Figure 7
Figure 7. Inactivation of Spry2 increases ERK1/2-dependent B-cell proliferation and survival. (A) Freshly isolated Spry2-null or WT splenic B cells from sex-matched littermates were assessed for ERK1/2 phosphorylation by Western blot. (B) Spry2-null or WT B cells were assayed for CD69 (ERK1/2-dependent) and CD25 (ERK1/2-independent) expression after 16 hours of costimulation with anti-CD40 and anti-IgM Abs. The mean fluorescence intensity for each B-cell activation marker from 3 separate experiments as measured by flow cytometry after costimulation is shown. (C) 3H-thymidine incorporation by Spry2-null or WT splenic B cells from sex-matched littermates, stimulated with serum alone or with serum plus anti-CD40 plus anti-IgM costimulation in the presence or absence of the MEK 1/2 inhibitor, U0126. 3H-thymidine was added to the medium at 44 hours of incubation for a final 16 hours of labeling (60-hour total culture time). (D) Spry2-null or WT splenic B cells from sex-matched littermates were anti-CD40 plus anti-IgM costimulated and evaluated for apoptosis by annexin V–FITC and propidium iodide staining with flow cytometry. (E) Percentage of CD19+B220+ B cells determined by flow cytometry from the bone marrows of 7 poly-dI:dC-injected Mx1-Cre × Spry2fl/fl (Spry2-null), 6 poly-dI:dC-injected Spry2fl/fl (WT), and 3 1× PBS, (pH 7.4)-injected Mx1-Cre × Spry2fl/fl mice (WT). (F) Model illustrating SPRY2-mediated regulation of the ERK signal transduction pathway in mature peripheral B cells. The diagram on the left (WT) shows that BCR/CD40 signaling results in activation of ERK1/2, which in turn stimulates B-cell proliferation. However, it also induces expression of SPRY2 protein, which subsequently represses ERK1/2 pathway signaling and prevents excessive cell proliferation. The diagram on the right (Spry2-null) illustrates the consequences of loss of SPRY2 function, by either dense promoter DNA hypermethylation or gene knockout: repression of ERK1/2 pathway signaling does not occur, resulting in increased B-cell proliferation. TCL1 also enhances the ERK signaling pathway in response to BCR stimulation in mature peripheral B cells, which results in an increase in Spry2 protein expression. Data are representative of 2 (A), 3 (C), or 4 (B,D) independent experiments.

Inactivation of Spry2 increases ERK1/2-dependent B-cell proliferation and survival. (A) Freshly isolated Spry2-null or WT splenic B cells from sex-matched littermates were assessed for ERK1/2 phosphorylation by Western blot. (B) Spry2-null or WT B cells were assayed for CD69 (ERK1/2-dependent) and CD25 (ERK1/2-independent) expression after 16 hours of costimulation with anti-CD40 and anti-IgM Abs. The mean fluorescence intensity for each B-cell activation marker from 3 separate experiments as measured by flow cytometry after costimulation is shown. (C) 3H-thymidine incorporation by Spry2-null or WT splenic B cells from sex-matched littermates, stimulated with serum alone or with serum plus anti-CD40 plus anti-IgM costimulation in the presence or absence of the MEK 1/2 inhibitor, U0126. 3H-thymidine was added to the medium at 44 hours of incubation for a final 16 hours of labeling (60-hour total culture time). (D) Spry2-null or WT splenic B cells from sex-matched littermates were anti-CD40 plus anti-IgM costimulated and evaluated for apoptosis by annexin V–FITC and propidium iodide staining with flow cytometry. (E) Percentage of CD19+B220+ B cells determined by flow cytometry from the bone marrows of 7 poly-dI:dC-injected Mx1-Cre × Spry2fl/fl (Spry2-null), 6 poly-dI:dC-injected Spry2fl/fl (WT), and 3 1× PBS, (pH 7.4)-injected Mx1-Cre × Spry2fl/fl mice (WT). (F) Model illustrating SPRY2-mediated regulation of the ERK signal transduction pathway in mature peripheral B cells. The diagram on the left (WT) shows that BCR/CD40 signaling results in activation of ERK1/2, which in turn stimulates B-cell proliferation. However, it also induces expression of SPRY2 protein, which subsequently represses ERK1/2 pathway signaling and prevents excessive cell proliferation. The diagram on the right (Spry2-null) illustrates the consequences of loss of SPRY2 function, by either dense promoter DNA hypermethylation or gene knockout: repression of ERK1/2 pathway signaling does not occur, resulting in increased B-cell proliferation. TCL1 also enhances the ERK signaling pathway in response to BCR stimulation in mature peripheral B cells, which results in an increase in Spry2 protein expression. Data are representative of 2 (A), 3 (C), or 4 (B,D) independent experiments.

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