Effects of Transforming Growth Factor-βl and Phorbol Ester on PALI-1 and PA Genes in Human Lung Cells

Abstract

Transforming growth factor-β (TGF-β) mediates the production of extracellular matrix proteins, proteases and protease inhibitors in epithelial cells. Both TGF-β and phorbol-12-myristate-13-acetate (PMA) exert both positive and negative effects on mitogenesis in these as well as other cell types. Phorbol esters act through stimulation of protein kinase C (PKC) and are among the most potent tumor promoters known. The present study was conducted to determine whether the effect of TGF-β in human non-small cell lung cancer (NSCLC) and normal human bronchial epithelial (NHBE) cells parallels that of the phorbol esters and whether this effect of TGF-β involves PKC. TGF-β1 and PMA increased expression of TGF-βl mRNA 24 hr after their addition to both NSCLC and NHBE cells. The effects of these agents on expression of the mRNAs for TGF-β2 and TGF-β3 were more complex; while TGF-β2 and TGF-p3β mRNAs increased transiently in response to TGF-p1 in NHBE cells and TGF-β3 mRNA increased transiently in some NSCLC cells, expression of these mRNAs decreased in most of these cells in response to PMA with the exception of the carcinoid NCLH727 where TGF-pZ mRNA increased dramatically. TGF-β1 and PMA both caused a persistent increase in expression of the mRNAs for both plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA) up to 24 hr in most NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. In contmst, while TGF-βl also increased expression of PAI-1 mRNA in NHBE cells, the expression of PA mRNA decreased simultaneously. The effect of PMA on PAI-1 and PA mRNAs was opposite of TGF-β1 in these cells, with expression of PAI-1 mRNA decreasing and PA mRNA increasing after addition of PMA. These data show that there is parallel regulation of the genes for TGF-βl, PAI-1 and PA by TGF-β1 and PMA in NSCLC, but differential regulation of the genes for PAL1 and PA by these agents in NHBE cells. The responses of the mRNAs and proteins of TGF-β1, PAI-1 and PA to TGF-βl and PMA were inhibited by the serind threonine kinase inhibitor H7 in NSCLC cells. Treatment of NSCLC cells with TGF-β1 and PMA resulted in a persistent increase in the expression of fibronectin mRNA and protein. This response was blocked by the addition of H7. Inhibition of these effects by H7 in NSCLC cells suggests that H7 blocks TGF-p responses by inhibiting a protein serindthmnine kinase(s). Because the effects of TGF-p and PMA on the different TGF-p isoforms, PA, PA1 and fibronectin in NHBE and NSCLC cells are complex, our data suggest that there are distinct mechanisms for controlling the different TGF-p isoforms, PA, PA1 and extracellular matrix proteins in normal lung and lung cancer cells.