Effect of Valproate on In Vitro Steroid Responsiveness in 2-Phase Erythroid Culture in Diamond Blackfan Anemia (DBA): Potential Role for Histone Deacetylation in the Mechanism of Action of Steroids in DBA.

We have previously adapted a 2-phase liquid erythroid culture system to demonstrate a consistent and profound failure in erythroid expansion in DBA cultures, which localizes to early in the second erythropoietin-dependent phase of culture. We have shown a partial corrective effect of steroids, which both enhance the clonogenicity of early erythroid colony forming cells, and increase erythropoietin sensitivity by a direct effect on erythroid progenitors at the later stage of CFU-E expansion. Following a report of the induction of remission by valproic acid in a patient with refractory DBA, we have extended our study to include the effect of added valproate at concentrations equivalent to therapeutic plasma levels. In preliminary studies of cultures from normal controls, sodium valproate at 50ng/ml markedly increased erythroid expansion in cultures without added dexamethasone (p<0.001, n=13). A similar effect was observed in DBA cultures, although this was not statistically significant (p=0.07, n=7). However, the enhancing effect of 10⁻⁷M dexamethasone in control cultures was inhibited by valproate 100 ng/ml, (n=13, p<0.05). A similar inhibition of the steroid effect was observed in both of 2 DBA cultures showing in vitro steroid responsiveness. In contrast, at low dose valproate (25ng/ml) and dexamethasone (10⁻⁸M), there was evidence of synergistic enhancement of erythroid expansion. Valproate is a known histone deacetylase (HDAC) inhibitor, and its interactions with dexamethasone in this culture system strongly suggest that dexamethasone influences erythroid histone acetylation status. In support of this, a similar dose-dependent interaction with dexamethasone was observed with trichostatin A, another HDAC inhibitor. This represents a potential molecular basis for the mechanism of action of the stimulatory effect of steroids in erythropoiesis, although the complex dose-dependent interaction between valproate and dexamethasone would be consistent with differential effects of steroids on histone deacetylation at different stages in erythropoiesis. Interestingly, the inhibition of the effect of dexamethasone at higher concentrations of valproate resembles our previously reported effect of prolactin in this culture system, suggesting that modification of histone acetylation also underlies the effect of prolactin. We will address this further by analysing the histone acetylation status of primary erythroid cells throughout the 2-phase culture system, especially at the time of divergence of DBA and control cultures, and the effect of different concentrations of steroids. This may elucidate the individual variation in steroid responsiveness in DBA, and possibly suggest steroid sparing agents for clinical use.