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Growth regulation of primary rat tracheal epithelial cell cultures by endogenous transforming growth factor-beta s
1Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Abstract:
Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to secrete transforming growth factor-beta (TGF beta) and to be growth inhibited by exogenous TGF beta. The purpose of the present studies was to determine whether the endogenous TGF beta(s) were regulating the growth of RTE cell cultures and, if so, which isoforms were involved. Neutralizing antibodies specific to TGF beta 1 and TGF beta 2 were added to cultures, and their effects on several growth parameters were measured. Addition of antibodies to early cultures (day 1), resulted in 1.8- and 3-fold increases in colony formation and cell number, respectively, above control IgG-treated cultures. Antibody dose-response experiments revealed that TGF beta 2 was the predominant isoform inhibiting early RTE cell growth. The antibody treatments resulted in similar stimulation of early growth at low and high seeding densities, suggesting that the endogenous TGF beta s were acting locally. Anti-TGF beta 1 treatment of cultures at various stages of growth resulted in 1.2-1.7-fold increases in DNA synthesis above controls, whereas anti-TGF beta 2 treatment resulted in increased DNA synthesis only in early and late cultures (1.7- and 2.5-fold, respectively), but not during midlogarithmic growth. Continuous treatment with a combination of both antibodies resulted in increased growth and decreased exfoliation in early cultures, but had no effect on the slow down of growth in late cultures. Thus endogenous TGF beta s inhibited primarily early growth and contributed to, but did not appear to be responsible for, plateau of growth in late stage cultures. Antibody treatment of secondary cultures resulted in 4-70-fold increases in colony formation, depending on the age of the primary cultures when replated, indicating that endogenous production of both TGF beta 1 and TGF beta 2 greatly inhibits the subculturability of primary RTE cells. Other experiments suggested that cholera toxin enhances RTE cell growth in part by counteracting the inhibitory effects of endogenous TGF beta s.
Insights
Endogenous transforming growth factor-beta (TGF-β) isoforms, particularly TGF-β2, inhibit primary rat tracheal epithelial (RTE) cell growth and subculturability. Counteracting TGF-β effects enhances RTE cell proliferation and colony formation.
Area of Science:
- Cell Biology
- Epithelial Cell Biology
- Growth Factor Signaling
Background:
- Primary rat tracheal epithelial (RTE) cells secrete transforming growth factor-beta (TGF-β) and are inhibited by exogenous TGF-β.
- The role of endogenous TGF-β in regulating RTE cell growth and proliferation is not fully understood.
Purpose of the Study:
- To determine if endogenous TGF-β regulates RTE cell growth.
- To identify which TGF-β isoforms are involved in this regulation.
Main Methods:
- Neutralizing antibodies specific to TGF-β1 and TGF-β2 were used to block endogenous TGF-β activity in RTE cell cultures.
- Growth parameters including colony formation, cell number, DNA synthesis, and exfoliation were measured.
- Effects were assessed at different culture stages (early, midlogarithmic, late) and seeding densities.
Main Results:
- Anti-TGF-β antibodies significantly increased colony formation and cell number in early cultures, with TGF-β2 identified as the predominant inhibitory isoform.
- Anti-TGF-β1 increased DNA synthesis across various stages, while anti-TGF-β2 primarily affected early and late cultures.
- Endogenous TGF-β isoforms were found to inhibit early growth and contribute to growth plateau in late cultures.
- Antibody treatment of secondary cultures showed substantial increases in colony formation, indicating TGF-β inhibits subculturability.
Conclusions:
- Endogenous TGF-β, predominantly TGF-β2, plays a significant role in inhibiting early RTE cell growth and subculturability.
- TGF-β contributes to, but is not solely responsible for, the growth slowdown observed in late-stage cultures.
- Cholera toxin may enhance RTE cell growth by counteracting endogenous TGF-β inhibition.