trichostatin-a and sphingosine-1-phosphate

RGS10 has emerged as a key regulator of proinflammatory cytokine production in microglia, functioning as an important neuroprotective factor. Although RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 levels in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is suppressed in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of Rgs10 expression in microglia. We demonstrate that endogenous RGS10 is present in spinal cord microglia, and RGS10 protein levels are suppressed in the spinal cord in a nerve injury-induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor trichostatin A blocks the ability of lipopolysaccharide (LPS) to suppress Rgs10 transcription in BV-2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of Rgs10 Furthermore, we used chromatin immunoprecipitation to demonstrate that H3 histones at the Rgs10 proximal promoter are deacetylated in BV-2 microglia following LPS activation, and HDAC1 association at the Rgs10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1-phosphate, an endogenous microglial signaling mediator that inhibits HDAC activity, enhances basal Rgs10 expression in BV-2 microglia, suggesting that Rgs10 expression is dynamically regulated in microglia in response to multiple signals.

Topics: Acetylation; Animals; Azacitidine; Cell Line; Chemokine CXCL2; Disease Models, Animal; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Inflammation; Lipopolysaccharides; Lysophospholipids; Methyltransferases; Mice, Inbred C57BL; Microglia; Promoter Regions, Genetic; Receptors, G-Protein-Coupled; RGS Proteins; RNA, Messenger; Signal Transduction; Sphingosine; Transcription, Genetic; Tumor Necrosis Factor-alpha

The histone deacetylase inhibitor, trichostatin A (TSA), and the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (Aza-dC), induced epigenetic regulation of sphingosine-1-phosphate (S1P) receptors in human melanoma cells, switching S1P from motility inhibitor to stimulator. Quantitative PCR revealed increased expression of S1P(1) and S1P(3), associated with S1P-induced chemotaxis, and decreased expression of S1P(2), associated with motility inhibition. Expression of lysophosphatidic acid (LPA) receptors was less affected. The TSA effect was reversible suggesting no mutational change, and Aza-dC treatment resulted in demethylation of a putative S1P(1) promoter. S1P receptors, therefore, appear to be susceptible to epigenetic regulation, accompanied by altered cellular functionality.

Topics: Azacitidine; Cell Line, Tumor; Cell Movement; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation; Humans; Hydroxamic Acids; Lysophospholipids; Melanoma; Nerve Tissue Proteins; Receptors, Lysophospholipid; Receptors, Lysosphingolipid; RNA-Binding Proteins; RNA, Messenger; Sphingosine