Regulation of myelopoiesis through syntenin-mediated modulation of IL-5 receptor output by Jeffrey M. Beekman, Liesbeth P. Verhagen, Niels Geijsen, and Paul J. Coffer Blood Volume 114(18):3917-3927 October 29, 2009 ©2009 by American Society of Hematology

Colocalization of IL-5Rα and syntenin in TF-1 cells. Colocalization of IL-5Rα and syntenin in TF-1 cells. (A) Subcellular distribution of IL-5Rα (indicated in red) and EGFP-tagged syntenin (green) in TF-1 cells maintained in IL-3. Colocalization of IL-5Rα and syntenin is indicated in yellow in the right column (merge). A representative cell from 3 independent experiments is shown in the top panel. The bottom 3 panels show colocalizing structures at higher magnification. (B) Representative examples of EGFP-syntenin–expressing TF-1 cells that were stained for various endocytic markers in red. (C) TF-1 cells ectopically expressing GFP-syntenin were starved for 4 hours in 0.5% serum and incubated with IL-5 and an IL-5Rα–specific monoclonal mouse IgG for 60 minutes at 4°C (top panel), or for different time points at 37°C (bottom panels). Cells were then fixed, and stained using a Cy3-conjugated mAb recognizing mouse IgG (red). Representative examples are shown (n = 3). Jeffrey M. Beekman et al. Blood 2009;114:3917-3927 ©2009 by American Society of Hematology

Syntenin selectively promotes IL-5–driven proliferation. Syntenin selectively promotes IL-5–driven proliferation. (A) Stable polyclonal TF-1 cell lines were generated that ectopically express HA-tagged full-length syntenin, syntenin lacking its N-terminal domain (PDZ1+2), or EGFP as control. All lines were more than 95% EGFP positive. Viable cell numbers of 6-day cultures in either IL-5–supplemented (■) or GM-CSF–supplemented (□) medium are displayed. An average of 4 independent experiments is depicted (± SD). (B) Anti–HA-tagged Western blot indicating the presence of HA-tagged syntenin or PDZ1+2 in selected TF-1 cell–derived clones. Two clones per group are shown. (C) Expression of IL-5Rα for each subclone was analyzed by flow cytometry. Histograms of isotype-stained (black) or IL-5Rα–stained (white) cells are indicated. (D) Proliferative responses of selected clones to IL-5 or GM-CSF after 72 hours. Viable cells (live gate, propidium iodide negative) were counted by flow cytometry after culturing the cells for 3 days without cytokine, or after addition of IL-5 or GM-CSF. Mean of triplicate samples (± SD) is indicated for 2 control EGFP-clones, 2 clones transduced with syntenin, and 2 clones transduced with PDZ1+2. An average of 4 independent experiments is depicted (± SD). (E) Surface levels of clones expressing syntenin, PDZ1+2, or EGFP were incubated with IL-5 (10nM) for various time points and remaining surface expression was analyzed using flow cytometry. Data represent mean ± SD of triplicates. Jeffrey M. Beekman et al. Blood 2009;114:3917-3927 ©2009 by American Society of Hematology

Syntenin increases IL-5–mediated signaling. Syntenin increases IL-5–mediated signaling. (A) TF-1 cells ectopically expressing syntenin and PDZ1+2 were starved overnight in 0.5% serum and stimulated for 15 minutes with IL-5 or GM-CSF. Cell lysates were prepared, proteins quantified, and subsequent Western blots assessed for phosphorylated ERK1/2 (pERK1/2), phosphorylated STAT5 (pSTAT5), and actin in the top panel. A representative example of 4 independent experiments is shown. The bottom panel shows a representative example of Western blots assessed for phosphorylated and total JAK2, and tubulin. (B) pERK1/2 and pSTAT5 levels of 4 independent experiments as outlined in panel A were quantified using ImageJ software. Mean integrated intensity ± SD is indicated. *P < .05; **P < .01. (C) EGFP or ectopic syntenin-expressing cells were starved overnight and stimulated with IL-5 for indicated time points. Protein lysates were quantified for protein amount, and Western blots were prepared in duplicate and analyzed for pERK1/2 and total ERK levels. A reference sample (R) was included to facilitate comparison between blots; a lower amount of reference was loaded on the blots that were probed for total ERK. (D) Cells ectopically expressing EGFP, PDZ1+2, and syntenin were stimulated with IL-5 for indicated time points, and protein lysates were prepared and quantified. Subsequent Western blots were incubated with antibody recognizing phosphorylated PKB (pPKB) substrates and tubulin as loading control. One of 3 representative experiments is shown. (E) Cells ectopically expressing EGFP, PDZ1+2, and syntenin were cultured for 3 days in the presence of IL-5 and an inhibitor (U0126) of MEK1/2, the upstream kinases of ERK1/2. Viable cell numbers are depicted (average ± SD), and were determined by flow cytometry in 3 independent experiments. Jeffrey M. Beekman et al. Blood 2009;114:3917-3927 ©2009 by American Society of Hematology

