Figure 5.

Regulation of TNW transcription by TGFβ1 and glucocorticoids. (a) The Smad4‐binding site (gcctAGACagg) was mutated by site‐directed mutagenesis and replaced with the scrambled sequence (Scr‐AGAGTGAT). Construct −79/+77 containing the normal (SMAD4) or modified Smad4 sequence (Scr‐control) was transfected in HT1080 cells and analyzed for SEAP activity normalized to the empty pSEAP as described in Fig. 3 b. (b) BMSCs were treated with SB‐431542‐DMSO (10 µM) or DMSO only for 1 h before the addition of TGFβ1 (5 ng/ml) for 24 h. TNW transcript levels were then analyzed by qRT‐PCR in comparison to the endogenous TBP. Relative mRNA levels were normalized to the nontreated control cells using the ΔΔCt method. (c) Constructs with the three intronic regions (I/II/III) cloned upstream the minimal TNW promoter (−79 bp/+77) were transfected in HT1080 cells in medium containing 3% FBS (light green bars) or glucocorticoid‐depleted 3% DCC‐FBS (dark green bars) for 24 hr. SEAP activity is shown in arbitrary units (a.u.) normalized to a co‐transfected secreted luciferase plasmid revealing that the intronic conserved region I inhibited the reporter activity in a glucocorticoid‐dependent manner. (d) BMSCs were cultured in untreated 3% FCS (light green bar) or in the presence of 3% charcoal/dextran‐treated FBS for 24 hr before measuring transcript levels of TNW by qRT‐PCR in comparison to the endogenous TBP. Relative mRNA levels were normalized to the cells grown in 3% FCS using the ΔΔCt method. All experiments were repeated at least three times (error bars = SEM). (e) Schematic model of the TNW gene regulation by a SMAD4 element preceding a TATA box in the proximal promoter region upstream of exon 1 (red box 1) and a negative glucocorticoid‐response element (nGRE) in the first conserved region within the first intron (green oval I). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]