Substance P modulates bone remodeling properties of murine osteoblasts and osteoclasts

Abstract

Clinical observations suggest neuronal control of bone remodeling. Sensory nerve fibers innervating bone, bone marrow and periosteum signal via neurotransmitters including substance P (SP). In previous studies we observed impaired biomechanical and structural bone parameters in tachykinin (Tac) 1-deficient mice lacking SP. Here, we aim to specify effects of SP on metabolic parameters of bone marrow macrophage (BMM)/osteoclast cultures and osteoblasts isolated from Tac1-deficient and wildtype (WT) mice. We demonstrated endogenous SP production and secretion in WT bone cells. Absence of SP reduced bone resorption rate, as we found reduced numbers of precursor cells (BMM) and multinucleated osteoclasts and measured reduced cathepsin K activity in Tac1-/- BMM/osteoclast cultures. However, this might partly be compensated by reduced apoptosis rate and increased fusion potential of Tac1-/- precursor cells to enlarged "super" osteoclasts. Contrarily, increased ALP enzyme activity and apoptosis rate during early osteoblast differentiation accelerated osteogenesis and cell death in the absence of SP together with reduced ALP activity of Tac1-/- osteoblasts during late osteogenic differentiation resulting in reduced bone formation at later stages. Therefore, we suggest that absence of SP presumably results in a slight reduction of bone resorption rate but concomitantly in a critical reduction of bone formation and mineralization rate.

Figure 1

Cell number (A,C) and vitality (B,D) of BMM (A,B) and osteoblasts (C,D). Cells were analysed after isolation from bone marrow and outgrowth from bone explants obtained from WT and Tac1−/− mice. N = 12–19.

Figure 2

Endogenous SP production and NK1R protein expression. SP concentration in cell culture supernatants of BMM/osteoclast cultures (A) and osteoblasts after 14 and 21 days of osteogenic differentiation (B), kept for 24 hours in differentiation medium; w/o stimulation with IL-1β (0.5 ng/ml) to mimic proinflammatory conditions. N = 5–8. Assay Detection Limit: 9.76 pg/ml. Endogenous SP (Molecular weight = 1350 g/L) production in BMM/osteoclast cultures and osteoblasts corresponds to about 1–2.5 × 10⁻¹¹ M. (C) Representative fluorescence image of NK1R staining (red fluorescence) in WT and Tac1−/− BMM/osteoclast cultures. Size bar = 50 µm. (D) Representative fluorescence image of NK1R staining (green fluorescence) in WT and Tac1−/− osteoblasts after 21 days osteogenic differentiation. Size bar = 50 µm Cell nuclei were counterstained with DAPI. (E) Representative Western Blot image of NK1R- and β-actin protein expression in WT and Tac1−/− BMM/osteoclast cell lysates, w/o stimulation with 10⁻⁸/10⁻¹⁰ M SP for 24 h. Image is cropped, full length images are provided in Supplementary file 4A. Ø = no stimulation. N = 4–8. (F) Representative Western Blot image of NK1R and β-actin protein expression in WT and Tac1−/− osteoblast cell lysates after 14 and 21 days of osteogenic differentiation, w/o stimulation with 10⁻⁸/10⁻¹⁰ M SP for 24 h. Image is cropped from two blots (black line), full length images are provided in Supplementary file 4B (osteoblasts after 14 days) and 4 C (osteoblasts after 21 days). Ø = no stimulation. N = 4–8. Quantification of NK1R protein expression of WT and Tac1−/− BMM/osteoclast cultures (G) and osteoblasts after 14 (H) and 21 days (I) in osteogenic medium, w/o stimulation with 10⁻⁸/10⁻¹⁰ M SP for 24 h, normalized to β-actin protein content. NK1R protein expression in Tac1−/− BMM/osteoclast cultures and osteoblasts was calibrated to WT controls (dotted line = 100%). Ø = no stimulation. N = 4–8. OC = osteoclasts; OB = osteoblasts; *p < 0.05.

Figure 3

Apoptosis and proliferation rate of BMM/osteoclast and osteoblast cultures. Proliferation of BMM cultures (A) and osteoblasts after 14 (B) and 21 (C) days in osteogenic medium isolated from WT and Tac1−/− mice. N = 8. Caspase 3/7 activity of BMM/osteoclast cultures (D) and osteoblasts after 14 (E) and 21 (F) days in osteogenic medium isolated from WT and Tac1−/− mice. N = 8. BMM/osteoclast cultures and osteoblasts from Tac1−/− mice were stimulated w/o SP 10⁻⁸/10⁻¹⁰ M either for the last 24 h or for the whole culture time (only osteoblast cultures; 14/21 days). Values of WT bone cells were set to 100% and results of Tac1−/− bone cells were calibrated to WT controls (dotted lines = 100%). Ø = no stimulation.

