β2-adrenergic receptors inhibit the expression of collagen type II in growth plate chondrocytes by stimulating the AP-1 factor Jun-B

The sympathetic nervous system can regulate both osteoblast and chondrocyte growth and activity through β(2)-adrenergic receptors (β(2)-AR). We have shown previously that β(2)-AR activate both adenylyl cyclase and mitogen-activated protein kinases ERK1/2 in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Here we examined β(2)-AR inhibition of collagen type II (Col II) expression in growth plate chondrocytes and the molecular pathways involved. Stimulation of β(2)-AR by isoproterenol inhibited Col II mRNA and protein levels by ～50% beginning at 2 h, with both remaining suppressed over 24 h. This inhibition was blocked by propranolol and inhibitors of either MEK1 or PKA. Isoproterenol stimulated an AP-1-luciferase reporter and increased the expression of AP-1 factors c-Fos, Fra-1, Fra-2, c-Jun, and Jun-B but had no effect on Jun-D. Stimulation of AP-1 activity was blocked by inhibitors of MEK1 or PKA. siRNA inhibition of AP-1 factors showed that depletion of only Jun-B attenuated isoproterenol-mediated inhibition of Col II. Transfection with jun-B or c-fos showed selective inhibition of Col II mRNA and a Col II luciferase reporter construct by jun-B. Isoproterenol as well as jun-B overexpression in the chondrocytes also inhibited the expression of Sox-6 mRNA and protein, and depletion of Jun-B abrogated β(2)-AR inhibition of Sox-6. Collectively, these findings demonstrate regulation of chondrocyte differentiation through β(2)-AR mediated by ERK1/2 and PKA stimulation of the AP-1 factor Jun-B that inhibits the expression of Sox-6 and Col II.     

Light-Induced Variations in AP-1 Binding Activity and Composition in the Rat Suprachiasmatic Nucleus

Abstract : Expression of immediate early genes, including fos -like and jun -like genes, in the suprachiasmatic nucleus is believed to be part of the mechanism for photic entrainment of circadian rhythms to the environmental light/dark cycle. However, the effects of a light stimulus on activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus remain unclear. The photic regulation of AP-1 DNA-binding activity and composition in the rat suprachiasmatic nucleus was evaluated by using an electrophoretic mobility shift assay. A light pulse given during subjective night induced an increase in AP-1 binding activity when either nuclear or whole-cell extracts from suprachiasmatic nuclei were used. Under constant dark conditions, proteins that are predominant components of AP-1 complexes are Fra-2 and Jun-D. Under light stimulation, c-Fos and Jun-B consistently increased, as expected, but this was also the case for Fra-2, Jun-D, and c-Jun, although to a lesser extent. An immunocytochemical study of the Fra-2 expression pattern demonstrated the presence of the protein in the ventrolateral as well as in the dorsomedial subdivisions of the suprachiasmatic nucleus. Light regulation of Fra-2 immunoreactivity, however, appeared to be restricted to the ventrolateral subdivision. It is concluded that light may be acting both by increasing constitutive AP-1 complexes and by inducing the expression of specific complexes.     

DNA binding activities of three murine Jun proteins: stimulation by Fos

Three members of the Jun/AP-1 family have been identified in mouse cDNA libraries: c-Jun, Jun-B, and Jun-D. We have compared the DNA binding properties of the Jun proteins by using in vitro translation products in gel retardation assays. Each protein was able to bind to the consensus AP-1 site (TGACTCA) and, with lower affinity, to related sequences, including the cyclic AMP response element TGACGTCA. The relative binding to the oligonucleotides tested was similar for the different proteins. The Jun proteins formed homodimers and heterodimers with other members of the family, and they were bound to the AP-1 site as dimers. When Fos translation product was present, DNA binding by Jun increased markedly, and the DNA complex contained Fos. The C-terminal homology region of Jun was sufficient for DNA binding, dimer formation, and interaction with Fos. Our general conclusion is that c-Jun, Jun-B, and Jun-D are similar in their DNA binding properties and in their interaction with Fos. If there are functional differences between them, they are likely to involve other activities of the Jun proteins.     

IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA

The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-alpha, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-alpha, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-alpha, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-gamma production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS.     

Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. Despite the advances in surgical and medical management, the mechanisms underlying human osteosarcoma progression and metastasis remain to be elucidated. Gene expression profiles were compared by the cDNA microarray technique between two different human osteosarcoma sublines, MNNG/HOS and 143B, which differ greatly in spontaneous pulmonary metastatic potential. Here we report an enhanced expression of matrix metalloproteinase (MMP)-1 in the highly metastatic human osteosarcoma cell line 143B. Moreover, the in vitro invasion activity of 143B cells was MMP-1-dependent. The activator protein (AP)-1 binding site in the MMP-1 gene promoter was required for the constitutive expression of MMP-1 in 143B cells. Two AP-1 components, c-Jun and Fra-1, were phosphorylated, and bound to the AP-1 binding site of the MMP-1 promoter in 143B cells. Activated c-Jun and Fra-1 were essential for MMP-1 gene expression in 143B cells. Mitogen-activated protein kinase pathways including the c-Jun NH₂-terminal kinase and the extracellular signal-regulated kinase activate c-Jun and Fra-1 and thereby regulate c-Jun/Fra-1 mediated events, establishing the mitogen-activated protein kinase/AP-1/MMP-1 axis as important in 143B cells. These data suggest that MMP-1 plays a central role in osteosarcoma invasion. Accordingly, MMP-1 might be a biomarker and therapeutic target for invasive osteosarcomas and pulmonary metastases.     

Response of nitric oxide production to CpG oligodeoxynucleotides in turkey and chicken peripheral blood monocytes

We evaluated the innate immune response to various synthetic CpG-containing oligodeoxynucleotides (CpG ODNs) by measuring nitric oxide production in the peripheral blood monocytes from turkey poults. The results indicate that the presence of the CpG dinucleotide in ODNs was a prerequisite for activation of turkey monocytes and induction of nitric oxide (NO) synthesis. CpG motifs and sequence structure of the ODNs were also found to influence stimulatory activity greatly. The most potent CpG ODN to induce NO synthesis in turkey monocytes was human-specific CpG ODN M362, followed by CpG ODN 2006 (human), CpG ODN#17 (chicken) and CpG ODN 1826 (mouse). The optimal CpG motif for NO induction was GTCGTT. Phosphorothioate modification of CpG ODNs also significantly increased stimulatory activity. Compared with chicken monocytes, turkey monocytes appeared to be less sensitive to CpG motif variation, whereas chicken monocytes were found to respond more strictly to human-specific CpG ODNs or ODNs that contain GTCGTT motifs.     

Surface Ig receptor-induced nuclear AP-1-dependent gene expression in B lymphocytes.

The relationship between signals generated via the sIgR complex of B lymphocytes and subsequent changes in gene expression is poorly understood at the molecular level. To illuminate mechanisms that may couple these events, we examined the expression and function of tetradecanoyl phorbol acetate-response element (TRE)-binding proteins (i.e., activator protein 1, (AP-1)) in the murine B lymphoma cell line BAL-17.7.1 (BAL-17), which models primary B lymphocyte responses in a number of respects. Cross-linking of sIgR led to substantial induction of nuclear AP-1, in BAL-17 B cells, that bound the TRE, as detected by electrophoretic mobility shift assay. The sIgR-induced TRE-binding activity consisted of both Jun and Fos proteins, on the basis of immunoreactivity of nucleoprotein complexes with specific antisera. In addition, immunoprecipitation with specific antisera showed that de novo synthesis of Jun-B and c-Jun proteins, accompanied by c-Fos, was stimulated after cross-linking of sIgR on BAL-17 B cells. Transient transfection of BAL-17 B cells with reporter gene constructs showed that B cell AP-1 failed to trans-activate the TRE-containing human collagenase gene promoter, for which activity is dependent upon functional expression of cellular c-Jun. In contrast, sIg-induced AP-1 trans-activated a HSV-tk promoter that contained three TRE; this pattern of gene expression is consistent with the presence of functional Jun-B-containing AP-1 in B lymphocytes. These results are the first to attribute a functional role to sIgR-mediated AP-1 in B lymphoid cells and suggest that AP-1 functions to couple the sIgR complex to changes in nuclear gene expression.     

Effects of a hexameric deoxyriboguanosine run conjugation into CpG oligodeoxynucleotides on their immunostimulatory potentials

CpG oligodeoxynucleotides (ODNs) are promising immunomodulatory agents for treating human diseases and vaccine development. Phosphodiester CpG ODNs were demonstrated to have poor immunostimulatory potentials for cytokine production. However, the conjugation of consecutive deoxyriboguanosine residues, called a dG run, at the 3' terminus of phosphodiester CpG ODNs significantly enhanced TNF-alpha and IL-12 production from mouse splenic dendritic cells (DCs). The optimal induction of cytokine production was achieved by the addition of a hexameric dG (dG6) run. In contrast, the existence of a dG6 run either at the 5' terminus of phosphodiester CpG ODNs or at the 3' terminus of phosphorothioate CpG ODNs diminished CpG-mediated cytokine induction, suggesting that the effects of a dG run depend on its location and the chemical property of the ODN backbone, respectively. In addition, we provided the evidence that the conjugation of a dG6 run caused the structural transformation of CpG ODNs, which facilitates their targeting into mouse APCs such as splenic DCs, B cells, and peritoneal macrophages with a scavenger receptor type A ligand specificity. Among primary APCs, DCs were the most potent for CpG ODN-mediated IL-12 production. Furthermore, we demonstrated that the conjugation of a dG6 run into the 3' terminus of phosphodiester CpG ODNs was crucial for their ability to generate Th1 immunity in vivo. Thus, the conjugation of a dG6 run into phosphodiester CpG ODNs would be an alternative way to optimize their immunostimulatory potentials in vitro and in vivo.     

Structure-dependent modulation of alpha interferon production by porcine circovirus 2 oligodeoxyribonucleotide and CpG DNAs in porcine peripheral blood mononuclear cells

DNA sequences containing CpG motifs are recognized as immunomodulators in several species. Phosphodiester oligodeoxyribonucleotides (ODNs) representing sequences from the genome of porcine circovirus type 2 (PCV2) have been identified as potent inducers (ODN PCV2/5) or inhibitors (ODN PCV2/1) of alpha interferon (IFN-alpha) production by porcine peripheral blood mononuclear cells (poPBMCs) in vitro. In this study, the IFN-alpha-inducing or -inhibitory activities of specific phosphodiester ODNs were demonstrated to be dependent on their ability to form secondary structures. When a poly(G) sequence was added to a stimulatory self-complementary ODN, high levels of IFN-alpha were elicited, and the induction was not dependent on pretreatment with the transfecting agent Lipofectin. In addition, the IFN-alpha-inducing ODN required the presence of an intact CpG dinucleotide, whereas the inhibitory activity of ODN PCV2/1 was not affected by methylation or removal of the central CpG dinucleotide. Of particular significance, the IFN-alpha inhibition elicited by ODN PCV2/1 was only effective against induction stimulated by DNA control inducers and not RNA control inducers, indicating activity directed to TLR9 signaling. The PCV2 genome as a whole was demonstrated to induce IFN-alpha in cultures of poPBMCs, and the presence of immune modulatory sequences within the genome of PCV2 may, therefore, have implications with regard to the immune evasion mechanisms utilized by PCV2.