Hispidulin attenuates bone resorption and osteoclastogenesis via the RANKL-induced NF-κB and NFATc1 pathways

European Journal of Pharmacology 715 (2013) 96–104

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Hispidulin attenuates bone resorption and osteoclastogenesis via the RANKL-induced NF-κB and NFATc1 pathways Manoj Nepal a,1, Hwa Jung Choi a,1, Bo-Yun Choi a, Moon-Shik Yang b, Jung-Il Chae a, Liang Li a, Yunjo Soh a,n a Department of Dental Pharmacology, School of Dentistry and Institute of Oral Bioscience, Brain Korea 21 project, Chonbuk National University, Jeonju 561-756, Republic of Korea b Division of Biological Sciences, Chonbuk National University, Jeonju 561-756, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 3 October 2012 Received in revised form 31 May 2013 Accepted 8 June 2013 Available online 19 June 2013

Hispidulin, a flavonoid that is known to have anti-inflammatory and anti-oxidant effects, attenuates osteoclastogenesis and bone resorption. To investigate the molecular mechanism of its inhibitory effect on osteoclastogenesis, we employed the receptor activator of the nuclear factor κB (NF-κB) ligand (RANKL)-induced murine monocyte/macrophage RAW 264.7 cells and bone marrow-derived macrophages (BMMs) for osteoclastic differentiation in vitro. The inhibitory effect on in vitro osteoclastogenesis was evaluated by counting the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and by measuring the expression levels of osteoclast-specific genes such as matrix metalloproteinase 9 (MMP9), TRAP and cathepsin K. Similarly, hispidulin significantly inhibited osteoclast activity in RAW 264.7 cell as well as stimulated the ALP activity of MC3T3E1 cells. Furthermore, the in vivo suppressive effect on bone loss was assessed quantitatively in a lipopolysaccharide (LPS)-induced mouse model using microcomputational tomography (μCT) and histochemical analyses. Hispidulin was found to inhibit RANKL-induced activation of Jun N-terminal kinase (JNK) and p38, in addition to NF-κB in vitro experiment. Additionally, hispidulin decreased NFATc1 transcriptional activity in RANKL-induced osteoclastogenesis. This study identifies hispidulin as a potent inhibitor of osteoclastogenesis and bone resorption and provides evidence for its therapeutic potential to treat diseases involving abnormal bone lysis. & 2013 Elsevier B.V. All rights reserved.

Keywords: Hispidulin Osteoclastogenesis Bone resorption RANKL μCT

1. Introduction Bone-remodeling imbalances induced by decreased osteoblastogenesis and increased osteoclastogenesis are known to cause various osteolytic bone diseases including osteoporosis, Paget's disease, bone metastatic diseases, erosive arthritis and aseptic bone loosening (Ang et al., 2011; Karsenty and Wagner, 2002). Understanding the molecular processes by which osteoclasts are formed and activated and identifying potential pharmacological interventions to perturb these processes might contribute to the prevention and treatment of abnormal bone lysis. RANK–RANKL interaction is essential to activate a variety of downstream signaling pathways required for osteoclast development. Osteoclast precursors that express RANK, a tumor necrosis factor (TNF) receptor family member, recognize RANKL and differentiate into osteoclasts in the presence of macrophage/monocyte

n

Corresponding author. Tel.: +82 63 270 4038; fax: +82 63 270 4037. E-mail address: [email protected] (Y. Soh). 1 The first two authors contributed equally to this work.

