Jin et al. 10.1073/pnas.0503137102. |
Fig. 6. Inhibition of EN Tyr phosphorylation by CEP-701. Cells were treated with CEP-701 for 2 h. Cell extracts were subjected to immunoprecipitation using anti-TrkC or IgG and Gamma-bind beads (Amersham Pharmacia Biosciences), followed by immunoblotting with anti-phospho-Tyr Ab.
Fig. 7. Expression of ETV6-NTRK3 (EN) represses TGF-b-mediated growth inhibition in HaCaT human keratinocytes. (a) To generate HaCaT cell lines stably expressing EN, HaCaT cells were infected with retroviruses containing control vector alone (murine stem cell virus; MSCV) or EN. Puromycin-resistant clones were pooled, and successful expression of EN was determined by using anti-TrkC (NTRK3) antibody. (b) EN inhibits TGF-b1-induced growth inhibitory activity. EN-expressing HaCaT cells and control cells were treated with TGF-b1 (5 ng/ml), and cell numbers were counted as indicated. (c) EN-expressing HaCaT and control cells were treated with varying concentrations of TGF-b1 as indicated. After 20 h of TGF-b1 treatment for the cells, the cells were pulsed with [3H]thymidine and harvested 2 h later. The experiments were repeated three times. All values represent the averages of the three determinations (mean ± SD). (d) EN-expressing HaCaT cells and vector control cells were incubated in the presence or absence of TGF-b1 for 24 h. p21 protein level on whole-cell lysate was examined by Western blot analysis. The extent of Smad2 phosphorylation induced by 30-min stimulation with TGF-b1 (5 ng/ml) also was examined. (e) The interaction between EN and type II TGF-b receptor (TbRII) also was examined in HaCaT cells expressing EN. Cell extracts were subjected to immunoprecipitation using anti-TrkC (NTRK3) antibody and Gamma-bind beads, followed by immunoblotting with anti-TbRII antibody.
Fig. 8. EN interacts with TbRII, but not with activin R-IIA (ActRIIA), activin R-IIB (ActRIIB), or BMP-RII. (a) EN was cotransfected into 293T cells with the Flag-tagged ActRIIA, Flag-tagged ActRIIB, Flag-tagged BMP-RII, or hemagglutinin (HA)-tagged TbRII constructs. Cell extracts were subjected to immunoprecipitation using anti-Flag antibody followed by immunoblotting with anti-V5 antibody. (b) NIH 3T3 cells were cotransfected with either BMP response reporter (BRE-lux) or TGF-b response reporter (SBE-luc) with or without EN. The transfected cells were cultured in the presence or absence of either BMP2 (50 ng/ml) or TGF-b1 (5 ng/ml). Cells were harvested, and luciferase activity was measured 20 h after transfection. (c) NIH3T3 cells expressing EN and EN-K380N or vector control cells were transfected with pAR3-lux (21) with or without pCMV-hFAST-1 (FAST-1). The transfected cells were cultured in the presence or absence of activin (100 ng/ml). Cells were harvested, and luciferase activity was measured 20 h after transfection. (d) BRE-luc was transfected into control NIH3T3 cells or NIH3T3 cells expressing EN and EN-K380N. The transfected cells were cultured in the presence or absence of BMP2 (50 ng/ml) for 20 h, and luciferase activity was measured. Bars and brackets represents the means and SDs calculated from triplicate transfections. (e) EN does not affect activin-induced SBE4-luc reporter activation. TGF-b-responsive reporter SBE4-luc was transfected into NIH3T3 cells together with EN or EN-K380N as well as HA-TbRII. Luciferase activity was measured 24 h after TGF-b1 or activin stimulation.
Fig. 9. TbRIID267-271 was transiently cotransfected into HaCaT cells with or without EN, and cells were treated with TGF-b1 (5 ng/ml). After 20 h of TGF-b1 treatment for the cells, the cells were pulsed with [3H]thymidine and harvested 2 h later. The experiments were repeated three times. All values represent the averages of the three determinations mean ± SD.
Supporting Text
Supporting Materials and Methods
RT-PCR Analysis of Primary Tumor Specimens.
RT-PCR was used to screen for ETV6-NTRK3 fusion transcripts in frozen tissue samples from banked primary tumors using methods described in ref. 1. Total RNA was used as starting material and was isolated by using standard procedures. The TEL541 forward primer (5'-CCTCCCACCATTGAACTGTT-3') and the TRKC2 reverse primer (5'-CCGCACACTCCATAGAACTTGAC-3') were used for PCR.Generation of Stable Cell Lines Expressing Either EN or EN-K380N.
