A Novel Model of Short Chain Fatty Acid (SCFA)- Mediated up-Regulation of Embryonic/Fetal Globin Genes during Definitive Erythropoiesis.

Persistence of fetal hemoglobin can ameliorate adult beta (β)-globin gene disorders such as sickle cell disease and β-thalassemia. Short chain fatty acids (SCFAs) up-regulate embryonic/fetal globin gene expression in vitro and in vivo and have great therapeutic potential. We first studied the SCFA-responsiveness of embryonic and fetal globin gene expression during short-term culture in a murine primary cell model. Pooled erythroid fetal liver cells (eFLCs) from wild-type (wt) and human β-globin gene locus-containing mice were examined after treatment with SCFAs. Cells were cultured in basal media alone or in basal media plus propionate (5mm), butyrate (1mM), and/or insulin (2 U/mL) and erythropoietin (10 mg/mL, ‘ins/EPO’). Murine embryonic β-type globin gene expression, ((β^H1+ε^Y)/( β^H1+ε^Y+β^MAJ)x100) was markedly increased at 72 hours in SCFA-treated wt eFLCs, from 1.7±1.2% at baseline to, at 72 hours, 4.88±2.21% in propionate, 5.40±3.39% in butyrate, 19.48±8.30% in butyrate & ins/EPO, compared with ins/EPO-only-treated wt eFLCs, at 0.3±0.3% (n ≥ 3 for each, p<.05). Human fetal γ-globin gene expression was also up-regulated in human transgenic eFLCs, at 29.0±5.0% in butyrate & ins/EPO vs. 3.07±1.08 in ins/EPO only (n=2, p<.05). PCR analysis of FACS-sorted individual eFLCs after culture confirmed that both ins/EPO- and butyrate & ins/EPO-treated cells express detectable α-globin (n=15 for both, p= n.s.). However, only butyrate & ins/EPO-treated cells also express detectable embryonic β^H1 globin (11/15, p<.005). Apoptosis was not increased in SCFA-treated eFLCs, with or without ins/EPO. Erythroid differentiation was analyzed by FACS quantitation of CD71 and TER119 co-immunostaining. At 72 hours, 21.5±3.5% of ins/EPO- vs. 65.5±9.9% of butyrate-only- and 77.5±4.0% of propionateonly-treated erythroid cells were highly differentiated (p<.005). Cytospins confirmed a marked increase in erythroid differentiation in SCFA-treated eFLCs.

We also investigated SCFA-associated phosphorylation of signaling molecules, notably STAT5 and p38, which had been described in other models of erythropoiesis. Western blotting of protein extracts from cultured eFLCs showed phosphorylation of STAT5 in ins/EPO- and butyrate & ins/EPO-, but not in butyrate-only-, treated cells. However, p38 was constitutively phosphorylated in all of these conditions. Inhibitors of p38 kinase activity, SB 203580 (5–100 μM) or PD 169316 (5–300 μM), added to each condition throughout culture, prevented SCFA-mediated induction of embryonic globin gene expression in a concentration dependent manner. Erythroid differentiation was also blocked by p38 inhibition. Our data suggest that SCFAs augment both pancellular embryonic globin gene expression and erythroid differentiation. However, in our model, p38 phosphorylation and kinase activity are not unique to SCFA treatment in eFLCs; p38-associated erythroid differentiation, common to eFLCs in all culture conditions tested, may be an essential precursor for additional, as yet uncharacterized, molecular triggers for SCFA-mediated reactivation of embryonic/fetal globin gene expression in eFLCs.