ABSTRACT
Temporal down-regulation of the CD8 co-receptor after receiving positive-selection signals has been proposed to serve as an important determinant to segregate helper versus cytotoxic lineages by generating differences in the duration of TCR signaling between MHC-I and MHC-II selected thymocytes. By contrast, little is known about whether CD8 also modulates TCR signaling engaged by the non-classical MHC-I-like molecule, CD1d, during development of invariant natural killer T (iNKT) cells. Here, we show that constitutive transgenic CD8 expression resulted in enhanced differentiation of innate memory-like CD8+ thymocytes in both a cell-intrinsic and cell-extrinsic manner, the latter being accomplished by an increase in the IL-4-producing iNKT2 subset. Skewed iNKT2 differentiation requires cysteine residues in the intracellular domain of CD8α that are essential for transmitting cellular signaling. Collectively, these findings shed a new light on the relevance of CD8 down-regulation in shaping the balance of iNKT-cell subsets by modulating TCR signaling.
Subject(s)
CD8 Antigens/metabolism , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/genetics , Immunity, Innate , Natural Killer T-Cells/immunology , Animals , CD8 Antigens/genetics , Cell Differentiation/immunology , Cells, Cultured , Mice , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/genetics , Thymocytes/immunology , TransfectionABSTRACT
During differentiation of CD4+CD8+ double-positive (DP) thymocytes into the CD4-CD8+ single-positive (CD8SP) thymocytes committed to the cytotoxic T cell lineage, Cd8a transcription is temporally terminated after positive selection and is subsequently reinitiated, a process known as coreceptor reversal. Despite the identification of a transcriptional enhancer in the Cd8a gene that directs reporter transgene expression specifically in CD8SP thymocytes, the molecular mechanisms controlling reactivation of the Cd8a gene are not fully understood. Here, we show that, after positive selection, hCD2 reporter expression from the Cd8a locus, which was generated by insertion of hCD2 cDNA into the first exon of the Cd8a gene, requires the incorporation of intron sequences into the hCD2 transcript. The presence of polyadenylation signals after hCD2 cDNA inhibited hCD2 expression in mature CD8+ T cells, whereas hCD2 expression in DP thymocytes recapitulated the Cd8a expression. Incorporation of the endogenous short intron structure and heterologous intron structure of the Cd4 locus restored hCD2 expression in mature CD8+ T cells in a variegated manner. Interestingly, stage-specific DNA demethylation was impaired in Cd8a reporter alleles that failed to express hCD2 in CD8+ T cells, and intron sequences lacking RNA splicing signals still restored hCD2 expression. These observations indicate that "intron-mediated enhancement" is involved in a stage-specific reactivation of the Cd8a locus harboring hCD2 cDNA. However, the Cd8a gene was transcribed in mature CD8+ T cells, albeit at a lower level, from a mutant Cd8a locus lacking intron structures, suggesting that protein-coding sequences in transcripts affect sensitivity to intron-mediated enhancement.
Subject(s)
CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD8 Antigens/metabolism , CD8-Positive T-Lymphocytes/metabolism , Introns , T-Lymphocytes, Cytotoxic/metabolism , Thymocytes/metabolism , Animals , CD4 Antigens/genetics , CD4-Positive T-Lymphocytes/cytology , CD8 Antigens/genetics , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Cells, Cultured , Mice , RNA Splicing , Thymocytes/cytologyABSTRACT
BACKGROUND: The Runt-related transcription factors (Runx) are a family of evolutionarily conserved transcriptional regulators that play multiple roles in the developmental control of various cell types. Among the three mammalian Runx proteins, Runx1 is essential for definitive hematopoiesis and its dysfunction leads to human leukemogenesis. There are two promoters, distal (P1) and proximal (P2), in the Runx1 gene, which produce two Runx1 isoforms with distinct N-terminal amino acid sequences, P1-Runx1 and P2-Runx1. However, it remains unclear whether P2-Runx specific N-terminal sequence have any specific function for Runx1 protein. RESULTS: To address the function of the P2-Runx1 isoform, we established novel mutant mouse models in which the translational initiation AUG (+1) codon for P2-Runx1 isoform was modulated. We found that a truncated P2-Runx1 isoform is translated from a downstream non-canonical AUG codon. Importantly, the truncated P2-Runx1 isoform is sufficient to support primary hematopoiesis, even in the absence of the P1-Runx1 isoform. Furthermore, the truncated P2-Runx1 isoform was able to restore defect in basophil development caused by loss of the P1-Runx1 isoform. The truncated P2-Runx1 isoform was more stable than the canonical P2-Runx1 isoform. CONCLUSIONS: Our results demonstrate that the N-terminal sequences specific for P2-Runx1 are dispensable for Runx1 function, and likely serve as a de-stabilization module to regulate Runx1 production.
Subject(s)
Core Binding Factor Alpha 2 Subunit/chemistry , Core Binding Factor Alpha 2 Subunit/metabolism , Animals , Core Binding Factor Alpha 2 Subunit/genetics , Flow Cytometry , Gene Expression Regulation, Developmental/genetics , Hematopoiesis/genetics , Hematopoiesis/physiology , Immunoblotting , Mice , Mice, Mutant Strains , Promoter Regions, Genetic/genetics , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
Cathepsin D is a normal and major component of lysosomes, it is found in almost all cells and tissues of mammals. Present review describes different events in cellular life of cathepsin D mainly its biosynthesis, co-translational and posttranslational modifications, targeting to lysosomes and proteolytic processing and maturation within lysosomes.
