Carbohydrate-dependent signaling from the phosphatidylglucoside-based microdomain induces granulocytic differentiation of HL60 cells

Abstract

Glycosphingolipids form glycosphingolipid signaling microdomains. Here, we report an unrecognized type of phosphatidylglucoside (PhGlc)-based lipid microdomain in HL60 cells. Treatment of cells with rGL-7, which preferentially reacts with PhGlc, induced differentiation of HL60 cells. This was manifested by the appearance of nitroblue tetrazolium-positive cells together with CD38 expression and c-Myc down-regulation. We determined the molecular mechanisms underlying early stages of signal transduction. rGL-7 treatment induced rapid tyrosine phosphorylation of Src family protein kinases Lyn and Hck. Reduction of endogenous cholesterol after application of methyl-beta-cyclodextrin suppressed rGL-7-stimulated tyrosine phosphorylation. Phosphorylated proteins and PhGlc colocalized in the Triton X-100 insoluble, light buoyant density fraction after sucrose gradient ultracentrifugation of HL60 cell lysates. This suggests PhGlc-based microdomain is involved in GL-7 signaling. Ligation of known components of microdomains, such as sphingomyelin and ganglioside GM1, with corresponding antibodies failed to induce differentiation and tyrosine phosphorylation. These results show that PhGlc constitutes a previously undescribed lipid signaling domain, and the glucose residue of PhGlc is critical for organization of the carbohydrate-dependent signaling domain involved in cellular differentiation of HL60 cells.

Fig. 1.

Deduced amino acid sequences of the variable regions within the heavy (H) and light (L) chains of the reconstructed Fab antibody rGL-7. FR, framework region; CDR, complementarity-determining region.

Fig. 2.

Occurrence of PhGlc in HL60 cells. (A) rGL-7 reacts with antigens from both cord RBCs and HL60 cells. PhGlc obtained from umbilical cord RBCs and HL60 cells (≈12.5–100 pmol) was separated via TLC, and spots were visualized through sequential incubation with biotinylated rGL-7, horseradish peroxidase-conjugated avidin, and the chromagen Ni-intensified DAB. Immunoreactive spots were measured by a dual wavelength TLC scanner (Shimadzu CS-930). The peak areas were plotted against amounts of PhGlc applied. (B)A single immunoreactive spot arose from the PBA-retained fraction from HL60 cells. PhGlc-containing spots were visualized by using the same method described in A.(C) Secondary ion mass spectrometry–collision-induced dissociation spectrum of PhGlc from HL60 cells. The immunoreactive spot shown in B was transferred onto a PVDF membrane and subjected to secondary ion mass spectrometry–collision-induced dissociation mass spectrometric analysis.

Fig. 3.

Immunofluorescence staining of HL60 cells with GL-2. Antibody binding was detected as described in the text. PhGlc (A and E), GM1 (B), sphingomyelin (F), and Nomarski images (C and G) are shown. D and H are merged images of A and B and E and F, respectively. (Scale bar, 5 μm.)

Fig. 4.

Phenotypic changes in HL60 cells resulting from long-term rGL-7 treatment. (A) NBT reduction by cells treated with rGL-7 or Fab fragments of Vj41 and DH59B. Cells in each treatment were reacted with 0.5% NBT solution at 37°C for 30 min and then stained with the May–Grünwald–Giemsa method. (B) Down-regulation of C-Myc expression. HL60 cells were treated with rGL-7, Vj41, DH59B, or ATRA for 36 h as described in the text. Nuclear fraction from HL60 cells was examined by Western blotting with the anti-c-Myc, 9E-10. (C) Surface expression of CD38 determined by mAb IOB-6 immunofluorescence. HL60 cells were stimulated with rGL-7 (1 μg per 10⁶ cells) or 0.1 μM ATRA for 48 h, stained with mAb IOB-6, followed by FITC-conjugated anti-mouse IgG, then analyzed by using a flowcytometer CytoAce 150. (D) Induction of NAD⁺ glycohydrolase activity by rGL-7. HL60 cells were stimulated with ≈0.4–4μg/ml rGL-7. Aliquots (10⁵ cells, 100 μl) of cell suspensions were used for analysis of NAD⁺ glycohydrolase activity.

Fig. 5.

Effects of antibody treatment on molecules that localize to the DIM fraction of HL60 cells. (A) Antibody stimulation affects HL60 cell growth. Cells were cultured in the presence of rGL-7 (≈5–80 pmol per 10⁶ cells), GL-2 (20 pmol per 10⁶ cells), or the Fab fragments of Vj41 or DH59B. To block Fc-R, cells were pretreated with an unrelated human IgM (for Vj41) or IgG (for DH59B), then treated with GL-2 (i.e., entire antibody). Results are means ± SEM, with statistical significance (P < 0.05) determined by Student's t test. (B) SM stimulation increased apoptosis. (Left) HL60 cells were treated with rGL-7 (≈5–80 pmol), GL-2, or Fab fragments of Vj41 or DH59B for 48 h, and annexin-positive cells were counted by using a flow cytometer. (Right) Cells treated with 20 pmol per 10⁶ cells of rGL-7, Vj41 (IgM) after pretreatment with human IgM and DH59B (IgG) after pretreatment with human IgG for 48 h were stained with the terminal deoxynucleotidyltransferase-mediated dUTP endlabeling method. Arrow points to a typical cell with apoptotic bodies.

Fig. 6.

(A) Short-term stimulation of HL60 cells with rGL-7 induces protein tyrosine phosphorylation. After incubation of HL60 cells (2 × 10⁷ cells per ml) for 4 days, 50-μl aliquots were transferred to microtubes, and an equal volume of rGL-7 (20 μg/ml) was added. After 10 s^–10 min, stimulation was terminated by adding 50 μl of SDS sample buffer. For the control, cells were treated with an unrelated Fab antibody (anti-HBs). Protein tyrosine phosphorylation was assessed by immunoblotting using horseradish peroxidase-conjugated PY20. (B) Lyn, Hck, and Cbp coexist with PhGlc in the DIM fraction. Lysates from rGL-7-treated HL60 cells were incubated with Lyn-, Hck-, and Cbp-conjugated protein A agarose gel or biotinylated rGL-7-conjugated avidin agarose gel. The resulting protein complexes were subjected to SDS/PAGE and analyzed by immunoblotting with antibodies against corresponding proteins.

Fig. 7.

Preparation and analysis of the DIM fraction. HL60 cells (10⁸ cells, 10⁶ cells per ml) were treated with rGL-7 (1 μg per 10⁶ cells) for 18 h and separated by sucrose density gradient centrifugation. The gradient solutions were separated into 12 fractions (fraction 12 was omitted in this figure). GM1 (CTx), DIM marker; TfR, non-DIM marker. For TLC analysis of PhGlc, lipids were visualized by exposure to iodine vapor. Arrowheads indicate PhGlc bands.