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Article Figures & Data

Figures

  • Figure 1.

    Identification of 4 novel genes induced in RA-treated NB4 cells. (A) Autoradiogram of 288/JAML, 438, 809, and 813 mRNA expression in NB4 cells either untreated or treated for 24 hours with 5 × 10-7 M 9-cis RA, and in HT-29 cells as a nonhematopoietic negative control. Positions of the 28S and 18S rRNAs are indicated on the left side. The lower part is methylene blue–stained 28S and 18S rRNAs on membranes after transfer as assessment of RNA quantities in each lane (5 μg total RNA).

  • Figure 2.

    Induction of JAML mRNA in RA-treated cells. (A) Time-response to ATRA in NB4 cells. Cells were treated for different times using 5 × 10-7 M ATRA. (B) JAML expression correlates with the capacity of NB4 cells to differentiate. NB4 and NB4.306 cells were cultured with 10-6 M ATRA and harvested after 0, 24, and 48 hours. Viable cells (i) and percent of NBT-positive cells (ii) were counted each day and JAML mRNA expression was analyzed (iii). (C) JAML mRNA is induced in ATRA-treated primary APL cells. Cells were purified from 3 untreated patients cultivated in the absence (–) or presence (+) of 10-7 M ATRA for 5 (APL no. 2)or6(APL no. 1 and APL no. 3) days. Northern blots were performed using 8 μg (A,Biii) or 2 μg (C) total RNA. Hybridization with a glyceraldehyde phosphate dehydrogenase (GAPDH) probe controlled for RNA quantities in each lane.

  • Figure 3.

    JAML is related to JAM proteins. (A) cDNA and deduced amino acid sequences of the human JAML. Nucleotide residues are numbered in the 5′ to 3′ orientation, and amino acids in the reading frame are designated by the one-letter code. Asterisk indicates the stop codon. The putative hydrophobic signal peptide (underlined) and transmembrane sequences (dotted underline) are marked. Potential N-linked glycosylation sites are shaded. Cysteines likely to form disulfide bonds in the 2 Ig domains are circled. (B) Dendogram of various IgSF family members containing 2 Ig domains (JAM-1, JAM-2, JAM-3, JAML, Coxsackie and adenovirus receptor (CAR), and endothelial cell-selective adhesion molecule (ESAM)). (C) Alignment of the amino-terminal region of the membrane distal Ig domains of these proteins and their mouse (JAM-1, JAM-2, JAM-3, CAR, ESAM) or bos taurus (JAML) homologs. Shaded regions represent residues identical between at least 4 proteins. The dimerization motif is boxed.

  • Figure 4.

    JAML is a transmembrane protein induced upon ATRA-treatment in NB4 cells. (A) COS-7 cells were mock-transfected (-) or transfected with the p513-JAML-HA vector (JAML). Cell lysates were separated by SDS-PAGE and subjected to immunoblotting with anti-JAML (i) or anti-HA (ii) antibodies. The arrowhead indicates the major specific band. Brackets indicate minor specific bands likely corresponding to glycosylated JAML protein. (iii) JAML protein was in vitro–translated in the absence (–) or presence (+) of canine microsomal membranes. Arrow indicates a glycosylated JAML protein. (B) JAML protein expression during ATRA-induced differentiation of NB4 cells. NB4 cells were treated with 5 × 10-7 M ATRA for different times. Protein lysates corresponding to 105 cells were analyzed by Western blot, using anti-JAML or anti–β-actin polyclonal antibodies. In panels A and B, asterisks indicate nonspecific bands. (C) Cellular localization of JAML. MDCK cells stably transfected with the pMT-JAML-HA or pMT-JAMLK54D-HA were either untreated (-Zn) or treated (+Zn) with 125 μM ZnSO4 for 40 hours. Cells were treated with anti-HA monoclonal antibody and stained with Cy3-conjugated goat anti-mouse antibody (ii,iii,vi, vii). In panels i, iv, v, and viii, the same cells as in panels ii, iii, vi, and vii, respectively, were counterstained with Hoechst 33258 reagent.

  • Figure 5.

    JAML mRNA is up-regulated during induced differentiation of myeloid leukemia cells and is expressed in normal hematopoietic tissues. (A) Up-regulation of JAML mRNA during induced differentiation of myeloid leukemia cells. Autoradiograms of JAML mRNA expression before and after treatment of HL-60 and PLB-985 cells with different inducers of differentiation. Northern blots were performed using 5 μg total RNA. Hybridization with a GAPDH probe controlled for RNA quantities in each lane. (B) Autoradiograms of JAML mRNA expression in immune tissues (i) and peripheral blood cells (ii). Northern blots were either obtained from Clontech (i) or were performed using 5 μg total RNA from granulocytes, monocytes, and lymphocytes (ii). In panels A and B, GAPDH was used as a probe for assessment of RNA quantities in each lane.

    U937/MT, U937/MT-JAML-HA, and U937/MT-JAMLK54D-HA cells stably transfected with the pMT, the pMT-JAML-HA, or pMT-JAMLK54D-HA respectively were untreated (-Zn) or treated (+Zn) with ZnSO4 for 18 hours prior to fluorescent labeling and subjected to a cell adhesion assay using human bone marrow endothelial cells either untreated (HBMECs) or treated (HBMECs + TNFα) as a target layer. Results are expressed in each condition as the amount of released fluorescence from adherent cells, normalized to unity for the corresponding -Zn/-TNFα condition. The adhesion level of U937/MT-JAML-HA cells onto untreated or TNFα-pretreated HBMECs, in the absence of Zn treatment (-Zn), was measured as 2.3% and 15.5% respectively, of the total number of incubated cells. Values shown are ± SD.

Tables

  • Table 1.

    Characteristics of the differential cDNAs identified

    CloneDescriptionGenBank accession no.
    3 5-lipoxygenase activating protein (FLAP) X52195
    13 Monocyte chemoattractant protein 1 (MCP-1) X14768
    16 Neutrophil cytosol factor 1 (p47phox) M25665
    20 G0S2 M69199
    26 Cellular transglutaminase homologue (TGase H) M98479
    301 CD11b J03925
    617 Plasminogen activator inhibitor-2 (PAI-2) J03603
    685 Lymph node homing receptor (L-selectin) M25280, X16070
    685 Leu-8 X17519
    685 LAM-1 X16150
    732 Interleukin-8 (IL-8) M17017
    748 Bfl-1/A1 U27467, U29680
    752 Defensin 1 M26602, M21130
    756 Corticostatin HP-4 (defensin 4) X65977
    781 Delta-aminolevulinate synthase (ALAS1) X56351
    796 CAMPATH-1 (CD52) X62466
    806 Coactosin-like protein (CLP) L54057
    808 ICAM-1 J03132
    820 c-fgr M19722
    832 GLUT5 glucose transporter M55531
    850 Src-like adapter protein (SLAP) D89077
    • Leu-8 indicates leukocyte surface antigen Leu-8; LAM-1, leukocyte adhesion molecule-1; Bfl-1/A1, Bcl-2—related gene expression in fetal liver-1/Bcl-2—related protein A1; ICAM-1, intercellular adhesion molecule-1; and c-fgr, Gardner-Rasheed feline sarcoma viral proto-oncogene.

  • Table 2.

    Comparison of gene structure between JAM family members

    HGNC exonNo.JAM-1 1q21.2-21.3iJAM-2 21q21.2iJAM-3 11q25iJAML 11q23.3i
    5 0 >73
      Intron 0-1 10 054
    ATG/signal sequence 1 103 >301 >76 64
      Intron 1-2 44 222 1 43 994 1 79 105 1 2262 1
    EC, Ig-fold 1 2 59 66 66
      Intron 2-3 167 1 5916 1 740 1
    EC, Ig-fold 1 3 98 108 114 153
      Intron 3-4 252 1 3782 1 3470 1 1694 1
    EC, Ig-fold 1 4 137 153 153 228
      Intron 4-5 282 1 4768 1 398 1 4493 1
    EC, Ig-fold 2 5 203 203 203 110
      Intron 5-6 167 0 3290 0 951 0 2217 0
    EC, Ig-fold 2 6 103 100 100 238
      Intron 6-7 128 1 3709 1 2511 1 2815 1
    Transmembrane 7 108 108 130 137
      Intron 7-8 231 1 3347 1 2384 1
    IC 8 13 16 95
      Intron 8-9 304 2 2890 2 87 2 1176 2
    IC 9 49 43 55 88
      Intron 9-10 136 0 2257 0 321 0 2318 0
    IC/STOP 10 >937 >283 >36 >452
    • HGNC indicates HUGO Nomenclature Committee; i, phase of introns; EC, extracellular domain; IC, intracellular domain; and Ig-fold, immunoglobulin-like fold.

  • Table 3.

    JAML expression enhances U937 cell adhesion to HBMECs

    HBMECsHBMECs + TNFα
    U937/MT
      - Zn 1.00 ± 0.36 5.29 ± 0.17
      + Zn 1.07 ± 0.33 4.79 ± 0.29
    U937/MT-JAML
      - Zn 1.00 ± 0.30 6.74 ± 0.52
      + Zn 2.65 ± 0.17 11.04 ± 1.39
    U937/MT-JAMLK54D
      - Zn 1.00 ± 0.13 4.83 ± 0.30
      + Zn 1.38 ± 0.47 4.23 ± 0.47
    • U937/MT, U937/MT-JAML-HA, and U937/MT-JAMLK54D-HA cells stably transfected with the pMT, the pMT-JAML-HA, or pMT-JAMLK54D-HA respectively were untreated (-Zn) or treated (+Zn) with ZnSO4 for 18 hours prior to fluorescent labeling and subjected to a cell adhesion assay using human bone marrow endothelial cells either untreated (HBMECs) or treated (HBMECs + TNFα) as a target layer. Results are expressed in each condition as the amount of released fluorescence from adherent cells, normalized to unity for the corresponding -Zn/-TNFα condition. The adhesion level of U937/MT-JAML-HA cells onto untreated or TNFα-pretreated HBMECs, in the absence of Zn treatment (-Zn), was measured as 2.3% and 15.5% respectively, of the total number of incubated cells. Values shown are ± SD.