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.06.002

colony stimulating factor (M-CSF) (Tao et al., 2011). On the other hand, osteoblasts express RANKL, which induces the signaling essential for osteoclast precursor cells to differentiate into osteoclasts, whereas M-CSF, secreted by osteoblasts, provides the survival signal to these cells (Takayanagi et al., 2002). The interaction of RANKL with RANK results in a cascade of intercellular events including the expressions of NF-κB, AKT, MAPKs, nuclear factor of activated T cells (NFAT), and ionic calcium and calcium/calmodulin-dependent kinase (Ang et al., 2007; Kim et al., 2011; Leibbrandt and Penninger, 2008; Sugatani et al., 2003). Terminal differentiation in the osteoclastic lineage is characterized by acquisition of mature phenotypic markers such as expression of tartrate resistant acid phosphatase (TRAP), matrix metalloproteinase 9 (MMP9), cathepsin K, and the morphological conversion into large multinucleated cells with the capability to form resorption lacunae on bone (Choi et al., 2010). Naturally occurring remedies have long been used in traditional medicine and provide small molecules that are generally recognized as safe. These substances are considered to have a greater likelihood than many synthetic compounds to exhibit specific bioactivities (Li and Vederas, 2009). Moreover, natural compounds represent a significant reservoir of unexplored chemical diversity for early-

M. Nepal et al. / European Journal of Pharmacology 715 (2013) 96–104

stage drug discovery (Koehn and Carter, 2005). Recently, many natural compounds have been shown to suppress osteoclast differentiation and bone-resorption and may reduce bone resorption without a concomitant decrease in bone formation via inhibition of specific signaling pathways. It is believed that the inhibition of osteoclastogenesis by those compounds may partially attribute to their anti-inflammatory and antioxidant properties (Bu et al., 2008; Choi et al., 2010; Lee et al., 2010). Accordingly, natural compounds and dietary components with antioxidant and anti-inflammatory activity may optimize bone health and stimulate bone formation. Hispidulin (5,7-dihydroxy-2-(4-hydroxyphenyl)6-methoxy4H-1-benzopyran-4-one) is a flavonoid, usually extracted from the Artemisia species, and has been traditionally considered to have anti-inflammatory effects. Hispidulin has also recently been shown to have antioxidant and antineoplastic properties (Dabaghi-Barbosa et al., 2005; He et al., 2011). In our search for natural compounds that exhibit pharmacologically relevant inhibitory effects on osteoclasts, we have found that hispidulin inhibits osteoclast formation via the MAPK and NF-κB signaling pathways. In this study, the effect of hispidulin on bone resorption was further investigated using an LPS-induced bone resorption model. Results strongly suggest that hispidulin may be useful as a therapeutic agent for bone lysis diseases.

97

factor (M-CSF) at 37 1C in CO2 for 1 day. The non-adherent cells were collected, plated in a 100 mm culture dish and incubated in the presence 30 ng of M-CSF for 3 days. Adherent cells were used as BMMs after washing and were replated on culture plates and incubated in the presence of M-CSF and RANKL until the cells differentiated into osteoclasts. Cells were cytochemically stained for TRAP to confirm differentiation into osteoclast cells. TRAP-positive multinucleated cells (MNCs) with more than 3 nuclei were counted. 2.4. Cell proliferation assay For the cell proliferation assay, RAW 264.7 cells were seeded onto 96-well plates at a density of 5
103 cells/well. After 16 h, various concentrations of hispidulin were added to the medium for 24 h, after which cells were washed with phosphate-buffered saline (PBS) and treated with medium containing 100 μg/ml of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] for 2 h at 37 1C. Cells were then washed with PBS and solubilized by the addition of 200 μl of DMSO. The resulting intracellular purple formazan was quantified with a spectrophotometer at an absorbance of 540 nm. 2.5. TRAP staining and TRAP activity assay

