Role Of Connexin 32 In Hematopoiesis: Maintaining Quiescence Of Hematopoietic Stem Cells and Their Proliferation

Previously, we reported the findings of our studies on the role of connexin (Cx) 32 during steady-state hematopoiesis and its potential protective role against leukemogenesis. Namely, in wild-type mice, Cx32 expression was solely detected in primitive hematopoietic stem/progenitor cells (HSCs/HPCs). Since Cxs are essential molecules for multicellular organisms, Cxs are surmised to be present in the hematopoietic tissue to facilitate cell-cell communication between HSCs/HPCs themselves rather than between HSCs/HPCs and stromal cells. In addition, Cx32-knockout (KO) mice showed the following characteristics: first, a prominent decrease in the number of peripheral mononuclear cells (PMCs) associated with various HPCs; second, a significant increase in the number of HSCs, at least until 20 weeks of age; and third, an apparently delayed regeneration of HPCs after chemical abrasion. Furthermore, the incidence of leukemogenicity induced by methylnitrosourea increased prominently. These possible leukemogenic propensities taken together imply that Cx32 plays an important role in maintaining steady-state hematopoiesis and in suppressing leukemogenesis.

In this study, first, we examined the cell kinetics of HPCs [CFU-GM, colony forming unit (CFU) granulomacrophage; CFU-S9/CFU-S13, CFU in spleen on day 9/13] by evaluating the percentage of cycling HPCs with continuous incorporation of bromodeoxyuridine (BrdUrd) in vivo for up to 3 months, followed by exposure to ultraviolet-A to eliminate cells that incorporated BrdUrd. Using this method with the continuous incorporation of BrdUrd in vivo in mice up to 1.5 years of age, we discovered the existence of a long-term and stable, dormant fraction in the HPCs of wild-type mice. Without Cx32, the percentage of the entire cycling fraction of CFU-S13 apparently increased continuously, which indicates that Cx32 could restore the quiescence of hematopoiesis and thereby maintain HSCs/HPCs. This is consistent with the findings that the number of HPCs increased and the number of HSCs decreased with aging in Cx32-KO mice. Next, we examined the bone-marrow reconstitution capability of HSCs of Cx32-KO mice by serial transplantation. Five hundred cells in the lineage-negative, c-kit-positive, and Sca1-positive (LKS) fraction isolated from wild-type and Cx32-KO mice (Ly5.2) were transplanted into lethally irradiated first recipients (Ly5.1) separately with 2x10⁵ freshly isolated bone marrow cells from wild-type F1(Ly5.1/Ly5.2) mice as rescuing cells, which prevent acute radiation injury. Two months after transplantation, both groups showed reconstituted hematopoiesis without any significant differences in various hematopoietic parameters, although mice reconstituted with cells in the LKS fraction without Cx32 showed a rather higher average percentage of donor-origin PMCs and a lower average percentage of the donor-origin cells in the LKS fraction than those with Cx32 (wild-type group vs. Cx32-KO group, average percentage of Ly5.2 with respect to the total ± standard deviations (s.d.); PMCs, 34.7±14.3% vs. 50.7±9.9 %, p=0.006; cells in the LKS fraction, 20.8±5.2% vs. 14.2±5.3%, p=0.110). Then, donor cells (Ly5.2) in the LKS fraction were isolated from the primary recipients of both groups separately and 400 cells were transferred into each secondary recipient (Ly5.1) with 2x10⁵ rescuing cells from F1(Ly5.1/Ly5.2) mice. Although secondary recipients from both groups showed reconstituted hematopoiesis without any significant differences in various hematopoietic parameters four months after reconstitution, similar to the primary recipients, cells of donor origin in the LKS fraction could be detected only in the wild-type group. Namely, four out of seven recipients in the wild-type group showed over 0.5% donor cells and the average percentage and s.d. for 4 mice was 25.1±27.9%, whereas none of the recipients out of five in the Cx32-KO group showed more than 0.5%. The above-mentioned findings in this study in addition to the previous findings imply that Cx32 plays an essential role in maintaining self-renewal proliferation of primitive hematopoietic stem cells to prevent their exhaustion, and also in suppressing neoplastic changes.