Molecular Characterization of trans -Golgi p230: a human peripheral membrane protein encoded by a gene on chromosome 6p12-22 contains extensive coiled-coil α-helical domains and a Granin Motif

Using autoantibodies from a Sjögren's syndrome patient, we have previously identified a 230-kDa peripheral membrane protein associated with the cytosolic face of the trans-Golgi (Kooy, J., Toh, B. H., Pettitt, J. M., Erlich, R. and Gleeson, P. A. (1992) J. Biol. Chem. 267, 20255-20263). Here we report the molecular cloning and sequence analysis of human p230 and the localization of its gene to chromosome 6p12 22. Partial cDNA clones, isolated from a HeLa cell cDNA library using autoantibodies, were used to obtain additional cDNAs, which together span 7695 base pairs (bp). The p230 mRNA is approximately 7.7 kilobases. Two alternatively spliced mRNAs for p230 were detected. These differed by 21- and 63-bp insertions in the 3'-sequence, resulting in differences in amino acid sequence at the carboxyl terminus. The predicted 261-kDa protein is highly hydrophilic with 17-20% homology with many proteins containing coiled-coil domains. Apart from two proline-rich regions (amino acids 1-117 and 239-270), p230 contains a very high frequency of heptad repeats, characteristic of alpha-helices that form dimeric coiled-coil structures. p230 also includes the sequence ESLALEELEL (amino acids 538-546), a motif found in the granin family of acidic proteins present in secretory granules of neuroendocrine cells. This is the first report of a cytosolic Golgi protein containing a granin motif. The structural characteristics of p230 indicate that it may play a role in vesicular transport from the trans-Golgi

EEA1, an early endosome-associated protein: EEA1 is a conserved α-helical peripheral membrane-protein flanked by cysteine fingers and contains a calmodulin-binding IQ motif

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly cu-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine ''finger'' motifs. The COOH-terminal fingers, Cys-X(2)-Cys-X(12)-Cys-X(2)-Cys and Cys-X(2)-Cys-X(16)-Cys-X(2)-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenarhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity

Depletion of B2 but Not B1a B Cells in BAFF Receptor-Deficient ApoE−/− Mice Attenuates Atherosclerosis by Potently Ameliorating Arterial Inflammation

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE−/− mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE−/− mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R−/− ApoE−/− (BaffR.ApoE DKO) and BAFF-R+/+ApoE−/− (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE−/− mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation

Amniotic Epithelial Cells from the Human Placenta Potently Suppress a Mouse Model of Multiple Sclerosis

Human amniotic epithelial cells (hAEC) have stem cell-like features and immunomodulatory properties. Here we show that hAEC significantly suppressed splenocyte proliferation in vitro and potently attenuated a mouse model of multiple sclerosis (MS). Central nervous system (CNS) CD3+ T cell and F4/80+ monocyte/macrophage infiltration and demyelination were significantly reduced with hAEC treatment. Besides the known secretion of prostaglandin E2 (PGE2), we report the novel finding that hAEC utilize transforming growth factor-β (TGF-β) for immunosuppression. Neutralization of TGF-β or PGE2 in splenocyte proliferation assays significantly reduced hAEC-induced suppression. Splenocytes from hAEC-treated mice showed a Th2 cytokine shift with significantly elevated IL-5 production. While transferred CFSE-labeled hAEC could be detected in the lung, none were identified in the CNS or in lymphoid organs. This is the first report documenting the therapeutic effect of hAEC in a MS-like model and suggest that hAEC may have potential for use as therapy for MS