Infections of NIH3T3 fibroblasts and HaCaT cells were carried out by using the murine stem cell virus (MSCV) retroviral expression system (Clontech) according to the manufacturer’s instructions. Puromycin-resistant colonies were isolated after 2 weeks of selection, expanded, and analyzed. For cell-proliferation assay, EN-expressing cells were plated in 24-well plates at a density of 5 × 104 cells per well in 0.5 ml of assay medium (MEM/0.2% FBS). After incubating for 22 h in the presence or absence of TGF-b1, cells were pulse-labeled with 0.5 mCi (1 Ci = 37 GBq) of [3H]thymidine for 2 h at 37°C.Supporting Results
EN Inhibits TGF-b1-Induced Smad2 or Smad3 Phosphorylation.
Smad signaling is believed to mediate the arrest of cell proliferation in the G1 phase of the cell cycle. We therefore examined whether EN blocks the ability of TGF-b1 to inhibit cell growth and DNA synthesis in HaCaT human keratinocytes infected with recombinant retroviruses carrying EN full-length cDNA (see Fig. 7a). As expected, TGF-b1 inhibited cell growth and DNA synthesis in the vector control HaCaT cells (HaCaT–MSCV). However, the expression of EN (HaCaT–EN) blocked the ability of TGF-b1 to inhibit both cell proliferation and DNA synthesis (see Fig. 7 b and c). To investigate the functional significance of this finding, we examined whether EN inhibits endogenous TGF-b target genes in these cells. Vector control or EN expressing HaCaT cells were analyzed for the expression of several TGF-b-responsive genes by Western blot (see Fig. 7d). Cells were treated with TGF-b1 for 20 h, and a Western blot of the known TGF-b1 target, p21Cip1/Waf1 (2), was performed. TGF-b1 treatment markedly increased the level of the p21 protein in the control HaCaT cells, whereas the induction of this protein by TGF-b1 was significantly inhibited in the EN-expressing HaCaT cells (see Fig. 7d). TGF-b1-stimulated endogenous Smad2 phosphorylation was also inhibited in EN-expressing HaCaT cells. These results suggest that EN can clock TGF-b1-mediated cell-cycle arrest in vivo.EN Does Not Interact with the Activin and BMP Type II Receptors.
To determine whether EN also interacts with type II receptors of other TGF-b family members, 293T cells were cotransfected with Flag-activin R-IIA (ActRIIA) (3), Flag-tagged activin R-IIB (ActRIIB) (3), or Flag-tagged BMP-RII (4) expression construct along with EN expression construct. Total cell extracts were subjected to immunoprecipitation with anti-Flag Abs followed by immunoblotting with the anti-V5 Ab. EN did not interact with ActRIIA, ActRIIB, or Flag-BMP-RII, demonstrating that the binding activity of EN is specific to TbRII among type II receptors of the TGF-b family (see Fig. 8a). To confirm the functional specificity of the EN/TbRII interaction, we used two other Smad-dependent reporter constructs, pAR3-lux, which is responsive to activin, and another tandemly repeated SBE reporter, BRE-lux, which is derived from the Jun B promoter that is responsive to BMPs. Introduction of EN had little or no effect on activin or BMP-2-mediated activity in NIH3T3 cells (see Fig. 8b). We also transfected these reporter constructs into NIH3T3 cells stably expressing EN or EN-K380N (see Fig. 8 c and d). Activin and BMP signaling were not affected in the presence of EN. To support this finding, we performed an experiment to see whether EN modulates the effect of activin on SBE4-luc reporter activity. Activin slightly induced SBE4-luc reporter activity in NIH3T3 cells, but EN did not affect activin-induced SBE4-luc reporter activity (see Fig. 8e). These findings further indicate that EN specifically suppresses TGF-b signaling.1. Knezevich, S. R., McFadden, D. E., Tao, W., Lim, J. F. & Sorensen, P. H. (1998) Nat. Genet. 18, 184–187.
2. Hu, P. P., Shen, X., Huang, D., Liu, Y., Counter, C. & Wang, X.-F. (1999) J. Biol. Chem. 274, 35381–35387.
3. Mathews, L. S. (1994) Endocrine Rev. 15, 310–325.
4. Yamashita, H., ten Dijke, P., Heldin, C. H. & Miyazono K. (1996) Bone 19, 569–574.