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W. J. O'Brien, T. Heimann, F. Rizvi, D. Conklyn; TIAM1/Rac/ERK Signal Transduction in Cell Survival of Serum Deprived/Cytokine Treated Rabbit Corneal Stromal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6207.
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These studies investigated the hypothesis that the Rac 1 activator, TIAM1, regulates the survival of rabbit corneal stromal (RCS) fibroblasts by controlling ERK phosphorylation.
RCS cells were grown as fibroblasts in DMEM containing 5% FBS and Mito+ serum extender. Cells were induced to undergo apoptosis by serum deprivation and were rescued from apoptosis by treatment with a mixture of IL-1β, TNF-α and rRaIFN-γ. Apoptosis was assessed by caspase 3 activity and TUNEL assays. Expression of TIAM1, Rac 1 and ERK as well as ERK phosphorylation were assessed by western blotting. Western blots were evaluated by densitometry and standardized relative actin. Amounts of TIAM1 and Rac mRNAs were evaluated by real time RT-PCR.
The addition of 5 to 10 µM PD98059, an inhibitor of MEK phosphorylation or U0126, an inhibitor of MEK activity significantly induced caspase 3 activity by 6 hr post-treatment (n =6, p<0.001). Both of these inhibitors significantly reversed these anti-apoptotic activity of the cytokine mixture as measured by caspase 3 activity (n=6, p< 0.001). The phosphorylation of ERK 1 and 2 was significantly enhanced during the first 2 hr following the addition of cytokines to serum deprived cells (n =3, p< 0.05). By 4 hr post-cytokine treatment the steady state levels of phosphorylated ERK 1 and 2 in serum starved/non-cytokine treated cells relative to amounts in cytokine treated cells were similar. During the initial 6 hrs post-serum deprivation both real time RT PCR and western blots indicated a transient increase in steady state levels of TIAM1, a GEF, that activates Rac1(n=3 p< 0.001). After 6 hr post-serum deprivation the levels of TIAM1 decreased to barely detectable. The turnover of TIAM1 appeared to be mediated by caspase 3 as assessed by a 75KDa degradation product known to be produced by the action of caspase 3 on TIAM1. The steady state levels of Rac 1 detected in the membrane fractions of cytokine treated serum deprived cells increased relative to serum starved cells that were not cytokine treated as documented in western blots.
The data indicate that the survival of RCS fibroblast following the stress of serum/growth factor deprivation was ERK 1 and 2 dependent. After the addition of cytokines rapid phosphorylation of ERK occured, steady state levels of TIAM1 and the membrane localization of Rac1 increased. These data suggest that a TIAM1/Rac1/ERK pathway may function in enhancing the survival of corneal stromal fibroblasts during inflammation.
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