Suppression of T-cell responsiveness by inducible cAMP early repressor (ICER).

Depending on the nature of the costimulation of T lymphocytes, expression of regulatory cytokines and chemokines is either susceptible or resistant to cyclic AMP (cAMP)-mediated inhibition. Our data show that cAMP-mediated inhibition of endogenously expressed cytokines, which is characteristic for T helper (Th) 1- and Th 2-like phenotypes, correlates with the induction of a potent transcriptional repressor, inducible cAMP early repressor (ICER), in both subsets of T cells activated under conditions of suboptimal interleukin-2 (IL-2) expression. Importantly, Th-specific expression of certain chemokines is also susceptible to cAMP-mediated transcriptional attenuation. To determine whether ICER per se, rather than forskolin-mediated elevation of intracellular cAMP, is responsible for the observed inhibitory effect, we generated transgenic mice expressing ICER under the control of a lymphocyte-specific lck promoter. On stimulation, transgenic thymocytes overexpressing ICER exhibited reduced levels of IL-2 and interferon (IFN)-gamma and failed to express the macrophage inflammatory protein (MIP)-1alpha and MIP-1beta genes. Splenic T cells from ICER-transgenic mice showed a defect in proliferation and lacked a mixed lymphocyte reaction response, implying that ICER-mediated inhibition of cytokine and chemokine expression might play an important role in T-cell inactivation.

Twisted gastrulation can function as a BMP antagonist.

Bone morphogenetic proteins (BMPs), including the fly homologue Decapentaplegic (DPP), are important regulators of early vertebrate and invertebrate dorsal-ventral development. An evolutionarily conserved BMP regulatory mechanism operates from fly to fish, frog and mouse to control the dorsal-ventral axis determination. Several secreted factors, including the BMP antagonist chordin/Short gastrulation (SOG), modulate the activity of BMPs. In Drosophila, Twisted gastrulation (TSG) is also involved in dorsal-ventral patterning, yet the mechanism of its function is unclear. Here we report the characterization of the vertebrate Tsg homologues. We show that Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late developmental stages. Our data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis.

Suppression of T cell function: a potential role for transcriptional repressor ICER.

In this article, we review the inducible cAMP early repressor (ICER) and its possible critical involvement in modulation of T cell responsiveness by its capacity to transcriptionally attenuate interleukin-2 (IL-2) gene expression. It seems clear that the failure to produce the IL-2 is an important determinant of anergy induction. It is important that the CD28-responsive element (CD28RE), a composite DNA binding element consisting of NFAT and cyclic AMP-responsive (CRE)-like motifs in position of -160 of IL-2 promoter has the high affinity for ICER binding as well as NFAT/ICER complex formation. Moreover, CD28RE with adjacent DNA sequences was also shown to be essential for conferring anergy in T lymphocytes. Because ICER does not possess a transactivation domain required for the recruitment of CBP/p300, the binding of ICER to CD28RE and/or composite motifs containing CRE-like DNA motifs may lead to uncoupling of CBP/p300 thus extinguishing IL-2 expression as well as expression of numerous other cytokines and chemokines.

Generation of monoclonal antibodies to integrin-associated proteins. Evidence that alpha3beta1 complexes with EMMPRIN/basigin/OX47/M6.

