ABSTRACT
The thymus is an organ that produces functionally competent T cells that protect us from pathogens and malignancies. Foxn1 is a transcription factor that is essential for thymus organogenesis; however, the direct target for Foxn1 to actuate thymic T-cell production is unknown. Here we show that a Foxn1-binding cis-regulatory element promotes the transcription of ß5t, which has an essential role in cortical thymic epithelial cells to induce positive selection of functionally competent CD8+ T cells. A point mutation in this genome element results in a defect in ß5t expression and CD8+ T-cell production in mice. The results reveal a Foxn1-ß5t transcriptional axis that governs CD8+ T-cell production in the thymus.
Subject(s)
CD8-Positive T-Lymphocytes/physiology , Forkhead Transcription Factors/metabolism , Gene Expression Regulation/physiology , Proteasome Endopeptidase Complex/genetics , Thymus Gland/physiology , Animals , Cell Differentiation/genetics , Cells, Cultured , Epithelial Cells/physiology , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis, Site-Directed , Point Mutation , Regulatory Elements, Transcriptional/physiology , Thymus Gland/cytology , Tissue Culture TechniquesABSTRACT
The present study aimed to investigate the involvement of the NMDA receptor (NMDAR) and/or nitric oxide (NO) pathway in ketamine-induced behavioral sensitization. Mice received repeated subcutaneous administration of ketamine (25mg/kg), once daily or once weekly for a total of five doses. Even three administrations of ketamine, daily or weekly, induced a rapid increase in locomotor activity in wild-type (WT), but not in GluN2D knockout (GluN2D-KO) mice. Furthermore, for WT mice receiving daily ketamine, elevated locomotor activity was maintained after a 1-month withdrawal period; however, this was not the case when ketamine was administered weekly. The effect of acute ketamine on nNOS activities was estimated with nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) histochemistry. Ketamine rapidly increased the number of NADPH-d activated cells and strongly stained dendrites in the dorsal striatum and prefrontal cortex of WT mice, but not GluN2D-KO mice. These results suggest that ketamine-induced locomotor sensitization and nNOS activation in the frontal cortex-striatum neuronal circuit are positively correlated and that the NMDAR GluN2D subunit plays an important role in the acquisition and maintenance of ketamine-induced behavioral sensitization.
Subject(s)
Corpus Striatum/drug effects , Ketamine/pharmacology , Motor Activity/drug effects , Nitric Oxide Synthase Type I/metabolism , Prefrontal Cortex/drug effects , Psychotropic Drugs/pharmacology , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Corpus Striatum/metabolism , Enzyme Activation , Male , Mice, Inbred C57BL , Mice, Knockout , NADP/metabolism , Neurons/metabolism , Nitric Oxide/biosynthesis , Organ Specificity , Prefrontal Cortex/metabolism , Protein Subunits/metabolism , Receptors, N-Methyl-D-Aspartate/geneticsABSTRACT
BACKGROUND: Epidermal-type transglutaminase (TGase 3) is involved in the cross-linking of structural proteins in the epidermis, which results in the formation of the cornified envelope. TGase 3 is activated by limited proteolysis of a 77 kDa zymogen during keratinocyte differentiation. OBJECTIVE: To characterize the expression of TGase 3 in human epidermis and cultured keratinocytes, we established specific monoclonal antibodies against the TGase 3. METHODS: Recombinant proteins for human TGase 3 produced in bacteria and baculovirus-infected insect cells were purified as an antigen. Hybridomas are established and used for characterization of expression in epidermis and keratinocytes. RESULTS: Four antibodies were generated against recombinant human TGase 3, which reacted with the 77 kDa zymogen and in some cases either the 47 or 30 kDa active proteolytic fragments. In human epidermis and cultured keratinocytes, only the zymogen form of TGase 3 was detected. Immunohistochemical analysis of the skin revealed that the enzyme is present in the cells of the granular and cornified layers consistent with its role in cornified envelope formation. In cultured keratinocytes, TGase 3 was expressed in differentiating cells coincident with profilaggrin and keratin 10 expressions. CONCLUSION: Using monoclonal antibody against human TGase 3, we showed the expression of TGase 3 in upper layers of epidermis. TGase 3 displayed a diffuse cytoplasmic distribution in vitro consistent with its proposed role in the early phase of cornified cell envelope assembly in the cytoplasm.
