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Hematopoiesis is dependent upon the bone marrow microenvironment, which is comprised of multiple mesenchymal cell types, including fibroblasts, endothelial cells, osteoblasts and stroma progenitors. The canonical Wnt signaling pathway, which relies on the β-catenin protein to mediate its signal, is necessary for the normal development of mesenchymal tissue. We hypothesized that canonical Wnt signaling regulates the cellular composition and function of the bone marrow microenvironment. We observed that a β-catenin-deficient bone marrow microenvironment maintained hematopoietic stem cells but exhibited a decreased capacity to support primitive hematopoietic cells. These results correlated with decreased numbers of osteoblasts and with decreased production of basic fibroblast growth factor, stem cell factor, and vascular cell adhesion molecule-1. From these data, we propose a model in which β-catenin in the microenvironment is required non-cell autonomously for long-term maintenance of hematopoietic progenitors.
Hematopoiesis is the process by which blood cells of all lineages are generated throughout the lifetime of an organism. In adult mammals, hematopoiesis primarily takes place within the marrow of the long bones and is initiated by the hematopoietic stem cell (HSC). HSCs are the only bone marrow cells capable of differentiating into all blood cell lineages in a clonal manner . In order to maintain their numbers throughout life, HSCs can also give rise to daughter cells that retain the function of the parent stem cell, a process called self-renewal.
The mechanisms by which the HSC decides between a cell fate of differentiation and self-renewal are unclear. For over 30 years, it has been known that the non-hematopoietic cells that comprise the bone marrow microenvironment, such as fibroblasts, osteoblasts, adipocytes, reticular cells and endothelial cells, have a functional role in regulating hematopoiesis . These cells support hematopoiesis through production of soluble (e.g., stem cell factor [SCF], stromal-derived factor-1α [SDF-1α], granulocyte-macrophage colony stimulating factor [GM-CSF], and interleukin-6 [IL-6]) and membrane-bound (e.g., vascular cell-adhesion molecule 1 [VCAM-1] and Jagged) factors [3–8]. Physically, HSCs interact with the microenvironment at biologically distinct cellular niches. HSCs preferentially reside within the trabecular bone area (TBA), and physically interact with osteoblasts and endothelial cells [9, 10]. Signaling pathways that regulate differentiation of mesenchymal tissues may be necessary for the development of functional niches that regulate hematopoietic stem cells and their progenitors.
The Wnt family of ligands are secreted glycoproteins that activate several distinct signaling pathways, the best characterized of which is the canonical Wnt pathway . Briefly, this pathway is activated by a Wnt ligand binding to its cognate Frizzled receptor (10 in the mouse) and one of two low-density lipoprotein receptor-related protein (LRP) 5/6 co-receptors [12, 13]. This ultimately leads to the stabilization and subsequent nuclear translocation of β-catenin, where it binds to T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors and induces target gene expression . In the absence of Wnt ligand binding, two kinases, GSK-3β and CK1α, phosphorylate β-catenin and target it for ubiquitination and degradation [15, 16].
β-catenin is necessary for the development of mesoderm, the germ layer from which the hematopoietic organs, the vasculature, and the long bones of the limbs are derived [17, 18]. We hypothesized that β-catenin is necessary for the microenvironment to support normal hematopoiesis. In this report, we show that a bone marrow microenvironment deficient in β-catenin exhibited a decreased capacity to support primitive hematopoietic cells in vitro. This decrease correlated with decreased numbers of osteoblasts in vitro and in vivo and with decreased production of the hematopoietic regulatory factors basic fibroblast growth factor (bFGF), SCF, and VCAM-1. From these observations, we propose a model in which β-catenin is necessary for generation of osteoblasts and the maintenance and expansion of hematopoietic progenitors in the adult bone marrow microenvironment.
Mice containing conditional null β-catenin alleles (CatnbC/C) were generated as described in Guo, et al., . Catnblox(ex3) mice were provided by Dr. Mark M. Taketo (Kyoto, Japan) . CatnbC/C and Catnblox(ex3) mice were crossed with Mx1-Cre mice to generate CatnbC/C Mx1-Crecre/+ and Catnblox(ex3) Mx1-Crecre/+ mice. CatnbC/C and Catnblox(ex3) mice were used as wild-type controls. B6.SJL-Ptprca/BoAiTac mice were obtained from Taconic (Germantown, NY). 129 x C57BL/6 F1 mice were obtained from the Jackson Laboratory (Bar Harbor, ME).