2. Material and methods 2.1. Reagents and animals Six-week-old ICR (Institute of Cancer Research) mice were purchased from Damool Science (Daejeon, Korea). Cell culture medium, fetal bovine serum (FBS), and horse serum were obtained from Invitrogen (Gaithersburg, MD, USA). RANKL was obtained from PeproTech (Rocky Hill, NJ, USA). M-CSF was obtained from R&D Systems (Minneapolis, MN, USA). Hispidulin was purchased from TOCRIS Bioscience (Ellisville, MO, USA), and the stock solution was prepared at a concentration of 20 mM in dimethyl sulfoxide (DMSO). A leukocyte acid phosphatase assay kit for TRAP staining was obtained from Sigma (St. Louis, MO, USA), and a luciferase assay kit was obtained from Promega (Madison, WI, USA). Specific antibodies, anti-phospho ERK, anti-phospho p38, anti-phospho JNK, anti-ERK, anti-JNK, anti-p38 and anti-c-Fos, were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NFATc1 and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma and/or the same as described (Choi et al., 2010), unless otherwise indicated. 2.2. Cell culture and osteoclastic differentiation The mouse monocyte/macrophage RAW 264.7 cell line was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured with DMEM containing 10% heatinactivated FBS, 2 mM glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate (growth medium) at 37 1C with a humidified atmosphere of 5% CO2. For osteoclastic differentiation, RAW 264.7 cells were suspended in α-MEM containing 10% FBS, 2 mM L-glutamate, 100 U/ml penicillin, and 100 μg/ml streptomycin, seeded at 3
103 cells/well in 96-well culture plates and cultured with 50 ng/ml soluble RANKL for 6 days. Cells were stained for TRAP, an osteoclast marker protein. 2.3. Isolation of bone marrow-derived macrophages Bone marrow-derived macrophages (BMMs) were obtained from tibia and femur bone marrow of six-week-old ICR mice and cultured in α-MEM with 10% FBS and 30 ng/ml macrophage colony-stimulating

For the TRAP staining, RAW 264.7 cells were washed with PBS and fixed with 3.7% formaldehyde for 10 min. After washing with PBS, cells were incubated with 0.1% (v/v) Triton X-100 for 1 min. Cells were washed, and then incubated for 40 min at 37 1C in the Table 1 Primer sequences for RT-PCR. Target genes (Accession number)

Primers forward/reverse

PCR condition Tm(1C)

Size (bp)

Cathepsin K (NM_007802)

5′-aggcggctatatgaccactg-3′ 3′-gatatgctctcttggctcgg-5′

55

403

TRAP (NM_007388)

5′-ctgctgggcctacaaatcat-3′ 3′-ggtagtaagggctggggaag-5′

55

400

MMP-9 (NM_013599)

5′-cgtcgtgatccccacttact-3′ 3′-tcctgggcaagcagtactct-5′

55

433

c-Fos (NM_010234)

5′-atgggctctcctgtcaacac-3′ 3′-tggagtttattttggcagcc-5′

55

480

NFATc1 (NM 198429)

5′-gggtcagtgtgaccgaagat-3′ 3′-ggaagtcagaagtgggtgga-5′

55

224

GAPDH (NM_008084)

5′- accacagtccatgccatcac-3′ 5′-tacagcaacagggtggtgga-3′

56

452

Hispidulin (25 μg/kg body weight) oral ad. Day -1

0

1

2

3

4

LPS (5 mg/kg body weight) i.p.

5

6

7

8 Sacrifice

Fig. 1. Animal model and treatment protocol. The LPS-induced bone resorption animal model was established by injecting LPS (5 mg/kg body weight/day) intraperitonially at two different time points (days 0 and 4). Animals in the treatment (n¼ 5) or the control group (n¼ 5) were administered oral hispidulin (25 μg/kg body weight/day) at 5 time points, respectively (Day −1 to 7). Mice were sacrificed on day 8.

M. Nepal et al. / European Journal of Pharmacology 715 (2013) 96–104

50 μl of cell lysates, and the reaction mixture was incubated for 1 h at 37 1C. Fifty microliter of a 3 N NaOH solution was added to stop the reaction and the absorbance was measured at 405 nm.

dark with a mixture of solutions of Fast Garnet GBC, sodium nitrite, naphthol AS-BI phosphoric acid, acetate, and tartrate of the Leukocyte Acid Phosphatase Assay kit (Sigma) according to the manufacturer's instructions. Cells were washed with distilled water and TRAP-positive multinucleated cells containing three or more nuclei were counted under a light microscope. To measure TRAP activity, the cells were harvested and then cell lysate was incubated in phosphatase substrate solution at 30 1C for 30 min. Absorbance at 405 nm was measured using a microplate reader and then trap activity was calculated.