Decreased IL-5R functions upon knockdown of syntenin in TF-1 cells. Decreased IL-5R functions upon knockdown of syntenin in TF-1 cells. (A) Scrambled control siRNa and syntenin directed siRNA were electroporated into TF-1 cells using an Amaxa system. TF-1 cells recovered after 24 hours in the presence of IL-3 (cells were ∼ 90% trypan blue negative), and were starved for 4 hours before cells were stimulated with IL-5 for 15 minutes. Cells were lysed in Laemmli buffer, proteins content was quantified, and Western blots were stained for syntenin and tubulin as loading control. (B) pERK1/2 levels (± SD) were assessed by quantifying Western blot from 3 independent experiments as described in panel A using ImageJ software. (C) TF-1 cells were transduced with bicistronic constructs that express nonfunctional shRNA (control) or syntenin-targeting shRNA in combination with EGFP. EGFP cells were sorted beyond 95% purity and syntenin levels were assessed by Western blot. (D) Control and syntenin-targeting shRNA-expressing TF-1 cells were cultured for 3 days in the presence of IL-5 or GM-CSF. Viable cell numbers were determined by flow cytometry. Results depicted are from 2 experiments with 4 samples per group per experiment (± SD). *P < .05. Jeffrey M. Beekman et al. Blood 2009;114:3917-3927 ©2009 by American Society of Hematology

Syntenin expression is induced during eosinophil differentiation. Syntenin expression is induced during eosinophil differentiation. (A) CD34+ cells were isolated from cord blood and maintained in FLT-3, SCF, IL-3, GM-CSF, and IL-5. At day 3, cells were transferred to medium containing IL-3 and IL-5 to induce eosinophil differentiation, or to G-CSF–supplemented medium to induce neutrophils. Protein lysates were prepared at the days indicated, and 30 μg of protein was loaded per lane. Blots were incubated with antibodies recognizing syntenin and tubulin as loading control. One of 4 representative Western blots is indicated. (B) Syntenin levels from 4 Western blots were quantified using ImageJ software and mean integrated density is displayed (± SD). **P < .01. (C) Northern blot analysis of HL-60 cells maintained at pH 7.7 for 2 months (HL-60 7.7) that were differentiated toward the eosinophilic lineage by 0.5 mM butyric acid (days of treatment are indicated). Total RNA (20 μg) was blotted and probed for IL-5Rα, syntenin, and GAPDH as loading control. (D) From 2 distinct donors, eosinophils and neutrophils were purified based on Ficoll centrifugation and CD16 selection (magnetic-activated cell sorting; Miltenyi Biotec). Thirty micrograms protein of whole-cell lysate was assessed by Western blotting for syntenin and actin levels. Jeffrey M. Beekman et al. Blood 2009;114:3917-3927 ©2009 by American Society of Hematology

