Connexin-43 Regulates the Cell Cycle Entry of Hematopoietic Stem Cells within the Stem Cell Niche.

Abstract 1500

Poster Board I-523

Bone marrow (BM) osteoblasts and stromal (O/S) cells are crucial in the establishment of the hematopoietic niches in the BM. Connexin 43 (Cx43) is expressed by BM stromal cells and by hematopoietic stem cells and progenitors (HSC/P) and is overexpressed in the BM endosteal space upon administration of chemotherapy or radiotherapy. We have previously reported that Cx43 is critical in fetal liver and in BM hematopoiesis. Since Cx43 is expressed by both HSC and the hematopoietic microenvironment, we dissected out the cellular mechanisms responsible for Cx43 function in the BM. We analyzed the hematopoiesis of mice deficient in Cx43 in the O/S cells (Collagen 1α-Cre^flox/flox; O/S-Cx43-deficient) or in the hematopoietic cells (Vav1-Cre^flox/flox; H-Cx43-deficient). Upon basal conditions, analysis of the HSC compartment of H-Cx43-deficient mice showed a ∼30% decreased content of immunophenotypically defined long-term HSC (LT-HSC) in BM of H-Cx43KO mice compared with their WT littermates, whereas there was not significant variation in the ST-HSC population content. The reduced LT-HSC population in H-Cx43KO mice was associated with a modest increased quiescence (∼12% increase of LT-HSC in G₀). Interestingly, the expression of cyclin D1 and p21^cip1 in the H-Cx43KO LT-HSC were 50% reduced and 4-fold increased, respectively, suggesting a decreased ability to enter cell cycle. While we found no significant engraftment difference in primary recipients of competitive repopulation assays, we found a marked reduction (>50%) in the engraftment ability of LT-HSC Cx43-deficient cells when transplanted into secondary recipients. When submitted to stress by 5-fluorouracil (5-FU) administration, H-Cx43KO mice showed a severely decreased hematopoietic recovery of peripheral blood (PB) counts for neutrophils and platelets accompanied with a marked reduction in the BM cellularity and hematopoietic progenitor content on day +14 after treatment. This defect was associated with a dramatic decreased (∼75 %) in the proliferation of the Cx43-deficient, LT-HSC population by 48 hours post-5-FU administration and a relative decrease of the expansion of the ST-HSC/MPP pool as early as 6 days post-5-FU administration. Interestingly, O/S-Cx43-deficient mice also showed severely delayed hematological recovery after 5-FU administration, with reduction in cellularity and hematopoietic progenitor content, suggesting that the increased hematopoietic toxicity induced by 5-FU in the context of Cx43 deficiency may depend on HSC-to-O/S Cx43 homotypic communication. This communication would be responsible of control of the G₁ restriction checkpoint in LT-HSC. In summary, our findings suggest that Cx43 expression plays a crucial role controlling the LT-HSC pool size and fitness in response to stress.