2.7. Isolation of total RNA and reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated using the TRIzol (invitrogen) reagent according to the manufacturer's instructions. The SuperScript™ First-Strand Synthesis System (Invitrogen) was used to reversetranscribe 2 μg of RNA. Reaction products were amplified with specific primers; a set of primers was designed by Bioneer (Daejeon, Korea) for each gene of interest, and primer sequences are listed in Table 1. RT-PCR was performed as previously described (Choi et al., 2010).

2.6. Alkaline phosphatase (ALP) activity The ALP activity was measured using the alkaline phosphatase yellow liquid substrate in the ELISA kit (sigma) according to manufacturer's instructions. Briefly, MC3T3-E1 cells were washed three times with PBS, sonicated with a lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA) and the protein concentration was measured using the Bradford reagent. The reaction was started by the addition of 200 μl of para-nitrophenylphosphate (Sigma) to

1μ Μ

10μ Μ

0μ Μ

1μ Μ

10μ Μ

#

70

140 120 100

*

80 60 40 20 0

The cells were washed with ice-cold PBS and then resolved in lysis buffer [20 mM Tris–HCl (pH 7.5), 137 mM NaCl, 10% glycerol, 1%

0μ Μ

0 0.1 1 10 Hispidulin (μM)

# of TRAP+ MNCs

# of TRAP+ MNCs

160

2.8. Cell lysate extraction and immunoblot analysis

#

140

60 50 40 30

* ** **

20

120 100 80 60 40 20 0

10 0

Cell viability (%)

98

0 0.1 1 10 Hispidulin (μM)

0 0.1 1 10 Hispidulin (μM)

Fig. 2. The inhibitory effect of hispidulin on RANKL-induced osteoclastogenesis. (A) BMMs were cultured with the indicated concentration of hispidulin in the presence of M-CSF (30 ng/ml) and RANKL (200 ng/ml). After 6 days of culture, cells were stained for TRAP. (B) Murine monocyte/macrophage RAW 264.7 cells were cultured with the indicated concentration of hispidulin in the presence of RANKL (50 ng/ml) for 6 days. The cells were stained for TRAP. (C) TRAP-positive multinucleated cells (# of TRAP+ MNCs) were counted in cultured BMMs. (D) TRAP-positive multinucleated cells were counted in cultured RAW 264.7 cells. (E) The effect of hispidulin on RAW 264.7 cell viability was measured by the MTT assay. The results are expressed as mean7 S.E.M. n¼5 per group. nPo 0.05 and nnPo 0.01 versus vehicle-treated cells (#), respectively.

M. Nepal et al. / European Journal of Pharmacology 715 (2013) 96–104

Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1
protease inhibitor cocktail]. After centrifugation at 16,000
g for 15 min at 4 1C, supernatants were used as cell extracts. The extracts were separated on an 8–10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad). An immunoblot assay was performed as previously described (Choi et al., 2010), and the antigen–antibody complexes were visualized with an ECL Plus kit (Amersham Biosciences, Piscataway, NJ, USA). In some experiments, the optical intensities of individual bands were normalized to the corresponding values of β-actin. 2.9. Transfection and luciferase reporter assay

99

the Chonbuk National University Laboratory Animal Center (Jeonju, Korea). The animal model of bone resorption was established by direct intraperitoneal injection of LPS as performed in a previous study (Park et al., 2007). Five 6-week-old ICR mice were twice injected intraperiotoneally with LPS (5 mg/kg body weight) or vehicle. Hispidulin (25 μg/kg body weight) was administered orally 5 times on every second day from day −1 to day 7 as scheduled (Fig. 1). One mouse in each group was sacrificed on day 8, and then bone resorption was analyzed with microcomputational tomography (μCT). 2.11. μCT and histopathology