Syntenin modulates eosinophil differentiation. Syntenin modulates eosinophil differentiation. (A) CD34+ cells were isolated from cord blood, and cultured for 3 days in FLT-3, SCF, IL-3, and GM-CSF before the medium was supplemented with IL-3 in the presence or absence of IL-5. Cells were transduced with EGFP control virus at days 1 and 2. At day 17, EGFP+ cells were sorted, cytospins were prepared, and cells were stained by May-Grünwald-Giemsa. Percentage of eosinophils was scored in 4 independent experiments using different donors (± SD). (B) Eosinophil differentiations of 3 independent donors upon transduction with EGFP control protein or syntenin and incubation with IL-5 as described in panel A. Eosinophil numbers after EGFP transduction were set at 100%, and the fold increase upon syntenin transduction was calculated per donor (± SD). (C) Representative cytospins at day 17 of EGFP or syntenin-transduced CD34+ cells that were differentiated toward eosinophils. EGFP-positive cells were sorted and stained by May-Grünwald-Giemsa. (D) CD34+ cells were transduced with retroviral vectors expressing control shRNA or syntenin-targeting shRNA and differentiated toward eosinophils. At day 17, fold increase of the percentage of eosinophils for each donor (n = 3, data show average ± SD) after control shRNA transductions (set at 100%) or syntenin-targeting shRNA transduction was determined. **P < .01. (E) Surface IL-5Rα expression of primary CD34+ cells transduced with EGFP or syntenin (3 donors) or control or syntenin-targeting shRNA (2 donors). Eosinophil cultures were analyzed at day 14 for IL-5Rα expression by flow cytometry (PE-channel). Nontransduced and transduced cells were indicated by EGFP transduction and IL-5Rα staining was compared with isotype staining. Numbers represent geometric mean fluorescent intensities of the boxed region in the PE channel. Identical results were obtained for all donors and 1 representative sample is shown. Jeffrey M. Beekman et al. Blood 2009;114:3917-3927 ©2009 by American Society of Hematology

Syntenin binds multiple IL-5Rα chains. Syntenin binds multiple IL-5Rα chains. (A) Schematic representation of the experimental design of the PCA. Venus, an improved version of yellow fluorescent protein (YFP), was fragmented into nonfunctional parts that were fused to the N-termini of the intracellular domains of IL-5Rα (YFP1–IL-5Rα and YFP2–IL-5Rα). When both of the fusion products bind syntenin, the nonfunctional Venus parts regain fluorescence. (i) As a control, expression of the fusion products with a single PDZ domain of syntenin should not result in functional restoration of the Venus protein. (ii) As an additional control, we fused YFP-1 to the C-terminus of IL-5Rα (IL-5Rα–YFP1) instead of its N-terminus. Because syntenin binds the C-terminus of IL-5Rα, no additional fluorescence is expected upon full-length syntenin or PDZ2 cotransfection (iii and iv, respectively). (B) HEK293 cells were transiently transfected with YFP1–IL-5Rα and YFP2–IL-5Rα together with empty vector, or syntenin's second PDZ domain (PDZ2), or full-length syntenin (left panel). The percentage of positive cells was determined by flow cytometry. At right, similar conditions were assayed but an IL-5Rα–YFP1 instead of YFP1–IL-5Rα fusion product was used. Empty vector transfections per experiments were set at 100%. Data depict 3 independent experiments (± SD). *P < .05. (C) YFP1–IL-5Rα and YFP2–IL-5Rα dose-dependent increase of fluorescence in the presence of full-length syntenin. Cells were transfected with fixed amounts of empty vector, syntenin PDZ2 or full-length syntenin (each 0.5 μg), and increasing concentrations of YFP1–IL-5Rα and YFP2–IL-5Rα. Cells were analyzed 48 hours after transfection for YFP fluorescence by flow cytometry. Percentage of YFP-fluorescent cells is indicated. One representative example of 2 identical experiments is indicated. (D) Representative dot plots of cells transfected with empty vector, syntenin PDZ2 or full-length syntenin (0.5 μg), together with 0.5 μg of YFP1–IL-5Rα and YFP2–IL-5Rα. Forward scatter is plotted against YFP fluorescence in the FL-1 channel. Geometric mean fluorescent intensities of the positive cells are indicated in the squares. (E) Immunoprecipitation (IP) of syntenin–IL-5Rα complexes from transfected HEK293 cells in the presence of the chemical cross-linker DSP. The left panels indicate expression of the myc–IL-5Rα (17.5 kDa) and HA-tagged syntenin (35 kDa) in whole-cell lysates (WCLs). The middle panel shows HA immunoprecipitates analyzed by nonreducing SDS-PAGE and myc-immunoblotting (IB), whereas the right panel shows HA immunoreactivity of the same samples blotted in duplicate. Jeffrey M. Beekman et al. Blood 2009;114:3917-3927 ©2009 by American Society of Hematology