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Upon antigen encounter epidermal Langerhans cells (LC) and dendritic cells (DC) emigrate from peripheral organs and invade lymph nodes through the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Part of this process is mimicked by metastasizing tumor cells. Since splice variants of CD44 promote metastasis to lymph nodes we explored the expression of CD44 proteins on migrating LC and DC. We show that following antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6 and v9. Antibodies against CD44 epitopes arrest LC in the epidermis, prevent the binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.

The neurofibromatosis 2 (NF2) tumor suppressor gene encodes an intracellular membrane-associated protein, called merlin (or schwannomin), that belongs to the band 4.1 family of cytoskeleton-associated proteins. Inactivating NF2 mutations occur in several sporadic tumor types and have been linked to the NF2 disease, whose hallmark is the development of bilateral Schwann cell tumors (schwannomas) of the eighth cranial nerve. Two major alternatively spliced NF2 variants are expressed in normal tissues: 'NF2-17' lacking exon 16 and 'NF2-16' that contains exon 16 and encodes a merlin protein truncated at the C-terminus. We report that overexpression of NF2-17 in rat schwannoma cells inhibits their growth in vitro and in vivo, while NF2-16 fails to influence schwannoma growth. Tumor growth inhibition by merlin depends on an interdomain association occurring either in cis or in trans between the N- and C-termini. This association does not occur in the truncated NF2-16 protein nor in a mutant NF2-17 protein lacking C-terminal sequences. These data indicate that merlin has a unique mechanism of tumor suppression, inhibiting cell proliferation via self-association.

Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.

Recent evidence has shown that the activation of receptor tyrosine kinases is not only dependent on binding of their ligands but in addition requires adhesion molecules as coreceptors. We have identified CD44v6 as a coreceptor for c-Met in several tumor and primary cells. The CD44v6 ectodomain is required for c-Met activation, whereas the cytoplasmic tail recruits ERM proteins and the cytoskeleton into a signalosome complex. Here we demonstrate that c-Met (and hepatocyte growth factor and Gab1) is haploinsufficient in a cd44-/- background, as the cd44-/-; met+/- (and cd44-/-; hgf+/- and cd44-/-; gab1+/-) mice die at birth. They have impaired synaptic transmission in the respiratory rhythm-generating network and alterations in the phrenic nerve. These results are the first genetic data showing that CD44 and c-Met collaborate in vivo and that they are involved in synaptogenesis and axon myelination in the central and peripheral nervous systems.

Activation of the mitogen-activated protein kinase (MAPK) cascade after Toll-like receptor stimulation enables innate immune cells to rapidly activate cytokine gene expression. A balanced response to signals of infectious danger requires that cellular activation is transient. Here, we identify the MAPK phosphatase dual specificity phosphatase 1 (DUSP1) as an essential endogenous regulator of the inflammatory response to lipopolysaccharide (LPS). DUSP1-deficient (DUSP1−/−) bone marrow–derived macrophages showed selectively prolonged activation of p38 MAPK and increased cytokine production. Intraperitoneal challenge of DUSP1−/− mice with LPS caused increased lethality and overshooting production of interleukin (IL)-6 and tumor necrosis factor α. Transcriptional profiling revealed that DUSP1 controls a significant fraction of LPS-induced genes, which includes IL-6 and IL-10 as well as the chemokines CCL3, CCL4, and CXCL2. In contrast, the expression of the important mediators of endotoxin lethality, interferon γ and IL-12, was not significantly altered by the absence of DUSP1. These data together demonstrate a specific regulatory role of DUSP1 in controlling a subset of LPS-induced genes that determines the outcome of endotoxin shock.

The proto-oncogene c-jun is the cellular homologue of v-jun, the transforming oncogene of the avian sarcoma virus 17 (ref. 1). c-jun encodes one major component of the AP-1 transcription factor complex and is expressed in many organs during mouse development and in the adult. Because of its rapid induction in cells following growth stimulation and the presence of AP-1 binding sites in the promoter regions of many genes, the c-Jun protein is thought to have important functions in cell proliferation and differentiation. But embryonic stem (ES) cells lacking c-Jun are viable and have a normal in vitro differentiation capacity, although c-Jun appears to be important for growth of teratocarcinomas in vivo. To define the function of c-jun better, targeted ES cells were used to generate mice lacking c-Jun. Here we report that heterozygous mutant mice appear normal, but embryos lacking c-Jun die at mid- to late-gestation and exhibit impaired hepatogenesis, altered fetal liver erythropoiesis and generalized oedema. Interestingly, c-jun-/- ES cells can participate efficiently in the development of all somatic cells in chimaeric mice except liver cells, further suggesting an essential function of c-Jun in hepatogenesis.

Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo.

The Wilms tumor suppressor gene WT1 ( wt1 in mouse) is unique among tumor suppressors because, in addition to its involvement in cancer [1,2] and various other diseases [3–6], it has an essential role in the development of certain organs. This is revealed by the phenotype of mice with inactivated wt1 alleles [7]. These animals exhibit a complete failure of kidney and gonad development as well as abnormalities of the heart and mesothelial structures. On a C57BL/6 genetic background, wt1 −/− animals die between day 13.5 (E13.5) and 15.5 (E15.5) of embryonic development [7]. We report here that crossing of the wt1 mutation onto different mouse backgrounds delayed embryonic lethality until birth. In wt1 −/− mice on these different genetic backgrounds, we observed a dramatic failure of spleen development, in addition to the well characterized phenotypic abnormalities. The spleen anlage formed at around E12 to E13 and involuted by the E15 stage, before the invasion of hematopoietic cells. The absence of proper spleen development in these wt1 −/− embryos correlated with enhanced apoptosis in the primordial spleen cells. The expression of hox11 , a gene that also controls development of the spleen [8,9], was not altered by the inactivation of wt1. In situ hybridization revealed that the two genes are regulated independently. These findings demonstrate that the penetrance of the wt1 −/− phenotype depends on the existence of one or more modifier gene(s) and that wt1 plays a pivotal role in the development of the spleen, thereby extending its role in organogenesis.

Using subtractive immunization to identify cell surface epitopes expressed in a metastasis-specific fashion on cells of the rat MT-W9 mammary carcinoma model, we generated a monoclonal antibody called M-N#1. This antibody binds specifically to metastasizing cells of the MT-W9 series and also to certain other metastasizing rat mammary carcinoma cell lines. We demonstrate that the M-N#1 antibody recognizes a fucosylated N-glycosyl sugar modification, and furthermore show that the epitope specificity of the M-N#1 antibody is for blood group antigen B subtypes 2, 3 and 4 with slight cross-reactivity with blood group antigen A subtype 2. The expression of these carbohydrate epitopes on MT-450 cells is functionally important, because the M-N#1 antibody efficiently inhibits MT-450 tumour growth in spontaneous metastasis assays. These results suggest that expression of the subtypes of blood group antigen B recognized by the M-N#1 antibody does not directly participate in the metastatic cascade but rather confers a growth or survival advantage on the tumour cells.

Nature 365, 179-181 (1993) FIGURE 2 of this letter compares the morphological appearance of c-jun–/– and c-jun+/+ embryos at 12.5 days of gestation, and not of c-jun–/- and c-jun–/- embryos as stated in the opening sentence of the figure legend.