Induction of Mx1-Cre was performed by injecting 300 mg polyinosinic-polycytosylic acid (pIpC; 3 to 5 injections; Invivogen, San Dieg, CA) intraperitoneally into Catnb C/C Mx1-Crecre/+ mice, Catnblox(ex3) Mx1-Crecre/+ mice, and their respective control mice every other day. All mice used in these experiments were between 6 to 8 weeks of age.
Confluent monolayers of bone marrow derived stroma cells were generated according to the principles established in Dexter, et al., . Briefly, bone marrow cells were harvested and plated in Myelocult M5300 media (Stem Cell Technologies, Vancouver, Canada) at a concentration of 3.75 × 106 cells/ml. The media was supplemented with 10−6 M hydrocortisone. The cultures were incubated at 33° C with 5% CO2 for 2 to 3 weeks. Half of the media was changed twice per week. Confluent stromal monolayers were then irradiated with 1500 cGy. For certain experiments, rat parathyroid hormone (PTH 1–34; Bachem Bioscience, King of Prussia, PA) or vehicle was added at each media change during stroma establishment.
Conditional excision of Catnb sequences was determined by PCR analysis of stroma cell genomic DNA. PCR was performed using the primers CatnbYC1F: 5-'CAGCAAGCCACCGATGGGATC-3' and either CatnbYC1B: 5'-CTGAAAATG CTACCTGAAGAAGC-3' or CatnbYC2B: 5'-CTCCCTCACCCTTAAGGTCCTT-3’ (Figure 1). 1F and 1B amplify a 150 bp fragment from a wild-type Catnb allele or a 202 bp fragment from the conditional Catnb C/C allele. 1F and 2B amplify a 240 bp fragment from the excised ΔCatnb allele. PCR was performed for 35 cycles at the following conditions: 94° for 1 min., 60° for 1 min. and 72° for 1 min. Real-time PCR was also performed on stroma DNA samples. 50 ng of genomic DNA isolated from wild-type and ΔCatnb stroma cells were amplified with primer sets 1F + 1B or 1F + 2B using a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). PCR reactions were carried out using the AB 7900 HT platform (Applied Biosystems).
Canonical Wnt signaling activity was measured in stroma cells after transient co-transfection of TOPFLASH or FOPFLASH (Millipore, Temecula, CA) at a 20:1 ratio with the internal control plasmid pGL4-hRluc (Promega, Madison, WI). Plasmid DNA was transfected using FuGENE reagent (Roche Diagnostics, Indianapolis, IN). Measurement of luciferase activity was performed 48 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.
To measure the ability of mesenchymal progenitors to form fibroblast colonies (CFU-F), 1 × 106 bone marrow cells were harvested from mice two days after the final pIpC treatment and cultured for 10 days in MesenCult medium (Stem Cell Technologies) according to the manufacturer's instructions. After 10 days, CFU-F were fixed in methanol, stained with Giemsa staining solution and scored.
Bone marrow cells were harvested from wild-type B6.SJL-Ptprc a/BoAiTac mice and depleted for hematopoietic lineage-positive cells as previously described . Lineage negative (Lin−) cells were then seeded onto irradiated stroma layers at a concentration of 4 × 104 cells/cm2 culture surface area. Media was changed twice per week.
For in vitro analysis of hematopoietic progenitors, cells were cultured in Methocult GF M3434 methylcellulose media (Stem Cell Technologies) after 1, 2, and 3 weeks in co-culture with stroma. Colony forming units-granulocyte, macrophage (CFU-GM) were scored after 7 days in culture. Unseeded and irradiated stroma cells were also cultured in methylcellulose media as a negative control.
To detect long-term culture-initiating cells (LTC-IC), 5 × 103 lin− cells were seeded onto stroma and co-cultured for 4 weeks. Adherent and non-adherent cells were harvested and hematopoietic cells counted. Cells were plated in methylcellulose at 5 × 104 cells per culture. CFU-GM were scored after 7 days. To determine the frequency of LTC-IC per 103 input lin− cells, we used the formula
For in vivo analysis of hematopoietic progenitors, between 1 and 5 ×105 hematopoietic cells that were co-cultured on stroma for 1 week were transplanted into irradiated 129 x B6 F1 recipients (850 cGy). Colony-forming units-spleen (CFU-S) were scored twelve days later after fixation of recipient spleens in Telly's fixative solution.
