Dysregulation of autocrine TGF-β isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts

Abstract

This study examined the autocrine production of TGF-β1, -β2 and -β3 in culture supernatants from tumour-derived (H series, n = 7; BICR series, n = 5), Ha-ras-transfected (n = 4) and normal (n = 2) human keratinocytes using a sandwich enzyme-linked immunosorbent assay (ELISA). Detection limits were 39.0 pg ml-1 for TGF-β1, 78.0 pg ml-1 for TGF-β2 and 1.9 ng ml-1 for TGF-β3. Tumour-derived oral keratinocytes predominantly produced less TGF-β1 than normal oral epithelial cells; the expression of endogenous TGF-β2 was variable. In keratinocytes containing mutant Ha-ras, TGF-β1 production was enhanced and TGF-β2 was undetectable. TGF-β3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) but the protein was not detected in conditioned media, most probably because of the low detection limits of the ELISA for this isoform. Neutralisation experiments indicated that the latent TGF-β peptide was secreted in keratinocyte conditioned medium. Seven tumour-derived keratinocyte cell lines (H series) and fibroblasts separated from normal (n = 1) and tumour-derived (n = 2) keratinocyte cultures were examined for their response to exogenous TGF-β1, -β2 and -β3. Six of seven tumour-derived keratinocyte cell lines were inhibited by TGF-β1 and TGF-β2 (-β1 > -β2); one cell line was refractory to both TGF-β1 and TGF-β2. Keratinocytes were inhibited (4 of 7), stimulated (1 of 7) or failed to respond (2 of 7) to TGF-β3. TGF-β1, -β2 and -β3 stimulated both normal and tumour-associated fibroblasts, bur the tumour-associated fibroblasts showed less response to the ligands than their normal counterparts following prolonged treatment with each isoform. The results demonstrate variable autocrine production of TGF-β isoforms by malignant keratinocytes, with loss of TGF-β1 generally associated with the tumour-derived phenotype and modification of endogenous isoform production dependent on the genetic background of the tumour cells. Further, the variable response of the tumour-derived keratinocytes and contiguous fibroblasts to the TGF-β isoforms suggests that dysregulation of TGF-β autocrine and paracrine networks are common characteristics of squamous epithelial malignancy.