Fig 1.
MCR expressions during erythroblast differentiation.
(A) Schematic diagram of an in vitro differentiation protocol for deriving erythroblasts from human HPCs. Human CD34+ HPCs were expanded for 7 days (E0–E7) and stocks were frozen in liquid nitrogen (LN2). The stock cells were differentiated for 3 days (D0–D3) before undergoing maturation (M0–M7). The number of enucleated erythrocytes increased from M5 to M7. ACTH, adrenocorticotropic hormone; EPO, erythropoietin; FL, flt-3 ligand; IL-3, interleukin-3; IL-6, interleukin-6; α-MSH, α-melanocyte stimulating hormone; SCF, stem cell factor; TPO, thrombopoietin. (B) May-Grunwald-Giemsa staining of control erythroblasts at day M0–5 day. Cells differentiated to Pro-E stage at day M0 and Baso-E stage at day M3. Arrows, Poly-E; Arrow heads, Orho-E. *, Reticulocyte. Bar 10 μm. (C) Conventional RT-PCR for MCRs during erythroblasts differentiation between M0 and M6 day.
Fig 2.
ACTH enhances enucleation of erythroblasts derived from human HPCs.
(A) The neutralization of melanocortins by addition of the neutralising antibodies (nAbs) for ACTH and α-MSH into culture media on the M0 day and the M3 day. The enucleation rate on the M5 day was measured. *, P < 0.01. n = 3; error bars, s.e.m. (B) Cell number between M0 and M4 day. ACTH39 has no significant effect on the proliferation of erythroblasts. n = 3; error bars, s.e.m. (C) The representative images of definitive erythroid differentiation induced by melanocortins on the M5 day. The glycophorin A (GPA)-positive cells without nuclei are increased in number by treatment with 10 nM ACTH39. Control, vehicle (0.1% BSA); ACTH39, 10 nM ACTH39; GPA, APC (red); nuclei, Hoechst33342 (blue). White arrows, enucleated cells; White arrowheads, enucleating cells; Green arrows, normal erythroblasts; Green arrowheads, excreted nuclei. Bar, 20 μm. The enucleation rates in definitive erythroid cells by treatments with (D) ACTH39, (E) ACTH24, and (F) α-MSH between the M5 and M7 days. All melanocortins showed effects on the induction of enucleation by erythroblasts. *, P < 0.01; **, P < 0.001; ***, P < 0.0001, each point versus control in the same day. n = 3; error bars, s.e.m.
Fig 3.
Blocking of MC1R, MC2R or MC5R causes a decrease in the enucleation ratio of erythrocytes.
(A) MC1–5R nAb were added to the culture medium on M0 and M3, and the enucleation ratio was analyzed at M7. The enucleation ratio was decreased in the erythroblasts treated with anti-MC1R nAb, anti-MC2R nAb or anti-MC5R nAb. *, P < 0.0001, each point versus control. n = 3; error bars, s.e.m. (B) The number of erythroblasts at the polychromatic erythroblast stage, the basophilic stage and the orthochromatic stage increase after treatment with the anti-MC1R nAb, anti-MC2R nAb and anti-MC5R nAb, respectively. Baso, basophilic erythroblasts; Poly, polychromatic erythroblasts; Ortho, orthochromatic erythroblasts; Re, reticulocyte. *, P < 0.001. Values are compared with cells treated with control IgG. (C) The effects of MC1R, MC2R or MC5R nAbs on erythroblast proliferation at M5 day. Cell number is significantly decreased after treatment with anti-MC2R nAb for 5 days; however, treatment with anti-MC1R nAb or anti-MC5R nAb does not have a significant effect (n = 3, ANOVA). Error bars, s.e.m.
Fig 4.
MCRs differentially regulate EPO-induced phosphorylation of ERK, STAT5 and AKT in erythroblasts during differentiation.
