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. 2008 Jun;14(6):914-25.
doi: 10.1016/j.devcel.2008.03.022.

Synaptotagmin VII regulates bone remodeling by modulating osteoclast and osteoblast secretion

Haibo Zhao  1 , Yuji Ito, Jean Chappel, Norma W Andrews, Steven L Teitelbaum, F Patrick Ross
Affiliations

Synaptotagmin VII regulates bone remodeling by modulating osteoclast and osteoblast secretion

Haibo Zhao et al. Dev Cell. 2008 Jun.

Abstract

Maintenance of bone mass and integrity requires a tight balance between resorption by osteoclasts and formation by osteoblasts. Exocytosis of functional proteins is a prerequisite for the activity of both cells. In the present study, we show that synaptotagmin VII, a calcium sensor protein that regulates exocytosis, is associated with lysosomes in osteoclasts and bone matrix protein-containing vesicles in osteoblasts. Absence of synaptotagmin VII inhibits cathepsin K secretion and formation of the ruffled border in osteoclasts and bone matrix protein deposition in osteoblasts, without affecting the differentiation of either cell. Reflecting these in vitro findings, synaptotagmin VII-deficient mice are osteopenic due to impaired bone resorption and formation. Therefore, synaptotagmin VII plays an important role in bone remodeling and homeostasis by modulating secretory pathways functionally important in osteoclasts and osteoblasts.

