The sphingosine 1-phosphate receptor 1 (S1P1) is abundant in endothelial cells, where it regulates vascular development and microvascular barrier function. In investigating the role of endothelial cell S1P1 in adult mice, we found that the endothelial S1P1 signal was enhanced in regions of the arterial vasculature experiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-1, was enhanced in mice with endothelial cell–specific deletion of S1pr1 and suppressed in mice with endothelial cell–specific overexpression of S1pr1, suggesting a protective function of S1P1 in vascular disease. The chaperones ApoM+HDL (HDL) or albumin bind to sphingosine 1-phosphate (S1P) in the circulation; therefore, we tested the effects of S1P bound to each chaperone on S1P1 signaling in cultured human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to ApoM+HDL-S1P, but not to albumin-S1P, promoted the formation of a cell surface S1P1–β-arrestin 2 complex and attenuated the ability of the proinflammatory cytokine TNFα to activate NF-κB and increase ICAM-1 abundance. Although S1P bound to either chaperone induced MAPK activation, albumin-S1P triggered greater Gi activation and receptor endocytosis. Endothelial cell–specific deletion of S1pr1 in the hypercholesterolemic Apoe−/− mouse model of atherosclerosis enhanced atherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM+HDL to act as a biased agonist on S1P1 inhibits vascular inflammation, which may partially explain the cardiovascular protective functions of HDL.

iRHOM2, encoded by the gene Rhbdf2, regulates the maturation of the TNF-α convertase (TACE), which controls shedding of TNF-α and its biological activity in vivo. TACE is a potential target to treat TNF-α-dependent diseases, such as rheumatoid arthritis, but there are concerns about potential side effects, because TACE also protects the skin and intestinal barrier by activating EGFR signaling. Here we report that inactivation of Rhbdf2 allows tissue-specific regulation of TACE by selectively preventing its maturation in immune cells, without affecting its homeostatic functions in other tissues. The related iRHOM1, which is widely expressed, except in hematopoietic cells, supported TACE maturation and shedding of the EGFR ligand TGF-α in Rhbdf2-deficient cells. Remarkably, mice lacking Rhbdf2 were protected from K/BxN inflammatory arthritis to the same extent as mice lacking TACE in myeloid cells or Tnfa-deficient mice. In probing the underlying mechanism, we found that two main drivers of K/BxN arthritis, complement C5a and immune complexes, stimulated iRHOM2/ TACE-dependent shedding of TNF-α in mouse and human cells. These data demonstrate that iRHOM2 and myeloid-expressed TACE play a critical role in inflammatory arthritis and indicate that iRHOM2 is a potential therapeutic target for selective inactivation of TACE in myeloid cells.

Summary Lipid mediators influence immunity in myriad ways. For example, circulating sphingosine 1-phosphate (S1P) is a key regulator of lymphocyte egress1,2. Although the majority of plasma S1P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle3, immunological functions of the ApoM-S1P complex are unknown. Here, we show that ApoM-S1P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the S1P1 receptor on bone marrow (BM) lymphocyte progenitors. Mice that lacked ApoM (Apom−/−) had increased proliferation of Lin−Sca1+cKit+ hematopoietic progenitor cells (LSK) and common lymphoid progenitors (CLP) in BM. Pharmacologic activation or genetic overexpression of S1P1 suppressed LSK and CLP proliferation in vivo. ApoM was stably associated with BM CLPs, which showed active S1P1 signaling in vivo4. Moreover, ApoM+HDL, but not albumin-bound S1P, inhibited lymphopoiesis in vitro. Upon immune stimulation, Apom−/− mice developed more severe experimental autoimmune encephalomyelitis5, characterized by increased lymphocytes in the central nervous system (CNS) and breakdown of the blood-brain barrier. Thus, the ApoM-S1P-S1P1 signaling axis restrains the lymphocyte compartment and subsequently, adaptive immune responses. Unique biological functions imparted by specific S1P chaperones could be exploited for novel therapeutic opportunities.

CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.

The fibroblast growth factor receptor 2-IIIb (FGFR2b) and the vascular endothelial growth factor receptor 2 (VEGFR2) are tyrosine kinases that can promote cell migration and proliferation and have important roles in embryonic development and cancer. Here we show that FGF7/FGFR2b-dependent activation of epidermal growth factor receptor (EGFR)/ERK1/2 signalling and cell migration in epithelial cells require stimulation of the membrane-anchored metalloproteinase ADAM17 and release of heparin-binding epidermal growth factor (HB-EGF). Moreover, VEGF-A/VEGFR2-induced migration of human umbilical vein endothelial cells also depends on EGFR/ERK1/2 signalling and shedding of the ADAM17 substrate HB-EGF. The pathway used by the FGF7/FGFR2b signalling axis to stimulate shedding of substrates of ADAM17, including ligands of the EGFR, involves Src, p38 mitogen-activated protein-kinase and PI3K, but does not require the cytoplasmic domain of ADAM17. Based on these findings, ADAM17 emerges as a central component in a triple membrane-spanning pathway between FGFR2b or VEGFR2 and EGFR/ERK1/2 that is required for cell migration in keratinocytes and presumably also in endothelial cells.

