Canadian Medical News

Like other fibroblasts, cultured NIH-3T3 fibroblasts produce fibronectin in a constitutive fashion, and we have previously demonstrated that nicotine greatly enhances fibronectin expression in these cells. To examine the effects of FP on fibronectin protein expression, NIH-3T3 fibroblasts were pretreated in the presence or absence of FP for 1 h, cultured with or without nicotine (50 pg/mL) for 48 h, followed by harvesting and processing for western blotting.

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FP pretreatment inhibited the production of fibronectin protein in both untreated and nicotine-stimulated fibroblasts. In addition, nicotine also stimulated an increase in fibronectin mRNA of > 2.5-fold (p < 0.03) as compared to untreated control cells. Preincubation of fibroblasts for 1 h with FP was associated with a significant reduction (p < 0.04) in fibronectin mRNA accumulation in nicotine-treated fibroblasts as determined by real-time PCR.

The observations described above suggested that FP might exert its effect by inhibiting fibronectin gene transcription. To test this, NIH-3T3 fibroblasts were stably transfected with pFN(1.2kb)LUC, a DNA construct containing the human fibronectin promoter fused to a luciferase reporter gene.

As expected, the transfected cells showed constitutive fibronectin gene transcription as determined by measuring luciferase activity. The transcription of the fibronectin gene was enhanced by treatment with nicotine alone . Of note, the exposure of fibroblasts to FP inhibited the transcription of the fibronectin gene at baseline and after nicotine stimulation .

The effect of FP pretreatment on nicotine-induced fibronectin gene transcription was exerted in a dose-dependent manner with maximum activity between 1 and 5 pmol/L FP . The same effect was observed in primary mouse lung fibroblasts containing pFN(1.2 kb)LUC, where FP pretreatment significantly inhibited transcription of the fibronectin gene after nicotine stimulation.

We and others have shown that fibronectin gene transcription is dependent on phosphorylation and DNA binding by the transcription factor CREB. Consequently, we postulated that FP might affect fibronectin expression by inhibiting CREB. Consistent with this idea, we found that nicotine-treated fibroblasts show increased CREB phosphorylation when compared to untreated fibroblasts. FP pretreatment diminished phosphorylated CREB after nicotine stimulation, whereas the amount of total CREB protein remained relatively unchanged.