TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition - PubMed (original) (raw)
TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition
Ulrich Valcourt et al. Mol Biol Cell. 2005 Apr.
Abstract
Epithelial-mesenchymal transition (EMT) contributes to normal tissue patterning and carcinoma invasiveness. We show that transforming growth factor (TGF)-beta/activin members, but not bone morphogenetic protein (BMP) members, can induce EMT in normal human and mouse epithelial cells. EMT correlates with the ability of these ligands to induce growth arrest. Ectopic expression of all type I receptors of the TGF-beta superfamily establishes that TGF-beta but not BMP pathways can elicit EMT. Ectopic Smad2 or Smad3 together with Smad4 enhanced, whereas dominant-negative forms of Smad2, Smad3, or Smad4, and wild-type inhibitory Smad7, blocked TGF-beta-induced EMT. Transcriptomic analysis of EMT kinetics identified novel TGF-beta target genes with ligand-specific responses. Using a TGF-beta type I receptor that cannot activate Smads nor induce EMT, we found that Smad signaling is critical for regulation of all tested gene targets during EMT. One such gene, Id2, whose expression is repressed by TGF-beta1 but induced by BMP-7 is critical for regulation of at least one important myoepithelial marker, alpha-smooth muscle actin, during EMT. Thus, based on ligand-specific responsiveness and evolutionary conservation of the gene expression patterns, we begin deciphering a genetic network downstream of TGF-beta and predict functional links to the control of cell proliferation and EMT.
Figures
Figure 1.
TGF-β induces EMT and growth inhibition in human and mouse normal epithelial cells. (A) Actin cytoskeleton direct fluorescence microscopy of HMEC, MCF-10A, NMuMG, HPL1 (clone D), NHEK, and HaCaT cells treated with vehicle (control), TGF-β1 (2.5 ng/ml), TGF-β2 (2.5 ng/ml), TGF-β3 (2.5 ng/ml), activin-A (10 ng/ml), or BMP-7 (150 ng/ml) for 36 h. Bars represent 10 μm and the magnification power of each series of photomicrographs is shown above the bars. (B) Dose-response growth inhibition assays performed in HMEC, NMuMG, NHEK, and HaCaT cells in response to TGF-β1 (black bars) and BMP-7 (gray bars). The percentage of radiolabeled thymidine incorporated after TGF-β1 or BMP-7 treatment is plotted relative to the level of vehicle-treated cells (set as 100%; open bars). Error bars represent SEs of the mean derived from triplicate determinations per experiment.
Figure 2.
All three type I receptors of the TGF-β branch are capable of eliciting EMT. (A) Expression of TGF-β superfamily type I and type II receptors in NMuMG cells. The expression of genes encoding the ALK and type II receptors was analyzed by RT-PCR. Lane M shows markers of molecular size (in kbp, kb). (B) Immunoblot analysis of phospho-Smad2 (Smad2-P) and α-SMA levels in NMuMG cells. Cells were stimulated with the indicated ligands for 36 h, under the same conditions as in Figure 1A, and total cell extracts were analyzed with antibodies for the indicated proteins (arrows). (C) Effect of constitutively active ALK-3, -4, and -5 receptors on EMT. NMuMG cells were infected with control adenovirus LacZ (Ad-LacZ; MOI 100) or adenoviruses (MOI 100) expressing constitutively active forms of the BMP, activin, or TGF-β type I receptors, and the localization of E-cadherin and ZO-1 was assessed 36 h later. (D) NMuMG cells were infected either with the control adenovirus (Ad-LacZ, MOI 200) or with the adenoviruses expressing the dominant-negative versions of the indicated type I receptors (Ad-ALK(KR), MOI 200) and were treated with vehicle (Control) or TGF-β1 (2.5 ng/ml) for 36 h. Direct fluorescence microphotographs of the actin cytoskeleton are shown. Bars, (C) and (D) 10 μm; the magnification power of each series of photomicrographs is shown above the bars. (E) NMe cells were infected with Ad-LacZ (MOI 100) or Ad-ALK-5(TD) (MOI 100) and then left unstimulated or stimulated with TGF-β1 (5 ng/ml), in the presence of vehicle (dimethylsulfoxide) or SB431542 inhibitor (10 μM), and the EMT process was analyzed 48 h later. The levels of endogenous α-SMA, E-cadherin (E-cadh.), phosphorylated Smad2 (Smad2-P) and Smad3 (Smad3-P), total Smad2 and Smad3, exogenous ALK5(TD), and β-tubulin were determined by immunoblotting. Relevant protein bands (arrows) and background bands (asterisk) are marked.
Figure 3.
Endogenous Smad pathways mediate EMT in NMuMG cells. (A) NMuMG cells were infected either with Ad-LacZ (MOI 300) or with increasing doses of Ad-Smad3(D407E) (triangle: MOI 20, 100, 300) and were treated with vehicle (Control) or TGF-β1 (2.5 ng/ml) for 36 h. Localization of ZO-1 was visualized by immunofluorescence. (B) Flag-tagged Smad3(D407E) levels measured by immunoblotting using extracts from cells treated as in A. (C) E-cadherin immunofluorescence microscopy of individual NMuMG stable clones transfected with empty vector pMEP4 (Mock), pMEP4-Smad2, and pMEP4-Smad2(SA). Transfected cells were induced (+) or not (-) with CdCl2 and subsequently treated with vehicle (Control) or 5 ng/ml TGF-β1 for 36 h. (D) Immunoblot analysis of endogenous α-SMA in pools of NMuMG cells stably transfected with empty vector (Mock) or Smad2(SA) and treated under identical conditions as in C. Control immunoblots of endogenous phospho-Smad2 (Smad2-P), exogenous Flag-tagged Smad2 (F-Smad2), and endogenous β-tubulin, which serves as loading reference are also shown. (E) Immunofluorescence microscopy of E-cadherin in NMuMG cells infected with Ad-LacZ (MOI 100) or with Ad-Smad7 (MOI 100) and treated with vehicle (Control) or TGF-β1 (5 ng/ml) for 36 h. Bars, (A, C, and E) 10 μm; the magnification power of each series of photomicrographs is shown above the bars. (F) Smad7 blocks α-SMA protein synthesis induced by TGF-β1. NMuMG cells were infected with Ad-LacZ (MOI 100) or increasing doses of Ad-Smad7 (triangle: MOI 30, 100), and then treated with vehicle (-) or with 5 ng/ml TGF-β1 (+) for 48 h. The levels of α-SMA, phosphorylated Smad2 (Smad2-P), exogenous Flag-Smad7 (F-Smad7), and total Smad2 and Smad3 were determined by immunoblotting. In B, D, and F the relevant protein bands are marked with arrows, and an asterisk marks background bands.
Figure 4.
Summary and clustering of highly regulated genes by TGF-β1 in NMuMG cells. (A) Venn diagrams of gene expression itemized in three ways, up-regulated genes (left), down-regulated genes (middle), and total regulated genes (right). The three time points (2, 8, and 36 h) are indicated outside each circle, and gene numbers are shown within each section. (B and C) Clustering of highly regulated genes. Up-regulated (B) and down-regulated (C) genes upon TGF-β1 stimulation were clustered according to their expression profile using the _K_-means algorithm. Graphs represent the average values of normalized ratios of all genes per cluster plotted with their SEs (ratios of gene expression have no unit). The number of genes belonging to each cluster is indicated.
Figure 5.
Differential response of selected gene targets to TGF-β1 versus BMP-7. Semiquantitative RT-PCR analysis showing the mRNA steady-state levels of 14 selected genes. NMuMG cells were treated (+) or not (-) with 5 ng/ml TGF-β1 for 2, 8, or 36 h (A and B) or cells were treated with 300 ng/ml BMP-7 for 2, 8, or 48 h (C and D). Expression of down-regulated (A) and up-regulated (B) genes is shown. The classification to down-regulated (C) and up-regulated (D) genes in the BMP-7 experiments is based on gene responsiveness to TGF-β1. Gapdh serves as a control of equivalent cDNA synthesis, and water and minus RT controls show the specificity of the reactions.
Figure 7.
Id2 inhibits BMP-7 from regulating α-SMA expression. (A and B) Semiquantitative RT-PCR analysis showing the mRNA steady state levels of Idb2 and control Gapdh. NMuMG cells were treated with vehicle (-), 5 ng/ml TGF-β1 for 2, 8, and 36 h (A) or 300 ng/ml BMP-7 (+) for 2, 8, and 48 h (B). Water and minus RT controls show the specificity of the reactions. (C) Knockdown of Id2 allows BMP-7 to cause EMT and enhance α-SMA protein synthesis in a time-dependent manner. NMe clonal cells were transfected with control (siRNA-luc) or specific (siRNA-Id2) and then treated with vehicle (0) or with 300 ng/ml BMP-7 for 8 or 36 h. The levels of endogenous α-SMA, Id2, and β-tubulin were determined by immunoblotting.
Figure 6.
Role of the L45 loop of the TGF-β type I receptor in EMT and gene regulation. (A) ALK5(TD)mL45 fails to induce α-SMA protein synthesis. NMuMG cells were infected with Ad-LacZ (MOI 100) or increasing doses of Ad-ALK5(TD) or Ad-ALK5(TD)mL45 (triangles: MOI 50, 100), or alternatively they were treated with vehicle (-) or with 5 ng/ml TGF-β1 (+) for 48 h. The levels of endogenous α-SMA, phosphorylated Smad2 (Smad2-P), and Smad2 and Smad3 and exogenous ALK5(TD) receptors were determined by immunoblotting. The two different receptors are detected using anti-HA and anti-Flag immunoblotting as shown. (B) ALK5(TD)mL45 reduces α-SMA protein synthesis. NMuMG cells were infected with Ad-LacZ (MOI 100) or increasing doses of Ad-ALK5(TD)mL45 (triangle: MOI 2, 5, 20, 50, 100) for 48 h. The levels of endogenous α-SMA, exogenous ALK5(TD)mL45 receptor and total Smad2 and Smad3 were determined by immunoblotting. In A and B relevant protein bands (arrows) and background bands (asterisk) are marked. (C) Semiquantitative RT-PCR analysis showing the mRNA steady state levels of nine selected genes from Table 1. NMuMG cells were infected with Ad-LacZ, Ad-ALK5(TD), or Ad-ALK5(TD)mL45 (MOI 100) and then treated with vehicle (-) or with 5 ng/ml TGF-β1 (+) for 36 h. Gapdh serves as a control of equivalent cDNA synthesis, and water and minus RT controls show the specificity of the reactions.
Figure 8.
Patterns of gene regulation by TGF-β1. (A) Expression patterns of 10 selected genes from those tested in Figure 5. Quantitative real-time RT-PCR analysis performed with HMEC and HaCaT cells stimulated (gray bars) or not (white bars) with TGF-β1 (5 ng/ml) for 8 and 36 h. Normalized values of relative gene expression (arbitrary units) are plotted in bar graphs as described in Materials and Methods. Smad2 phosphorylation status in HMEC (B) and HaCaT (C) cells in response to TGF-β1 under the identical conditions used for the RT-PCR analysis. Immunoblots for phospho-Smad2 (Smad2-P) and total Smad2 and Smad3 are shown. The relevant protein bands are marked with arrows and an asterisk marks background bands.
References
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