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Tmem30a in erythropoiesis and EPOR signaling
TMEM30A is required for human erythroid differentiation
To examine whether TMEM30A also plays a role in human erythropoiesis, we used shRNA-mediated knock- down approach in human cord blood CD34+ cells.30-32 Online Supplementary Figure S6A shows efficient knock- down of TMEM30A. As demonstrated by the decreased expression of GPA (Online Supplementary Figure S6B), delayed upregulation expression of band 3/downregula- tion of α4 integrin (Online Supplementary Figure S6C), TMEM30A knockdown impaired erythroid differentia- tion. Knockdown of TMEM30A also led to reduced cell growth (Online Supplementary Figure S6D). Moreover, sim- ilar to the murine data, TMEM30A knockdown also induced a significantly increase in the frequency of Annexin V positive cells (Online Supplementary Figure S6E and F). We further detected apoptosis of control and TMEM30A knockdown human erythroid cells by TUNEL assay. TMEM30A knockdown led to increased apoptosis (Online Supplementary Figure S6G and H). Finally, we exam- ined the effect of TMEM30A on EPOR-mediated signal transduction in human erythroid cells. TMEM30A knock- down resulted in attenuated phosphorylation of EPOR downstream target STAT5 (Online Supplementary Figure S6I and J). Thus, TMEM30A plays a conserved function in both human and murine erythropoiesis.
Discussion
Studies over the past decade have clearly documented that Tmem30a is required for the flippase activity of P4- ATPases.33 However, the function of Tmem30a in vivo remains largely unexplored. In the present study, we found that, unexpectedly, selective deletion of Tmem30a in hematopoietic cells severely impaired fetal liver erythro-
poiesis which contributes to the embryonic lethality of the mice. Our findings have, therefore, uncovered a novel role for Tmem30a in erythropoiesis.
In exploring the underlying mechanisms for the impaired erythropoiesis, we found that, while Tmem30a deletion did not affect cell cycle, it led to increased apoptosis of ery- throid cells. Interestingly, the increased apoptosis is due to significantly impaired activation of JAK2-STAT5 signal transduction pathway, the essential pathway for survival of erythroid cells. Further examination revealed that Tmem30a deletion impaired lipid rafts formation accompa- nied with impaired EPOR clustering. Our findings provide new insights into the mechanisms by which EPO/EPOR signal transduction pathway is regulated.
The type 4 subfamily of P-type adenosine triphos- phatases (P4-ATPases) actively transports phospholipids across the membrane bilayer. There are 14 P4-ATPases (ATP1-14) in eukaryotes whereas only three Tmem30 (termed Tmem30a, Tmem30b, Tmem30c) homologs are identified, each Tmem30 protein interacting with multiple P4-ATPases.21,34 It is very interesting to note that, of the three Tmems, only Tmem30a is expressed in both murine and human erythroid cells. Since members of the Tmem family can compensate each other, the lack of expression of other Tmem family members in erythroid cells may explain the severe phenotypic changes of erythroid cells when Tmem30a is depleted. These findings imply the important role of flippase activity in erythropoiesis. Together with previous findings that ATP11C mutated mice showed a lower rate of PS translocation in pre-B cells and defective differentiation of B lymphocytes,35 we sug- gest that flippase activity may play important roles in hematopoiesis in general, and this warrants future studies. It is likely that members of P4-ATPases family and Tmem family may contribute to hematopoiesis in a lineage-spe- cific manner.
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haematologica | 2019; 104(10)
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Figure 5. Tmem30a deletion impairs erythropoietin receptor (EPOR) clustering. (A) Isolated Ter119 low erythroid cells were stained with DAPI (blue) and with an anti-EPOR antibody (red). Lipid rafts were detected using GM1 ganglioside with FITC conjugated CTB (green). (B) Proportions of cells that showed lipid raft clustering are indicated as cluster+. Values are presented as Mean±Standard Error of Mean. ***P<0.001. N=3 slides per group.