Andrew Wakeham

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyers patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

Lymphoproliferative Disorders with Early Lethality in Mice Deficient in Ctla-4

The role of the cell-surface molecule CTLA-4 in the regulation of T cell activation has been controversial. Here, lymph nodes and spleens of CTLA-4-deficient mice accumulated T cell blasts with up-regulated activation markers. These blast cells also infiltrated liver, heart, lung, and pancreas tissue, and amounts of serum immunoglobulin were elevated. The mice invariably became moribund by 3 to 4 weeks of age. Although CTLA-4-deficient T cells proliferated spontaneously and strongly when stimulated through the T cell receptor, they were sensitive to cell death induced by cross-linking of the Fas receptor and by gamma irradiation. Thus, CTLA-4 acts as a negative regulator of T cell activation and is vital for the control of lymphocyte homeostasis.

Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.

The multiple biological activities of tumor necrosis factor (TNF) are mediated by two distinct cell surface receptors of 55 kd (TNFRp55) and 75 kd (TNFRp75). Using gene targeting, we generated a TNFRp55-deficient mouse strain. Cells from TNFRp55-/-mutant mice lack expression of TNFRp55 but display normal numbers of high affinity TNFRp75 molecules. Thymocyte development and lymphocyte populations are unaltered, and clonal deletion of potentially self-reactive T cells is not impaired. However, TNF signaling is largely abolished, as judged by the failure of TNF to induce NF-kappa B in T lymphocytes from TNFRp55-deficient mice. The loss of TNFRp55 function renders mice resistant to lethal dosages of either lipopolysaccharides or S. aureus enterotoxin B. In contrast, TNFRp55-deficient mice are severely impaired to clear L. monocytogenes and readily succumb to infection. Thus, the 55 kd TNFR plays a decisive role in the hosts defense against microorganisms and their pathogenic factors.

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome—a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies—the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

Early Lethality, Functional NF-κB Activation, and Increased Sensitivity to TNF-Induced Cell Death in TRAF2-Deficient Mice

TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.

High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice

BACKGROUND Germ-line and sporadic mutations in the tumor suppressor gene PTEN (also known as MMAC or TEP1), which encodes a dual-specificity phosphatase, cause a variety of cancers such as Cowden disease, glioblastoma, endometrial carcinoma and prostatic cancer. PTEN is widely expressed, and Cowden disease consistently affects various organ systems, suggesting that the PTEN protein must have an important, although as yet poorly understood, function in cellular physiology. RESULTS Homozygous mutant mice lacking exons 3-5 of the PTEN gene (mPTEN3-5) had severely expanded and abnormally patterned cephalic and caudal regions at day 8.5 of gestation. Embryonic death occurred by day 9.5 and was associated with defective chorio-allantoic development. Heterozygous mPTEN3-5 mice had an increased incidence of tumors, especially T-cell lymphomas; gamma-irradiation reduced the time lapse of tumor formation. DNA analysis of these tumors revealed the deletion of the mPTEN gene due to loss of heterozygosity of the wild-type allele. Tumors associated with loss of heterozygosity in mPTEN showed elevated phosphorylation of protein kinase B (PKB, also known as Akt kinase), thus providing a functional connection between mPTEN and a murine proto-oncogene (c-Akt) involved in the development of lymphomas. CONCLUSIONS The mPTEN gene is fundamental for embryonic development in mice, as mPTEN3-5 mutant embryos died by day 9.5 of gestation, with patterning defects in cephalic and caudal regions and defective placentation. Heterozygous mice developed lymphomas associated with loss of heterozygosity of the wild-type mPTEN allele, and tumor appearance was accelerated by gamma-irradiation. These lymphomas had high levels of activated Akt/PKB, the protein product of a murine proto-oncogene with anti-apoptotic function, associated with thymic lymphomas. This suggests that tumors associated with mPTEN loss of heterozygosity may arise as a consequence of an acquired survival advantage. We provide direct evidence of the role of mPTEN as a tumor suppressor gene in mice, and establish the mPTEN mutant mouse as an experimental model for investigating the role of PTEN in cancer progression.

Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4

Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5–8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.

Role of the NF-ATc transcription factor in morphogenesis of cardiac valves and septum

In lymphocytes, the expression of early immune response genes is regulated by NF-AT transcription factors, which translocate to the nucleus after dephosphorylation by the Ca2+-dependent phosphatase, calcineurin. We report here that mice bearing a disruption in the NF-ATc gene fail to develop normal cardiac valves and septa and die of circulatory failure before day 14.5 of development. NF-ATc is first expressed in the heart at day 7.5, and is restricted to the endocardium, a specialized endothelium that gives rise to the valves and septum. Within the endocardium, specific inductive events appear to activate NF-ATc: it is localized to the nucleus only in endocardial cells that are adjacent to the interface with the cardiac jelly and myocardium, which are thought to give the inductive stimulus to the valve primordia. Treatment of wild-type embryos with FK506, a specific calcineurin inhibitor, prevents nuclear localization of NF-ATc. These data indicate that the Ca2+/calcineurin/NF-ATc signalling pathway is essential for normal cardiac valve and septum morphogenesis; hence, NF-ATc and its regulatory pathways are candidates for genetic defects underlying congenital human heart disease.

Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b.

The signalling thresholds of antigen receptors and co-stimulatory receptors determine immunity or tolerance to self molecules. Changes in co-stimulatory pathways can lead to enhanced activation of lymphocytes and autoimmunity, or the induction of clonal anergy. The molecular mechanisms that maintain immunotolerance in vivo and integrate co-stimulatory signals with antigen receptor signals in T and B lymphocytes are poorly understood. Members of the Cbl/Sli family of molecular adaptors function downstream from growth factor and antigen receptors. Here we show that gene-targeted mice lacking the adaptor Cbl-b develop spontaneous autoimmunity characterized by auto-antibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Resting cbl-b -/- lymphocytes hyperproliferate upon antigen receptor stimulation, and cbl-b-/- T cells display specific hyperproduction of the T-cell growth factor interleukin-2, but not interferon-γ or tumour necrosis factor-α. Mutation of Cbl-b uncouples T-cell proliferation, interleukin-2 production and phosphorylation of the GDP/GTP exchange factor Vav1 from the requirement for CD28 co-stimulation. Cbl-b is thus a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity.

The Tumor Suppressor Gene Brca1 Is Required for Embryonic Cellular Proliferation in the Mouse

Mutations of the BRCA1 gone in humans are associated with predisposition to breast and ovarian cancers. We show here that Brca1+/- mice are normal and fertile and lack tumors by age eleven months. Homozygous Brca1(5-6) mutant mice die before day 7.5 of embryogenesis. Mutant embryos are poorly developed, with no evidence of mesoderm formation. The extraembryonic region is abnormal, but aggregation with wild-type tetraploid embryos does not rescue the lethality. In vivo, mutant embryos do not exhibit increased apoptosis but show reduced cell proliferation accompanied by decreased expression of cyclin E and mdm-2, a regulator of p53 activity. The expression of cyclin-dependent kinase inhibitor p21 is dramatically increased in the mutant embryos. Buttressing these in vivo observations is the fact that mutant blastocyst growth is grossly impaired in vitro. Thus, the death of Brca1(5-6) mutant embryos prior to gastrulation may be due to a failure of the proliferative burst required for the development of the different germ layers.