Abstract
Hematopoietic stem cells (HSCs) comprise only ~0.02% of the whole bone marrow cells but possess the capacity to extensively proliferate in order to restore hematopoietic homeostasis. Under homeostasis, HSCs are relatively quiescent with a slow cell cycle progression rate. However, upon stimulation, HSCs are able to promptly proliferate and undergo self-renewal to regenerate HSCs as daughter cells. While regulatory mechanisms involved in cell cycle progression are well characterized to be essential for HSC self-renewal, the mechanisms that facilitate the return of proliferating HSC to their quiescent state have been largely overlooked. The expression of CD81 (also called TAPA-1), a transmembrane protein that belongs to the Tetraspanin family, has been found associated with HSC proliferation. While CD81 is normally absent on HSC, it becomes markedly upregulated during HSC proliferation (Figure 1).
To understand the function of CD81 in regenerating HSCs, we utilized a murine stem cell retroviral vector to deliver genes into 5-FU treated bone marrow progenitors to test the effect of enforced CD81 overexpression on HSC. The CD81-transduced proliferating progenitors were found to give rise to an increased number of phenotypically-defined HSC (SP-KLS) without significantly affecting the homeostasis in peripheral organs. In addition, we also characterized the HSCs from CD81 knock-out mice. We discovered that CD81-null HSC failed to engraft in peripheral blood of secondary recipients in serial transplantation assays (Figure 2), suggesting a role of CD81 in preserving a functional HSC compartment during proliferation-induced stress.
When investigating further, we discovered that CD81 is a cell cycle suppressor for HSC, as the CD81KO HSCs are delayed in returning quiescence. In addition, clustering of CD81 on the HSC cell membrane using a monoclonal antibody rapidly induced a quiescent phenotype. This was found to be associated with an altered phosphorylation level of AKT, an inhibitor of the transcription factor FOXO1a and FOXO3a, which have been reported to be essential for HSC self-renewal through suppressing HSC proliferation. Taken together, these results demonstrate an essential role of CD81 in HSC self-renewal, and a novel mechanism that advances quiescence from a proliferating state.
Figure 1. CD81 expression is upregulated at the time when HSCs (SPKLS) are proliferating in response to 5FU stimulation, a chemotheraputic agent that induces HSC to proliferate. The expression of CD81 is found at a background level in quiescent stages (5FU-Day0 and 5FU-Day11), and is upregulated during proliferation stages (starting 5FU-Day2) Figure 1. CD81 expression is upregulated at the time when HSCs (SPKLS) are proliferating in response to 5FU stimulation, a chemotheraputic agent that induces HSC to proliferate. The expression of CD81 is found at a background level in quiescent stages (5FU-Day0 and 5FU-Day11), and is upregulated during proliferation stages (starting 5FU-Day2) Figure 2. CD8KO HSCs fail to engraft in the secondary competitive transplantation assay, indicating a self-renewal defect. In this assay, 300 donor-derived HSCs (CD45.2 SPKLS) were purified from the primary recipients and transplanted along with 2×105 competitors into lethally irradiated mice (**p<0.01). Figure 2. CD8KO HSCs fail to engraft in the secondary competitive transplantation assay, indicating a self-renewal defect. In this assay, 300 donor-derived HSCs (CD45.2 SPKLS) were purified from the primary recipients and transplanted along with 2×105 competitors into lethally irradiated mice (**p<0.01).
Chronic myeloid leukaemia cells require the bone morphogenic protein pathway for cell cycle progression and self-renewal