For the rescue experiments described in Figure 6B, recombinant mouse bFGF (10 ng/ml), SCF (100 ng/ml), and osteopontin (OPN; 1 µg/ml) (R & D Systems, Minneapolis, MN) were added with the lin− cells and at subsequent media changes. The amounts of recombinant factors used in this experiment were based on previously published data that demonstrated biological effects of these factors on hematopoietic cells (bFGF, ; SCF, ; OPN ).
Analysis of hematopoietic progenitors by flow cytometry was performed on hematopoietic progenitors harvested from stroma co-cultures 3 days after seeding. Cells were depleted for lineage-positive cells as previously described . For all experiments, cells were stained with monoclonal antibodies to c-kit (APC-Cy 5.5-conjugated, clone 2B8, Invitrogen, Carlsbad, CA), Sca-1 (Pacific Blue-conjugated, clone D7, BioLegend, San Diego, CA), and IL-7Rα (Biotin-conjugated, clone A734R, eBioscience, San Diego, CA). Subsequent detection of biotinylated IL-7Rα was performed using APC-conjugated streptavidin (BD Biosciences, San Diego, CA).
To analyze apoptosis of hematopoietic progenitors, cells were harvested from stroma co-cultures 3 and 7 days after seeding and stained with FITC-conjugated anti-Annexin V (eBioscience) and propidium iodide according to the manufacturer's instructions. To analyze cell cycle status, cells were fixed using Cytofix/Cytoperm solution according to the manufacturer’s instructions (BD Biosciences) and then incubated for 2 hours on ice in a solution of 50 µg/ml propidium iodide with 0.05 mg/ml RNase A. To analyze cellular proliferation, cells were fixed and stained with a FITC-conjugated anti-BrdU antibody using the FITC BrdU Flow Kit (BD Biosciences) according to the manufacturer’s instructions. Analysis for this and subsequent experiments was performed on a LSRII Aria (BD Biosciences) using 488-nm argon and 633-nm helium neon lasers. P values were generated using Student’s t-test.
1 × 107 whole bone marrow cells were harvested from B6.SJL-Ptprca/BoAiTac mice and transplanted into lethally irradiated CatnbC/C and CatnbC/C Mx-1-Cre mice as above. Eight weeks later, recipients were treated with 3 −5 doses of pIpC. Four weeks later, bone marrow cells from surviving recipients were analyzed by flow cytometry and used in competitive repopulation assays (Figure 4A).
To analyze the ability of the in vivo microenvironments to support hematopoietic progenitors, bone marrow cells were harvested from CatnbC/C and CatnbC/C Mx1-Cre mice 4 weeks after their final treatment with pIpC and stained with APC-conjugated rat anti-mouse antibodies CD4, (clone GK1.5), CD8 (53-6.7), B220 (6B2), Mac-1 (M1/70), Gr-1 (8C5), and Ter119, FITC-conjugated rat anti-mouse Sca-1, PE-conjugated rat anti-mouse c-kit (BD Biosciences), and biotin-conjugated rat anti-mouse IL-7Rα. Secondary staining was performed using streptavidin-conjugated PE-Cy7 (BD Biosciences). P-values were generated by Student’s t-test.
For competitive repopulation assays, 1 × 106 bone marrow cells from the primary recipients (CD45.1+) were mixed with 1 × 106 bone marrow cells from C57Bl/6 primary whole bone marrow cells (CD45.2) and transplanted into lethally irradiated 129Sv x C57Bl/6 F1 recipients (950 cGy). Hematopoietic repopulation was measured after sixteen weeks by staining whole bone marrow cells with APC-conjugated rat anti-mouse CD45.1 (clone A20, eBioscience), FITC-conjugated rat anti-mouse CD45.2 (clone 104), and PE-conjugated rat anti-mouse CD4, CD8, B220, Mac-1, Gr-1, and Ter119 (BD Biosciences)
Secondary transplants were performed by transplanting 2 × 106 bone marrow cells from primary recipients into lethally irradiated 129Sv x C57Bl/6 F1 recipients. Hematopoietic repopulation was measured after sixteen weeks by staining whole bone marrow cells with APC-conjugated rat anti-mouse CD45.1, FITC-conjugated rat anti-mouse CD45.2, and PE-conjugated rat anti-mouse B220 and Gr-1.
In vitro histochemical staining for alkaline phosphatase activity was performed as described in Calvi, et al., . For histological analysis, hind tibias were isolated from 6 to 8 week old wild type and ΔCatnb mice (past the age at which growth plate closure occurs) four weeks after the final injection with pIpC. Hind tibias were fixed in 4% paraformaldehyde and decalcified using sodium citrate/formic acid. Samples were embedded in paraffin and cut into 5 µm-thick sections, which were attached to slides. Slides were then stained with hematoxylin and eosin. All steps after the initial fixation were performed by Histoserv, Inc. (Gaithersberg, MD). Histomorphometric analysis of sectioned tibias was performed using the OsteoMeasure system (Osteometrics Inc., Atlanta, GA). Four samples from tibias of wild type or ΔCatnb mice were used. Areas of trabecular bones proximal to the hypertrophic chondrocytes were selected for the measurements as shown from the figure (Figure 5C). The results generated from the software were analyzed using Student t-test.
Custom mouse protein antibody arrays were developed using the services of RayBiotech (Norcross, GA). For a list of the proteins tested, please see Supplemental Table 1. Isolation and hybridization of whole cell extract from pooled stroma cultures of wild type and β-catenin deficient cells was performed according to the manufacturer's instructions. Briefly, equal amounts of whole cell extracts from wild type and β-catenin deficient stroma cells were hybridized to membranes bound with antibodies to 30 different proteins. After hybridization, the membrane was then probed with a set of biotin-labeled antibodies specific to the 30 proteins. Bound secondary antibody was detected through streptavidin-conjugated horseradish peroxidase, allowing signals to be detected and quantified through chemiluminescence. Chemiluminescent signals were detected with a Typhoon scanner and the relative protein levels were normalized to the positive control.
Stromal monolayers were stained with rat anti-mouse monoclonal antibodies to VCAM-1 (FITC-conjugated, clone 429, BD Biosciences) and either polyclonal mouse anti-alkaline phosphatase (APC-conjugated, clone B4–78, R & D Systems) or rat anti-mouse plasmalemma vesicle associated protein (PVLAP, clone MECA-32, Abcam, Cambridge, MA). Secondary staining using APC-conjugated goat-ant-rat IgG was performed to detect PVLAP. P-values were generated by Student’s t-test.
To inactivate β-catenin in the bone marrow microenvironment, we crossed transgenic mice that contained loxP sites flanking the third through sixth exons of the gene encoding β-catenin (CatnbC/C) with mice that expressed Cre recombinase under the control of the Mx1 promoter (Mx1-Cre) [19, 26]. Injection of pIpC into CatnbC/C; Mx-Crecre/+ mice induced the deletion of exons 3 through 6, resulting in mice homozygous for the null allele (ΔCatnb) (Figure 1A). As described previously, deletion of Catnb resulted in the majority of ΔCatnb mice dying within four weeks after the final treatment and 100% mortality after 8 weeks (Figure 1B) with no gross hematologic defects .
We harvested bone marrow cells from pIpC-treated wild-type mice (CatnbC/C) and ΔCatnb mice 2 days after the final pIpC treatment to establish confluent monolayers of bone marrow stroma cells to test whether β-catenin is necessary for normal support of hematopoiesis in vitro . These stroma cultures consist of the adherent cells of the bone marrow and therefore contain adherent hematopoietic cells as well as mesenchymal cells. Deletion of Catnb was analyzed by PCR analysis of genomic DNA isolated from stroma cells and was nearly 100% (Figure 1C and Supplemental Figure 1). We then determined whether Canonical Wnt signaling was absent in ΔCatnb stroma cultures. Transfection of stroma cells with a luciferase reporter cassette controlled by TCF/LEF binding elements (TOPFLASH) resulted in a significant decrease in canonical Wnt-mediated gene expression in ΔCatnb stroma compared to wild-type (Figure 1D, p = .001). Furthermore, there was no difference in luciferase activity between wild-type and ΔCatnb stroma when transfected with a reporter cassette containing mutated TCF/LEF binding sites (FOPFLASH). These data indicate that canonical Wnt signaling is active in in vitro wild-type stroma cultures and that the loss of β-catenin reduces canonical Wnt signaling to a background level. We also observed no difference in the average number of stroma cells in wild type and ΔCatnb cultures or in fibroblast colony formation (Figure 1E and 1F).
Stroma cultures were irradiated and seeded with wild-type hematopoietic progenitors. After one week, there was a 50% reduction (p = 0.04) in the number of hematopoietic cells in cultures with ΔCatnb stroma cells relative to control (Figure 2A and Table 1). The reduction in viable cell numbers correlated with an initial 1.5-fold increase in the percentage of hematopoietic cells cultured on ΔCatnb stroma undergoing apoptosis compared to cells cultured on wild-type stroma (Figure 2B and 2C; p < .01). The increase in apoptosis of cells cultured on ΔCatnb stroma was observed across different populations of progenitors (HSC, common lymphoid progenitors (CLP), and common myeloid progenitors (CMP); Supplemental Figure 2) [28, 29]. We observed no difference between the percentages of hematopoietic progenitor populations in S/G2/M phases (Supplemental Figure 2). We did observe a significant 3-fold decrease in the percentage of CMPs, but not HSCs or CLPs, which progressed through the cell cycle when cultured on ΔCatnb stroma (Figure 2D and 2E, Supplemental Figure 2). After the initial decline, cells cultured on ΔCatnb stroma expanded at the same rate as cells cultured on wild-type stroma (Table 1) and after 1 week in culture, we observed no difference between the percentages of actively apoptotic or proliferating hematopoietic cells cultured on wild-type or ΔCatnb stroma (data not shown).
We tested the ability of ΔCatnb stroma to support hematopoietic progenitor cells. After 1 week in culture, we observed a significant decrease in the total number (7.7-fold, p = 0.03) and frequency (3.5-fold, p = 0.05) of CFU-C in cultures with ΔCatnb stroma compared to wild-type (Figure 3A and 3B and Table 1, Top Section, Experiments 1 through 4). We then tested the ability of wild-type and ΔCatnb stroma to support formation of hematopoietic colonies in long-term cultures. In two independent experiments, we observed decreases in both the total number and frequency of CFU-C after three weeks in culture with ΔCatnb stroma compared to wild-type (Figure 3C and 3D and Table 1, Top Section, Experiments 3 and 4). These results indicated that the loss of β-catenin inhibited the ability of stroma cells to maintain hematopoietic progenitors. Based on these data, we determined whether β-catenin was necessary for the stroma to maintain more primitive hematopoietic cells. The total number of primitive multilineage hematopoietic progenitor cells (CFU-S) significantly declined (3.0-fold, p = 0.03) when cultured on ΔCatnb stroma (Figure 3E and Table 1, Bottom Section). We also observed a 7-fold decrease (p < 0.001) in the ability of ΔCatnb stroma to support long-term culture-initiating cells (LTC-IC) compared to wild-type (Figure 3F).
To determine whether the levels of β-catenin in the stroma directly regulated hematopoietic progenitor function, we established a model of constitutive stabilization of β-catenin in stroma cells. We crossed transgenic mice that contained loxP sites flanking the third exon of Catnb (Catnb lox(ex3)/+) with Mx1-Cre mice. Treatment of Catnb lox(ex3)/+ Mx1-Crecre/+ mice with pIpC resulted in excision of the third exon and a constitutively stable β-catenin protein. (Catnb Δex3/+). In two independent experiments using our in vitro experimental system, we observed no difference in the ability of hematopoietic progenitors to form myeloid colonies when cultured on wild-type or Catnb Δex3/+ stroma for 1 week (Figure 3G). However, after 2 and 3 weeks, CFU-C frequency was higher among hematopoietic progenitors cultured on Catnb Δex3/+ stroma compared to wild-type. Together, these data suggest that β-catenin regulates the ability of stroma cells to maintain normal numbers and function of hematopoietic progenitor cells.
To determine whether a β-catenin deficient microenvironment would support fewer HSCs in vivo, untreated wild-type and ΔCatnb mice were transplanted with control CD45.1+ bone marrow (Figure 4A). Eight weeks later, wild-type and ΔCatnb recipients were treated with pIpC, establishing a mouse model of a β-catenin deficient microenvironment and wild-type hematopoietic system. Because of the lethality associated with Catnb excision, bone marrow cells were harvested from surviving mice and analyzed for HSC content and function 4 weeks after the final pIpC treatment.
