Short Chain Fatty Acids and p38 in the Induction of Embryonic/Fetal Globin Genes During Definitive Erythropoiesis.

Abstract 460

Augmentation of fetal hemoglogin (HbF) is therapeutically desirable in β –globin gene disorders, such as sickle cell anemia and β-thalassemia. Short chain fatty acids (SCFAs) can augment embryonic/fetal β-type globin gene expression in vivo and in vitro during adult definitive erythropoiesis (EryD), but the underlying molecular mechanisms are not well understood. p38 signaling, triggered by stress erythropoiesis, has been implicated in the induction of fetal globin gene expression. Here, we examined p38 signaling and its effects using a murine primary cell model of EryD, isolated from fetal liver. This model overcomes some limitations of previously used transformed or long-term primary cell models, and has afforded complementary data. E14.5 erythroid fetal cells (eFLCs) were examined at harvest and after culture in insulin and erythropoietin (‘ins/EPO') with or without the SCFA butyrate, and in the presence or absence of p38 inhibitors SB 203580, SB 202190, PD 169316 at concentrations of 0-50 μM for up to 72 hours. Western blots showed that p38 phosphorylation was constitutive at harvest and preserved in cultures with ins/EPO, but was diminished by half after concurrent culture in PD169316. Embryonic globin gene expression, ((β^H1+ε^Y)/(β^H1+ε^Y+β^MAJ) × 100), was analyzed by real-time PCR, normalized to 18S expression. Embryonic globin gene induction, relative to maximal expression at 72 hours in butyrate with ins/EPO, was blocked in a dose dependent manner by all inhibitors of p38 signaling, at 10-30% of maximal induction when cultured in 50 mM of any inhibitor. Primary transcripts from the adult β-globin gene, β^maj, were likewise diminished by p38 inhibition, with a median level of 8.3% and 4.0% of maximal transcript in ins/EPO and butyrate & ins/EPO respectively after culture for 72 hours in 50 μM PD 169316 (n=3, p<.01 relative to non-inhibited cultures). Erythroid differentiation, analyzed by FACS quantitation of CD71 and TER119 co-immunostaining, was delayed by p38 inhibitors, with 22-38% of ins/EPO +/- SCFA-treated cells comprised of orthochromatophilic cells after 72 hours, compared with 4-8% of parallely treated cells to which p38 inhibitors had been added. To further explore stress erythropoesis, cell divisions were analyzed using carboxyfluorescein succinimidyl ester (a fluorescent cytoplasmic dye that is diluted with each cell division) as monitored by FACS. Cell divisions were modestly slowed, but not blocked, by SCFAs. A median of 60% of ins/EPO-, vs. 29% of butyrate & ins/EPO-, treated cells had undergone 2 cell divisions at 48 hours (n=2, p<.05), but by 72 hours a median of 56% and 51% of treated cells, respectively, had completed 2 divisions (n=2, p=n.s.). In contrast, cell division was perturbed by p38 inhibitors, with <26% of eFLCs having undergone a 2^nd division by 72 hours. Our studies suggest that p38 signaling is required for, but is not specific to, the SCFA-mediated induction of embryonic/fetal globin gene expression. We do not find that p38 phosphorylation is stimulated by SCFAs in short-term primary EryD. The central role that p38 signaling plays in the induction of embryonic and fetal globin gene expression, as manifested by lost induction with p38 inhibition, may instead reflect p38's constitutive function in cell division and differentiation.