Abnormal development of urogenital organs in Dlgh1-deficient mice

Dlgh1 (discs large homolog 1) is a mammalian homolog of the Drosophila tumor suppressor Discs large 1, and is a member of the membrane-associated guanylate kinase (MAGUK) scaffolding proteins that contain three PSD-95/Dlg/ZO-1 (PDZ) domains. Discs large 1 is involved in epithelial polarization and cell-cell adhesion complex formation during Drosophila development. However, the functions of Dlgh1 during mammalian development remain to be elucidated. We generated Dlgh1-knockout mice and found that homozygous Dlgh1-knockout mice developed various abnormalities in their renal and urogenital organs. The kidneys and ureters were hypoplastic and the lower ends of the ureters were ectopic. In addition, the vagina and seminal vesicle, which are derived from the lower part of the Müllerian and Wolffian duct, respectively, were absent. Unexpectedly, loss of Dlgh1 function in the developing ureters did not disrupt cell-cell junctional complexes, but did impair cellular proliferation in the epithelium. These results suggest a novel role for Dlgh1 in regulating epithelial duct formation and morphogenesis during mammalian development. Although congenital absence of the vagina associated with other variable Müllerian duct abnormalities has been reported in humans, its mechanism has not yet been clarified. Our findings might contribute to a better understanding of such abnormalities.     

The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog 1

Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.     

Cd81 Interacts with the T Cell Receptor to Suppress Signaling

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling.     

T-cell signalling and immune system disorders

T-cell receptor (TCR) engagement initiates intracellular signalling cascades that lead to T-cell proliferation, cytokine production and differentiation into effector cells. These cascades comprise an array of protein-tyrosine kinases, phosphatases, GTP-binding proteins and adaptor proteins that regulate generic and specialised functions. The integration of these signals is essential for the normal development, homeostasis and function of T cells. Defects in a single mediator can produce T cells that are unable to participate fully in an immune response and/or that mount an inappropriate response, which leads to immunodeficiency, autoimmunity or leukaemia/lymphomas. This review highlights some of the key players in T-cell signalling and their involvement in the development of various clinical disease states. Some of these immune-specific signalling proteins are attractive potential targets in the development of therapies to augment T-cell responses to antigen or tumours, and to treat immune cell disorders.     

Differential Ras signaling via the antigen receptor and IL-2 receptor in primary T lymphocytes

Ras can become activated via multiple distinct receptors in T lymphocytes. However, mechanistic studies of Ras signaling in normal T cells have been hampered by the lack of an efficient technology for gene transfer into resting post-thymic cells. We have overcome this limitation by utilizing adenoviral transduction of T cells from Coxsackie/adenovirus receptor transgenic mice. Unexpectedly, dominant negative Ras17N blocked activation of Ras and ERK in response to IL-2R engagement but not TCR/CD3 ligation. However, TCR-induced ERK activation was suppressed by inhibitors of PKC and PLC-gamma. This first biochemical study of DN Ras in normal quiescent T cells reveals a striking contrast in Ras signaling via two receptors, and suggests that the principal mechanism of TCR-induced Ras activation in normal T cells may be distinct from that utilized in T-lineage tumor cell lines.     

Cutting edge: Rac GTPases sensitize activated T cells to die via Fas

In activated CD4(+) T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4(+) T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.     

Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.     

HIP-55 is important for T-cell proliferation, cytokine production, and immune responses

Engagement of the T-cell receptor (TCR) triggers a series of signaling events that lead to the activation of T cells. HIP-55 (SH3P7 or mAbp1), an actin-binding adaptor protein, interacts with and is tyrosine phosphorylated by ZAP-70, which is a crucial proximal protein tyrosine kinase for TCR signaling. HIP-55 is important for JNK and HPK1 activation induced by TCR signaling. In this study, we report the generation and characterization of HIP-55 knockout mice. We found that HIP-55 knockout mice were viable and fertile but showed decreased body weight and increased occurrence of death within the first 4 weeks after birth. The lymphoid organs in HIP-55 knockout mice showed cellularity and T-cell development comparable to that of the wild-type mice. HIP-55 knockout T cells displayed defective T-cell proliferation, decreased cytokine production, and decreased up-regulation of the activation markers induced by TCR stimulation. TCR internalization was slightly increased in HIP-55 knockout T cells. These phenotypes were accompanied by reduced immune responses, including antigen-specific antibody production and T-cell proliferation in HIP-55 knockout mice. The TCR-induced signaling events, including LAT/phospholipase Cgamma1 phosphorylation and HPK1/JNK activation, were partially defective in HIP-55 knockout T cells. These results demonstrate the importance of HIP-55 as an adaptor protein in the TCR signaling and immune system.     

