We are exploring the role of transforming growth factors alpha and beta (TGFalpha, TGFbeta) in the regulation of growth of normal and transformed airway epithelial cells. Normal rat tracheal epithelial cells (RTE) highly sensitive to the growth inhibitory effects of TGFbeta treatment early in culture but become nearly unresponsive late in culture. However, studies with TGFbeta1 and TGFbeta2 neutralizing antibodies indicate that DNA synthesis in both early and late cultures is negatively affected by the autocrine TGFbeta secretion. Cultured normal as well as transformed RTE cells, express 3 different TGFbeta1 transcripts, 2.5kb, 1.9kb and 1.4kb in size, respectively. In contrast, A549 cells, a human lung cancer cell line expresses only the 2.5kb TGFbeta mRNA. Since the 1.4kb transcript had previously not been described, we decided to investigate it's structure, regulation and function. Northern analysis, using TGFbeta subclones, indicates that the 1.4kb transcript contains the complete TGFbeta coding sequence. PCR cloning of the 3'end of the molecule suggests that it contains the same sequence as the 2.5kb transcript. The 1.4kb transcript is not detectable on northern blots of intact adult tracheas but is expressed during tracheal regeneration in vivo. The small TGFbeta transcript may be more readily translated and may play a role in regeneration and wound healing. Normal RTE cells are highly epidermal growth factor (EGF) dependent; in contrast, most transformed cell variants are not. Both normal and transformed RTE cells secrete TGFalpha, a functional equivalent of EGF, which binds to the EGF receptor. Studies with neutralizing TGFalpha antibody, with the EGFR tyrosine kinase inhibitor Tyrphostin 8 and with TGFalpha antisense oligonucleotides indicate that both normal and transformed RTE cells are at least partially dependent on autocrine TGFalpha for growth. Normal cells drastically down regulate TGFalpha production in late stages of growth while transformed cells do so only to a small extent. Interestingly, transformed cells show reduced levels of EGFR message in northern analysis but express significantly increased numbers of EGF binding sites at the level of the cell membrane. These studies are being continued since functional abnormalities of the EGFR could play a major role in the EGF independence typical of many transformed RTE cell variants.