There was no significant difference in bone marrow cellularity between mice with a wild-type or ΔCatnb microenvironment (Supplemental Figure 3). Similar to our ex vivo results, we observed a significant reduction in the percentage of lin−, c-kitHI, Sca-1HI (LSK) cells (which includes both day-12 CFU-S and HSCs) in mice with a ΔCatnb microenvironment compared to control (Figure 4B and 4C) [30, 31]. We observed no difference in the percentage of bone marrow cells with either a CLP or CMP phenotype (Supplemental Figure 3). In competitive repopulation analysis, we observed no difference in mean repopulation between HSCs from the wild-type microenvironment compared to the ΔCatnb microenvironment (Figure 4D). HSCs from a ΔCatnb microenvironment exhibited multilineage differentiation (Figure 4E) and long-term self-renewal (as measured in a secondary transplantation assay) (Figure 4F).
An important component of the bone marrow microenvironment is the osteoblast and several groups have used transgenic models to show that elimination of canonical Wnt signaling during embryogenesis inhibits development of the osteoblast lineage [18, 32, 33]. We hypothesized that the loss of β-catenin in the bone marrow microenvironment would result in decreased numbers of osteoblasts, and as a consequence, decreased support of hematopoiesis. Histochemical analysis for alkaline phosphatase (ALP), a marker for osteoblasts, on confluent monolayers of ΔCatnb and wild-type stroma cells demonstrated a significant reduction in the percentage of ALP+ cells in ΔCatnb stroma compared to wild-type (Figure 5A and 5B, p < .05). To determine whether a similar effect occurred in vivo, we isolated tibias from wild-type and ΔCatnb mice 4 weeks after the final pIpC treatment. Hematoxylin and eosin staining of decalcified tibia sections revealed no difference between wild-type and ΔCatnb mice in the thickness of cortical bone or in chondrocyte formation. There was a significant decrease in the number of osteoblasts within the trabecular bone area in ΔCatnb mice (Figure 5C and 5D, p< .01). These data indicate that the loss of β-catenin function can significantly alter the cellular composition of the microenvironment, primarily by regulating osteoblast formation.
Parathyroid hormone (PTH), a positive regulator of osteoblast formation, can enhance the ability of the bone marrow microenvironment to support hematopoiesis and activate the canonical Wnt pathway [7, 34, 35]. To test whether the effects of PTH on hematopoiesis required β-catenin, we established ΔCatnb stroma cultures that had been treated with recombinant PTH and then seeded them with wild-type lin− progenitors. When PTH was added during the establishment of ΔCatnb stroma, CFU-C frequency was 3-fold higher than in ΔCatnb stroma cultures without PTH (p < .001) (Figure 5E). PTH also increased the percentage of ALP+ cells in ΔCatnb stroma (Supplemental Figure 4). These data indicate that PTH can rescue the defect in hematopoiesis due to the loss of β-catenin in bone marrow stroma.
We surveyed hematopoietic regulatory protein levels using mouse antibody arrays that were probed with whole cell extract from cultures of wild-type and ΔCatnb stroma cells (Figure 6A). Out of the 30 proteins analyzed (Supplemental Table 1), we observed that ΔCatnb stroma cell extract contained significantly decreased levels of bFGF, osteopontin (OPN), SCF, and VCAM-1 (Figure 6B). All of these factors have been shown to regulate proliferation, apoptosis, and adhesion of hematopoietic cells [25, 36–38]. We then tested whether addition of these factors could rescue the defect in hematopoietic support capability of ΔCatnb stroma. Addition of soluble bFGF, SCF, and OPN at pharmacologic doses significantly increased the total number and frequency of CFU-GM in cells cultured on ΔCatnb stroma (Figure 6C and Supplemental Table 2). In contrast, addition of these factors individually had no effect on CFU-GM number or frequency (data not shown).
To determine whether the decreased levels of VCAM-1 could be caused by either a reduction in the number of cells producing the protein or by a reduction in the level of VCAM-1 per cell, we performed flow cytometry analysis of VCAM-1 expression on cultured stroma cells. We observed a significant reduction in the percentage of ΔCatnb VCAM-1+ ALP+ osteoblasts and VCAM-1+ MECA-32+ endothelial cells (Figure 6D and 6E; p < .01), indicating that the loss of β-catenin affects VCAM-1 levels in multiple cell types.
