Regulation of cell cycle progression in astrocytes by tumor necrosis factor-alpha
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Astrocytes that are quiescent in the cell division cycle can react to brain injury or disease by cell cycle re-entry and active proliferation, with eventual formation of a neuroinhibitory gliotic scar, the mechanisms of which remain unclear. The central hypothesis of this project was that astrocyte progression through G1 and G1/S transition is shortened by inflammatory mediators. To better understand mechanisms regulating astrocyte cell cycle progression this project examined synchronized astrocytes through G1 and the G1/S transition, the effects of LPS and TNF-α, and expression of cell division regulators. Results include a G1/S phase synchrony curve of DNA synthesis that established the G1/S transition at approximately 12 hours following G1 re-entry, utilizing incorporation into DNA. LPS-stimulated synchronized astrocytes exhibited greatest DNA synthesis 24 hours following G1 re-entry at [100 pg/ml]. LPS (100 pg/ml) shifted the DNA synthesis kinetic curve up and left with significant differences at later time points, which were significantly reduced by anti-TNF-α antibody, determined by tritiated-thymidine incorporation. TNF-α activity in culture medium increased mid G1 then returned to baseline upon entering S phase; whereas LPS stimulation produced the greatest increase in TNF-α activity 24 hours following G1 re-entry at [1 μg/ml] and continual increases in activity over the 24 hours following G1 re-entry, determined utilizing a TNF-α-sensitive cytolytic assay. Intracellular and released TNF-α were greater in long-term LPS-stimulated versus non-LPS-stimulated cultures, by Western blot analysis. GFAP expression increased minimally over the 24 hours following G1 re-entry, by Western blot, though were >85% GFAP-immunofluorescent by day 4. Recombinant TNF-α increased BrdU-positive S phase nuclei, and had no effect on GFAP, 24 hours following G1 re-entry utilizing immunocytochemistry. Cyclin D1 expression increased during G1/S, peaked late S phase, and degraded by 36 hours following G1 re-entry, while incubation with rTNF-α shifted the cyclin D1 kinetic curve left by the end of 36 hours, determined by Western blot. These results provide important information about the astrocyte cell cycle and inflammatory mediators under controlled culture conditions to better understand astrocyte cell cycle progression. Understanding regulators of astrocyte proliferation may provide strategies to minimize damage associated with brain injury or disease.