Limits...
Association of C-Type Lectin Mincle with Fc ε RI β γ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk

View Article: PubMed Central - PubMed

ABSTRACT

Macrophage-inducible C-type lectin (Mincle) interacts with the γ-subunit of high-affinity IgE receptor (FcεRIγ) and activates Syk by recognizing its specific ligand, trehalose-6,6′-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only FcεRIγ but also FcεRIβ in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of FcεRIβγ subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase Cγ2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the FcεRIβγ complex.

No MeSH data available.


Engagement of Mincle induces protein tyrosine phosphorylation and ERK phosphorylation in RBL-2H3 cells.(a) Time course. Cell lines expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb (anti-myc) for the indicated periods of time or preincubated overnight with anti-DNP IgE mAb and then stimulated with 300 ng/ml DNP-BSA for 10 min (DNP). (b) Dose dependency. Cell lines expressing WT Mincle or the R42I mutant were stimulated with the indicated concentrations of the anti-myc mAb for 30 min. (a and b) Detergent-soluble lysates were analyzed by immunoblotting with the indicated antibodies. Molecular size markers are indicated at the left in kDa. Data are representative of three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines. Similar results were obtained from the other cloned cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5385489&req=5

f2: Engagement of Mincle induces protein tyrosine phosphorylation and ERK phosphorylation in RBL-2H3 cells.(a) Time course. Cell lines expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb (anti-myc) for the indicated periods of time or preincubated overnight with anti-DNP IgE mAb and then stimulated with 300 ng/ml DNP-BSA for 10 min (DNP). (b) Dose dependency. Cell lines expressing WT Mincle or the R42I mutant were stimulated with the indicated concentrations of the anti-myc mAb for 30 min. (a and b) Detergent-soluble lysates were analyzed by immunoblotting with the indicated antibodies. Molecular size markers are indicated at the left in kDa. Data are representative of three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines. Similar results were obtained from the other cloned cell lines.

Mentions: Using these stable cell lines, we next examined whether stimulation with Mincle could induce signaling in RBL-2H3 cells. In addition to ERK phosphorylation, engagement of Mincle with an anti-myc monoclonal antibody (mAb) increased the tyrosine phosphorylation level of proteins in cells expressing WT Mincle, but not the R42I mutant (Fig. 2a). Dose-response experiments showed that the levels of protein tyrosine phosphorylation reached a plateau at 3 μg/ml anti-myc mAb (Fig. 2b). The pattern of tyrosine phosphorylation of cellular proteins was comparable but not identical to that induced by stimulation with FcεRI. These results suggest a Mincle-mediated signaling pathway in RBL-2H3 cells, which may share FcεRI-mediated signaling that uses FcεRIβγ subunits to trigger activation of Syk.


Association of C-Type Lectin Mincle with Fc ε RI β γ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk
Engagement of Mincle induces protein tyrosine phosphorylation and ERK phosphorylation in RBL-2H3 cells.(a) Time course. Cell lines expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb (anti-myc) for the indicated periods of time or preincubated overnight with anti-DNP IgE mAb and then stimulated with 300 ng/ml DNP-BSA for 10 min (DNP). (b) Dose dependency. Cell lines expressing WT Mincle or the R42I mutant were stimulated with the indicated concentrations of the anti-myc mAb for 30 min. (a and b) Detergent-soluble lysates were analyzed by immunoblotting with the indicated antibodies. Molecular size markers are indicated at the left in kDa. Data are representative of three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines. Similar results were obtained from the other cloned cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385489&req=5

f2: Engagement of Mincle induces protein tyrosine phosphorylation and ERK phosphorylation in RBL-2H3 cells.(a) Time course. Cell lines expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb (anti-myc) for the indicated periods of time or preincubated overnight with anti-DNP IgE mAb and then stimulated with 300 ng/ml DNP-BSA for 10 min (DNP). (b) Dose dependency. Cell lines expressing WT Mincle or the R42I mutant were stimulated with the indicated concentrations of the anti-myc mAb for 30 min. (a and b) Detergent-soluble lysates were analyzed by immunoblotting with the indicated antibodies. Molecular size markers are indicated at the left in kDa. Data are representative of three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines. Similar results were obtained from the other cloned cell lines.
Mentions: Using these stable cell lines, we next examined whether stimulation with Mincle could induce signaling in RBL-2H3 cells. In addition to ERK phosphorylation, engagement of Mincle with an anti-myc monoclonal antibody (mAb) increased the tyrosine phosphorylation level of proteins in cells expressing WT Mincle, but not the R42I mutant (Fig. 2a). Dose-response experiments showed that the levels of protein tyrosine phosphorylation reached a plateau at 3 μg/ml anti-myc mAb (Fig. 2b). The pattern of tyrosine phosphorylation of cellular proteins was comparable but not identical to that induced by stimulation with FcεRI. These results suggest a Mincle-mediated signaling pathway in RBL-2H3 cells, which may share FcεRI-mediated signaling that uses FcεRIβγ subunits to trigger activation of Syk.

View Article: PubMed Central - PubMed

ABSTRACT

Macrophage-inducible C-type lectin (Mincle) interacts with the γ-subunit of high-affinity IgE receptor (FcεRIγ) and activates Syk by recognizing its specific ligand, trehalose-6,6′-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only FcεRIγ but also FcεRIβ in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of FcεRIβγ subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase Cγ2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the FcεRIβγ complex.

No MeSH data available.