Can aqueous humor protein levels activate signal transduction pathways in human trabecular meshwork cells?
Purpose: Since the protein content of aqueous humor (AH) can vary, we investigated the effect of changing serum levels on human trabecular meshwork (HTM) cells. Methods: Cultured HTM cells were shifted from 20% to 0% fetal bovine serum. After 24 hours, the levels of the most prominent proteins phosphorylated on tyrosine (P-Y) residues, ccB-crystallin, and c-fos were estimated by chemiluminescent western blot analysis. The level of c-fos mRNA was determined by quantitative PCR and the activity of MAP kinase was measured by the phosphorylation of myelin basic protein. Results: Surprisingly, little change was noted in the levels of the P-Y on the major proteins in the HTM cells when the serum was reduced to 0%. This result was in sharp contrast to the 18 fold increase in the aB-crystallin, a small heat shock protein. The activity of the MAP kinase, an enzyme implicated in the signal transduction pathway, increased 3.3 fold. The mRNA of c-fos, an immediate early protein, increased about 4.5 fold and this was mirrored in the level of protein detected by western blot (5.6 fold). Conclusions: The observed increase in the aB-crystallin indicated that there is substantial stress to a cultured cell that is deprived of serum. The increase in the MAP kinase activity suggests that there is an activation of a signal transduction pathway as a result of the shift in serum level, and this is consistent with the changes observed in the c-fos. The unusual feature in these results is the apparent lack of change in the levels P-Y on the major proteins in the HTM cells. Our results point to an activation of a signa] transduction pathway that may be of physiological importance to HTM when AH protein content is altered.
Russell, P; Tumminia, SJ; Mitton, KP; Arora, J; Epstein, DL
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