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The postnatal age of rat lung fibroblasts influences G1/S phase transition in vitro
1Department of Pediatrics, University of Kentucky Medical School, Lexington 40536, USA.
Insights
Neonatal rat lung fibroblast proliferation peaks around postnatal Day 4, with significant age-dependent differences observed in cell cycle regulation and cyclin E levels, impacting alveolar development.
Area of Science:
- Cell Biology
- Developmental Biology
- Pulmonary Research
Background:
- Alveolar development in neonatal rats occurs between postnatal Days 4-13, involving a significant increase in interstitial fibroblasts.
- Understanding factors that regulate fibroblast proliferation during septal elongation is crucial but challenging due to in vivo study limitations.
Purpose of the Study:
- To investigate the age-dependent proliferation rates of neonatal rat lung fibroblasts.
- To characterize the cell cycle kinetics and identify molecular regulators of fibroblast proliferation during lung development.
Main Methods:
- Flow cytometry was used to analyze DNA content and cell cycle phases (S, G2/M) of freshly isolated and in vitro cultured lung fibroblasts from rats of different postnatal ages.
- 3H-thymidine incorporation assays assessed fibroblast proliferation rates.
- Serum starvation and release protocols synchronized cells for cell cycle transit time analysis.
- Western blotting or similar techniques were used to measure cyclin E levels.
Main Results:
- Fibroblast proliferation, measured by S plus G2/M phase percentages, peaked on postnatal Day 4 and decreased by Days 9-10.
- In vitro proliferation rates mirrored in vivo findings, with fibroblasts from 4-5-day-old pups showing higher proliferation and 3H-thymidine incorporation.
- Cells from 5-day-old pups exhibited faster entry into S phase and higher cyclin E levels, indicating enhanced G1/S transition.
- Age-dependent differences in proliferation were linked to events preceding S-phase entry, as hydroxyurea treatment abolished these variations.
Conclusions:
- Neonatal rat lung fibroblast proliferation is highest in early postnatal life (around Day 4-5) and declines significantly thereafter.
- The proliferative capacity observed in vivo is maintained in vitro, providing a reliable model for studying fibroblast behavior.
- Age-specific differences in cell cycle regulation, particularly at the G1/S transition, are critical determinants of lung fibroblast proliferation during development.
- These findings provide insights into the cellular mechanisms governing lung alveolarization and identify potential targets for therapeutic interventions.
Abstract:
In the neonatal rat lung, alveolar development occurs from postnatal Days 4-13, during which time there is a fourfold increase in interstitial fibroblasts. Factors influencing emergence of new septa and cell proliferation associated with septal elongation have yet to be identified, in part because of difficulties inherent in studying this process in vivo. Using flow cytometric analysis of the DNA content of freshly isolated lung fibroblasts, we found that proliferation, as indicated by the percentage of cells in S plus G2/M phases, peaked on postnatal Day 4 (P < 0.04). By Days 9-10 the proliferation rate was lower than on Days 3, 4, 5, or 6 (P < 0.005). We then evaluated rates of in vitro proliferation as a function of postnatal age in first passage fibroblasts and found that the proliferative phenotype expressed in vivo persists in vitro. Fibroblasts from 4-5-d-old pups increased in number and incorporated 3H-thymidine at a faster rate than did fibroblasts obtained from pups at other postnatal ages (P < 0.0001). Age-dependent differences in cell cycle transit time were compared in fibroblasts synchronized by serum starvation and analyzed by flow cytometry at 2-h intervals from 13-21 h after release from serum starvation. A greater percentage of cells from 5-d-old pups entered S phase during this period than was seen for cells obtained from 2-, 9-, 13-, or 23-d-old rat pups (P = 0.0001). Cells from 5-, 9-, and 13-d-old pups reentered G0/G1 by 21 h after release from serum starvation, in contrast to fibroblasts from 2- and 23-d-old rats which did not. Throughout the 15-h period after release from serum starvation, levels of cyclin E, which peaks at the G1/S border, were highest in the 5-d-old cells (P < 0.025). Synchronization with 2.5 mM hydroxyurea which inhibits DNA synthesis completely abolished age-related differences in cell cycle transit time, implying that age-dependent differences in lung fibroblast proliferation rates are the result of events occurring before S-phase entry.