Eosinophil as a cellular target of the ocular anti-allergic action of mapracorat, a novel selective glucocorticoid receptor agonist

Abstract: Purpose: Glucocorticoids can either suppress gene transcription (transrepression) or activate it (transactivation). This latter process may contribute to certain side effects caused by these agents. Mapracorat (also known as BOL-303242-X or ZK 245186) is a novel selective glucocorticoid receptor agonist that maintains a beneficial anti-inflammatory activity but seems to be less effective in transactivation, resulting in a lower potential for side effects; it has been proposed for the topical treatment of inflammatory skin disorders. This study assessed the anti-allergic activity of mapracorat at the ocular level and whether eosinophils and mast cells are targets of its action

Identification of the N-Linked Glycosylation Sites of the Transcription Factor Rest and Effect of Glycosylation on DNA Binding and Transcriptional Activity

REST is a zinc-finger transcription factor implicated in several processes such as maintenance of embryonic stem cell pluripotency and regulation of mitotic fidelity in non-neuronal cells [Chong et al., 1995]. The gene encodes for a 116-kDa protein that acts as a molecular platform for co-repressors recruitment and promotes modifications of DNA and histones [Ballas, 2005]. REST showed different apparent molecular weights, consistent with the possible presence of post-translational modifications [Lee et al., 2000]. Among these the most common is glycosylation, the covalent attachment of carbohydrates during or after protein synthesis [Apweiler et al., 1999] My thesis has ascertained, for the first time, the presence of glycan chians in the transcription factor REST. Through enzymatic deglycosylation and MS, oligosaccharide composition of glycan chains was evaluated: a complex mixture of glycans, composed of N-acetylgalactosamine, galactose and mannose, was observed thus confirming the presence of O- and N-linked glycan chains. Glycosylation site mapping was done using a 18O-labeling method and MS/MS and twelve potential N-glycosylation sites were identified. The most probable glycosylation target residues were mutated through site-directed mutagenesis and REST mutants were expressed in different cell lines. Variations in the protein molecular weight and mutant REST ability to bind the RE-1 sequence were analyzed. Gene reporter assays showed that, altogether, removal of N-linked glycan chains causes loss of transcriptional repressor function, except for mutant N59 which showed a slight residual repressor activity in presence of IGF-I. Taken togheter these results demonstrate the presence of complex glycan chians in the transcription factor REST: I have depicted their composition, started defining their position on the protein backbone and identified their possible role in the transcription factor functioning. Considering the crucial role of glycosylation and transcription factors activity in the aetiology of many diseases, any further knowledge could find important and interesting pharmacological application

Therapeutic Targeting of Eosinophil Adhesion and Accumulation in Allergic Conjunctivitis

none5Considerable evidence indicates that eosinophils are important effectors of ocular allergy. Increased worldwide prevalence of allergic eye pathologies has stimulated the identification of novel drug targets, including eosinophils and adhesion molecules. Accumulation of eosinophils in the eye is a key event in the onset and maintenance of allergic inflammation and is mediated by different adhesion molecules. Antihistamines with multiple mechanisms of action can be effective during the early and late phases of allergic conjunctivitis by blocking the interaction between β1 integrins and vascular cell adhesion molecule (VCAM)-1. Small molecule antagonists that target key elements in the process of eosinophil recruitment have been identified and reinforce the validity of α4β1 integrin as a therapeutic target. Glucocorticoids are among the most effective drugs for ocular allergy, but their use is limited by adverse effects. Novel dissociated glucocorticoids can prevent eosinophil accumulation and induce apoptosis of eosinophils, making them promising candidates for ophthalmic drugs. This article reviews recent understanding of the role of adhesion molecules in eosinophil recruitment in the inflamed conjunctiva along with effective treatments for allergic conjunctivitis.mixedBaiula M; Bedini A; Carbonari G; Dattoli SD; Spampinato SBaiula M; Bedini A; Carbonari G; Dattoli SD; Spampinato

REST is up-regulated by epidermal growth factor in HeLa cells and inhibits apoptosis by influencing histone H3 acetylation

none6noREST (repressor element 1-silencing transcription factor) is a transcription factor that recruits histone deacetylases to silence gene transcription. REST appears to play a paradoxical role in cancer cells: it exhibits tumor suppressor activity or promotes tumorigenesis, depending upon the setting. The extracellular signaling molecules that control REST gene expression in cancer cells remain poorly understood. In this study, we report that REST expression in HeLa cells is elevated in cells exposed to epidermal growth factor or serum, whereas the rate of cell apoptosis is low. Apoptosis induced by serum withdrawal is significantly increased in HeLa cells treated with an antisense phosphorothioate oligodeoxynucleotide (AS ODN) capable of down-regulating REST expression, whereas in HeLa cells transfected with a REST expressing plasmid, REST overexpression reduces the marked apoptosis caused, in absence of serum, by exposure to an anti-Fas receptor antibody imitating the Fas ligand activity plus PD 98059, a blocker of extracellular signal-regulated kinase 1/2 activation. REST knockdown also reduces mRNA levels of the antiapoptotic protein Bcl-X(L) whereas in HeLa cells overexpressing REST, the reduction of Bcl-X(L) mRNA caused by the anti-Fas receptor antibody plus PD 98059 is significantly decreased. Finally, we report that acetylation of histone H3 is increased in HeLa cells exposed to AS ODN or anti-Fas receptor antibody, whereas it is reduced in cells transfected with the REST expressing plasmid. Our findings indicate that REST is a novel gene regulated by EGF in HeLa cells that potentially contributes to the modulation of apoptosis via epigenetic mechanisms.mixedBaiula M; Carbonari G; Dattoli SD; Calienni M; Bedini A; Spampinato S.Baiula M; Carbonari G; Dattoli SD; Calienni M; Bedini A; Spampinato S