The intracellular domain of CD44 promotes the fusion of macrophages - PubMed (original) (raw)

The intracellular domain of CD44 promotes the fusion of macrophages

Weiguo Cui et al. Blood. 2006.

Abstract

Macrophages seed all tissues in which they have the ability, in specific and rare instances, to fuse with themselves and to differentiate into osteoclasts in bone or into giant cells in chronic inflammatory reactions. Although these cells play a central role in osteoporosis and in foreign body rejection, respectively, the molecular mechanism used by macrophages to fuse remains poorly understood. Macrophages might also fuse with somatic and tumor cells to promote tissue repair and metastasis, respectively. We reported that CD44 expression is highly induced in macrophages at the onset of fusion in which it plays a role. We report now that the intracellular domain of CD44 (CD44ICD) is cleaved in macrophages undergoing fusion and that presenilin inhibitors prevent the release of CD44ICD and fusion. We also show that CD44ICD promotes the fusion of tissue macrophages and bone marrow-derived macrophages. Finally, we report that CD44ICD is localized in the nucleus of macrophages in which it promotes the activation of NF-kappaB. These observations open avenues to study the role of CD44ICD in blood cells and tumors.

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Figures

Figure 1.

Figure 1.

The PS inhibitors DAPT and DupE inhibit the fusion of macrophages and prolong the induced expression of PS2. (A) Rat alveolar macrophages were cultured in fusogenic conditions and treated with increasing concentrations of DAPT or DupE for 3 days. Whereas control cells fused efficiently into multinucleated macrophages, as reflected by the extensive plasma membrane of multinucleate macrophages (*), DAPT and DupE inhibited fusion of macrophages dose dependently, as illustrated by the lack of plasma membrane extension (bar represents 1 mm; *P < .001 versus control; SD; n = 6). (B) Rat alveolar macrophages were cultured in fusogenic conditions in 96-well dishes for the indicated times in the absence or presence of 10 μM DAPT, 3 μM DupE, or 50 nM JLK2. Cells were subjected to Western blotting analysis (0.2 × 106 cells/lane) using antibodies directed against PS1, PS2, or GAPDH. Note that PS1 and PS2 migrated as 23-kDa cleaved amino-terminal fragments, whereas intact PS2 ran as an intact protein of 50 kDa. Also, note the lower molecular weight band of about 15 kDa that is detected in all samples, but at time zero only.

Figure 2.

Figure 2.

PS inhibitors DAPT and DupE, not JLK2, decrease the expression of CD44 and inhibit the release of CD44ICD. (A) Rat alveolar macrophages (5 × 106 cells/35-mm dish) were cultured for the indicated times, treated or not with DAPT (10 μM), DupE (0.5 μM), or JLK2 (20 μM), then lysed and subjected to Western blot analysis. In parallel wells, 24-hour cell supernatants from macrophages were collected and analyzed for CD44 content using Western blotting. (B) Schematic diagram of the CD44 constructs generated for this study. (C) Rat alveolar macrophages were transduced with the retroviral vector MigR1 empty (control) or encoding CD44ΔE, and then treated or not with DAPT (10 μM), DupE (0.5 μM), or JLK2 (20 μM) for 24 hours. The cell lysates were subjected to Western blotting analysis using an mAb directed against myc. The cleavage of CD44 ΔE into CD44ICD was inhibited by DAPT and DupE, but not by JLK2.

Figure 3.

Figure 3.

CD44ICD promotes the fusion of macrophages and the differentiation of TRAP+ multinucleated osteoclasts. (A) Rat alveolar macrophages were transduced with the retroviral vector MigR1 empty or encoding CD44FL, CD44ΔE, or CD44ICD, plated in fusogenic conditions, and then cultured for only 2 days. Whereas macrophages transduced with empty vector, CD44FL, or CD44ΔE had barely initiated fusion by day 2, those transduced with MigR1 encoding CD44ICD were well fused (bar represents 3 mm; *P < .001 versus MigR1; SD; n = 5). (B) Mouse bone marrow cells were transduced with the retroviral vector MigR1 empty or encoding CD44FL, CD44ΔE, or CD44ICD, treated with M-CSF supplemented with RANKL for only 2 days to differentiate into osteoclasts, then reacted for TRAP, an osteoclast marker. Whereas very few macrophages transduced with empty vector or vector encoding CD44FL or CD44ΔE were fused by day 2, macrophages transduced with CD44ICD were highly differentiated into TRAP+ multinucleated osteoclasts (bar is 2 mm; *P < .001 versus MigR1; SD; n = 6). (C) CD44ICD promotes the fusion of peritoneal macrophages but does not induce the fusion of 3T3 cells. Rat peritoneal macrophages and 3T3 cells were cultured in fusogenic conditions for 18 hours and 48 hours, respectively, following transduction with MigR1 either empty or encoding CD44ICD. CD44ICD strongly promoted the fusion of rat peritoneal macrophages (bar represents 1 mm; *P < .001 versus MigR1; SD; n = 4).

