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First published online 12 December 2006
doi: 10.1242/jcs.03310

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Hyaluronan inhibits osteoclast differentiation via Toll-like receptor 4

Eun-Ju Chang^1,*, Hyon Jong Kim^1,*, Jeongim Ha¹, Hyung Joon Kim¹, Jiyoon Ryu¹, Kwang-Hyun Park², Uh-Hyun Kim², Zang Hee Lee¹, Hyun-Man Kim¹, David E. Fisher³ and Hong-Hee Kim^1,

¹ Department of Cell and Developmental Biology and Brain Korea 21 Program, DRI, Seoul National University, Seoul 110-749, Korea
² Department of Biochemistry and Institute of Cardiovascular Research, Chonbuk National University Medical School, Jeonju 561-182, Korea
³ Division of Pediatric Hematology/Oncology and Melanoma Program in Medical Oncology, Dana-Farber Cancer Institute and Children's Hospital, Harvard Medical School, Boston, MA 02115, USA

View larger version (45K): [in this window] [in a new window]   Fig. 1. Inhibition of osteoclast differentiation from BMMs and PBMCs by HA. (A,B) Effect of HA on osteoclastogenesis from BMMs. Mouse primary BMMs were cultured for 6 days with RANKL (100 ng/ml), M-CSF (30 ng/ml) and indicated concentrations of HA. After TRAP staining, TRAP+ multinuclear cells (TRAP+ MNCs) with more than three nuclei were scored as osteoclasts. *P<0.05 and **P<0.01 compared with vehicle-treated control. (C) No effects of HA on osteoclastic differentiation of RAW264.7 cells. Cells were cultured for 4 days with RANKL (100 ng/ml) and HA (0.1-5 µg/ml) and stained for TRAP, and TRAP+ MNCs were counted. (D) Effect of HA on human PBMC differentiation to osteoclasts. Human PBMCs were cultured for 9 days with RANKL (200 ng/ml), M-CSF (30 ng/ml) and indicated concentrations of HA. Cells were stained for TRAP and TRAP+ MNCs were counted. *P<0.05 and **P<0.01 compared with vehicle-treated control.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Requirement of HMM HA for osteoclastogenesis suppression. (A) Hyaluronidase treatment abolishes the effect of HA. HA preparation from umbilical cord (1 µg/ml) was digested with 150 U/ml hyaluronidase (HYAL) or pronase for 30 minutes followed by boiling for 10 minutes. Digested and undigested HA was added to BMMs and osteoclastogenesis was induced by culturing the cells with RANKL (100 ng/ml) and M-CSF (30 ng/ml) for 6 days. After TRAP staining, TRAP+ MNCs were scored. **P<0.01 compared with vehicle-treated control. (B) Stimulatory effects of LMM-HA on BMM osteoclastogenesis. BMMs were cultured for 4.5 days with RANKL (100 ng/ml), M-CSF (30 ng/ml), and indicated concentrations of different sizes of HA. Cells were stained for TRAP and TRAP+ MNCs were counted. *P<0.05 and **P<0.01 compared with vehicle-treated control.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Independence of HA inhibition of osteoclastogenesis on CD44. (A) HA does not suppress CD44 expression. BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 days in the presence or absence of HA (1 µg/ml). At each day, cells were collected and surface expression of CD44 was examined by flow cytometry. Red line, -HA; blue line, + HA; black line, secondary (2°) Ab control. MFI, mean fluorescence intensity. (B) CD44 independence of osteoclastogenesis inhibition by HA. BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 6 days. Anti-CD44 Ab (1 µg/ml) and HA (0.5-1 µg/ml) were included for the whole culture period where indicated. Cells were stained for TRAP, and TRAP+ MNCs were counted. NS, no significant difference. (C) Effectiveness of the anti-CD44 Ab to neutralize the CD44-OPN-dependent response of BMMs. BMMs were placed on the top chamber of a transwell plate. Anti-CD44 Ab (1 µg/ml) and HA (1 µg/ml) were added to the top chamber and OPN (a CD44 ligand, 2 µg/ml) was added to the bottom chamber. The plate was incubated for 8 hours. The number of migrated cells was determined after hematoxylin staining. **P<0.01 compared with OPN-treated control.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Requirement of TLR4 for HMM-HA inhibition of osteoclast differentiation. (A) Blockade of HA effects by a TLR4-neutralizing Ab. Human PBMCs were cultured for 9 days with RANKL (200 ng/ml), M-CSF (30 ng/ml), HA (1 µg/ml) and a TLR4 Ab (1-10 µg/ml) or an isotype-matched control Ab (10 µg/ml). Cells were stained for TRAP, and TRAP+ MNCs were counted. **P<0.01 compared with HA-treated control. (B) Lack of HA effects in TLR4-mutant cells. BMMs from TLR4-mutant mice were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) together with HA (0.1-5 µg/ml) for 6 days. TRAP+ MNCs were counted. (C) Binding of HA to TLR4. HEK-293 cells were transfected with TLR2, TLR4, TLR5, TLR9, or a control (mock) plasmid. At 24 hours after transfection, cells were incubated with anti-CD44 (1 µg/ml, 1 hour) to block potential HA binding to CD44. Cells were then incubated with FITC-HA for 30 minutes and analyzed in a flow cytometer. Red line, TLR-transfected cells; black line, mock-transfected cells.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Suppression of M-CSF signaling by HA. (A) BMMs were serum-starved, pretreated with HA (1 µg/ml) for 30 minutes and stimulated with M-CSF (300 ng/ml). Cell lysates were immunoblotted with phosphorylation-specific antibodies to detect the activation of ERK (a), JNK (b), and p38 (c). The same membranes were stripped and reprobed to detect total levels of each MAPK. The relative levels of phosphorylated forms were determined by densitometry. (B) BMMs were serum-starved and pretreated with HA (1 µg/ml) for 30 minutes. The cells were loaded with DCF-DA and stimulated with M-CSF (300 ng/ml) for 5 minutes. The DCF fluorescence was detected by confocal microscopy. The inbox in the upper left panel shows single-cell fluorescence. The average of the mean fluorescence intensity of several fields is presented as a histogram. *P<0.05 compared with HA-untreated group. (C) BMMs were serum-starved, pretreated with HA (1 µg/ml) for 30 minutes and stimulated with M-CSF (300 ng/ml) for 5 minutes. Total cell lysates were prepared and subjected to the Rac activity assay (top). In the bottom panel, stimulated cells were lysed and cytosolic and membrane fractions were immunoblotted with anti-Rac antibody. The same membranes were reprobed with anti-ß-actin and anti-flotillin antibodies to ensure comparable amounts of loading.