In this study, we have demonstrated that the loss of β-catenin within the microenvironment is associated with decreased maintenance of hematopoietic progenitors but not for HSCs. Our data indicate that β-catenin directly regulates the cellular composition of the microenvironment, specifically the osteoblast lineage. This observation is in agreement with those reported in Scheller, et al., that production of constitutively active β–catenin correlated with increased trabecular bone . It has been reported that inhibition of canonical Wnt signaling early in embryonic development and in mesenchymal stem cells is necessary for differentiation of the osteoblast lineage [18, 32, 33, 40]. Conversely, Glass, et al., used an osteoblast-specific Col1a1-Cre transgenic system in which β-catenin is deleted at later stages of development and observed no decrease in osteoblast numbers . It is unclear in our system whether the loss of β-catenin inhibits differentiation of osteoblasts from mesenchymal progenitors or survival of mature osteoblasts and may have different functions at different stages at osteoblast development and maintenance.
Decreased amounts of bFGF, SCF, OPN, and VCAM-1 in ΔCatnb stroma also correlated with decreased hematopoietic cell number and increased apoptosis of hematopoietic progenitors. These observations are consistent with previous studies that have shown that bFGF, SCF, and VLA4/VCAM-1 signaling inhibit apoptosis in hematopoietic cells [23, 24, 42]. Together, our data suggest a model in which β-catenin is necessary for regulating the function of the established bone marrow microenvironment in addition to its cellular composition. In addition to its role as the central mediator of canonical Wnt signals, β-catenin also interacts with either E- or N-cadherin to form part of the adherens junction complex. [43, 44]. Therefore, it is possible that the decreased ability of the microenvironment to support hematopoiesis due to the loss of β-catenin is caused by disruption of adherens junctions in stroma cells in addition to loss of canonical Wnt signaling.
Osteoblasts support hematopoietic progenitors in vitro and in vivo models of osteoblast deficiency exhibit compromised hematopoiesis [7, 45–49]. Visnijic, et al, reported that the reversible deletion of osteoblasts led to decreased bone marrow cellularity and an increase in extramedullary hematopoiesis in vivo . However, we did not detect a decrease in bone marrow cellularity or a corresponding increase in the number of LSK cells within the spleen (Supplemental Figure 2). We attempted to determine the effects of deletion of β-catenin specifically within osteoblasts using the Col1a1-Cre model. However, similar to previous reports, we were unable to observe complete osteoblast-specific deletion of β-catenin using this system and were unable to detect any differences in either LSK numbers or in HSC repopulating ability (data not shown) . Therefore, it is still unclear as to whether osteoblast-specific loss of β-catenin affects the normal function of the microenvironment.
Our data show a correlation between reduced numbers of osteoblasts in ΔCatnb stroma and reduced numbers of hematopoietic progenitors. The effect that the loss of β-catenin has on HSC function is still unclear. It is possible that the loss of β-catenin has a reduced effect on the largely quiescent HSC population as opposed to the more actively cycling hematopoietic progenitors [29, 51, 52]. We were unable to detect any difference in the percentage of LSK cells in the G0 phase of the cell cycle (Supplemental Figure 3). Conversely, Fleming, et al. demonstrated that osteoblast-specific production of the soluble factor Dikkopf-1, which inhibits canonical Wnt signaling, induces cycling of HSCs and the eventual loss of HSC repopulating activity . One explanation for this apparent discrepancy is that we would observe a similar outcome in our model if not for the intervening mortality. Alternatively, their observations may be due to the combination of soluble Dikkopf-1 factor acting on HSCs and the microenvironment in contrast to our system in which removal of β-catenin is targeted to the microenvironment.
We propose that the decrease in the numbers of LSK cells in the ΔCatnb microenvironment is due to a reduction in the number of CFU-S rather than HSCs. This finding is in agreement with the results of Kiel, et al., in which biglycan-deficient mice exhibited loss of trabecular bone and osteoblasts but not a corresponding loss of HSC frequency or function . We propose that β-catenin in the microenvironment does not directly regulate HSC function but is necessary to support actively proliferating hematopoietic progenitors.
We thank Dr. Mark M. Taketo for his generous gift of the Catnblox(ex3)/+ transgenic mouse line. MJN was supported in part by institutional funds provided by Roswell Park Cancer Institute
Author Contribution:MJN: Conception and design, financial support, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript
KKM: Collection and assembly of data and data analysis
YY: Conception and design, provision of study material, final approval of manuscript
DMB: Conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript