Objective:

Granulocyte colony-stimulating factor (G-CSF) is the critical regulator of the proliferation, differentiation, and survival of granulocytes. Recently, it has been shown that G-CSF can adversely affect bone health in both animal models and patients. Here, the authors aimed to investigate whether G-CSF could inhibit the growth of osteoblasts and osteocytes by regulating nitric oxide.

Methods:

The C57BL/6 mice were divided into the control group, G-CSF treatment group and recovery group (G-CSF+ L-NAME). The morphology of femurs was assessed by histology and immunohistochemistry. The expression of apoptosis-related molecules in femurs was detected by immunohistochemistry and quantitative RT-PCR, respectively. To examine if neutrophil-secreted factors can induce apoptosis in osteoblasts, Gr1-positive (Gr1+) neutrophils from the bone marrow of wild-type mice were sorted and co-cultured with MC3T3 pre-osteoblasts for 2 days.

Results:

The number of osteoblasts and newly embedding osteocytes significantly decreased and markers related to osteoblasts and osteocytes were downregulated in the G-CSF treatment compared to the control group. Moreover, G-CSF treatment did not change proliferation markers but induced apoptosis in osteoblast-lineage cells. The combined treatment of mice with G-CSF and a nitric oxide inhibitor partially restored the number of osteoblasts and osteocyte parameters.

Conclusions:

The G-CSF can inhibit osteoblasts and osteocytes by upregulating nitric oxide.

Granulocyte-colony stimulating factor (G-CSF) has emerged as an important drug to mobilize hematopoietic stem and progenitor cells for human bone marrow (BM) transplantation. 1–3 Increased G-CSF levels have been proved associated with disease conditions such as myeloproliferative disorders and bacterial infections. 4 However, elevated G-CSF has adverse effects on skeletal health. Long-term G-CSF treatment significantly reduces bone mineral density and induces vertebral compression fractures. 5, 6 The overexpression of G-CSF in mice can reduce trabecular and cortical bone thickness. 7 Therefore, it is necessary to understand the mechanism how G-CSF influence bone cell properties. Interestingly, the expression of G-CSF receptors (G-CSFRs) is negative in osteoblastic cell lines and primary osteoblasts. 8, 9 This suggests that the osteopenic effects of G-CSF are mediated by non-osteoblastic cells types. G-CSFRs are expressed on many other cell types including hematopoietic cells, endothelial cells, neurons, and muscle cells. 10, 11 Using a bone marrow transplantation model, a previous study has shown that the inhibitory effects of G-CSF on osteoblasts are definitively mediated via hematopoietic cells. 12 These effects of G-CSF are mediated by neutrophils, because depletion of neutrophils abrogates the inhibitory effects of G-CSF on osteoblasts. 13 Neutrophils are the target as well as the source of various cytokines, including nitric oxide (NO). 14 The NO is a short-lived gas synthesized intracellularly by either of the 3 nitric oxide synthase (NOS) enzymes (NOS1, NOS2, and NOS3), depending on the cell type. In osteolineage cells, NO induces apoptosis and reduces adaptation to mechanical signals. 15