiles of H receptor expression in animal models used to study inflammatory diseases are unknown.
Methods Histamine receptor expression and electro‐mechanical responses to H were tested in simian and murine colonic smooth muscle using qualitative and quantitative PCR, isometric force measurements, microelectrode recordings and patch clamp techniques.
Key Results H1, H2, and H4 receptor transcripts were expressed at similar levels in simian colonic tissue whereas only the H2 receptor transcript was detected in murine colonic tissue. Stimulation of simian colonic muscles with H caused depolarization and contraction in the presence of tetrodotoxin. Histamine activated non‐selective cation channels in simian SMC. In contrast, H caused hyperpolarization and inhibited contractions of murine colon. The hyperpolarization was inhibited by the KATP channel blocker, glibenclamide. Histamine‐activated K+ currents were inhibited by glibenclamide in murine colonic SMC.
Conclusions & Inferences Histamine receptor expression in simian SMC was similar to that reported in humans. However, H receptor profile and responses to H were considerably different in mice. Thus, monkey colon may be a more suitable model to study how inflammatory mediators affect the gain of smooth muscle excitability.
Estrogen treatment has been found to have suppressive activity in several models of autoimmunity. To investigate the mechanism of 17β-estradiol (E2) suppression of experimental autoimmune ...encephalomyelitis, we evaluated E2 effects on TNF-α expression in the central nervous system (CNS) and spleen of C57BL/6 mice immunized with MOG 35-55/CFA. Kinetic analysis demonstrated that E2 treatment drastically decreased the recruitment of total inflammatory cells as well as TNF-α+ macrophages and T cells into the CNS at disease onset. In contrast, E2 had only moderate effects on the relatively high constitutive TNF-α expression by resident CNS microglial cells. E2 treatment also had profound inhibitory effects on expression of TNF-α by splenic CD4+ T cells, including those responsive to MOG 35–55 peptide. We propose that the mechanism of E2 protection may involve both systemic inhibition of TNF-α expression and local (CNS) recruitment of inflammatory cells, with modest effects on TNF-α expression by resident CNS microglial cells.
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