Fbxw11 Variants Control the Quiescence of HSCs

Ubiquitination is a posttranslational mechanism that controls diverse cellular processes. Fbxw11, a constituent of the SCF (Skp1-Cul1-F-box) ubiquitin ligase complex, targets for degradation of several important transcription factors, including NF-κB, β-cantenin and ATF4. Fbxw11 may play pivotal roles in many aspects of hematopoiesis through regulating various signal transduction pathways. However, the role of Fbxw11 on HSCs quiescence remains largely unknown. In this study, we cloned three transcript variants (Fbxw11a, Fbxw11c and Fbxw11d) to study the biological function of Fbxw11 in hematopoiesis.

To elucidate Fbxw11 distribution in hematopoietic system, hematopoietic cell populations at different stages of differentiation were isolated from bone marrow of 8 week-old mice and Fbxw11 expression was studied by real-time PCR. Expression of Fbxw11 were lower in purified long term hematopoietic stem cells (LT-HSC, LSK CD34^- Flt3^-), but higher in short term hematopoietic stem cells (ST-HSC, LSK CD34⁺ Flt3^-), hematopoietic stem and progenitor cells (LSK), and various hematopoietic progenitor cells. The results reveal that Fbxw11 is preferentially expressed in more mature progenitor cells. The expression of Fbxw11 in mature blood cells was also studied showing that Fbxw11 was expressed at lower level in neutrophils, higher level in B and T lymphocytes, and moderate level in monocytes.

To assess the impact of Fbxw11 on reconstitution capacity of LT-HSCs, we cloned Fbxw11a, Fbxw11c and Fbxw11d into retrovirus system, respectively. LSK cells were infected with MSCV-Fbxw11a/Fbxw11c/Fbxw11d-IRES-GFP or the blank control vector MSCV-GFP. Competitive repopulation assays we performed 48h later after infection, and reconstitution in peripheral blood (PB) was analyzed every 4 weeks. Repopulation of donor cells expressing high level of Fbxw11 variants was significantly lower than those infected with control vector at 1 and 4 months in PB and at 4 months in BM after transplantation. These data indicate that Fbxw11 is negative for the long-term repopulating capacity of HSCs.

To further confirm the effects of Fbxw11 variants in hematopoiesis, the effect of Fbxw11 variants on the growth and enumeration of hematopoietic progenitor cells was detected by colony-forming cell assay (CFC). The number of CFU-G, CFU-GM, CFU-GEMM and the total number of CFU were lower in LSK over-expressing Fbxw11 variants when compared with LSK control. To determine the cell-cycle distribution of HSC cells, Hoechst 33342 and Ki67 staining were performed showing that G0 phase LSK cells were decreased when they over-expressing Fbxw11 variants.

In conclusion, our data reveal unrecognized roles for Fbxw11 in the regulation of HSPCs. Our findings suggest that Fbxw11 variants have negative effect on reconstitution capacity of LT-HSCs. Fbxw11 variants decrease the reconstitution capacity through promoting cell proliferation, which results in loss of hematopoietic stem cell quiescence. We anticipate that our experiments will facilitate the understanding of hematopoiesis through which Fbxw11-mediated signals control HSC quiescence and functions.

The work was supported by the Grants 81300376, 81370634, 81570153 from the National Natural Science Foundation of China (NSFC); 14JCQNJC10600 from the Tianjin Science and Technology Programs;