The alpha3beta1 integrin forms complexes with other cell-surface proteins, including transmembrane-4 superfamily (TM4SF) proteins (e. g. CD9, CD53, CD63, CD81, and CD82). To identify additional cell-surface proteins associated with alpha3beta1 integrin, a monoclonal antibody selection protocol was developed. Mice were immunized with integrin alpha3beta1-containing complexes isolated from HT1080 fibrosarcoma cells, and then 712 hybridoma clones were produced, and 95 secreted antibodies that recognized the HT1080 cell surface. Among these, 12 antibodies directly recognizing integrin alpha3 or beta1 subunits were eliminated. Of the remaining 83, 16 co-immunoprecipitated proteins that resembled integrins under non-stringent detergent conditions. These 16 included 15 monoclonal antibodies recognizing EMMPRIN/basigin/OX-47/M6, a 45-55-kDa transmembrane protein with two immunoglobulin domains. The EMMPRIN protein associated with alpha3beta1 and alpha6beta1, but not alpha2beta1 or alpha5beta1, as shown by reciprocal immunoprecipitation experiments. Also, association with alpha3beta1 was confirmed by cell-surface cross-linking and immunofluorescence co-localization experiments. Importantly, EMMPRIN-alpha3beta1 complexes appear not to contain TM4SF proteins, suggesting that they are distinct from TM4SF protein-alpha3beta1 complexes.

NAG-2, a novel transmembrane-4 superfamily (TM4SF) protein that complexes with integrins and other TM4SF proteins.

Transmembrane-4 superfamily (TM4SF) proteins form complexes with integrins and other cell-surface proteins. To further characterize the major proteins present in a typical TM4SF protein complex, we raised monoclonal antibodies against proteins co-immunoprecipitated with CD81 from MDA-MB-435 breast cancer cells. Only two types of cell-surface proteins were recognized by our 35 selected antibodies. These included an integrin (alpha6beta1) and three different TM4SF proteins (CD9, CD63, and NAG-2). The protein NAG-2 (novel antigen-2) is a previously unknown 30-kDa cell-surface protein. Using an expression cloning protocol, cDNA encoding NAG-2 was isolated. When aligned with other TM4SF proteins, the deduced amino acid sequence of NAG-2 showed most identity (34%) to CD53. Flow cytometry, Northern blotting, and immunohistochemistry showed that NAG-2 is widely present in multiple tissues and cell types but is absent from brain, lymphoid cells, and platelets. Within various tissues, strongest staining was seen on fibroblasts, endothelial cells, follicular dendritic cells, and mesothelial cells. In nonstringent detergent, NAG-2 protein was co-immunoprecipitated with other TM4SF members (CD9 and CD81) and integrins (alpha3beta1 and alpha6beta1). Also, two-color immunofluorescence showed that NAG-2 was co-localized with CD81 on the surface of spread HT1080 cells. These results confirm the presence of NAG-2 in specific TM4SF.TM4SF and TM4SF-integrin complexes.

A study of the structure, function and distribution of beta 5 integrins using novel anti-beta 5 monoclonal antibodies.

Here, we have utilized six new anti-human beta 5 monoclonal antibodies to perform a detailed investigation of the structure, function and distribution of beta 5 integrins. Monoclonal anti-beta 5 specificity was confirmed by reactivity with beta 5-transfected CHO cells, by direct binding to the beta 5 subunit (immunoblotting), and by immunodepletion experiments using polyclonal anti-beta 5 serum. The beta 5 subunit was predominantly associated with the alpha v subunit, although on some cell lines, the level of beta 5 exceeded that of alpha v for unknown reasons. Cell adhesion studies showed that the adhesive function of beta 5 could be stimulated, inhibited or unaltered by different anti-beta 5 monoclonal antibodies. The beta 5 subunit was involved in adhesion to both vitronectin and fibronectin and, at least for K562 cells, fibronectin appeared to be the preferred ligand. Flow-cytometry studies showed that the beta 5 subunit was expressed at moderate to high levels on all adherent cell lines examined, was absent from all lymphoid cell lines, and was only weakly expressed on myeloid cell lines. Staining of thymic sections showed the distribution of beta 5 on blood vessels, Hassal's corpuscles, cortical and medullary stromal cells, and basement membranes. Skin sections showed beta 5 on the basal layer of the epidermis and on some dermal blood vessel walls, and kidney sections showed staining of glomerular regions, juxta glomerular apparatus, proximal convoluted tubules and collecting tubules, and at least one anti-beta 5 antibody also stained epithelial cells of proximal tubules.