(A) Phosphorylation of ERK, STAT and AKT in erythroblasts. Erythroblasts at M0, M3 and M6 were starved for 3 h in HPGM without cofactors and subsequently incubated for 15 min with or without EPO. (B)–(E), Synergistic effects on EPO downstream signaling of ACTH, and the signal inhibition by nAbs. After starvation with HPGM without cofactors for 3 h, the cells were incubated for 15 min with nAbs and reacted with EPO for 15 min. EPO-induced phosphorylation of ERK is not altered by the addition of 0.1 nM ACTH39 to erythroblasts at M0. n = 3; error bars, s.e.m (B). The phosphorylation of ERK is decreased by nAbs of MC2R and MC5R (C). ACTH39 enhances EPO-induced phosphorylation of STAT5 in erythroblasts at M3 (D). Phosphorylation of STAT5 is inhibited by nAbs of MC1R (E). (F) and (G), AKT phosphorylation by ACTH and, the signal inhibition by nAbs. Treatment with ACTH39 for 15 min without starvation causes phosphorylation of AKT in erythroblasts at M6 (F). The phosphorylation of AKT is inhibited by MC5R-nAb (G). EPO, 3 U/ml EPO; ACTH, 0.1 nM ACTH39. n = 3; error bars, s.e.m. IgG, 10 μg/ml normal IgG; MC1R, 10 μg/ml anti-MC1R nAb; MC2R, 5 μg/ml anti-MC2R nAb; MC5R, 10 μg/ml anti-MC5R nAb. n = 3; error bars, s.e.sm.
Fig 5.
The MC5R signal is required for enucleation.
(A) MC5R-positive cell ratio during maturation. The percentage of MC5R-positive cells is increased between M1 and M5 day by flow cytometric analysis. n = 3; error bars, s.e.m. (B) and (C), Expression of MC5R increased by ACTH treatment. (B) Changes in MC5Rs mRNA expression are induced by treatment with 0.1 nM ACTH39 at M4. The expression levels MC5R is significantly elevated by ACTH39 treatment. The MCR expression levels in the ACTH39-treated groups represent the levels relative to the controls. n = 3; error bars, s.e.m. (C) The ratio of MC5R-positive cells increased by ACTH treatment. n = 3; error bars, s.e.m. (D)The addition of 0.1 nM ACTH39 (A39) and 5 nM ACTH24 (A24) causes phosphorylation of MLC2 in erythroblasts at M6 without starvation. (E) ACTH39-dependent phosphorylation is inhibited by anti-MC5R nAb. n = 3; error bars, s.e.m. (F) and (G), Confocal laser scanning microscopic images of enucleating and enucleated cells at M6. (F) Phosphorylated MLC2 co-localizes with MC5R at the plasma membrane (white arrows). Bar, 5 μm. (G) MC5R is localized on the membrane (arrowheads, in panel b, c, e) with actin. White arrows, MC5R co-localizes with an actin-accumulating locus (panel b, c, e). Panel f or g is an enlarged image from I or II of panel e, respectively. Panel f, MC5R co-localizes with the contractile actomyosin ring in an enucleating cell. Panel g showed cross-section surface of contractile ring. Panel h (schema) is obtained schematizes f and g. Panel a, Nuclei, DAPI (blue); Panel b, MC5R, Alexa488 (green); Panel c, F-actin, Phalloidin-Alexa596 (red); Panel d, GPA, APC (light blue). Bars, 2 μm.
Fig 6.
A hypothetical model of the MCR system that regulates erythropoiesis.
MCR-induced activation of ERK, STAT5 and AKT are required for proliferation, differentiation, polarization and enucleation, respectively. We propose that MC1R and MC2R are involved in the modulation of responses to EPO. AKT and MLC2 are directly activated by an ACTH signal via MC5R. Baso-E, basophilic erythroblasts; Poly-E, polychromatic erythroblasts; Ortho-E, orthochromatic erythroblasts; Reti reticulocyte; RBC, red blood cell.