Bidirectional interactions between bone metabolism and hematopoiesis.

International audienceInteractions between hematopoiesis and bone metabolism have been described in various developmental and pathological situations. Here we review this evidence from the literature with a focus on microenvironmental regulation of hematopoiesis and bone metabolism. Our hypothesis is that this process occurs by bidirectional signaling between hematopoietic and mesenchymal cells through cell adhesion molecules, membrane-bound growth factors, and secreted matrix proteins. Examples of steady-state hematopoiesis and pathologies are presented and support our view that hematopoietic and mesenchymal cell functions are modulated by specific microenvironments in the bone marrow

DAP12 overexpression induces osteopenia and impaired early hematopoiesis.

ITAM-bearing transmembrane signaling adaptors such as DAP12 and FcRγ are important players in bone homeostasis, but their precise role and functions are still unknown. It has been shown that osteoclast differentiation results from the integration of the RANK and of the DAP12 and FcRγ signaling pathways. DAP12-deficient mice suffer from a mild osteopetrosis and culture of their bone marrow cells in the presence of M-CSF and RANKL, fails to give rise to multinucleated osteoclasts. Here, we report that mice overexpressing human DAP12 have an osteopenic bone phenotype due to an increased number of osteoclasts on the surface of trabecular and cortical bone. This enhanced number of osteoclasts is associated with an increased number of proliferating myeloid progenitors in Tg-hDAP12 mice. It is concomitant with an arrest of B cell development at the Pre-Pro B/Pre B stage in the bone marrow of Tg-hDAP12 mice and important decrease of follicular and marginal B cells in the spleen of these animals. Our data show that the overexpression of DAP12 results in both increased osteoclastogenesis and impaired hematopoiesis underlining the relationship between bone homeostasis and hematopoiesis

IR-induced senescent MSC failed to generate bone in vivo.

<p>(<b>A</b>) Schematic of the experiment. Control or IR-induced senescent MSC were mixed with HA/TCP particles along with collagen and injected subcutaneously to the flank of mice. 10 weeks post injection, implants were retrieved from the animals, embedded in plastic, sectioned and stained with Goldner’s trichrome to detect bone formation. (B) Representative images from n= 6 implants per group showing mineralization (Goldner’s trichrome in green) from control or IR-induced senescent MSC. Implants were counterstained with hematoxylin eosin. Scale bar: 300µm.</p

Abrogation of osteogenic differentiation potential following irradiation is limited to stromal progenitor cells.

<p>(<b>A</b>) MSC and osteoblasts (OB–SC) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown for each population. Scale bar: 2mm. Phase contrast photograph showing the presence of senescent MSC in absence of mineralization is also shown. (<b>B</b>) Quantification of mineralization was determined by the extraction of Alizarin Red S and detection by spectrophotometry. (<b>C</b> and <b>D</b>) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC and OB–SC populations cultured or not in osteogenic differentiation media. Mean ± standard error; *: <i>p</i> value < 0.05.</p

Loss of osteogenic but not adipogenic potential in senescent MSC is p53 dependent.

<p>(<b>A</b>) MSC derived from p53 knockout mice (MSC-p53KO) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later stained for the expression of SAβ-gal activity. (<b>B</b>) The proliferation capacity of MSC-p53KO was determined using a CFU assay one week post-exposure or not to IR. (<b>C</b>) One week post exposure or not to IR, MSC-p53KO were placed in adipogenic differentiation media for 7 to 14 days. Representative photographs showing lipid accumulation stained with Oil Red O is shown. Scale bar: 200µm. (<b>D</b>) Quantification of lipid accumulation has determined by the extraction of Oil Red O staining and detection by spectrophotometry. (<b>E</b>) Expression of PPARγ was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC-p53KO cultured in adipogenic differentiation media. (<b>F</b>) One week post exposure or not to IR, MSC-p53KO were placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown. (<b>G</b>) Quantification of mineralization was determined by the extraction of Alizarin Red S staining and detection by spectrophotometry. (<b>H</b>) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC-p53KO populations placed in osteogenic differentiation media. Mean ± standard error of at least 3 individual experiments is shown; *: <i>p</i> value < 0.05.</p

Senescence of multipotent and committed stromal lineages following exposure to IR.

<p>(<b>A</b>) Murine bone marrow-derived multipotent stromal cells (MSC), osteoblasts (OB–SC) and pre-adiopocytes (3T3-L1) were exposed (IR) or not (CTRL) to 10 Gy IR and 7 days later stained for the expression of the senescence-associated β-galactosidase (SAβ-gal). (<b>B</b>) Quantification of the proportion of SAβ-gal positive cells in each population. (<b>C</b>) Sustained activation of the DNA damage response in stromal populations was measured by staining for the presence of 53BP1 DNA damage foci (in red) one week post exposure to IR. Nuclei were counterstained with DAPI. (<b>D</b>) The proliferation capacity of MSC, OB–SC and 3T3-L1 cell population was determined using a CFU assay one week post-exposure or not to IR. Mean ± standard error of at least three individual experiments is shown. p values were obtained by performing a Student’s t-test.</p

Ability of Tg-hDAP12 Bone Marrow-Derived Osteoblasts to Promote Osteoclastogenesis.

<p>Four conditions of co-culture were tested: WT bone-marrow-derived osteoblasts co-cultured with either WT or Tg-hDAP12 spleen cells (respectively WTOC/WTOB, TgOC/WTOB) and Tg-hDAP12 bone marrow-derived osteoblasts co-cultured with either WT or Tg-hDAP12 spleen cells (respectively WTOC/TgOB and TgOC/TgOB). 35×10³bone marrow-derived osteoblasts of either 3-month-old WT or Tg-hDAP12 female mice were co-cultured with 12×10⁵ cells, isolated from the spleen of 3-month-old WT or Tg-hDAP12 mice. After 6 days of co-culture in presence of ascorbic acid and 1α,25-dihydroxyvitamin D3 at 10⁻⁸ M, osteoclasts with nuclei ≥3 were counted after fixation and TRAP staining. Results are expressed as mean values of three independent experiments ± SE. ***<i>p</i><0.001.</p

Proliferation of spleen and bone marrow osteoclast precursors.

<p>Spleen cells of three 3-month-old WT and Tg-hDAP12 mice were seeded in triplicate in wells of 96-well plates at 78×10³ cells/well. At each time point, cells were incubated with BrdU during 3 hours. Incorporated BrdU was measured at 370 nm with a 492 nm reference wavelenght, using a colorimetric immunoassay. White circles: WT cells, black squares: Tg-hDAP12 cells. Results are means ± SE. <i>p</i><0,001 (***);<i>p</i><0,01 (**).</p

RANKL dose-dependent response of spleen and bone marrow osteoclast precursors.

<p>Bone marrow and spleen cells of three 3-month-old WT and Tg-hDAP12 mice were seeded in triplicate in wells of 96-well plates. 12,5×10³ cells/well for bone marrow cells and 78×10³per well for spleen cells. Bone marrow and spleen cells were grown in presence of M-CSF and increasing concentrations of RANKL either 20, 30, 50 and 100 ng/ml for bone marrow cells (A) or 20, 30, 40, 50 and 100 ng/ml for spleen cells (B). After 4 days in culture for bone marrow cells or 6 days in culture for spleen cells, TRAP-positive osteoclasts with ≥3 nuclei were counted. White bars:WT cells; Grey bars:Tg-hDAP12 cells. Results are expressed as mean number of osteoclasts with nuclei ≥3 present on the total surface of each of the three wells ± SE. In (B), ***indicated <i>p</i><0.001.</p