A Novel Role of LKB1 in Erythroblast Enucleation

During terminal erythropoiesis, mammalian red blood cells (RBCs) undergo an unique process called enucleation, where the orthochromatic erythroblast expels the condensed nucleus.Establishment of nuclear polarization is required for orthochromatic erythroblast to expel its nucleus, yet the underlying mechanisms are not well understood. As a structurally polarized organelle, the Golgi apparatus is implicated in maintaining cell polarity. However, whether the Golgi apparatus is involved in erythroblast nuclear polarization and enucleation remains unknown. Here we show that LKB1, a serine-threonine protein kinase implicated in cell polarity, was abundantly expressed in erythroblasts. Knockdown of LKB1 in human erythroid cells impaired nuclear polarization and enucleation along with fragmentation of the Golgi apparatus.Monensin impaired nuclear polarization and enucleation. Phosphoproteomics analyses revealed altered vesicle transport in LKB1-knockdown orthochromatic erythroblasts.

To further define the molecular mechanisms for LKB1 knockdown-induced impairment in nuclear polarization and enucleation, we performed proteomics analyses on the control and LKB1-knockdown orthochromatic erythroblasts. Notably, the top 20 downregulated proteins in LKB1-knockdown cells included several kinesin complex proteins such as KIF3B. Furthermore, monensin treatment also led to decreased KIF3B. We found that the enucleation of KIF3B-knockdown erythroblasts was significantly impaired. We aslo treated the erythroid cells with vacuolin-1, a small chemical that induces rapid vesicle formation. Vacuolin-1 partially rescued the enucleation of LKB1 knockdown-erythroblasts. The partial rescue by vacuolin-1 suggests that LKB1 may regulate enucleation through other mechanisms besides vesicle transport.

We also found that the culture supernatant of LKB1-knockdown erythroblasts significantly inhibited the enucleation of normal orthochromatic erythroblasts. However, culture supernatant of control erythroblasts had no effect on the enucleation of LKB1-knockdown orthochromatic erythroblasts. Interestingly, pathways involved in lipid metabolism, including the lipoprotein metabolic process, were elevated in culture supernatants of LKB1-knockdown erythroblasts compared to controls. Notably, both proteomics and secretomics analyses revealed that APOE was increased in LKB1-knockdown erythroblasts as well as in its supernatant. Furthermore, treatment with monensin, but not BFA, significantly increased APOE levels in erythroblasts, indicating the regulation of APOE by the Golgi apparatus.

To examine the direct effect of APOE on enucleation, we added exogenous APOE to the erythroid culture system in vitro. APOE3, but not APOE4, inhibited enucleation. Then, we analyzed lipid raft by labeling endogenous G_M1 ganglioside. Interestingly, co-staining of APOE and G_M1-ganglioside showed that APOE colocalized with lipid rafts at the perinuclear site in orthochromatic erythroblasts. We found that total and free cholesterol were both elevated by shLKB1. However, immunofluorescence analysis showed that LKB1 knockdown significantly inhibited lipid raft clustering, indicating that increased cholesterol affects the mobility of specialized raft microdomains and thus inhibits the formation of lipid rafts during enucleation. Furthermore, as in LKB1-knockdown erythroblasts, monensin treatment significantly promoted the fluorescence intensity of G_M1 ganglioside, but inhibited lipid raft clustering. Then we performed combinational treatment of vacuolin-1 and cholesterol-lowering drugs. Compared to vacuolin-1 treatment alone, enucleation of LKB1-knockdown erythroblasts was significantly increased after combination of vacuolin-1 and MCD, without any difference when combined with Atorvastatin or Ezetimibe. Together, our findings suggest that another mechanism by which LKB1 regulates enucleation is via the LKB1-Golgi apparatus-APOE-cholesterol-lipid raft axis.

Our findings have uncovered a previously unrecognized role of LKB1 in nuclear polarization and enucleation of human erythroblasts through maintaining the integrity of the Golgi apparatus, the disruption of which led to impaired KIF3B-mediated vesicle traffic and APOE-mediated impairment in lipid raft clustering.