Figure 4.

Figure 4.

CD44ICD rescues the fusion of macrophages treated with DAPT. (A) Rat alveolar macrophages were transduced with MigR1 empty or encoding CD44FL, CD44ΔE, or CD44ICD, and treated or not with DAPT (10 μM) for 4 days (bar represents 2 mm; *P < .01 versus MigR1; SD; n = 6). (B) Cells from panel A were subjected to Western blotting analysis using an mAb directed against myc. Overexposed blots revealed CD44ICD in CD44FL- and CD44ΔE-transduced cells, but not in the presence of DAPT.

Figure 5.

Figure 5.

CD44ICD localizes to the nucleus. (A) Raw 264.7 cells were transduced with pBMN-eGFP or pBMN-CD44ΔE-eGFP and treated or not with JLK2 (20 μM) or DAPT (10 μM) for 16 hours. Note that in DAPT-treated cells that express CD44ΔE-eGFP, not JLK2-treated cells, the nucleus does not fluoresce. Also, DAPT affects the shape of macrophages and induces the clustering of CD44ΔE-eGFP in the plasma membrane (bar is 7 μm). (B) Rat alveolar macrophages were transduced with the retroviral vector MigR1 encoding CD44ΔE and then subjected to subcellular fractionation. Fractions were analyzed by Western blotting using antibodies directed against myc, MFR (plasma membrane), p38 (cytosol), and Sm (nucleus).

Figure 6.

Figure 6.

CD44ICD activates NF-κB. (A) Raw 264.7 cells were cotransfected with pcDNA3-CD44ICD and NF-κB, AP-1 or NFAT luciferase reporter constructs, grown for 2 days, and then either assayed for luciferase activity (*P < .05 and **P < .001 versus 0 μg CD44ICD; SD; n = 3) or subjected to Western blot analysis using anti-myc antibody. RANKL was used as a positive control. (B) Raw 264.7 cells were cotransfected with pcDNA3-CD44FL or pcDNA3-CD44ΔE and NF-κB luciferase reporter construct, grown for 2 days, and either assayed for luciferase activity (**P < .001 versus 0 μg CD44FL or CD44ΔE; SD; n = 3) or subjected to Western blot analysis using anti-myc antibody. RANKL was used as a positive control. (C) Raw 264.7 cells were transduced or not with MgR1-CD44ICD and stimulated or not for 30 minutes with LPS (100 μg/mL) or RANKL (100 ng/mL). Nuclear proteins were extracted and incubated for 20 minutes with radiolabeled NF-κB consensus oligonucleotides. Some cells were preincubated with NEMO-binding peptide (NBD; wt; 50 μM) or NBD mutant peptide (mut; 50 μM) for 1 hour at room temperature. The samples were resolved by electrophoresis. (D) Raw 264.7 cells were transduced with MgR1-CD44ICD and stimulated or not with RANKL (100 ng/mL) and then nuclear extracts were analyzed as in panel B, except that some nuclear extracts were preincubated with an antibody directed against p65, myc, or with both p65 and myc. (E) Raw 264.7 cells were transduced with MgR1 empty or encoding CD44ICD and stimulated with RANKL (100 ng/mL) for the indicated times. Total cell extracts were analyzed by Western blotting using antibodies directed against I-κB, phosphorylated I-κB (P-I-κB), and GAPDH.

Figure 7.

Figure 7.

Inhibition of NF-κB prevents fusion of macrophages. Rat alveolar macrophages (5 × 106 cells/well in 24-well dishes) were cultured for 3 days in the absence or presence of increasing concentrations of aspirin or curcumin. Note that 10 μM curcumin induced macrophages to detach from the plate (Bar represents 2 mm; *P < .01 and **P < .001 versus control; SD; n = 5).

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