Opposing roles of B lymphocyte subsets in atherosclerosis

Atherosclerosis is initiated by cholesterol entry into arteries that triggers chronic immune-inflammatory lesions in the vessels. Early lesions are clinically insignificant but advanced complex lesions and vulnerable rupture prone lesions impact on quality of life and can be life threatening. Rupture of vulnerable atherosclerotic lesions initiates thrombotic occlusion of vital arteries precipitating heart attacks and strokes that remain major killers globally despite therapeutic use of statins to lower blood cholesterol levels. Conventional B2 cells are proatherogenic whereas peritoneal Bla cells are atheroprotective. Depletion of B2 cells by administration of mAb to CD20 or to BAFF receptor or in BAFF receptor-deficient mice ameliorates atherosclerosis. B2 cells may promote atherosclerosis by production of IgG, secretion of proinflammatory cytokine TNFα and activation of CD4 T cells. Together these B2 cell mechanisms contribute to generation of rupture-prone vulnerable atherosclerotic plaques characterised by large necrotic cores. In contrast, peritoneal Bla cells protect against atherosclerosis by secretion of natural IgM that scavenges apoptotic cells and oxidised LDL and reduces necrotic cores in atherosclerotic lesions. These atheroprotective effects can be further increased by stimulating Bla cells by administration of apoptotic cells, liposomes of phosphatidylserine abundant on surfaces of apoptotic cell, by mAb to TIM1, a phosphatidylserine receptor expressed by B1a cells and by TLR4-MyD88 activation. Experimental studies of atherosclerosis in mouse models indicate that reductions in atherogenic B2 cells and/or activation of atheroprotective B1a cells protects against atherosclerosis development, findings which have potential for clinical translation to reduce risks of deaths from heart attacks and strokes

Role of Cell Division Autoantigen 1 (CDA1) in Cell Proliferation and Fibrosis

Cell Division Autoantigen 1 (CDA1) was discovered following screening a human expression library with serum from a patient with Discoid Lupus Erythematosus. CDA1, encoded by TSPYL2 on the X chromosome, shares anti-proliferative and pro‑fibrotic properties with TGF-b. It inhibits cell growth through p53, pERK1/2 and p21‑mediated pathways and is implicated in tumorigenesis and the DNA damage response. Its pro-fibrotic property is mediated through cross-talk with TGF-b that results in upregulation of extracellular matrix proteins. The latter properties have identified a key role for CDA1 in diabetes associated atherosclerosis. These dual properties place CDA1 as an attractive molecular target for treating tumors and vascular fibrosis including atherosclerosis and other vascular disorders associated with enhanced TGF-β action and tissue scarring

Potently immunosuppressive 5-fluorouracil-resistant mesenchymal stromal cells completely remit an experimental autoimmune disease

We treated mice with 5-fluorouracil (5-FU) to isolate a quiescent and undifferentiated mesenchymal stromal cell (MSC) population from the bone marrow. We examined these 5-FU–resistant MSCs (5-FU–MSCs) free from hematopoietic components for CFU fibroblasts (CFU-Fs) and assessed their immunosuppressive potential in vitro and in vivo. We differentiated fibroblastic CFU-Fs (Fibro–CFU-Fs) from mixed CFU-Fs, based on the absence of in situ expression of CD11b and CD45 hematopoietic markers, as well as on their differentiation capacity. Fibro–CFU-Fs were associated with increased numbers of large-sized Fibro–CFU-Fs (≥9 mm2) that displayed enhanced capacity for differentiation into adipogenic and osteogenic mesenchymal lineages. Administration of these 5-FU–resistant CD11b−CD45− MSCs 6 d after myelin oligodendrocyte glycoprotein (MOG) immunization completely remitted MOG-induced experimental autoimmune encephalomyelitis after initial development of mild disease. The remission was accompanied by reduced CNS cellular infiltration and demyelination, as well as a significant reduction in anti-MOG Ab and splenocyte proliferation to MOG. MOG-stimulated splenocytes from these mice showed elevated levels of Th2 cytokines (IL-4, IL-5, and IL-6) and decreased IL-17. Compared with untreated MSCs, 5-FU–MSCs demonstrated potent immunosuppression of Con A-stimulated splenocytes in vitro, even at a 1:320 MSC/splenocyte ratio. Immunosuppression was accompanied by elevated IL-1ra, IL-10, and PGE2. Blocking IL-1ra, IL-10, and PGE2, but not IL-6, heme oxygenase-1, and NO, attenuated 5-FU–MSC–induced immunosuppression. Together, our findings suggested that immunosuppression by 5-FU-MSC is mediated by a combination of elevated IL-1ra, IL-10, and PGE2, anti-inflammatory Th2 cytokines, and decreased IL-17. Our findings suggested that 5-FU treatment identifies a population of potently immunosuppressive 5-FU–MSCs that have the potential to be exploited to remit autoimmune diseases