Erythropoietin-derived Non-erythropoietic Peptides Conferring Oxidative Stress Resistance to Keratinocytes and Fibroblasts

Abstract

Erythropoietin (EPO) exerts tissue-protective effects; however, its erythropoietic activity limits broader use. Three EPO-derived peptides (ML1-C1/C2/C3) were designed from the C-helix of EPO to remove erythropoietic activity while retaining cell-protective activity. Circular dichroism and nuclear magnetic resonance spectroscopies were used to assess the solution structures of ML1-C1/C2/C3 peptides. The peptide activities for cytoprotection and growth support were assessed using skin-relevant cells, HaCaT cells and 3T3-L1 cells, which proposes an effect on skin epithelial keratinocytes and pre-adipocytic fibroblasts, respectively. Also, an erythroid-precursor cell line, TF-1, was used to evaluate the erythropoietic function of the three peptides. Spectroscopic analyses of ML1-C1/C2/C3 peptides revealed similar secondary structures and different flexibilities between the peptides. While ML1-C1 and ML1-C3 had highly flexible loop-like structures, ML1-C2 had less flexible loop-like structures. Also, their cellular effects vary in a cell type-dependent manner. The EPO-derived peptides can attenuate H2O2-induced loss of viability in HaCaT cells and 3T3-L1 cells. Under low-serum conditions, the three peptides promoted HaCaT proliferation, whereas only ML1-C1 improved 3T3-L1 proliferation. In TF-1 cells, none of the peptides increased cell viability or hemoglobin staining, whereas recombinant human EPO did, indicating the lack of erythropoietic activity of the peptides under experimental conditions. These findings support the potential of EPO-derived peptides as skin-protective agents and motivate future work for skin therapeutics or cosmetic purposes.