Granulocyte colony-stimulating factor induces osteoblast apoptosis and inhibits osteoblast differentiation

Abstract

Long-term treatment of mice or humans with granulocyte colony-stimulating factor (G-CSF) is associated with a clinically significant osteopenia characterized by increased osteoclast activity and number. In addition, recent reports have observed a decrease in number of mature osteoblasts during G-CSF administration. However, neither the extent of G-CSF's suppressive effect on the osteoblast compartment nor its mechanisms are well understood. Herein, we show that short-term G-CSF treatment in mice leads to decreased numbers of endosteal and trabecular osteoblasts. The effect is specific to mature osteoblasts, because bone-lining cells, osteocytes, and periosteal osteoblasts are unaffected. G-CSF treatment accelerates osteoblast turnover in the bone marrow by inducing osteoblast apoptosis. In addition, whereas G-CSF treatment sharply increases osteoprogenitor number, differentiation of mature osteoblasts is impaired. Bone marrow transplantation studies show that G-CSF acts through a hematopoietic intermediary to suppress osteoblasts. Finally, G-CSF treatment, through suppression of mature osteoblasts, also leads to a marked decrease in osteoprotegerin expression in the bone marrow, whereas expression of RANKL remains relatively constant, suggesting a novel mechanism contributing to the increased osteoclastogenesis seen with long-term G-CSF treatment. In sum, these findings suggest that the hematopoietic system may play a novel role in regulating osteoblast differentiation and apoptosis during G-CSF treatment.

FIG. 1

Loss of osteoblast number and function during G-CSF treatment. (A) Mice were treated with G-CSF (250 μg/kg/d) for 5 days, and osteoblast surface per bone surface (Ob.S/BS) was determined. (B) Immunohistochemistry showing GFP⁺ (brown) osteoblasts (arrows), bone-lining cells (arrowheads), and osteocytes in untreated or day 5 G-CSF treated pOBCol2.3-GFP transgenic mouse femurs. Insets show enlargement of area enclosed by dotted line. Original magnification ×100. (C) Quantification of mature osteoblasts, bone-lining cells, and osteocytes in transgenic mice treated with G-CSF for 5 days or untreated (n = 4 each group). (D) Representative scatter plots showing GFP expression (bottom panels) in the stromal (CD45 negative, Ter119 negative) cell population (top panels) isolated from nontransgenic and pOBCol2.3-GFP mice (left and right, respectively). (E) Number of GFP⁺ cells recovered from the femurs of transgenic mice after treatment with G-CSF (n = 2–10 each time point). Data represent the mean ± SE. ^a p < 0.01 vs. all other groups; ^b p < 0.05.

FIG. 2

Loss of endosteal and trabecular, but not periosteal, osteoblast activity during G-CSF treatment. Osteoid and mineralization were measured in untreated mice or mice treated for 7 days with G-CSF (n = 2–3 each group). (A) Percent osteoid surface was calculated in Masson trichrome–stained tibial sections from untreated and treated wildtype mice. (B) Mineral apposition rate (MAR) and percent mineralizing surface (Md.S/BS) were calculated on endosteal and periosteal surfaces from calcein-labeled calvaria. (C) Osteocalcin RNA in situ hybridization of long bones harvested from untreated mice or mice treated for 5 days with G-CSF. Shown are representative photomicrographs of three independent experiments. Periosteal surfaces (arrows), endosteal surfaces (arrowheads), bone (B), and bone marrow (BM) are indicated. Original magnification ×100. Data represent the mean ± SE. ^a p = 0.057; ^b p = 0.10.

FIG. 3

G-CSF receptor–deficient bone marrow chimeras. (A) G-CSFR^−/− CD45⁺ cKit⁺ lineage⁻ hematopoietic cells (KL) cells were transplanted into wildtype recipients (n = 4–5 each group). After hematopoietic reconstitution (6–8 wk), chimeric mice were treated with G-CSF (or left untreated), and osteocalcin mRNA expression in the bone marrow was measured by real-time RT-PCR. (B) Wildtype KL cells were transplanted into irradiated G-CSFR^−/− recipients (n = 6–7, each group) and analyzed in a similar fashion. Data represent the mean ± SE. ^a p < 0.05.

FIG. 4

Osteoblast turnover during G-CSF treatment. (A) Transgenic pOBCol2.3-GFP mice (n = 5–6, each group) were administered BrdU for 14 days and either treated for 5 days with G-CSF or left untreated. Mice were analyzed just before G-CSF treatment, after 5 days of G-CSF treatment, or after a 5 day recovery period (arrowheads). Shown is the percent of GFP⁺ cells in the bone marrow that were labeled with BrdU. (B) Representative scatter plots showing activated caspase 3 staining in the GFP⁺ cell population from untreated (left) or G-CSF–treated pOBCol2.3-GFP mice (right). (C) Shown is the percentage of GFP⁺ cells that express activated caspase 3 from untreated and day 3 G-CSF–treated mice (n = 4 each group). Data represent the mean ± SE. ^a p < 0.01, ^b p < 0.05.

FIG. 5

Analysis of early osteoblast lineage cells during G-CSF treatment. (A) Real-time RT-PCR for the indicated genes (BSP, bone sialoprotein; ALP, alkaline phosphatase; OC, osteocalcin) was performed on total bone marrow RNA isolated after 5 days of G-CSF treatment. RNA expression relative to β-actin mRNA was calculated and compared with untreated bone marrow (assigned a value of 1; n = 5–12). (B) Shown is the number of alkaline phosphatase negative (AP negative, left) and positive (AP positive, right) CFU-F generated from the bone marrow of untreated or G-CSF–treated mice (n = 5–6 each group). (C) Mice (n = 2–4 each time point) were treated with G-CSF for the indicated period up to 22 days. Mice were killed at the indicated time points and analyzed for osteocalcin mRNA by real-time RT-PCR. (D) Mice (n = 6, each group) were treated 5 days with G-CSF or left untreated and administered BrdU for 5 days during the recovery period. Shown is the percent of GFP⁺ cells in the bone marrow that were labeled with BrdU. Data represent the mean ± SE. ^a p < 0.05 vs. untreated; ^b p < 0.01 vs. untreated, p < 0.05 vs. runx2 and OC; ^c p < 0.0001 vs. untreated, p < 0.05 vs. other groups; ^d p < 0.01; ^e p < 0.05 vs. untreated.

FIG. 6

Osteoclastogenesis during G CSF treatment. (A and B) Wildtype mice (n = 2–6 each group) were treated with G-CSF for the indicated time or left untreated. Osteoclast number (A) and surface (B) were estimated by enumerating TRACP⁺ cells in paraffin-embedded sections of mouse long bones. (C) RANKL and OPG mRNA expression in the bone marrow of untreated or 5-day G-CSF–treated mice (N = 5–8 each group) was measured by real-time RT-PCR. (D) GFP⁺ cells were sorted from G-CSF–treated pOBCol2.3-GFP mice. RANKL and OPG mRNA was measured within this fraction. Data represent the mean ± SE. ^a p < 0.01 vs. other groups; ^b p < 0.001; ^c p < 0.05.