Connexin-43 Expression Regulates the Migration of Hematopoietic Stem Cells and Progenitors towards and From Bone Marrow.

Abstract 562

In the bone marrow (BM) cavity, the migratory traffic of hematopoietic stem cells and progenitors (HSC/P) from the endosteal niches to circulation and viceversa depends on their response to chemokine gradients and interaction with endothelial and mesenchymal pre-osteoblastic cells located at the endosteal niches, forming the hematopoietic microenvironment (HM). Several lines of evidence have pointed out the possible role of the gap junction-forming protein connexin-43 (Cx43) in the control of stem cell and progenitor migration. Our group previously demonstrated that Cx43 expression in the hematopoietic microenvironment (HM) is critical in the fetal liver and BM hematopoietic regeneration after administration of 5-fluorouracil (5-FU) and other investigators have shown that Cx43 is crucial controlling the migration of neural progenitors along radial glial during brain development. We hypothesized that Cx43 could regulate the bidirectional migration of HSC/P in the BM stroma. Since Cx43 is expressed by mesenchymal cells, endothelial cells and hematopoietic stem cells and progenitors, we decided to analyze the Cx43 contribution in the control of HSC/P migration in cell-specific conditional knock-out mice. To achieve this objective, we have used mice that were selectively deficient for Cx43 in the osteoblast/stromal cells (Collagen 1a-Cre^flox/flox; O/S-Cx43-deficient), in endothelial cells (Tek-Cre^flox/flox; E-Cx43-deficient) or in hematopoietic cells (Vav1-Cre^flox/flox; H-Cx43-deficient). O/S-Cx43-deficient mice have been shown to be a model of osteoblast loss of function (Chung DJ et al., J. Cell. Sci., 2006) and E-Cx43-deficient mice have been shown to be a model of arterial hypotension induced by both increase nitric oxide and angiotensin levels (Liao Y et al, PNAS 2001). Analysis with reporter crossings with Rosa-loxP-Stop-LoxP-LacZ mice showed anatomical specificity of the Cre recombinase expression in different cell types of BM, and western-blot and RT-PCR expression indicated practical abolishment of the expression of Cx43 in each of the specific cell types. First, we analyzed whether there were changes in the levels of circulating progenitors in O/S-, E- or H-Cx43-deficient mice. While H-Cx43-deficient mice did not show any change in the levels of circulating HSC/P, E-Cx43-deficient mice showed a 3.5-fold and 4.7-fold, respectively, increase of circulating CFU-C and competitive repopulating units while maintaining normal repopulation ability of BM HSC. O/S-Cx43-deficient mice showed a 30% reduction in basal conditions which was more accentuated when administered G-CSF (50% reduction on day +6), compared with their WT counterparts. Interestingly, while osteoblast loss-of-function was induced in O/S Cx43-deficient mice, the intramarrow expression levels of CXCL12a/b and mesenchymal progenitor content (CFU-F) were increased (4- and 2-fold, respectively). In correlation with the increased levels of CXCL12, the distance to endosteum of transplanted CFSE+/lin-/c-kit+ BM cells into non-myeloablated O/S-Cx43-deficient mice was dramatically decreased (36.1±4.3 vs 23.2±2.1 mm, p<0.01), suggesting a major change in the cellular composition and chemokinesis within the hematopoietic microenvironment “in vivo”. Interestingly, the 16-hour homing of HSC/P transplanted into lethally irradiated O/S-Cx43KO recipient mice showed a ∼60% reduction and a significantly decreased survival in a limiting-dose transplantation radioprotection assay (50% survival in WT mice vs 0% survival in O/S Cx43-deficient recipients). The homing/engraftment defect of these mice correlated with a reversal of the increased levels of CXCL12 in irradiated BM and a 50% reduction of the migration of WT HSC/P through O/S-Cx43-deficient stroma in response to CXCL12. Altogether, these data indicate that intercellular communication through Cx43 shares distinct functions between the different cell components of the hematopoietic microenvironment, and mediates CXCL12-dependent and CXCL12-independent mechanisms in control of the BM homing and retention of HSC/P.