Etoposide-induced Smad6 expression is required for the G1 to S phase transition of the cell cycle in CMT-93 mouse intestinal epithelial cells

Abstract

The inhibitory Smad6 and Smad7 are responsible for cross-talk between TGF-betabone morphogenic protein (BMP) signaling and other cellular signaling pathways, as well as negative feedback on their own signaling functions. Although inhibitory Smads are induced by various stimuli, little is known about the stimuli that increase Smad6 transcription, in contrast to Smad7. Here we demonstrate that etoposide, which induces double strand breaks during DNA replication, significantly up-regulates the transcription of the Smad6 gene in CMT-93 mouse intestinal cells by increasing specific DNA binding proteins. In addition, endogenous inhibition of the Smad6 gene by RNAi interference led to transient accumulation of G1 phase cells and reduction in incorporation of bromodeoxyuridine (BrdU). These findings strongly suggest that Smad6 plays a distinct role in the signaling of etoposide-induced DNA damage.

Figure 1

Expression of the Smad6 gene in CMT-93 mouse intestinal epithelial cells is induced by treatment with etoposide. (A) After treatment with etoposide, quantitative real-time RT-PCR analysis of the Smad6 gene was performed as described in the Materials and Methods for the indicated time. GAPDH expression was used for normalization. The results are shown as the mean values ± SD of three independent experiments. (B) Western blot analysis of Smad6 protein after treatment with etoposide. Actin was used for the loading control. The inset shows a representative result from at least three independent experiments.

Figure 2

The region from -1,812 to -1,785 of the Smad6 promoter is required for etoposide-induced transcriptional activation of the Smad6 gene. (A) Schematic representations of serial deletion mutants of the Smad6 promoter. (B) The luciferase activities of serial deletion mutants from the region -9.5 kb to -1,035 of the Smad6 promoter. EV: empty vector. (C) Schematic representations of subdivided mutants from the region -1,911 to -1,035 of the Smad6 promoter and their luciferase activities in the presence or absence of etoposide. EV: empty vector. (D) Sequences of the DNA damage-responsible element in the Smad6 promoter (DDRE-S6) and schematic representations of the deletion mutants of the region -1,812 to -1,761 of the Smad6 promoter. (E) The luciferase activities mediated by DDRE-S6, L region and R region in the presence or absence of etoposide. EV: empty vector. All plasmids in this figure were transiently transfected into CMT-93 cells and treated with or without etoposide for 12 h. The luciferase activities were normalized on the basis of TK-Renilla luciferase expression to adjust for variation in transfection efficiency. All data represent the mean value ± SD of three independent experiments.

Figure 3

Etoposide increases the specific DNA-binding proteins bound to the region from -1,812 to -1,785 (L region). Electrophorectic mobility shift assays (EMSA) were performed with the labeled double-stranded oligonucleotides, designated DDRE-S6 (A), L region (B), and R region (C), respectively, and nuclear extracts of CMT-93 cells treated without (lane 2) or with (lane 3) etoposide. Lane 1: free probe. Competition assays were performed with the labeled DDRE-S6 (D) and L region (E) and nuclear extracts of etoposide-treated CMT-93 cells according to the indicated molar ratios of cold competitors. The unlabeled oligonucleotides with the DDRE-S6 and L region were used as specific competitors (S), respectively, whereas double-stranded oligonucleotides containing NF-κB binding sites were used as non-specific competitors (N). Lane 1; free probe. Lane 2; no competitor. The data are representative results of at least three independent experiments.

Figure 4

Endogenous inhibition of the Smad6 gene by Smad6 siRNA induces changes in etoposide-induced G1-S cell cycle transition and BrdU incorporation. (A) Cell cycle analysis of Smad6-expressing CMT-93 wild type cells upon treatment with etoposide. The inset shows representative results of at least three independent experiments. (B) Analysis of the etoposide-induced accumulation of G1 phase cells in CMT-93 cells either expressing Smad6 (CMT-93-pU6PL) or stably expressing Smad6 siRNA (CMT-93-siS6). Cells were synchronized by double thymidine block, released into etoposide and harvested at the indicated time points. Cell cycle distribution was checked by flow cytometry after staining with propidum iodide. (C) Analysis of etoposide-induced BrdU incorporation into CMT-93 cells either expressing Smad6 (CMT-93-pU6PL) or stably expressing Smad6 siRNA (CMT-93-siS6). BrdU incorporation was analyzed as described in the Materials and Methods. The results (B and C) are shown as the mean ± SD of three independent experiments. (D) Western blot analysis of etoposide-induced p21 gene expression in CMT-93 cells either expressing Smad6 (CMT-93-pU6PL) or stably expressing Smad6 siRNA (CMT-93-siS6). Actin was used for the loading control. The insets show representative results of at least three independent immunoblotting experiments.