c-Cbl-mediated regulation of LAT-nucleated signaling complexes

The engagement of the T-cell receptor (TCR) causes the rapid recruitment of multiple signaling molecules into clusters with the TCR. Upon receptor activation, the adapters LAT and SLP-76, visualized as chimeric proteins tagged with yellow fluorescent protein, transiently associate with and then rapidly dissociate from the TCR. Previously, we demonstrated that after recruitment into signaling clusters, SLP-76 is endocytosed in vesicles via a lipid raft-dependent pathway that requires the interaction of the endocytic machinery with ubiquitylated proteins. In this study, we focus on LAT and demonstrate that signaling clusters containing this adapter are internalized into distinct intracellular compartments and dissipate rapidly upon TCR activation. The internalization of LAT was inhibited in cells expressing versions of the ubiquitin ligase c-Cbl mutated in the RING domain and in T cells from mice lacking c-Cbl. Moreover, c-Cbl RING mutant forms suppressed LAT ubiquitylation and caused an increase in cellular LAT levels, as well as basal and TCR-induced levels of phosphorylated LAT. Collectively, these data indicate that following the rapid formation of signaling complexes upon TCR stimulation, c-Cbl activity is involved in the internalization and possible downregulation of a subset of activated signaling molecules.     

Genetic analysis of SH2D4A, a novel adapter protein related to T cell-specific adapter and adapter protein in lymphocytes of unknown function, reveals a redundant function in T cells

T cell-specific adapter (TSAd) protein and adapter protein in lymphocytes of unknown function (ALX) are two related Src homology 2 (SH2) domain-containing signaling adapter molecules that have both been shown to regulate TCR signal transduction in T cells. TSAd is required for normal TCR-induced synthesis of IL-2 and other cytokines in T cells and acts at least in part by promoting activation of the LCK protein tyrosine kinase at the outset of the TCR signaling cascade. By contrast, ALX functions as a negative-regulator of TCR-induced IL-2 synthesis through as yet undetermined mechanisms. In this study, we report a novel T cell-expressed adapter protein named SH2D4A that contains an SH2 domain that is highly homologous to the TSAd protein and ALX SH2 domains and that shares other structural features with these adapters. To examine the function of SH2D4A in T cells we produced SH2D4A-deficient mice by homologous recombination in embryonic stem cells. T cell development, homeostasis, proliferation, and function were all found to be normal in these mice. Furthermore, knockdown of SH2D4A expression in human T cells did not impact upon their function. We conclude that in contrast to TSAd and ALX proteins, SH2D4A is dispensable for TCR signal transduction in T cells.     

A molecular framework for two-step T cell signaling: Lck Src homology 3 mutations discriminate distinctly regulated lipid raft reorganization events

Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require "partial" TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained "complete" TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.     

TRPM8 channel augments T-cell activation and proliferation

The transient receptor potential melastatin 8 (TRPM8) is an ion channel that has been widely studied as a cold-sensitive nociceptor. However, its importance in nonneuronal cells is mostly unexplored. Here, we describe the presence and functional significance of endogenous TRPM8, a nonselective Ca²⁺-channel in T cell functions. The major pool of TRPM8 resides at the T cell surface and its surface accumulation significantly increases in activated T cells. TRPM8 activation synergizes with T-cell receptor (TCR) stimulation to increase CD25, CD69 levels and enhances secretion of proinflammatory cytokine tumor necrosis factor. However, TRPM8 inhibition does not restrict TCR stimulation mediated activation of T cells, indicating that unlike the heat-sensitive TRPV1 and TRPV4 channels, the cold-sensitive TRPM8 channel may be dispensable during T-cell activation, at least in mice. In this study, we demonstrate that TRPM8 promotes TCR-induced intracellular calcium increase. TRPM8 activation is beneficial for T-cell activation and differentiation into effector cells. TRPM8 inhibition during the T-cell activation process may lead to altered phenotype and reduced proliferation, without affecting cell viability. These results collectively establish TRPM8 as a functional calcium channel whose activation may be utilized for mounting an effective immune response. The findings of this study will be relevant to the regulation and response of T cells during cell-mediated immunity. These results will likely further our understanding on the role of ion channels in T-cell activation.     

The cGMP/protein kinase G pathway contributes to dihydropyridine-sensitive calcium response and cytokine production in TH2 lymphocytes

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.