Bone Marrow Monocyte/Macrophages Provide Signals Necessary for Osteoblast Maintenance: Potential Role for Insulin-Like Growth Factor Signaling

Abstract There is considerable evidence showing that osteoblast lineage cells play a key role in regulating hematopoiesis. Less clear is the contribution of hematopoietic cells to osteoblast maintenance. We and others previously showed that treatment with granulocyte-colony stimulating factor (G-CSF) results in a striking loss of osteoblast number and function. Bone marrow transplantation experiments demonstrated that G-CSF does not directly suppress osteoblast function but acts through a transplantable hematopoietic intermediary. Earlier work from our lab suggested that mature neutrophils and B- and T-lymphocytes are dispensable for G-CSF-induced osteoblast loss. We therefore hypothesized that bone marrow monocytes play a role in regulating osteoblasts both during G-CSF treatment and at steady state. To begin to test this hypothesis, primary murine bone marrow stromal cultures—consisting of both adherent macrophages and mesenchymal-lineage stromal cells—were established and cultured three weeks in osteogenic media. Removal of macrophages from these cultures resulted in a striking reduction in osteoblast differentiation, as evidenced by reduced alkaline phosphatase and sharply reduced expression of osteocalcin (92% reduction, p<.0001, n=6), a mature osteoblast marker. To investigate whether bone marrow monocytes play a role in supporting osteoblasts in vivo, we made use of transgenic mice expressing an inducible suicide gene in the monocyte compartment under control of the c-fms promoter. Induction of the suicide gene by administration of an exogenous ligand resulted in efficient deletion of monocytes and macrophages from the bone marrow and peripheral blood. Histomorphometry of long bones showed a striking decrease in osteoblast number in monocyte-ablated mice compared to controls, and osteocalcin mRNA decreased to nearly undetectable levels (n=7 each group). The loss of osteoblasts after monocyte ablation does not result from bystander killing, as other boneassociated cells such as osteoclasts and osteoprogenitors are not decreased. Monocyte ablation and osteoblast loss were associated with a 15-fold mobilization of hematopoietic progenitors (n=5 each group), comparable to levels attained after G-CSF treatment. Taken together, these in vitro and in vivo findings suggest that monocytes provide factors necessary for osteoblast survival or development and raise the possibility that G-CSF administration modulates these factors. As a first step toward identifying these factors, expression profiling of monocytes harvested from the bone marrow of untreated or G-CSF treated mice was performed. These data show that insulin-like growth factor 1 (IGF1) is produced at high levels in resting bone marrow monocytes and is downregulated more than 2.5-fold by G-CSF. Moreover, expression of IGF binding protein 6 (IGFBP6), an IGF binding-protein family member that negatively regulates IGF signaling, is upregulated more than 6-fold in monocytes by G-CSF. Quantitative real time PCR confirmed that IGF- 1 mRNA is reduced >11-fold in bone marrow from monocyte-ablated transgenic mice (p<.05, n=2–3 each group) and >7-fold in wild type mice treated with G-CSF (p<.05, n=3 each group). In addition, IGFBP6 expression was increased ~100-fold in bone marrow during G-CSF treatment. Fractionation of hematopoietic cell populations confirmed that IGFBP6 mRNA and protein is induced in monocyte and granulocyte populations in the bone marrow during G-CSF treatment. Collectively, these data support a model in which monocytes provide signals required for osteoblast maintenance in adult bone marrow. IGF1 signaling is known to play a vital role in osteoblast development and maintenance. Thus, these data also suggest that G-CSF may modulate osteoblast number and function (and secondarily induce hematopoietic stem cell mobilization) through alteration of IGF1 and IGFBP6 expression by monocytes. Studies are underway to directly assess the affect of IGFBP6 on osteoblast survival and differentiation.