Incorporation of Ortho- and Meta-Tyrosine Into Cellular Proteins Leads to Erythropoietin-Resistance in an Erythroid Cell Line

Abstract

Background/Aims: Erythropoietin-resistance is an unsolved concern in the treatment of renal anaemia. We aimed to investigate the possible role of ortho- and meta-tyrosine - the hydroxyl free radical products of L-phenylalanine - in the development of erythropoietin-resistance. Methods: TF-1 erythroblast cell line was used. Cell concentration was determined on day 1; 2 and 3 by two independent observers simultaneously in Bürker cell counting chambers. Protein concentration was determined with colorimetric method. Para-, ortho- and meta-tyrosine levels were measured using reverse phase-HPLC with fluorescence detection. Using Western blot method activating phosphorylation of STAT5 and ERK1/2 were investigated. Results: We found a time- and concentration-dependent decrease of erythropoietin-induced proliferative activity in case of ortho- and meta-tyrosine treated TF-1 erythroblasts, compared to the para-tyrosine cultured cells. Decreased erythropoietin-response could be regained with a competitive dose of para-tyrosine. Proteins of erythroblasts treated by ortho- or meta-tyrosine had lower para-tyrosine and higher ortho- or meta-tyrosine content. Activating phosphorylation of ERK and STAT5 due to erythropoietin was practically prevented by ortho- or meta-tyrosine treatment. Conclusion: According to this study elevated ortho- and meta-tyrosine content of erythroblasts may lead to the dysfunction of intracellular signaling, resulting in erythropoietin-hyporesponsiveness.

Role of Tyrosine Isomers in Acute and Chronic Diseases Leading to Oxidative Stress - A Review

“…In the background, incorporation of o-Tyr into erythroid precursors and inhibition of proliferative signaling may by present, this has indeed been verified by a further study of our group, where supplementation of m- and o-Tyr to the media of erhythroblasts lead to the incorporation of these abnormal amino acids into cellular proteins and to a nearly 50% inhibition of cell proliferation [78]. …”

“…p-Tyr, on the other hand, when administered in an 2.6x or 1.8 abundance to media containing m- or o-Tyr, respectively, could restore proliferation [78]. This suggests a subacute and reversible effect of m- and o-Tyr, that seems to be competitively inhibitable using p-Tyr.…”

Roles of the tyrosine isomers meta- tyrosine and ortho- tyrosine in oxidative stress

“…The addition of 110 μM of either m - or o -tyrosine to the culture media of TF-1 erythroblasts significantly reduced erythropoietin-induced cell proliferation. This effect is likely mediated by the uptake of these isomers into cells because supplementing the culture with additional p -tyrosine counteracted this proliferative decline (Mikolas et al, 2013). …”

“…Mikolás et al reported similar results when TF-1 erythroblasts were incubated with either m - or o -tyrosine (110 μM). Treatment with tyrosine isomers inhibited the usual phosphorylation of the ERK1/2 and STAT5 proteins after stimulation with erythropoietin (Mikolas et al, 2013). It is worthwhile to note that the cell lines used for both studies have inherently high proliferative potential, and the reduction of mitogenic signaling may be characteristic only of the effects of m - and o -tyrosine on these types of cells.…”

“…Experiments testing whether phenylalanine hydroxylase is repressed in response to oxidative stress have not been conducted. It should, however, be noted that in a few studies supplementation of p -tyrosine limited the adverse effects of the tyrosine isomers (Mikolas et al, 2013; Molnar et al, 2016; Selley et al, 2015). While an explanation for this is unclear, it could contradict the rationale that reducing phenylalanine hydroxylase activity may also be protective.…”