OSM induces an increased rate of TF gene transcription.
The transcription rate of the TF gene was assessed by nuclear run-off assays. (A) Nuclear extracts were harvested from SMCs treated with or without OSM (10 ng/mL) for 1 hour. Equal amounts of32P-labeled in vitro transcribed RNA probes were hybridized to 5 μg of denatured TF, G3PDH, and pGEM-4Z cDNA. Results are representative of 2 separate experiments. (B) Activation of TF promoter by OSM was analyzed by studying the effect of OSM on TF gene promoter fused to luciferase. pTF(−2106)LUC, which contains 2.1 kilobase (kb) of the 5′ flanking region of the TF gene promoter, and pSV–β-galactosidase control vector were transfected into SMCs. The cells were treated with OSM (10 ng/mL) for 5 hours before harvesting. Luciferase activity was determined and normalized for β-galactosidase activity. Results are expressed as mean ± SEM (n = 4 experiments). *P < .05 compared to unstimulated SMCs. (C) SMCs were exposed to vehicle (control, ■) or OSM (10 ng/mL, ▪ and ●) for 1 or 24 hours before addition of actinomycin D (5 μg/mL). Total RNA was extracted at the indicated times after addition of actinomycin D. Northern blots were performed and probed with TF and G3PDH. The signal density of each RNA sample hybridized to TF was divided by that hybridized to the G3PDH. The corrected density was then plotted as a percentage of the 0-hour value (log scale) against time. Results are representative of 3 experiments.
