The F-Box Protein NIPA Limits Hematopoietic Stem Cell Survival and Transplantation Efficiency

Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase and contributes to the timing of mitotic entry. To examine the function of NIPA in vivo, we generated Nipa deficient animals, which are viable but sterile due to a defect in testis stem cell maintenance.

To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in Nipa deficient animals. FACS analyses of spleen cells and bone marrow (BM) showed differences in Leucocyte subpopulations. Measuring the CD4 and CD8 positivity within all Thy1.2⁺ cells, the balance in NIPA^-/- T-lymphocytes is destabilised in favour of CD4 positive cells. Besides CD43/CD19 positive as well as CD43/B220 positive cells within all leukocytes are increased in NIPA deficient spleen cells. Analysing more primitive cells, FACS data of bone marrow showed significantly decreased numbers of Lin^-Sca1⁺cKit⁺ (LSK) cells in NIPA^-/- mice (age > 20 month), where LSKs were reduced to 40% of wildtype (wt) littermates (p=0,0171). Additionally, in such older NIPA^-/- mice, only half the number of multipotent myeloid progenitors were detected in comparison to wt mice. To examine efficient response of stem cells to myeloid depression, mice were treated with 5-FU four days before BM harvest. We found that in NIPA^-/- mice, both the number of myeloid progenitors as well as the number of LSKs were severely reduced compared to those in wt levels after 5-FU treatment (p<0.001). Interestingly, the reduction of progenitors and LSK cells was not dependent on age of the NIPA ko mice, suggesting a role for NIPA in stem cell activation or regeneration. This statement was studied in vitro by methylcellulose assays with 10 000 BM cells seeded in methylcellulose with cytokines and replated for three times after 10 days. Nipa deficient hematopoietic progenitors showed a reduced ability to proliferate and differentiate into colonies compared to their controls with an increasing difference after each replating (p(third replating) < 0.0001). Dynamic cell cycle analysis of seeded BM cells with BRDU and PI uncovered delayed cell cycle progress and mitotic entry in NIPA^-/- BM cells in contrast to wt BM cells. Using competitive BM transplantation assay we investigated the role of NIPA for hematopoietic reconstitution in vivo. These experiments showed that NIPA^-/- BM cells were severely deficient in hematopoietic recovery as recipient mice of NIPA^-/- BM cells showed only half the amount of donor-derived peripheral blood cells in contrast to recipient mice of wt BM cells after 4, 11, 17 and over 23 weeks after transplantation. Furthermore NIPA^-/- cells contributed only 7% in BM of transplanted mice 6 month after transplantation compared to 33% in recipients transplanted with wt BM cells (p<0.005). To further explore this defect in hematopoietic repopulation capacity and apply to more primitive progenitors serial transplantation assays were conducted with LSK cells transplanted together with support BM cells.

Taken together our results demonstrate a critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion.