Mechanistic Study on the Functions of SYK and TMCC2 in Enucleation during Terminal Erythropoiesis

Abstract

Erythropoiesis refers to developing hematopoietic stem cells to mature red blood cells (RBCs), during which erythropoietin (EPO) initiates sequential differentiation. The differentiation from erythroid precursors to erythrocytes is terminal erythropoiesis, involving chromatin condensation, cell cycle exit, enucleation, and reticulocyte maturation. However, as the critical step in terminal erythropoiesis, the regulatory mechanism of enucleation needs to be better understood. Spleen tyrosine kinase (SYK) is recognized as a crucial mediator of immunoreceptor signaling inflammatory cells. The former researcher has revealed that Syk deficiency led to anemia using a mouse model, indicating that SYK also plays a vital role in erythropoiesis. As reported, transmembrane and coiled-coil domain family 2 (TMCC2) is identified as a regulator in terminal human erythropoiesis, and TMCC family proteins can regulate ER recruitment for endosome fission.

Notably, anemia is a common adverse event in clinical trials of SYK inhibitors. Previous researchers have demonstrated that Syk deletion led to anemia with extramedullary erythropoiesis in the spleen. Syk deletion disturbed in vivo terminal erythropoiesis of mouse bone marrow and fetal liver and ex vivo differentiation of erythroid progenitors with decreased enucleation rate. Moreover, Syk deficiency caused enhancement of E2F targeted genes and acceleration of retinoblastoma 1 (RB1) degradation. Cell cycle analysis showed improper cell cycle exit during erythropoiesis in Syk deficiency conditions.

In this thesis, we further investigate the mechanism of the role of SYK in regulating cell cycle exit and enucleation during terminal erythropoiesis using mouse fetal liver cells, murine erythroleukemia (MEL) cell lines, and human CD34⁺ cells. Mechanistically, upon EPO activation, SYK phosphorylates Src homology 2 (SH2) domain-containing inositol-5-phosphatase-1 (SHIP1), causing the delayed activation of E3 ligase Casitas B-lineage lymphoma (C-CBL). The activated C-CBL facilitates the degeneration of RB1 by ubiquitination. Besides, RB1 degradation can be rescued using the SHIP1 chemical activator, AQX1125. Our study proves that SYK regulates cell cycle exit and enucleation by affecting RB1 ubiquitination through C-CBL.

This study also indicates that TMCC2 regulates the enucleation of mouse terminal erythropoiesis, leading to anemia and defective erythropoiesis. In the Tmcc2 total knockout mouse model study, homozygous mice showed chronic anemia from 4-week to 16-week age, and the anemia phenomenon was severe in 8-week. Moreover, 8-week and 16-week homozygous mice both showed obvious extramedullary hematopoiesis in the spleen. Tmcc2 knockout disrupted the terminal erythropoiesis of mouse bone marrow, leading to the failure of enucleation in the last stage. Tmcc2 knockout also disturbed in vivo terminal erythropoiesis of fetal livers and ex vivo differentiation of erythroid progenitors, resulting in a significantly decreased enucleation rate. Notably, Tmcc2 knockout decreased reticulocyte size in the adult and embryonic stages. The average size of homozygous RBCs was smaller than that of wild-type (WT) ones in E18.5 embryos.

Surprisingly, Wright-Giemsa staining of blood smear showed protrusions in homozygous RBCs’ surface, indicating the effect of Tmcc2 deletion on the RBC membrane. This study shows that the Tmcc2 deletion mouse had decreased cholesterol content in RBC. The previous researcher’s analysis by immunoblotting and immunofluorescence revealed that TMCC2 is located on the endoplasmic reticulum (ER). I further studied the localization of cholesterol, ER, and RBC morphology during enucleation by immunofluorescence. It is implied that TMCC2 regulates enucleation by affecting cholesterol and ER distribution in terminal erythropoiesis. TMCC2 may function in mechanical membrane fission during enucleation, facilitating the separation of cytoplasm and the nucleus. In the next stage, I will continue investigating the detailed mechanism of TMCC2 function in enucleation during terminal erythropoiesis.

Collectively, this study reveals the underlying mechanisms of SYK and TMCC2 functions in terminal erythropoiesis. This study provides new insights into cell cycle exit and enucleation in erythroid differentiation.

Date of Award	18 Jan 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Xin DENG (Supervisor) & Jiahai SHI (Co-supervisor)