title: PTEN regulates the BMI1-mediated self-renewal of long-term hematopoietic stem cells in developmental and stress conditions
Creators: name: Zheng, Wen
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: PTEN
subject: BMI1
subject: Self-renewal
subject: Hematopoietic stem cells (HSCs)
subject: Stress conditions

description: In the hematopoietic system, self-renewing long-term hematopoietic stem cells (LT-HSCs) maintain stem cell pools and differentiate into hematopoietic progeny. In addition to its critical role in the daily production of blood cells, LT-HSCs are able to resist the threats posed by various types of stress. However, our understanding of LT-HSCs under stress is still limited. It has been observed in our laboratory that the deletion of Pten in the LT-HSCs of VE-cadherin-cre+;Ptenfl/fl mice does not lead to the loss of HSC self-renewal, raising the possibility that PTEN-mediated self-renewal regulation may be more complicated than previously reported. To understand the molecular regulation of HSC homeostasis, I crossed Bmi1GFP/+ knock-in reporter mice, in which the intact Bmi1 promoter drives the transcription of the GFP transgene instead of the self-renewal regulator gene Bmi1, with Pten-deficient mice (Vav-icre+;CD2-C3+;Ptenfl/fl, or vCPΔ/Δ) to generate vCBPΔ/Δ (vCPΔ/Δ;Bmi1GFP/+) mice. The intact GFP signals in vCBPΔ/Δ mice, which represent the expression of Bmi1, indicated that the transcription of Bmi1 was not affected by PTEN loss. However, vCBPΔ/Δ mice surprisingly suffered from the exhaustion of LT-HSCs and eventually died between 2 and 4 months after birth, which was reminiscent of the HSC defects in Bmi1-/- mice. The similar phenotypes between vCBPΔ/Δ mice and Bmi1-/- mice raised the possibility that PTEN plays an important role in the protein stability of BMI1 and the BMI1-mediated self-renewal of LT-HSCs. To understand the role of PTEN in the regulation of BMI1, the protein levels of BMI1 in vCBPΔ/Δ LT-HSCs were measured by protein-flow analysis. Indeed, the BMI1 protein in vCBPΔ/Δ LT-HSCs was decreased by more than 50%. In contrast, the protein levels of BMI1 in vCPΔ/Δ and Bmi1GFP/+ mice, where the HSC pools remained normal, were slightly reduced by less than 50%. Consistently, the stem cell exhaustion marker p16Ink4a, which is typically repressed by BMI1, was strikingly upregulated in vCBPΔ/Δ LT-HSCs. These observations indicate that the protein levels of BMI1 are critical for the expression of p16Ink4a and the BMI1-mediated self-renewal regulation. At the molecular level, the loss of PTEN or its phosphatase function led to the activation of the stress-responsive kinase p38α MAPK. The activated p38α phosphorylated BMI1 at T275, which resulted in the degradation of BMI1. However, the T275 phosphorylation of BMI1 was suppressed by the phosphatase-independent function of PTEN. The T275A mutant of BMI1, which was unable to be phosphorylated by p38α, rescued the protein levels of BMI1 and the HSC pool in vCBPΔ/Δ mice. These molecular and genetic findings suggest that the T275 phosphorylation of BMI1 is associated with its protein stability and self-renewal regulation. To investigate the molecular regulation of BMI1 by PTEN and p38α under stress, an X-ray irradiation murine model was employed. The 3 Gy irradiation led to a decrease in the BMI1 protein levels in response to irradiation, which was accompanied by the increased reactive oxygen species at 14 days post-irradiation. The irradiated HSCs with increased ROS exhibited compromised self-renewal activity in serial transplantation assays. Conversely, HSCs with the Bmi1T275A mutation rescued the self-renewal ability of irradiated HSCs in those serial transplantation assays. These observations highlight the BMI1 T275 as a critical checkpoint in HSCs in response to stress. Taken together, the regulators p38α, PTEN and BMI1 form a new circuit to regulate HSC self-renewal in developmental and stress conditions. PTEN and p38α play an essential role in regulating the protein levels of BMI1 and the self-renewal of HSCs via the BMI1 T275. The T275 locus in BMI1 is a critical checkpoint in response to stress.