Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Abstract

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Figure 1

Cx43 deficiency in OB/P compartments does not modify steady-state hematopoiesis but impair radioprotection in primary recipient mice. (A) BM cellularity (2 femora, 2 tibiae, and pelvis) in WT and OB/P Cx43-deficient mice. (B) BM content of HSC/P subpopulations in WT and OB/P Cx43-deficient BM. LT indicates long-term; ST, short-term; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, glanulocyte/macrophage progenitors; MEP, megakarycyte/erythroid progenitor; and CLP, common lymphoid progenitors. (C) Hematopoietic progenitor content (CFU-granulocyte-macrophage [GM], erythroid burst-forming units [BFU-E], and CFU-Mix) in the BM of WT and OB/P Cx43-deficient mice. (D) Survival curve of lethally irradiated WT (dashed line) or OB/P Cx43-deficient (solid line) recipient mice transplanted with 1 × 10⁴ WT BM cells (low dose). (E) Percentage of chimerism in WT (empty circles) and OB/P Cx43 deficient mice (solid circles) transplanted with 3 × 10⁵ WT CD45.1⁺ BM cells. Data are shown as mean ± SEM, of 2 independent experiments, with a minimum of 7 mice per group.

Figure 2

OB/P Cx43 expression is required for progenitor-dependent radioprotection and serial transplantation engraftment of WT HSCs. (A) Survival curve of secondary recipients of WT hematopoiesis from WT (dashed line) or OB/P Cx43-deficient primary recipients (solid line), which had been transplanted with 1 × 10⁶ WT BM cells (high dose). BM from primary recipient mice were pooled and transplanted into secondary recipients and analyzed. The remaining secondary recipient mice were killed on day 71 after transplantation and the experiment was terminated (arrow). (B) Representative micrographs (original magnification ×10; bar = 0.25 mm) of longitudinal femoral sections (H&E staining) of WT in WT secondary recipients and WT in OB/P Cx43-deficient secondary recipients. Migrographs were obtained with an Olympus CKX41, objectives ×10, ×40, and ×100. The images were acquired with a motican 2500 color camera (5.0 MPixel, USB2.0), Motich China Group Co Ltd, and processed using Motic Images Plus 2.0 software. (C-E) PB counts of control and WT hematopoiesis in OB/P Cx43-deficient secondary recipients on day 71 after transplantation. Empty bars represent data from WT controls; solid bars represent data from secondary recipients of WT hematopoiesis into OB/P Cx43-deficient hematopoiesis in primary recipients. (F-H) Quantification of BM cell populations from secondary recipients receiving WT chimeric BM from a primary recipient HM that was either WT (empty bar) or OB/P Cx43-deficient HM (solid bar). (F) Total CD45.1⁺ BM cellularity. (G) CD45.1⁺ Lin⁻/c-kit⁺ BM cells. (H) CD45.1⁺ LSK BM cells. Values shown are mean ± SEM n ≥ 5 mice per group (**P < .01).

Figure 3

BM homing of WT hematopoietic progenitor cells (HPCs) is impaired in myeloablated OB/P Cx43-deficient mice. Impaired migration of HSC/Ps through irradiated Cx43-deficient stromal cell lines in a vitro assay. (A) Homing of BM CFU-C in the BM of either irradiated control (empty bar) or OB/P Cx43-deficient (solid bar) recipient mice at 16 hours after transplantation. A dramatic reduction in the ability of HPCs to home into the BM was seen when Cx43 is not expressed in the HM. (B) HSC homing assay in WT or OB/P Cx43-deficient mice. Graph represents homing (%) 3 hours after transplantation of CRUs as analyzed 20 weeks after transplantation into congenic recipients. The secondary recipients were injected with BM cells from primary WT or OB/P Cx43 deficient recipient mice that were previously transplanted with WT 25 × 10⁶ CD45.1⁺ BM cells. Harvesting of BM cells from primary recipients was performed 3 hours after transplant. (C) Similarly to OB/P Cx43-deficient mice, homing of WT CFU-C to the BM of polyI:C treated Mx1-Cre-Cx43^flox/flox mice was drastically reduced compared with homing of either WT or Cx43-deficient HSC/P into Mx1-Cre;WT mice at 16 hours after transplantation. Data represent n = 4 independent experiments with 5 mice per group and experiment. (D) Experimental scheme of the transstromal migration assay. LDBM cells were incubated on a monolayer of BM stromal cells previously layered on a transwell membrane. A Cxcl12 gradient was established from bottom to top of the transwell. (E-F) Transstromal migration of WT (E) or Cx43-deficient (F) HSC/Ps through WT or Cx43-deficient stromal cells. Migration of WT or Cx43-deficient HSC/Ps was similarly impaired when assayed in presence of WT or Cx43-deficient stromal cells. (G) Transstromal migration was restored on reintroduction of rat-Cx43 expression (gray bar) into Cx43-deficient stromal cell lines. Results from WT controls (empty bars) and mock-transduced, Mx1-Cre;Cx43-deficient stromal cells (solid bar) are also depicted. Data for homing experiments are shown as mean ± SEM of 3 independent experiments, with a minimum of 7 mice per group. Data for transstromal migration are shown as mean ± SEM n = 4 independent experiments (*P < .05; **P < .01; ***P < .001; #P = .08).

Figure 4

HSC/Ps are retained basally or after G-CSF administration, and located closer to the endosteum in the BM of nonmyeloablated OB/P Cx43-deficient mice. (A) PB CFU-C counts in WT (empty bar) and OB/P Cx43 deficient mice (solid bar). N = 4 independent experiments, n = 5 mice per group. (B) Percentage of chimerism in WT CD45.1⁺ lethally irradiated recipient mice, which were transplanted with a mixture of 5 × 10⁵ WT CD45.1⁺ BM cells (competitor cells) and 400 μL PB from CD45.²⁺ WT or OB/P Cx43-deficient donors. N = 2 independent experiments, n = 5 mice per group. (C) Count of circulating HSC/Ps after G-CSF administration in WT and OB/P Cx43-deficient mice. N = 3 independent experiments, n = 5 mice per group. (D) Hematopoietic progenitor content of BM of G-CSF treated WT and OB/P Cx43-deficient mice. N = 3 independent experiments, n = 5 mice per group. (E) Nonirradiated WT or OB/P Cx43-deficient mice were transplanted with WT BM-EGFP⁺ cells. Percentage of EGFP⁺ LSK and LK⁺S⁻ cell populations, determined by flow cytometry, in the PB 16 hours after transplantation. N = 2 independent experiments, n = 7-8 mice per group. Data are shown as mean ± SEM. (F-G) In vivo imaging using multiphoton microscopy technique of transplanted CFSE-labeled hematopoietic progenitors (Lin⁻c-kit⁺ BM cells average n > 25) clustered closer to the endosteum in OB/P Cx43-deficient mice than in controls (empty circles; n = 2 independent experiments). HSC/Ps were stained with CFSE directly after isolation and transplanted into WT or OB/P Cx43-deficient mice. After 24 hours intravital multiphoton imaging was performed in the tibiae of the recipient animals. (F) HSC/Ps (displayed in green) located distantly from the bone (brown, detected by its SHG signal) in the tibia of a WT recipient. (ii) In addition, the blood vessels could be detected after an intravenous injection of rhodamine-dextran (displayed in red). IVM was performed using a Zeiss LSM-710 microscope with simultaneous detection via external nondescanned detectors and Zeis ZEN software (2009 release). Illumination was performed at 800 or 850 nm using a MaiTai TiSa laser via a 20× water-dipping Ins with 1.0 NA. Images were recorded every 60 seconds. Raw data were reconstructed using Volocity 4.0 software (PerkinElmer/Improvision). The dimensions of the original Z-stack were: X = 85.53 μm; Y = 85.53 μm; and Z = 108 μm. The image resolution (XYZ) was 512 × 512 × 28 μm. The pixel size was 0.167 μm in X and Y and 4 μm in Z. (iii) HSC/Ps (displayed in green) in close contact to the endosteum (brown, detected by its SHG signal) in the tibia of an OB/P Cx43-deficient mouse. (iv) After an injection of rhodamine-dextran also the blood vessels could be detected (displayed in red). The dimensions of the original Z-stack were: X = 227.36 μm and Y = 227.36 μm; Z = 92 μm. Image resolution (XYZ) was 512 × 512 × 28 μm. One pixel in X and Y are equivalent to 0.445 μm and in Z = 4 μm. (G) Distance to endosteum of individual HSC/P analyzed in vivo. Transversal bars denote mean ± SEM (*P < .05; **P < .01).

Figure 5

Cxc12 expression and secretion are increased in OB/P Cx43-deficient BM. (A-C) Western blot of whole BM lysates analyzed for Cxcl12 expression basally (A) and after G-CSF administration (C). (B-D) Extracellular BM Cxcl12 levels determined by ELISA in WT and OB/P Cx43-deficient BM from steady-state and lethally irradiated mice (16 hours after irradiation; B) and in nonmyeloablated mice after G-CSF administration (D). Empty bars represent control (WT) values and solid bars represent OB/P Cx43-deficient values. Data are shown as mean ± SEM of 3 independent experiments with a minimum of 5 mice per group (*P < .05; **P < .01).

Figure 6

Cx43 regulates the cellular composition in nonmyeloablated BM. (A-B) Content of BM mesenchymal (CFU-F; A) and osteoblastic progenitors (CFU-OB; B) in BM of WT and OB/P Cx43-deficient mice. (C-E) Quantification of immunophenotypically identified nonhematopoietic cells (C) and cadherin-11 negative (D) or positive (E) subpopulations. (F-G) OB/P-Cx43–deficient mice showed an increased number of Cxcl12⁺ cells among both CD45⁻/Ter119⁻/cadherin-11⁺ and CD45⁻/Ter119⁻/cadherin-11⁻ cell subpopulations. (H) Representative example of flow cytometry analysis of 5-bromo-2'-deoxyuridine (BrdU⁺) cells on different mesenchymal cell subpopulations. (I) In vivo proliferation of BM CD45⁻/Ter119⁻ and Cxcl12⁺ mesenchymal cells. (J) Normalized RNA expression of sclerostin in cortical bones from WT or OB/P Cx43-deficient mice. Results for sclerostin expression are presented as average of 2 mice per group where expression was analyzed in femora, tibiae, and pelvic cortical bones. Empty bars present control data; solid bars represent data from OB/P Cx43 deficient mice. Analysis of OB/P populations are shown as mean ± SD. N = 3 different experiments n = 6 mice per group (*P < .05; **P < .01).