LKB1 Coordinates Metabolic Pathways to Maintain Homeostasis of Haematopoietic Stem Cells and Regulate Erythropoiesis

Erythropoiesis is a highly regulated multistage process by which hematopoietic stem cells (HSCs) proliferate, differentiate and eventually form mature erythrocytes. Stem-cell maintenance and cell differentiation require homeostasis and coordination of multiple metabolisms. However, underlying mechanism of this coordination is poorly identified. Liver kinase B1 (LKB1) acts as evolutionarily conserved regulator to control cellular metabolism, cell polarity and proliferation during development and stress response. Considering that all the fundamental cellular processes regulated by LKB1 are present in erythropoiesis, we hypothesize that LKB1 may involve in orchestrating this coordination.

To explore the role of LKB1 in entire erythropoiesis, we firstly crossed mice carrying loxP-flanked LKB1 alleles with EpoR-tdTomato-Cre mice, which can lead to cleavage in from HSCs to erythroblasts. LKB1^fl/fl EPOR^cre mice developed exhaustion of HSCs, progressive severe anemia and ultimately lethality. Intriguingly, HSCs of LKB1^fl/fl EPOR^cre mice exhibited a swiftly skewness toward the erythoid lineage when LKB1 is deleted. Nevertheless, erythroblasts of LKB1^fl/fl EPOR^cre mice were significantly reduced. Further analysis showed that the colony forming ability of the colony-forming unit-erythroid (CFU-E) cells and subsequent terminal erythoid differentiation in LKB1^fl/fl EPOR^cre mice were seriously impaired.

In order to study erythropoiesis more specifically without the influence of stem cells, we next assessed the impact of deletion of LKB1 in the mouse erythropoietic system using GYPA-eGFP-Cre mice. LKB1^fl/flGYPA^cre mice showed mild anemia but had the normal lifespan.The Ter119 ⁺ cells were decreased while CFU-E cells were significantly increased. However, the colony forming ability of sorted CFU-E cells from LKB1^fl/flGYPA^cre mice were drastically reduced. In addition, LKB1^fl/flGYPA^cre mice exhibited disordered terminal erythropoiesis. Taken together, these results indicate that LKB1 may play multinomial roles in regulating stem-cell homeostasis and effective erythoid differentiation.

Previous studies have shown that LKB1 controls energy metabolism via phosphorylating AMPK and regulating PGC-1 transcription to maintain homeostasis of HSCs. We found that both of p-AMPK and PGC-1 of LKB1^fl/fl EPOR^cre mice were down-regulated in Lin ^- cells while Ter119 ⁺ cells have comparable p-AMPK and PGC-1. In this regard, LKB1^fl/flGYPA^cre mice, whether in Lin ^- cells or in Ter119 ⁺ cells, exhibited similar p-AMPK and PGC-1 level. Moreover, administration of ZLN005, an activator of PGC-1, partly rescued the anemia in LKB1^fl/fl EPOR^cre mice but not in LKB1^fl/flGYPA^cre mice. These data imply that there is other underlying mechanism of LKB1 in erythropoiesis.

To gain further mechanistic insight, we carried out proteomics analysis. Gene ontology analysis of differentially expressed proteins revealed that cholesterol metabolism related genes were significantly altered under LKB1 deficiency. We then found that LKB1 ablation led to a reduction of cholesterol level and diminishment of expression levels of primary cholesterol biosynthesis related genes. Moreover, the mature active form of SREBP2, the master transcriptional regulator of cholesterol biosynthesis, was prominently reduced. Golgi apparatus, in which SREBP2 is cleaved for activation, is intumescent or dispersed in erythroid cells of LKB1^fl/fl EPOR^cre mice and LKB1^fl/flGYPA^cre mice. These results supported that loss of LKB1 impaired the cholesterol metabolism in erythropoiesis.

To demonstrate Golgi apparatus-dependent cholesterol metabolism is essential for erythropoiesis, we treated LKB1^fl/flGYPA^cre mice with 2,3-oxidosqualene, the important intermediate in cholesterol synthesis and found that the phenotypes of LKB1^fl/flGYPA^cre mice were effectively restored. In parallel, 2,3-oxidosqualene treatment just slightly alleviated the anemia of LKB1^fl/fl EPOR^cre mice. However, combination treatment with 2,3-oxidosqualene and ZLN005 was more effective in restoring phenotypes in LKB1^fl/fl EPOR^cre mice, in contrast to ZLN005 alone.

Thus, LKB1 serves as an essential metabolic regulator to coordinate energy metabolism in hematopoietic stem cells and lipid metabolism in erythoid cells, thereby maintaining homeostasis of haematopoietic stem cells and functional fitness of erythropoiesis.