Transforming growth factor-β signaling enhancement by long-term exposure to hypoxia in a tumor microenvironment composed of Lewis lung carcinoma cells.

Transforming growth factor-β (TGF-β) is a potent growth inhibitor in normal epithelial cells. However, a number of malignant tumors produce excessive amounts of TGF-β, which affects the tumor-associated microenvironment by furthering the progression of tumorigenicity. Although it is known that the tumor-associated microenvironment often becomes hypoxic, how hypoxia influences TGF-β signaling in this microenvironment is unknown. We investigated whether TGF-β signaling is influenced by long-term exposure to hypoxia in Lewis lung carcinoma (LLC) cells. When the cells were exposed to hypoxia for more than 10 days, their morphology was remarkably changed to a spindle shape, and TGF-β-induced Smad2 phosphorylation was enhanced. Concomitantly, TGF-β-induced transcriptional activity was augmented under hypoxia, although TGF-β did not influence the activity of a hypoxia-responsive reporter. Consistently, hypoxia influenced the expression of several TGF-β target genes. Interestingly, the expressions of TGF-β type I receptor (TβRI), also termed activin receptor like kinase-5 (ALK5), and TGF-β1 were increased under the hypoxic condition. When we monitored the hypoxia-inducible factor-1 (HIF-1) transcriptional activity by use of green fluorescent protein governed by the hypoxia-responsive element in LLC cells transplanted into mice, TGF-β-induced Smad2 phosphorylation was upregulated in vivo. Our results demonstrate that long-term exposure to hypoxia might alter responsiveness to TGF-β signaling and affected the malignancy of LLC cells.