Silencing Sca-1 expression decreases colony formation and tumorigenic potential, and increases GDF10 expression. 34T cells were transduced with a lentivirus expressing either GFP shRNA as a control or a Sca-1 shRNA (Fig. S2). D8 cells expressing Sca-1 shRNA cells exhibited a 90% reduction in Sca-1 expression compared with control 34T cells, as assessed by FACS analysis (A) or Western blot analysis (B). (C) Down-regulation of Sca-1 expression reduces spheroid cluster morphology. 34T cells grew as spheroid clusters, whereas D8 cells grew as a flattened monolayer with contact-inhibited growth. (D) Silencing Sca-1 expression reduces colony formation. Colony-formation assay in D8 cells vs. 34T cells showed that growth was reduced by 70% in D8 cells vs. 34T cells. (E) Down-regulation of Sca-1 expression markedly reduces tumorigenicity. 34T cells and D8 cells were implanted s.c. at an inoculum of 100,000 cells into opposite flanks of eight syngeneic C57BL/6 mice. Tumor formation was monitored over 25 d, and tumor volume was calculated. Isografts from D8 cells exhibited little growth compared with isografts from 34T cells (P < 0.001, two-tailed Student t test). (F) Conditioned medium (CM) from 34T cells enhanced colony formation of D8 cells, whereas conditioned medium from D8 cells markedly reduced colony formation of 34T cells. The bar graph shows quantification of colony growth; the number in each bar corresponds to the plate number in the left panel. (G) Real-time qPCR analysis of TGF-β ligands. The fold increase in expression of each ligand in D8 cells relative to 34T cells is shown. D8 cells expressed >100-fold higher levels of GDF10 mRNA compared with 34T cells. (H) 34T/GFP shRNA cells stably expressing GDF10 (34T/GDF10) exhibit a >100-fold increase in GDF10 mRNA expression compared with control cells (34T). (I) D8 cells transfected with a GDF10 shRNA (D8/GDF10sh) show 90% reduced GDF10 mRNA expression compared with cells expressing a control shRNA (D8). (J) Modulation of GDF10 expression results in changes in colony formation. Expression of GDF10 in 34T cells (34T/GDF10) reduced colony formation by 80%, whereas silencing GDF10 expression in D8 cells (D8/GDF10sh) increased colony formation. (K) GDF10 modulates tumorigenicity. Cells were implanted into eight syngeneic C57BL/6 mice, and tumor growth was assessed as in E. Tumor volume was reduced in isografts of 34T/GDF10 cells compared with tumors from 34T cells (P < 0.001, two-tailed Student t test). In contrast, D8/GDF10sh cells showed increased tumor volume compared with D8 cells (P < 0.001, two-tailed Student t test).

GDF10 activates Smad2/3 signaling through the TGF-β pathway. (A) GDF10 increases Smad3 nuclear localization. Treatment of NMuMG cells for 30 min with 50 ng/mL of GDF10 increased nuclear localization of Smad2/3. Treatment with 5 ng/mL of TGF-β1 served as a positive control. The bar graph quantitates nuclear Smad2/3 and Smad1/5. (Scale bar: 10 μm.) (B) GDF10 induces Smad6 and PAI1 mRNA expression. NMuMG cells treated overnight with GDF10 and TGF-β as in A expressed increased levels of mRNAs for the TGF-β target genes Smad6 and PAI1, as determined by real-time qPCR. (C) GDF10 treatment activates Smad3. 34T and D8 cells were treated with either GDF10 or TGF-β1, and pSmad3 levels were determined by Western blot analysis. pSmad3 was increased at 10 min in 34T cells to a greater extent than in D8 cells due to the higher basal level in the latter cells; pSmad3 at 30 min was equivalent in both cell lines. TGF-β1 increased pSmad3 to a greater extent than GDF10 in both 34T and D8 cells. (D) TGF-β receptor expression in 34T and D8 cells. Receptor mRNA was quantified by real-time qPCR. Only TβRII mRNA was significantly different in D8 and 34T cells (P < 0.05, two-tailed Student t test). (E) GDF10 competes with TGF-β1 for receptor binding. Mv1Lu cells were surface-labeled with 5 ng/mL of [¹²⁵I]TGF-β1 and competed with either 100 ng/mL of unlabeled TGF-β1 or 100–200 ng/mL of unlabeled GDF10. Subsequent chemical cross-linking of [¹²⁵I]TGF-β1 to its receptors, SDS/PAGE, and autoradiography indicate that GDF10 competes with TGF-β1 for binding to TβRI (55 kDa), TβRII (70 kDa), and betaglycan (type III receptor). Competition with 100 ng/mL of cold TGF-β served as a positive control. (Right) An identical gel was stained with Coomassie blue to determine protein loading. (F) GDF10 activation of Smad3 is TβRI-dependent. Expression of type I TGF-β receptor mRNAs in WT and Tgfbr1^−/− MEFs was quantified by real-time qPCR and confirmed the expression of ALK4 and ALK7 and loss of TβRI/ALK5 in Tgfbr1^−/− MEFs. (Right) Treatment with 50 ng/mL GDF10 increased pSmad3 levels in WT, but not Tgfbr1^−/−, MEFs. (G) GDF10 activation of Smad3 is TβRII-dependent. pSmad3 was quantified by Western blot analysis in WT and Tgfbr2^−/− MEFs. Treatment with 50 ng/mL of GDF10 increased pSmad3 in WT, but not Tgfbr2^−/−, MEFs.

