Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junction channels (connexons) are formed by polypeptides (connexins) arranged in hexamers and represent the best described intercellular communication system. Connexin-43 (Cx43) is expressed by BM OS cells and has been associated with the cadherin/beta -catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Here, we employed an inducible gene-targeted murine approach to study the role of Cx43 in HSC proliferation and differentiation in vivo. Mx-Cre/Cx43+/+ and Mx-Cre/Cx43flox/flox littermates have been analyzed after gene deletion induced in vivo by the interferon-inducer poly (I)-poly (C), generating control (Cx43+) and Cx43-deficient (Cx43-/-) mice. After one week, Cx43+ and Cx43-/- mice were treated with 5-fluorouracil (5-FU). Cx43 expression in Cx43-/- BM was markedly reduced (> 90%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5- to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units (CFU-GM-), erythroid burst forming units (BFU-E) and mixed colony forming units (CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin-/c-kit+/Sca1+ cells in Cx43-/- BM was maintained when compared to Cx43+ BM. Short-term competitive repopulation ability of Cx43-/- BM cells was diminished as compared to Cx43+ mice, specifically for myeloid and B lymphoid cells, but showed spared long-term competitive repopulation ability with roughly normal hematopoietic differentiation. These data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.
Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junctions (GJs) represent the best described intercellular communication (IC) system, and they are characterized by the existence of plaques of narrow channels between contacting cells. Each cell contributes with one hemichannel, which is composed of six proteins, called connexins. Connexin 43 (Cx43) is expressed by BM OS cells. Cx43 has been associated with the cadherin/β-catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Multiple osteogenic defects have been reported in human Cx43 mutations and Cx43 has been shown to be essential in controlling osteoblast functions. BM Cx43 expression is up-regulated up to 100-fold by 5-fluorouracil (5-FU) treatment. Due to the perinatal death of Cx43 germline null mice, a conditional genetic approach was employed to study the role of Cx43 in stem cell proliferation and differentiation. The interferon-inducible Mx1 gene is expressed by both hematopoietic and stromal BM cells. Therefore, we crossed Cx43flox/+ mice with Mx1-Cre transgenic (Mx1-CreTg/−) mice. Cx43+/+:Mx1-CreTg/−and Cx43flox/flox:Mx1-CreTg/− littermates have been analyzed. Gene deletion was induced in vivo by injecting the interferon-inducer polyI:C (8 injections of 300 μg every other day), generating control (Cx43+) and Cx43-deficient (Cx43KO) mice. After one week, Cx43+ and Cx43KO mice were injected with 5-FU (150 mg/Kg i.v.). Cx43 expression in Cx43KO BM was markedly reduced (>80%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5 to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units -CFU-GM-, erythroid burst forming units (BFU-E) and mixed colony forming units -CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin−/c-kit+/Sca1+ cells in Cx43KO BM was maintained when compared to Cx43+ BM (139±19 vs 117±32 x 103 Lin−/c-kit+/Sca1+ cells per femur, respectively). Short-term competitive repopulation ability of Cx43KO BM cells was diminished as compared to Cx43+ mice (5.9±0.35% vs 22.2±4.6%, respectively, p<0.01), specifically for myeloid (9.2±1.4% vs 35.8±4.3%, respectively, p<0.001) and B lymphoid (0.4±0.2% vs 3.0±1.0%, respectively, p<0.01) cells, but showed spared long-term (6-month) competitive repopulation ability with roughly normal hematopoietic differentiation. Altogether, these data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.
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*
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