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. 2013 Sep 15;126(Pt 18):4187-94.
doi: 10.1242/jcs.127571.

Fibrillin-1 directly regulates osteoclast formation and function by a dual mechanism

Kerstin Tiedemann  1 , Iris Boraschi-Diaz, Irina Rajakumar, Jasvir Kaur, Peter Roughley, Dieter P Reinhardt, Svetlana V Komarova
Affiliations

Fibrillin-1 directly regulates osteoclast formation and function by a dual mechanism

Kerstin Tiedemann et al. J Cell Sci. .

Abstract

Mutations in the fibrillin-1 gene give rise to a number of heritable disorders, which are all characterized by various malformations of bone as well as manifestations in other tissues. However, the role of fibrillin-1 in the development and homeostasis of bone is not well understood. Here, we examined the role of fibrillin-1 in regulating osteoclast differentiation from primary bone-marrow-derived precursors and monocytic RAW 264.7 cells. The soluble N-terminal half of fibrillin-1 (rFBN1-N) strongly inhibited osteoclastogenesis, whereas the C-terminal half (rFBN1-C) did not. By contrast, when rFBN1-N was immobilized on calcium phosphate, it did not affect osteoclastogenesis but modulated osteoclast resorptive activity, which was evident by a larger number of smaller resorption pits. Using a panel of recombinant sub-fragments spanning rFBN1-N, we localized an osteoclast inhibitory activity to the 63 kDa subfragment rF23 comprising the N-terminal region of fibrillin-1. Osteoclastic resorption led to the generation of small fibrillin-1 fragments that were similar to those identified in human vertebral bone extracts. rF23, but not rFBN1-N, was found to inhibit the expression of cathepsin K, matrix metalloproteinase 9 and Dcstamp in differentiating osteoclasts. rFBN1-N, but not rF23, exhibited interaction with RANKL. Excess RANKL rescued the inhibition of osteoclastogenesis by rFBN1-N. By contrast, rF23 disrupted RANKL-induced Ca(2+) signaling and activation of transcription factor NFATc1. These studies highlight a direct dual inhibitory role of N-terminal fibrillin-1 fragments in osteoclastogenesis, the sequestration of RANKL and the inhibition of NFATc1 signaling, demonstrating that osteoclastic degradation of fibrillin-1 provides a potent negative feedback that limits osteoclast formation and function.

Keywords: Calcium signaling; Fibrillin; NFATc1; Osteoclastogenesis; RANKL.

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Figures

Fig. 1
Fig. 1. The N-terminal half of fibrillin-1 affects osteoclastic resorption and inhibits osteoclast differentiation
(A–C) Bone marrow cells were plated on calcium phosphate that was uncoated (positive control, PC) or coated with 50 μg/ml rFBN1-N (1N) or rFBN1-C (1C) and differentiation was induced with MCSF (50 ng/ml) and RANKL (50 ng/ml). After 5–6 days the samples were fixed and stained for TRAP. Osteoclasts were quantified, removed and resorption was assessed. (A) Representative images of osteoclasts (top) and resorption pits (bottom) formed under the indicated conditions. Scale bars apply to all images in the corresponding rows. (B,C) Average numbers of differentiated osteoclasts (B, top), total area resorbed per osteoclast (B, bottom), average pit size (C, top), and numbers of resorption pits per osteoclast (C, bottom). Data are means ± s.e.m., n=3 experiments; *P<0.05 assessed by Student’s t-test. (D,E) Bone marrow cells [white bars, all conditions treated with MCSF (50 ng/ml)] or RAW 264.7 (black bars) were cultured for 5 days without RANKL (negative control, NC); with RANKL (50 ng/ml, positive control, PC) or with RANKL and soluble rFBN1-N or rFBN1-C, fixed and stained for TRAP. (D) Representative images of osteoclastic cells formed from RAW 264.7 under the indicated conditions. Scale bars apply to all images. (E) Average numbers of osteoclasts. Data are means ± s.e.m., n=4 experiments for bone marrow cells, n=5–6 experiments for RAW 264.7; **P<0.01, ***P<0.001 assessed by Student’s t-test.
Fig. 2
Fig. 2. A small N-terminal subfragment of fibrillin-1 inhibits osteoclastogenesis
(A) Schematic of recombinant N-terminal (rFBN1-N) and C-terminal (rFBN1-C) halves of fibrillin-1, and recombinant sub-fragments of rFBN1-N – rF23, rF51, rF1F and rF18. (B–D) Bone marrow cells (white bars) and RAW 264.7 (black bars) were cultured for 5 days untreated (negative control, NC), treated with RANKL only (50 ng/ml, positive control, PC) or treated with RANKL and 50 μg/ml of soluble fibrillin-1 fragments rFBN1-N, rFBN1-C, rF23, rF51, rF1F or rF18. (B) Average numbers of osteoclasts formed under the indicated conditions. Data are means ± s.e.m., n=3–8 experiments; *P<0.05, **P<0.01, ***P<0.001 compared with PC assessed by Student’s t-test. (C,D) mRNA was isolated and the expression of cathepsin K (Ctsk, C) and Dcstamp (D) was quantified. Data are means ± s.e.m., n=3–9 experiments; *P<0.05, ***P<0.001, compared with PC assessed by Student’s t-test.
Fig. 3
Fig. 3. Fragments of fibrillin-1 are released during resorption and inhibit osteoclasts in vivo
(A) Bone marrow cells were plated on calcium phosphate coated with biotinylated rFBN1-N (80–155 μg/cm2) and after mature osteoclasts developed (5–7 days), the cultures were maintained for an additional 24 hours in serum free medium. Lane B, Biotinylated rFBN1-N before coating. Lane OC, conditioned medium from wells cultured with osteoclasts. Lane M, conditioned medium from wells cultured identically, but without cells. Arrowheads indicate degradation products in the range of 35–85 kDa. (B) Proteins were extracted from human vertebral bone and immunoblotted for fibrillin-1. Arrows indicate multimeric (FBN1 mult.) and monomeric full length fibrillin-1 (FBN1); arrowheads indicate fibrillin-1 degradation products. (C) Schematic of recombinant sub-fragments rF23 and rF31 (compare with Fig. 2 for position within full length fibrillin-1). (D) Bone marrow cells (white bars) and RAW 264.7 (black bars) were cultured in parallel for 5 days with RANKL only (50 ng/ml, positive control, PC), or with RANKL and 50 μg/ml of rF23 or rF31. Data are means ± s.e.m., n=2–3 experiments; *P<0.05, compared with PC assessed by Student’s t-test. (E) Healthy mice were injected with vehicle (PBS) or recombinant rF31 (50 μg/kg) intra-peritoneally every other day for 1 week and osteoclast numbers were assessed 1 week after the last injection. Left, representative images of upper tibial metaphyseal area of vehicle- or rF31-treated mice. Right, osteoclast number (OCN) per bone perimeter (BP) in vehicle- and rF31-injected mice. Data are means ± s.d., n=11 mice for vehicle-injected and n=4 mice for rF31-injected; *P<0.05, assessed by Student’s t-test.
Fig. 4
Fig. 4. The N-terminal half of fibrillin-1 binds and sequesters RANKL
(A) rFBN1-N (circles), but not rFBN1-C (squares) or rF23 (triangles), binds RANKL. RANKL (20 μg/ml) was immobilized and rFBN1-N, rFBN1-C or rF23 (0–150 μg/ml) were added as soluble ligands, in Ca2+-containing (open symbols) or Ca2+-free (filled symbols) buffer, and bound proteins were detected. Non-specific binding to milk proteins without coated RANKL was subtracted from all values. Data are representative of three independent experiments. (B,C) RAW 264.7 cells were incubated with 50 μg/ml of rFBN1-N (B) or rF23 (C) and increasing concentrations of RANKL (0.1, 1, 3 and 10 μg/ml), and osteoclast numbers were assessed. Data in B are means ± s.e.m., n=3–5 experiments, *P<0.05 and **P<0.01 indicate significance compared with PC; #P<0.05 indicates significance compared with the rFBN1-N treated sample including 0.1 μg/ml RANKL, as analyzed by ANOVA. Data in C are means ± s.e.m., n=4 experiments; **P<0.01 compared with PC, as assessed by ANOVA.
Fig. 5
Fig. 5. Fibrillin-1 fragments act through the Ca2+–NFATc1 pathway
RAW 264.7 cells were cultured untreated (negative control, NC), treated with RANKL (positive control, PC) or RANKL and 50 μg/ml of rFBN1-N (1N), rFBN1-C (1C), rF23 (23) or rF18 (18) for 48 hours. (A) NFATc1 localization was determined by immunofluorescence (green). Nuclei were counterstained using DAPI (blue). Scale bar: 100 μm. (B) The percentage of cells exhibiting nuclear NFATc1 of the total number of cells. Data are means ± s.d.; n=220 cells for positive control, n=137 cells for rFBN1-N-treated, n=282 cells for rFBN1-C-treated, n=135 cells for rF23-treated and n=117 cells for rF18-treated samples; *P<0.05 assessed by Student’s t-test. (C–E) Osteoclast precursors were loaded with Fura-2AM, and [Ca2+]i was monitored for 120 seconds. (C) Representative traces demonstrate changes in [Ca2+]i in eight individual cells per experimental condition. (D) For each cell, the average [Ca2+]i (top) and variation in baseline [Ca2+]i, measured as a standard deviation of basal levels (bottom) were determined and presented in an ascending order of the average [Ca2+]i; n=59 cells for positive control, n=65 cells for rF23-treated samples. (E) Average [Ca2+]i (top) and variation in [Ca2+]i (bottom). Data are means ± s.d.; n=59 cells for PC, n=65 for rFBN1-C-treated cells, n=85 for rFBN1-N-treated cells, n=65 for rF23-treated cells; *P<0.5, **P<0.01 indicates significance compared with PC, as assessed by Student’s t-test.
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