View larger version (40K): [in this window] [in a new window]   Fig. 6. Effects of HA on RANKL signaling. (A) BMMs were serum-starved, pretreated with HA (1 µg/ml) for 30 minutes, and stimulated with RANKL (500 ng/ml) for indicated times. Cell lysates were immunoblotted. (B) BMMs were cultured for 2 days with M-CSF (30 ng/ml) and RANKL (100 ng/ml) in the presence or absence of HA (1 µg/ml). The pre-conditioned cells were serum-deprived, and cells pre-conditioned in the presence of HA were treated with HA (1 µg/ml) for 30 minutes, whereas cells pre-conditioned in the absence of HA were treated with the vehicle. Cells were then stimulated with RANKL (500 ng/ml) for indicated times. Cell lysates were subjected to western blot analyses.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Reduction of RANK expression by HA. (A) BMMs with wild-type (WT) or mutant TLR4 were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) in the absence or presence of HA (1 µg/ml) for 1 or 2 days. Expression levels of RANK mRNA were measured by RT-PCR. (B) BMMs from wild-type mice were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 1 or 2 days. The surface levels of RANK were determined by FACS using TRITC-conjugated RANKL. Black line, -HA; red line, + HA; MFI, mean fluorescence intensity. (C) Effects of HA on the phosphorylation of JUN and MITF by M-CSF. BMMs were serum-starved, pretreated with HA (1 µg/ml) for 30 minutes, and stimulated with M-CSF (300 ng/ml) for indicated times. Cell lysates were immunoprecipitated with an Ab against phosphorylated MITF (P-MITF) and western blotted with a regular MITF Ab (third panel). All other protein levels [phosphorylated JUN (P-c-Jun), unphosphorylated JUN (c-Jun) and ß-actin] were determined with cell lysates. (D) Nuclear extracts were prepared from cells stimulated as in C and subjected to EMSA analyses with an AP-1-binding site oligonuleotide or an E-box sequence from RANK promoter (left). The DNA binding activity of M-CSF-stimulated nuclear extract to mutant probes (mut.) and to wild-type probes in the presence of 50-fold excess unlabeled probes (cold) was compared to the one with wild-type probes (WT).

View larger version (54K): [in this window] [in a new window]   Fig. 8. In vivo effects of HA on bone resorption. (A-C) Mouse calvarias were implanted with collagen sheets soaked with RANKL alone or with RANKL and HA. Five days later, calvarias were collected and analysed using micro-computed tomography (µ-CT). (A,B) 3D configurations of whole TRAP-stained calvarias (A); boxed areas of A are shown in B. (C) Relative percentages of calvarial bone volume. *P<0.05 and **P<0.01 between indicated groups. (D,E) Mouse calvarias treated as above were decalcified and embedded in paraffin. Coronal sections were stained with hematoxylin and eosine. Representative sections (D) and their respective percentage of marrow area (E) are shown. **P<0.01 between indicated groups.

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