The MHC class II transactivator (CIITA), the master regulator of MHC class II (MHC II) expression, is a co-activator that controls MHC II transcription. Human B lymphocytes express MHC II constitutively due to persistent activity of CIITA promoter III (pIII), one of the four potential promoters (pI-pIV) of this gene. Although increases in MHC II expression in B cells in response to cytokines have been observed and induction of MHC II and CIITA by IFN-gamma has been studied in a number of different cell types, the specific effects of IFN-gamma on CIITA expression in B cells have not been studied. To investigate the regulation of CIITA expression by IFN-gamma in B cells, RT-PCR, in vivo and in vitro protein/DNA binding studies, and functional promoter analyses were performed. Both MHC II and CIITA type IV-specific RNAs increased in human B lymphocytes in response to IFN-gamma treatment. CIITA promoter analysis confirmed that pIV is IFN-gamma inducible in B cells and that the GAS and IRF-E sites are necessary for full induction. DNA binding of IRF-1 and IRF-2, members of the IFN regulatory factor family, was up-regulated in B cells in response to IFN-gamma and increased the activity of CIITA pIV. In vivo genomic footprint analysis demonstrated proteins binding at the GAS, IRF-E and E box sites of CIITA pIV. Although CIITA pIII is considered to be the hematopoietic-specific promoter of CIITA, these findings demonstrate that pIV is active in B lymphocytes and potentially contributes to the expression of CIITA and MHC II in these cells.

The MHC Class II transactivator (CIITA) acts in the cell nucleus as the master regulator of MHC class II (MHC II) gene expression. It is important to study CIITA regulation in multiple myeloma since MHC expression is central to ability of myeloma cells to present antigen and to the ability of the immune system to recognize and destroy this malignancy. Regulation of CIITA by IFN-γ in B lymphocytes occurs through the CIITA type IV promoter (pIV), one of the four potential promoters (pI-pIV) of this gene. To investigate regulation of CIITA by IFN-γ in multiple myeloma cells, first the ability of these cells to respond to IFN-γ was examined. RTPCR analyses show that IFN-γR1, the IFN-γ-binding chain of the IFN-γ receptor, is expressed in myeloma cells and IRF-1 expression increases in response to IFN-γ treatment. Western blotting demonstrates that STAT1 is activated by phosphorylation in response to IFN-γ. RT-PCR and functional promoter analyses show that IFN-γ up regulates the activity of CIITA pIV, as does ectopic expression of IRF-1 or IRF-2. In vivo protein/DNA binding studies demonstrate protein binding at the GAS, E box and IRF-E sites. In vitro studies confirm the binding of IRF-1 and IRF-2 to CIITA pIV. Although multiple myeloma cells express PRDI-BF1/Blimp-1, a factor that represses both the CIITA type III and IV promoters, they retain the capability to up regulate CIITA pIV and MHC II expression in response to IFN-γ treatment. These findings are the first to demonstrate that although PRDI-BF1/Blimp-1 diminishes the constitutive ability of these cells to present antigen by limiting CIITA and MHC II expression, it is possible to enhance this expression through the use of cytokines, like IFN-γ.

MHC class II transactivator (CIITA), a co-activator that controls MHC class II (MHC II) transcription, functions as the master regulator of MHC II expression. Persistent activity of the CIITA type III promoter (pIII), one of the four potential promoters of this gene, is responsible for constitutive expression of MHC II by B lymphocytes. In addition, IFN-gamma induces expression of CIITA in these cells through the type IV promoter (pIV). Positive regulatory domain 1-binding factor 1 (PRDI-BF1), called B lymphocyte-induced maturation protein 1 (Blimp-1) in mice, represses the expression of CIITA pIII in plasma and multiple myeloma cells. To investigate regulation of CIITA pIV expression by PRDI-BF1 in the B lymphocyte lineage, protein/DNA-binding studies, and functional promoter analyses were performed. PRDI-BF1 bound to the IFN regulatory factor-element (IRF-E) site in CIITA pIV. Ectopic expression of either PRDI-BF1 or Blimp-1 repressed this promoter in B lymphocytes. In vitro binding and functional analyses of CIITA pIV demonstrated that the IRF-E is the target of this repression. In vivo genomic footprint analysis demonstrated protein binding at the IRF-E site of CIITA pIV in U266 myeloma cells, which express PRDI-BF1. PRDI-BF1beta, a truncated form of PRDI-BF1 that is co-expressed in myeloma cells, also bound to the IRF-E site and repressed CIITA pIV. These findings demonstrate for the first time that, in addition to silencing expression of CIITA pIII in B lymphocytes, PRDI-BF1 is capable of binding and suppressing CIITA pIV.

Transient receptor potential vanilloid receptor 4 (TRPV4) is a calcium-permeable non-selective cation channel implicated in numerous physiological and pathological functions. This study aimed to investigate the effect of TRPV4 activation on respiration and to explore the potential involvement of bronchopulmonary sensory neurons. Potent TRPV4 agonist GSK1016790A was injected into right atrium in anesthetized spontaneously breathing rats and the changes in breathing were measured. Patch-clamp recording was performed to investigate the effect of GSK1016790A or another TRPV4 activator 4α-PDD on cultured rat vagal bronchopulmonary sensory neurons. Immunohistochemistry was carried out to determine the TRPV4-expressing cells in lung slices obtained from TRPV4-EGFP mice. Our results showed, that right-atrial injection of GSK1016790A evoked a slow-developing, long-lasting rapid shallow breathing in anesthetized rats. Activation of TRPV4 also significantly potentiated capsaicin-evoked chemoreflex responses. The alteration in ventilation induced by GSK1016790A was abolished by cutting or perineural capsaicin treatment of both vagi, indicating the involvement of bronchopulmonary afferent neurons. The stimulating and sensitizing effects of GSK1016790A were abolished by a selective TRPV4 antagonist GSK2193874 and also by inhibiting cyclooxygenase with indomethacin. Surprising, GSK1016790A or 4α-PDD did not activate isolated bronchopulmonary sensory neurons, nor did they modulate capsaicin-induced inward currents in these neurons. Furthermore, TRPV4 expression was found in alveolar macrophages, alveolar epithelial, and vascular endothelial cells. Collectively, our results suggest that GSK1016790A regulates the respiration through an indirect activation of bronchopulmonary sensory neurons, likely via its stimulation of other TRPV4-expressing cells in the lungs and airways.

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