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F. Yang et al.
The fact that EPO/EPOR mediated signal transduction pathway in erythroid cells is essential for erythropoiesis has been well documented.36 However, how the pathway is initiated remains unclear. One striking finding of the current study is that EPO induces the clustering of EPOR at the areas of lipid raft domains on the plasma mem- brane. Importantly, disruption of lipid rafts formation due to Tmem30a deletion led to failure of EPOR clustering as well as impaired activation of JAK2-STAT5. These find- ings imply that initiation of EPOR signal pathway requires lipid rafts-mediated EPOR clustering. It has been shown that SCF receptor is essential for erythropoiesis because
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mutation of c-kit caused severe anemia.37 Therefore, in addition to EPO receptor, other receptors may also be affected after Tmem30a deletion. Moreover, a recent study showed that Tmem30a plays an essential role in ensuring the survival of hematopoietic cells in adult mice,38 suggest- ing that Tmem30a play different functions between embryo and adult hematopoiesis.
In summary, our study has uncovered a critical role of Tmem30a in erythropoiesis and identified the underlying mechanisms. As Tmem30a is required for the flippase activity, our findings suggest the role of flippase in ery- thropoiesis. Together with other findings, our study has
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haematologica | 2019; 104(10)
Figure 6. Tmem30a deficiency leads to decreased STAT5 phosphorylation. (A) STAT5 activation with or without erythropoietin (EPO) exposure, decreased STAT5 phos- phorylation at Tyr694 which was demonstrated by western blotting of protein of fetal liver cells isolated from E14.5 cKO fetal livers compared with protein samples of fetal liver control cells. (B) Expression of STAT5 responsive genes in E14.5 control and cKO fetal liver cells measured by qualitative polymerase chain reaction (n=3 for each genotype). Data are presented as Mean±Standard Error of Mean (SEM) of fold-expression relative to control; expression normalized versus actin. *P<0.05; **P<0.01; ***P<0.001. (C) Decreased expression level of BCL-XL after EPO exposure in cKO cells compared to control. (D) Levels of caspase-3/7 activity in total fetal liver cells were assessed. N=5 embryos for each group. (E) Enriched Ter119 negative fetal liver cells were cultured for 48 hours in vitro and stained with anti- CD71 and anti-Ter119 markers. (F) The pan-caspase inhibitor Q-VD-OPh partially reversed Tmem30a erythroid cell differentiation defects as analyzed by FACS.