Figure 4

Immunohistological detection of TRAP-positive cells on paraffin sections of WT and Tac1−/− femora and in vitro differentiation capacity and activity of BMM/osteoclast cultures. (A) Representative image of the region of interest (ROI) set in TRAP stained sections and used to count TRAP-positive cells. (B) Representative images of TRAP-positive osteoclasts in paraffin sections of WT femora. Scale bar = 20 µm. N = 4. TB = Trabecular bone; GP = Growth plate; BM = Bone marrow; B = Bone. (C) Number of TRAP-positive cells/mm² counted in ROI of femoral paraffin sections of WT and Tac1−/− animals. N = 4. Number (D) and pixel area (number of pixel) (E) of TRAP positive osteoclasts (≥3 nuclei) after 5 days of M-CSF and RANKL mediated differentiation under cell culture conditions, w/o stimulation of Tac1−/− BMM/osteoclast cultures with SP 10⁻⁸/10⁻¹⁰ M for the last 24 h of culture time. Results of Tac1−/− bone cells were calibrated to WT controls (dotted line = 100%). N = 7–8. (F) Cathepsin K enzyme activity after 5 days of differentiation, w/o stimulation of Tac1−/− BMM/osteoclast cultures with SP 10⁻⁸/10⁻¹⁰ M for the last 24 h of culture time. Results of Tac1−/− bone cells were calibrated to WT controls (dotted line = 100%). N = 5–8. Ø = no stimulation.

Figure 5

Immunohistological detection of Runx2-positive cells on paraffin sections of WT and Tac1−/− femora and in vitro activity of osteoblast cultures. (A) Representative image of the regions of interest (ROIs) set in Runx2 stained sections. Runx2-positive cells were counted only in cortical bone regions. (B) Representative images of Runx2-positive cells and isotype control in paraffin sections of WT femora. Scale bar = 20 µm. N = 3–4. M = Muscle; CB = Cortical bone. (C) Number of Runx2-positive cells/mm² counted in ROIs of femoral paraffin sections of WT and Tac1−/− animals. N = 3–4. Comparison of osteoblast ALP activity (4 minute time point) after 7 (D) and 21 (E) days in osteogenic differentiation medium, w/o short-term (24 h) or long-term (7/21 days) stimulation of Tac1−/− osteoblasts with SP 10⁻⁸/10⁻¹⁰ M. Results of Tac1−/− animals were calibrated to WT controls (dotted line = 100% line). N = 8. Comparison of matrix mineralization (calcium deposition ability) by quantification of alizarin red staining of osteoblasts after 28 days in osteogenic differentiation medium, w/o long-term (28 days) stimulation of Tac1−/− osteoblasts with SP 10⁻⁸/10⁻¹⁰ M. Results of Tac1−/− animals were calibrated to WT controls (dotted line = 100% line). N = 6. Ø = no stimulation.

Figure 6

Osteoclast- and osteoblast-specific marker gene expression. Comparison of RANK (A), MMP-9 (B), CTSK (cathepsin K; C) and NFATC1 (D) gene expression in Tac1−/− to WT BMM/osteoclast cultures after 5 days of M-CSF and RANKL mediated differentiation. N = 5–11. Comparison of TNFRSF11B (OPG Day 14; E), TNFSF11 (RANKL Day 21; F), BGLAP (osteocalcin day 21; G), RUNX2 (Day 21; H) gene expression in Tac1−/− to WT osteoblasts after 14 (E) and 21 (F–H) days in osteogenic medium. N = 8–16. Results of RNA isolated from Tac1−/− cells were calibrated to RNA isolated from WT cells ( = x-axes/0-line). Ø = no stimulation.

Figure 7

Proposed mechanism of how absence of SP affects metabolic activities of BMM/osteoclasts and osteoblasts. Macrophage/osteoclast precursor cultures: Loss of SP does not affect vitality and proliferation rate of macrophage cultures but results in reduced initial macrophage cell numbers under culture conditions. BMM/osteoclast cultures: Absence of SP does affect precursor fusion and differentiation to multinucleated osteoclasts resulting in less mature TRAP-positive osteoclasts with a larger size. Apoptosis rate is reduced in BMM/osteoclast cultures in the absence of SP but also bone resorption correlating to cathepsin K activity is negatively affected. Osteoblast cultures: Absence of SP mediated signaling has no effect on osteoblast proliferation during both differentiation time points. Loss of SP increases osteoblast apoptosis rate during early differentiation (14 days) time point whereas apoptosis rate was reduced by trend during late differentiation time point (21 days). ALP-activity was increased during very early differentiation time point (7 days) whereas ALP-activity in SP-deficient osteoblast cultures was reduced by trend at the late differentiation stage (21 days). Matrix mineralization (calcium deposition ability) of mature osteoblasts (28 days) was not affected in the absence of SP. Conclusion: Loss of SP mediated signaling presumably results in a slightly reduced bone resorption rate but also in a slightly reduced bone formation rate. As bone resorption is faster than bone formation, we suggest a net bone loss in the absence of SP.