Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1.

We have identified the PDZ domain protein AF-6 as an intracellular binding partner of the junctional adhesion molecule (JAM), an integral membrane protein located at cell contacts. Binding of AF-6 to JAM required the presence of the intact C terminus of JAM, which represents a classical type II PDZ domain-binding motif. Although JAM did not interact with the single PDZ domains of ZO-1 or of CASK, we found that a ZO-1 fragment containing PDZ domains 2 and 3 bound to JAM in vitro in a PDZ domain-dependent manner. AF-6 as well as ZO-1 could be coprecipitated with JAM from endothelial cell extracts, demonstrating the association of the endogenously expressed molecules in vivo. Targeting of JAM to sites of cell contacts could be affected by the loss of the PDZ domain-binding C terminus. Full-length mouse JAM co-distributed with endogenous AF-6 in human Caco-2 cells at sites of cell contact independent of whether adjacent cells expressed mouse JAM as an extracellular binding partner. In contrast, truncated JAM lacking the PDZ domain-binding C terminus did not co-distribute with endogenous AF-6, but was restricted to cell contacts between cells expressing mouse JAM. Our results suggest that JAM can be recruited to intercellular junctions by its interaction with the PDZ domain-containing proteins AF-6 and possibly ZO-1.

Gene regulation after Fc epsilon RI stimulation in the murine mast cell line CPII.

BACKGROUND: Mast cells produce a number of lymphokines and chemokines upon Fc epsilonRI stimulation. However, signal cascades and transcription factors involved in the induction of the corresponding genes are still poorly understood. METHODS: We addressed this issue using transient transfections of a TNF alpha promoter-driven reporter gene and corresponding 5' successive deletions, the two phosphoinositol-3 kinase inhibitors demethoxyviridin and wortmannin, ELISAs and Western and Southwestern blots. RESULTS: Nuclear factor of activated T cells (NF-AT) and AP1 transcription factors together mediate the activation of TNF alpha transcription in mast cells upon IgE plus antigen stimulation which, in contrast to the degranulation reaction and leukotriene synthesis, is independent of phosphoinositol-3-kinase. CONCLUSIONS: TNF alpha regulation in mast cells provides an experimental system for direct comparison of the regulation of this cytokine in T cells. In the context of our recent findings on IL-5 gene regulation in mast cells, a picture emerges in which NF-AT, dependent on the cytokine and not on cell type, interacts with a specific cofactor (AP1 for TNF alpha, GATA for IL-5). Multiple NF-AT family members found to be expressed in mast cells provide the structural basis for these different interactions with cofactors. The insensitivity of TNF alpha gene activation and release to inhibitors of phosphoinositol-3 kinase demonstrates that the activation of NF-AT and/or AP1 transcription factors in mast cells is not triggered along this signaling cascade.

Effects of phosphatidylinositol-3-kinase inhibitors on degranulation and gene induction in allergically triggered mouse mast cells.

BACKGROUND: Phosphatidylinositol-3-kinase (PI3-kinase) comprises an essential component in a number of signaling cascades, primarily of the growth factor type. Two specific inhibitors, wortmannin and demethoxyviridin (DMV), are widely used to block signaling via this molecule and link certain receptors to the PI3-kinase pathway. METHODS: We have studied the extent of involvement of PI3-kinase in signaling events by Fc epsilonRI in mast cells using a mouse mast cell line as a model system. This was done using beta-hexosaminidase release assays, a leukotriene ELISA, transient transfections with reporter gene constructs of TNF alpha and MARC, and in addition a TNF alpha ELISA. RESULTS: Consistent with previously published data in the rat basophilic cell line RBL-2H3, we find that wortmannin as well as DMV prevent the degranulation reaction in the mouse mast cell line CPII. DMV also inhibits the release of leukotrienes, leading to the conclusion that Fc epsilonRI activates PI3-kinase which then mediates these reactions. On the contrary, however, lymphokine and chemokine induction at the gene and protein level is not inhibited, suggesting that the activation of this gene set in mast cells is independent of PI3-kinase. CONCLUSION: PI3-kinase is activated in our mast cell model system via cross-linking of the Fc epsilonRI. This reaction is clearly necessary for the degranulation process and the release of leukotrienes. Activation of lymphokine and chemokine genes as well as secretion of their gene products are not triggered along the PI3-kinase signaling pathway. This is in agreement with our previous findings, showing that the MAP kinase pathway and Ca2+ influx are both involved in gene activation in this cell type.

Induction of the TNF-alpha promoter in the murine dendritic cell line 18 and the murine mast cell line CPII is differently regulated.

While it was recently shown that activation of dendritic cells (DC) results in the production of a number of cytokines, the signal pathways and transcription factors involved in this process have not been described. To address this issue we compared the events resulting in the activation of the human TNF-alpha promoter occurring in the fetal dendritic cell line 18 (DC18) with those in the well-characterized murine mast cell line CPII. As stimuli we employed the protein kinase C inducer, PMA, and the Ca2+ ionophore, ionomycin, both of which are known to activate a large variety of intracellular signaling pathways. In the DC18 cells, PMA alone induces the TNF-alpha promoter in a macrolide-insensitive manner. In contrast, in the mast cell line CPII, both stimuli (PMA plus ionomycin) are necessary for promoter activation which, in addition, is sensitive to immunosuppressive drugs. Mapping of the TNF-alpha promoter showed that in both cell types the so-called kappa factor binding site is the crucial promoter element for the induction. We show that in DC18 cells, this sequence is bound to and controlled by NF-kappaB proteins p50 (NF-kappaB1) and p65 (ReIA), whereas in CPII mast cells, NF-AT and AN factors are the predominant proteins that bind to and control the kappaB element of the TNF-alpha promoter. These and further experimental data indicate that in DC, NF-kappaB factors play a predominant role in the activation of the TNF-alpha promoter and, possibly, of other cytokine promoters.

The murine homolog of TB2/DP1, a gene of the familial adenomatous polyposis (FAP) locus.

The cDNA of the murine counterpart of the human TB2/DP1 (deleted in polyposis) gene, one of the six genes deleted in severe cases of familial adenomatous polyposis (FAP) disease, was isolated and analyzed. The murine transcript is 734-bp long and thereby considerably shorter than the 3100-bp human counterpart. This is due to a completely different 3' untranslated region in mouse which starts immediately after the translational stop codon, thereby deleting a RFLP (restriction-fragment length polymorphism) marker for this disease. The amino acid sequence, however, is 92% conserved between mouse and man. Triggering of murine mast cells by IgE plus antigen results in a decrease of TB2/DP1 mRNA up to 60% after 2 h implying a possible role of this gene in regulation of the allergic effector cell. Reverse transcription-polymerase chain reaction (RT-PCR) analysis shows an ubiquitous expression pattern in a number of mouse cell lines and tissues.

p21ras links Fc epsilon RI to NF-AT family member in mast cells. The AP3-like factor in this cell type is an NF-AT family member.

Very recently, an AP3-like transcription factor regulating the chemokine gene MARC and an NF-AT family member regulating IL-5 were the first components of the transcription factor repertoire to be described as activated in mast cells after an allergic triggering. In this study, we show that with respect to cross-competition in a gel shift analysis using an NF-AT consensus oligonucleotide binding site, the antigenicity to a recently generated serum against T cell NF-AT, and the sensitivity to macrolide immunosuppressants, the AP3-like activity on the MARC promoter is indistinguishable from that described for NF-AT in T cells. Additionally, we show that this factor functions on the MARC chemokine promoter without the AP1 cofactor, a situation reminiscent of the function of NF-AT in Th2-type T cells. In all of these aspects, and strengthened further by a gel shift competition analysis, the AP3-like transcription factor is identical to the NF-AT family member recently described by an analogous set of experiments as regulating IL-5 in mast cells. Our finding that p21ras, but probably not protein kinase C, is necessary to activate this factor after Fc epsilon RI triggering indicates a situation in which a common transcription factor denominator in mast cells induces chemokine (MARC) and lymphokine (IL-5) gene expression in a manner closely similar to Th2-type T cells, which are induced along the ras/raf signal pathway.