Abstract
Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be critical in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. BM cells are directly connected through gap junctions (GJs) which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM OS cells. Multiple osteogenic defects have been reported in human Cx43 mutations and Cx43 has been shown to be essential in controlling osteoblast functions. Due to the perinatal death of Cx43 germline null mice, an interferon-inducible, conditional genetic approach (Mx1-Cre), expressed by both hematopoietic and stromal BM cells, was used to study the role of Cx43 in stem cell function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration (Presley C, et al., Cell Comm. Adh., 2005). Here, we observed that after 5-FU administration, Cx43 expression is predominantly located in the endosteum. To study the role of stroma-dependent Cx43 in hematopoiesis, we developed hematopoietic chimeras by BM transplantation of wild-type Cx43 HSC into stromal Cx43-deficient mice. Stromal Cx43 deficiency induced a severe impairment of blood cell formation during the recovery phase after 5-FU administration compared to stromal Mx1-Cre-Tg wild-type controls (Table 1), as well as a significant decrease in BM cellularity (~60% reduction) and progenitor cell content (~83% reduction). Cell cycle analysis of 5-FU-treated BM progenitors from stromal Cx43-deficient mice showed an S-phase arrest (S phase: 63.5%; G2/M phase: <1%) compared to wild-type chimeric mice (S phase: 38.6%, G2/M phase: 7.8%, p=0.01) suggesting a cell division blockade. Unlike Cx43-deficient primary mice, a differentiation arrest at the HSC compartment was observed in 5-FU-treated, stromal Cx43-deficient mice, since the content of competitive repopulating units (CRU) at 1 month, of 14-day post-5-FU BM of stromal Cx43-deficient mice was increased (27.7 ± 0.67) compared to recipients of HSC from stromal wild-type counterparts (26.5 ± 0.92 CRU, p < 0.01). Interestingly, wild-type hematopoietic progenitor homing in stromal Cx43-deficient BM was severely impaired with respect to wild-type BM (5.1% vs10.4 %, respectively, p < 0.01), while hematopoietic Cx43-deficient BM progenitors normally homed into the BM, suggesting a differential role for Cx43 in stromal and HSC. In conclusion, expression of Cx43 in osteoblasts and stromal cells appears to play a crucial role in the regulation of HSC homing in BM and hematopoietic regeneration after chemotherapy.
Peripheral blood counts of WT and stromal Cx43-deficient chimeric mice after 5-FU administration (150 mg/Kg) Neutrophil counts (×10e9/L) Reticulocyte count (%) Day post-5-FU WT Cx43-deficient WT Cx43-deficient * p < 0.05 Day +8 2.89 ± 0.06 0.81 ± 0.02* 2.0 ± 0.6 3.0 ± 0.9 Day +11 9.11 ± 2.5 3.13 ± 0.8* 6.1 ± 0.6 2.7 ± 0.3* Day +14 6.22 ± 5.7 7.58 ± 8.2 7.5 ± 0.5 2.5 ± 0.5*
Bypass of an epigenetic S-phase transcriptional module by convergent nutrient stress signals