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Multiple myeloma (MM) is a B-cell neo IntroductionMultiple myeloma (MM) is a B-cell neoplasm, characterized by the clonal expansion of malignant plasma cells, and frequently associated with chromosomal translocations placing an oncogene under the control of the immunoglobulin heavy chain enhancer. 1 These translocations typically occur during immunoglobulin heavy chain switch recombination, but they may also occur as a result of errors in somatic hypermutation. Recurrent translocation partners deregulated in MM include cyclin D1 and cyclin D3, the transcription factors c-musculoaponeurotic fibrosarcoma (c-MAF) and myeloma SET (single exon transfer) domain (MMSET), and fibroblast growth factor receptor 3 (FGFR3). Despite these pathogenic translocations, malignant plasma cells retain their dependency on external signals for survival. Such signals include interleukin 6 (IL-6), insulin-like growth factor-1 (IGF-1), hemopoietic growth factor, basic fibroblast growth factor, B-lymphocyte stimulator (BLyS), and integrin-mediated cellular contact. [2][3][4][5][6][7] We present evidence that neurotrophin-receptor interactions, involving brainderived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), contribute to the survival signals necessary for MM tumor progression.The neurotrophin family of growth factors includes BDNF, NGF (nerve growth factor), and NT-4/5 (neurotophin-4/5). Each neurotrophin binds to a specific Trk: NGF binds to TrkA, BDNF and NT-4/5 bind to TrkB, and NT-3 binds to TrkC. In addition, neurotrophins share a common receptor, p75 NTR , a member of the tumor necrosis factor (TNF) receptor superfamily. 8 Although structurally related to TNF, neurotrophins are secreted as homodimers rather than trimers and induce receptor dimerization rather than trimerization. Trk receptor dimerization triggers kinase activation and the initiation of signaling cascades, including MEK/MAPK (mitogen-activated protein kinase kinasemitogen-activated protein kinase) and PI3K/Akt (phosphatidylinositol-3 kinase-a PI3K target), that favor cellular survival. 9 In contrast, p75 NTR engagement triggers divergent pathways to modulate survival, cell cycle regulation, and cytoskeletal rearrangement. 9 Thus, while p75 NTR binding can increase the affinity and specificity of Trk-neurotrophin interactions, p75 NTR can also induce apoptosis when Trk activation is reduced or absent. 10,11 The overall cellular response to neurotrophin exposure reflects a balance between p75 NTR and Trk engagement.Neurotrophins are synthesized as proneurotrophins and are enzymatically cleaved to yield mature proteins. Cleavage can occur either intracellularly, by the action of furin or proconvertase, or extracellularly, by the action of plasmin, matrix metalloproteinase 7 (MMP-7) or 12 Both mature neurotrophins and proneurotrophins are biologically active, with divergent roles that reflect differing receptor affinities: proneurotrophins display greater affinities for p75 NTR , whereas mature neurotrophins displa...

CP2, a transcription factor that binds the murine oa-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with MrS of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-i.We have previously described the purification and characterization of several o-globin promoter transcription factors present in nuclear extracts prepared from murine erythroleukemia (MEL) cells (1, 2, 9-11). These factors include the tissue-restricted transcription factor GATA-1, the heterotypic CCAAT factor CP1, and a third factor termed CP2. Purified CP2 (a polypeptide doublet with Mrs of 64,000 and 66,000 on sodium dodecyl sulfate [SDS]-polyacrylamide gels [9]) interacts with a region of the a-globin promoter that extends from -85 (where it overlaps with the binding site of the a-globin CCAAT factor, CP1) to -160, just downstream from the GATA-1 binding site (1, 11). The factor binds a consensus sequence (GCN[C/A]NANCCAG) repeated four times within this region (11). Factor binding activity is observed in a wide variety of cell types, including the human cell lines HeLa (4) and CEM. Our previous results have shown that CP2 binding activity is modestly induced (two-to threefold) during erythroid differentiation of MEL cells (1). Additionally, in vitro transcription experiments have shown that CP2 is present at functionally limiting amounts in MEL cell nuclear extracts (11) and that addition of DNA sequence a...