Silencing Sca-1 expression activates GDF10-dependent signaling through Smad3. (A) D8 cells exhibit increased Smad3 (TGF-β), but not Smad1/5 (BMP) reporter activity. D8 cells expressed a >20-fold increase in Smad3 activity compared with 34T cells. (B) GDF10 expression increases Smad3 activity. Reporter activity was increased in GDF10-overexpressing 34T cells (34T/GDF10), whereas silencing GDF10 expression in D8 cells (D8/GDF10sh) reduced the activity. (C and D) Expression of the TGF-β target genes Smad6 (C) and PAI1 (D) is regulated by GDF10 expression. Both Smad6 and PAI1 expression were increased by overexpression of GDF10 in 34T cells and decreased by down-regulation of GDF10 expression in D8 cells. (E) GDF10 expression regulates Smad3 activation, as assessed by immunoblotting for phosphoSmad3 (pSmad3). The levels of Smads and pSmads were determined by Western blot analysis. pSmad3 was increased in 34T/GDF10 cell, compared with 34T cells and was reduced in D8/GDF10sh cells compared with D8 cells. No difference in pSmad1/5 was observed. (F) GDF10 expression correlates with Smad2/3 nuclear localization. Fluorescence was measured by confocal microscopy using a Smad2/3 or Smad1/5 antibody and an Alexa Fluor 594-conjugated secondary antibody; nuclei were stained with DAPI and pseudo-colored in green. The merged image shows nuclear localization of Smad2/3 in 34T/GDF10 and D8 cells, but not in 34T and D8/GDF10sh cells. The bar graph quantifies Smad2/3 and Smad1/5 nuclear localization. *P < 0.01, Student two-tailed t test. (Scale bar: 10 μm.)

Sca-1 interacts with cell surface TGF-β receptors to inhibit Smad3 activation. (A and B) NMuMG cells were transfected with either a Sca-1 or control plasmid. Treatment of cells with either 50 ng/mL of GDF10 or 5 ng/mL of TGF-β1 resulted in increased pSmad3 (A) and transcription from a Smad3-responsive (TGF-β) reporter (B) in control cells, but not in Sca-1-expressing cells. (C) Sca-1 colocalizes with TβRI and TβRII in 34T cells. Fluorescence was visualized by confocal microscopy using either a TβRI or a TβRII mAb and an Alexa Fluor 488-conjugated secondary antibody or an Sca-1 mAb and an Alexa Fluor 594-conjugated secondary antibody. The merged images indicate that Sca-1 colocalizes with TβRI (Upper) and TβRII (Lower). Arrowheads indicate the cell membrane regions magnified in the insets. (Scale bar: 10 μm.) (D) Cell surface TβRI, but not TβRII, associates with Sca-1. Cell surface proteins in 34T cells were labeled with a membrane-impermeable biotin conjugate, and Sca-1–interacting proteins were isolated by immunoprecipitation with an Sca-1 antibody or IgG isotype control. Immunoprecipitated proteins were analyzed by Western blot using either a streptavidin-conjugated antibody for biotinylated proteins or TβRI, TβRII, and Sca-1 antibodies. Sca-1 associated with a biotinylated band of the same mobility as TβRI, but not with TβRII. (E) Silencing of Sca-1 expression facilitates TβRI and TβRII receptor complex formation. TβRI was immunoprecipitated from lysates of 34T and D8 cells, and associated TβRII was detected by Western blot analysis. D8 cells exhibited greater association of TβRI with TβRII compared with 34T cells. (F) Sca-1 reduces TβRI and TβRII receptor complex formation and Smad3 activation. D8 cells were transfected with a Sca-1 or control plasmid and cell lysates were immunoprecipitated with a TβRI antibody, and TβRI and TβRII were detected by Western blot analysis. (Left) Overexpression of Sca-1 inhibited the association of TβRI with TβRII. (Right) It also reduced pSmad3 levels. (G) Association kinetics of Sca-1 with TβRI. 34T cells were treated with either 50 ng/mL of GDF10 or 5 ng/mL of TGF-β1, and TβRI was immunoprecipitated. Sca-1, TβRI, pSmad3, and Smad2/3 were detected by Western blot analysis. Both GDF10 and TGFβ1 increased pSmad3, although phosphorylation was greater in the presence of TGFβ1. Sca-1 was found to be associated with TβRI in the absence of ligand, which was reduced after treatment with GDF10 or TGF-β1, whereas dissociation occurred more rapidly in the presence of TGF-β1. (H) Schematic representation of the regulation of TGF-β signaling by Sca-1. Sca-1 is depicted as interacting with TβRI to interfere with TβRI–TβRII complex formation and the subsequent activation of Smad3. When Sca-1 expression is reduced, GDF10 expression is increased, leading to stabilization of the receptor complex and Smad3 activation.