The effects of plasma gelsolin on human erythroblast maturation for erythrocyte production

Abstract

Gelsolin is an actin binding protein present in blood plasma and in cytoplasm of cells including macrophages. Gelsolin has important functions in cell cycle regulation, apoptotic regulation, and morphogenesis. Even though bone marrow macrophages and serum factors are critical for regulating erythropoiesis, the role of gelsolin on human erythroblasts has not been studied. Here, we investigated the effects of human recombinant plasma gelsolin (pGSN) on human immature erythroblasts. CD34+ cells isolated from cord blood were differentiated into erythroid cells in serum-free medium. When pGSN was applied to the culture medium, it accelerated basophilic and polychromatic erythroblast maturation and increased the enucleation rate with highly expressed erythropoiesis-related mRNAs. Also, pGSN was effective in reducing dysplastic changes caused by vincristine, suggesting its role in cell cycle progression at G2/M checkpoints. Also, pGSN activated caspase-3 during maturation stages in which caspase-3 functions as a non-apoptotic maturational signal or a pro-apoptotic signal depending on maturation stages. Our results suggest that pGSN has a pivotal role in maturation of erythroblasts and this factor might be one of the way how bone marrow macrophages and previously unknown serum factors work to control erythropoiesis. pGSN might be used as additive for in vitro production of erythrocytes.

CAP1, a target of miR‐144/451, negatively regulates erythroid differentiation and enucleation

“…In either case, high level expression of CAP1 at early erythroid differentiation may serve as a gatekeeper to prevent erythroblasts from premature enucleation via regulating actin dynamics. Notably, in addition to CAP1, a few proteins were identified to regulate erythroblast enucleation through modifying actin dynamics, including RAC1 50 and RAC2 GTPases, 45 dynein, 51 Gelsolin 52 and mDIA2 45 . It remains to be determined whether CAP1 interplays with these proteins to precisely regulate the actin dynamics of erythroblasts.…”

Role of Plasma Gelsolin Protein in the Final Stage of Erythropoiesis and in Correction of Erythroid Dysplasia In Vitro

“…Mononuclear cells were cultured in 2D culture plates and cytokines (erythropoietin 3 U/mL) were added to serum-free media (Stemline II hematopoietic stem cell expansion medium, Sigma) supplemented with 150 μg/mL holotransferrin (Sigma), 90 ng/mL ferric nitrate, 30.8 μM vitamin C, 160 μM 1-thioglycerol, 50 μg/mL insulin (Sigma), 4 μM L-glutamine, and 2 μg/mL cholesterol. The medium was changed every 2 days [ 16 , 62 ].…”

“…The plasmid was transformed into E. coli Rosetta2 (DE3) pLysS cells. Protein expression, and purification was performed as previously described [ 16 ].…”

“…For immunofluorescence staining, cells were treated as previously described [ 16 ] with His-Tag (D3I1O) XP ® Rabbit mAb (Alexa Fluor ® 647 conjugate; Cell Signaling, Beverly, MA, USA), Alexa Fluor 633–phalloidin (Thermo Fisher Scientific, Waltham, MA, USA), GPA (BioLegend, San Diego, CA, USA), and 4,6-diamidine-2-phenylindole dihydrocloride (DAPI).…”

“…We recently demonstrated that plasma gelsolin protein (pGSN) can enter immature erythroid cells at the stage of basophilic erythroblasts and reduces dysplastic changes by recovering the blocked G2/M cell cycle [ 16 ]. However, the roles of pGSN have not been known in late stage erythroblasts (polychromatic and orthochromatic erythroblasts) before enucleation.…”