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Figures

Figure 1
Figure 1. Syt VII−/− osteoclasts have defects in cathepsin K secretion and ruffled border formation
(A) Syt VII+/+ and Syt VII−/− BMMs were cultured with M-CSF and RANKL on bone slices for 5 days. F-actin and nuclei were stained with Alexa-488 phalloidin and Hoechst 33258 respectively. (B) Cathepsin K (Cat K) was co-stained with F-actin. The secretion of Cat K into the resorption lacunae, which are circumscribed by actin-rings, was detected by confocal microscopy. (C) Total and cathepsin K-containing cells in (A) and (B) were counted. Data are presented as mean ± SD n=5 in each group. ** p< 0.01 versus WT. (D) Osteoclasts were removed from bone slices and resorption pits were labeled with peroxidase-conjugated wheat germ agglutinin. (E) Medium CTx-I level was measured by ELISA. Lane 1, positive control in the kit; lane 2, medium alone control; lane 3, Syt VII+/+ cultures; lane 4, Syt VII−/−. Data are presented as mean ± SD n=6 in each group. ** p< 0.01 versus WT. (F) Transmission electron microscopic images. White arrows and an enlarged insert in the upper panel identify the convoluted ruffled border in a Syt VII+/+ osteoclast. This membrane structure is not observed in a Syt VII−/− osteoclast (white arrows and an enlarged insertion in the lower panel). White rectangles show the area enlarged in the inserts. Scale bars = 6.8 µm.
Figure 2
Figure 2. Syt VII associates with lysosomes in osteoclasts and translocates to the ruffled border
(A) and (B) BMMs transduced with Syt VII-GFP were cultured with MCSF and RANKL on bone slices for 5 days. Syt VII-GFP was co-stained for F-actin (A) or cathepsin K (Cat K). Cells transduced with empty vector served as staining controls. (C) and (D) Cytosol of mature osteoclasts was fractionated by iodixanol ultracentrifugation. The distribution of Syt VII, lysosomal proteins and organelle markers was detected by western blots and the data displayed graphically. (E) Syt VII immunoprecipitates were assessed for the presence of the lysosomal v-SNARE TI-VAMP. TCL, 5% of total cell lysates.
Figure 3
Figure 3. Syt VII−/− osteoblasts differentiate normally but form fewer bone nodules
(A) Syt VII+/+ and Syt VII−/− primary calvarial osteoblasts were cultured with differentiation media containing 50 µg/ml ascorbic acid and 2 mM β-glycerophosphate for 21 days. Cells were fixed at different days and stained for alkaline phosphatase (ALP). (B) Expression of ALP and osteocalcin (OC) mRNA was analyzed by RT-PCR. GAPDH served as loading control. (C) Bone nodule formation was visualized with time by Alizarin Red staining. (D) The intracellular pro-alpha chains of type I collagen in total cell lysates were detected by western blot. Actin served as loading control. (E) The extracellular mature form of type I collagen alpha chains in pepsin-digested extracts was detected by western blot with the same antibody as in (D). The density of α1 bands in (D) and (E) was quantified using NIH Image J programme and expressed as a ratio.
Figure 4
Figure 4. Syt VII associates with bone matrix proteins in osteoblasts
(A) The localization of Syt VII-GFP was visualized at different days of osteoblast differentiation cultures under fluorescent microscopy. (B) and (C) Syt VII-GFP was co-stained with osteopontin (OPN) or osteocalcin (OC) in day 14 osteoblast cultures by fluorescent microscopy. Empty vector transduced cells served as staining controls. Arrows in (B) and (C) overlays indicate co-localization of Syt VII with OPN and OC, respectively. (D) and (E) The cytosol of day 14 cultured osteoblasts was fractionated by iodixanol ultracentrifugation. The distribution of Syt VII, organelle markers and bone matrix proteins was detected by western blot analysis.
Figure 5
Figure 5. Retroviral transduction of WT Syt VII reconstitutes the function of Syt VII−/− osteoclasts and osteoblasts
Syt VII−/− BMMs and primary osteoblasts were isolated and transduced with empty vector or FLAG-tagged WT Syt VII. Transduced BMMs were cultured with M-CSF and RANKL on bone slices for 5 days. Osteoblasts were cultured with differentiation media for 21 days. (A) co-staining of cathepsin K (Cat K) and F-actin. (B) Pit staining. (C) Medium CTx-I assay. Data are presented as mean ± SD n=6 in each group. ** p<0.01 versus vector pMX. (D) Bone nodule staining. * p< 0.05 versus empty vector cultures. (E) The level of intracellular pro-alpha chains of type I collagen in total cell lysates was detected by western blot. Actin served as loading control. (F) Western blot of extracellular mature form of type I collagen alpha chains in pepsin-digested extracts from Syt VII−/− and wild type osteoblast cultures. The density of α1 bands in (E) and (F) was measured using NIH Image J programme and expressed as a ratio.
Figure 6
Figure 6. Syt VII−/− mice are osteopenic
(A) Representative 3D reconstruction of µCT images of distal femoral metaphysis from three different mice of each genotype. (B) The percentage of trabecular bone volume/tissue volume (BV/TV). (C) Trabecular thickness (Tb.Th). (D) Trabecular separation (Tb.Sp). (E) Bone mineral density (BMD). Data presented in B–E are present as mean ± SD, n=5 in each group of mice. * p<0.05 40 versus WT.
Figure 7
Figure 7. Decreased bone resorption and bone formation in Syt VII−/− mice
(A) and (B) Bone resorption and bone formation rates were quantified at sacrifice by serum CTx-I and osteocalcin levels, respectively. (C) Osteoclast number/bone perimeter (OC/B.Pm). (D) Osteoblast number/bone perimeter (OB/B/Pm). (E) Osteoid surface/bone perimeter (OS.S/B.Pm). (F) Representative fluorescent micrographs of calcein labeling. Data in (B) to (F) are presented as mean ± SD, n=5 in each group of mice. * p<0.05 versus WT. (G) and (H) Mineral apposition rate (MAR) and bone formation rate (BFR) in Syt VII−/− mice calculated from the data in G. Results are presented as mean ± SD, n=5 in each group of mice. * p<0.05 versus WT. (I) and (J) Systemic PTH levels of 6-week old (I) and 4-month old (J) wild type and Syt VII−/− mice. Data are presented as mean ± SD, n=4 in each group of mice.
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