Changes in responsiveness of rat tracheal epithelial cells to transforming growth factor‐β1 with time in culture

Abstract

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor-beta 1 (TGF beta 1) when treated within 1-2 days after plating. The purpose of the present studies was to examine the effects of TGF beta 1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGF beta 1 decreased dramatically as the cultures aged. The IC50 for inhibition of colony forming efficiency was 0.18 pM when TGF beta 1 was added 24 h after cell plating. When TGF beta 1 treatment was begun on day 5 of culture, the IC50 was 3-4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGF beta 1 was begun on day 19, the IC50 was 65 pM or greater than 500 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGF beta 1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGF beta 1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGF beta 1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGF beta 1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGF beta 1 down-regulated the expression of two proliferation-related genes, c-myc and transforming growth factor-alpha, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGF beta 1 by about twofold at both early and late times in culture. This indicates that the changes in TGF beta 1 responsiveness with time in culture are selective, apparently affecting primarily proliferation-related events.

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

“…Treatment with 10 pM TGF[3~ for 24 h resulted in a 40% inhibition of DNA synthesis. This is similar to the result of Rundhaug et al (1992) who reported that in 5-d-old cultures of RTE cells grown on plastic, TGF[3~ inhibited DNA synthesis with an ICso of 3-4 pM. Thus, RTE cells are sensitive to TGFl3~-induced growth arrest whether cultured submerged on plastic or at an ALI on a collagen gel.…”

“…Previous studies have demonstrated that primary-RTE cells in monolayer culture produce and respond to TGFI3s (Rundhaug et al, 1992;Rundhaug and Nettesheim, 1993). In these experiments, TGFI3s inhibited DNA synthesis and cell growth, and also increased terminal (squamous) differentiation (Rundhaug et al, 1992). TGF]3~ also induced squamous differentiation in cultures of human (Masui et al, 1986) and rabbit (Jetten et ah, 1986) tracheobronchial cells.…”

“…This suggested that growth arrest may be causally linked to differentiation into ciliated cells. TGF[3 treatment of RTE cells grown as a monolayer on plastic was previously shown to inhibit DNA synthesis and also to increase terminal differentiation, as measured by formation of cornified envelopes (Rundhaug et al, 1992). In vivo, the ciliated cell is believed to be the terminally differentiated cell type of the airways (Harkema et al, 1991;Randell, 1992).…”

“…Following administration of retinoic acid to vitamin A deficient rats, TGF[3 immunoreactivity was again observed, first in areas of squamous metaplasia and then in newly differentiated ciliated cells (Glick et al, 1991). Previous studies have demonstrated that primary-RTE cells in monolayer culture produce and respond to TGFI3s (Rundhaug et al, 1992;Rundhaug and Nettesheim, 1993). In these experiments, TGFI3s inhibited DNA synthesis and cell growth, and also increased terminal (squamous) differentiation (Rundhaug et al, 1992).…”

Growth regulation of primary rat tracheal epithelial cell cultures by endogenous transforming growth factor‐βs

“…Cells were then plated at the indicated densities in 2 mliwell in 12-well dishes (secondary cultures). Since we knew the replating efficiency of RTE cells decreases with time in primary culture Rundhaug et al, 1992), the seeding density of the replated cells was adjusted according to the age of the primaries in order to obtain a minimum of 1-2 colonieskulture in the controls. The day after replating, the media were changed (1 ml/well) and antibodiesiIgGs added.…”