4

RAW 264.7 cells were seeded at 5
10 /well in a 24-well dish and grown to 90–95% confluence in complete growth media. For each well, 1.0 μg of luciferase reporter plasmid construct harboring the NFATc1 binding site and 0.5 μg of pCMV-β-galactosidase control vector were co-transfected into cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After transfection for 12 h, the medium was changed, and the cells were treated with RANKL and hispidulin. The cells were then washed with PBS and lysed in 1
reporter lysis buffer (Promega). The activities of firefly luciferase in the cellular extracts were measured using the luciferase reporter assay system according to the manufacturer's instructions (Promega). Relative luciferase activity was obtained by normalizing the firefly luciferase activity to the β-galactosidase activity. 2.10. LPS-induced bone resorption The animal study was conducted in compliance with the principles of the Institutional Animal Care and Use Committee of

The femurs on the left sides of the animals were scanned with a Skyscan1076 μCT scanner (Aartselaar, Belgium). The voltage and current of the X-ray tube were 100 kV and 100 μA, respectively, with 240 ms of exposure time. X-ray projections were obtained at 0.61 intervals with a scanning angular rotation of 3601. The femurs of the other sides were fixed with 4% paraformaldehyde and decalcified in 12% EDTA (pH 7.4) at 4 1C for 14 days before being embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin Y (H&E), followed by microscopic evaluation. 2.12. Statistical analysis All the experimental data shown are expressed as mean 7 S.E. M. of at least three replicates, unless otherwise indicated. Statistical analyses were performed by Dunnett's multiple comparison test using SPSS ver. 12.0 software and P-values less than 0.05 were considered significant.

#

+

+

+

+

μM ) Hispidulin(μ

0

0.1

1

10

TRAP Specific Activity (unit/mg)

0.3 RANKL(50ng/ml)

Cathepsin K TRAP MMP-9

RANKL + 0 RANKL + 0.1 μM Hispidulin RANKL + 1 μM Hispidulin RANKL + 10 μM Hispidulin

80

# *

#

60 40

**

**

**

20

**

0 Cathepsin K

0.1

TRAP

MMP-9

-

800

ALP Activity (%)

mRNA level (%)

100

#

**

0.2

0.0 RANKL (50ng) Hispidulin (μM)

GAPDH

120

*

+ -

+ 1

+ 10

Control β-Gly + Asc Acid βGs+Hispidulin 1μM βGs+Hispidulin 10μM

600

** *

400

#

200 0 0

7

14

21

Days

Fig. 3. Suppression of RANKL-induced gene expression by hispidulin in RANKL-induced osteoclastogenesis with RAW 264.7 cells. (A) RAW 264.7 cells were cultured with the indicated concentrations of hispidulin in the presence of RANKL (50 ng/ml). After 6 days of culture, the mRNA expression levels of genes were determined by RT-PCR and compared with that of GAPDH as a control. (B) The histogram represents the mRNA level (%) compared with that of the control. The results are expressed as mean 7S.E.M. n¼ 5 per group. nPo 0.05 and nnP o 0.01 versus vehicle-treated cells (#), respectively. (C) RANKL stimulated TRAP specific activity in RAW 264.7 was calculated. The cell lysate was obtained from the cells treated in absence or presence of RANLK (50 ng/mg) for 6 days. Then incubated in phosphate substrate solution for 30 min and absorbance was taken at 405 nm. (D) For ALP activity, murine osteoblast cells MC3T3E1 were grown in absence and presence of 10 mM disodium β-glycerophosphate and 0.15 mM ascorbic acid in the presence of 1 μM and 10 μM of hispidulin for 21 days with 7 day's interval. Cell lysate was obtained and equal protein was used to obtain ALP activity. The absorbance was taken at 405 nm. These results are expressed as mean 7 S.E.M. n¼ 3 per group. nPo 0.05 and nnPo 0.01 versus positive control (#), respectively.

100

M. Nepal et al. / European Journal of Pharmacology 715 (2013) 96–104

3. Results 3.1. Hispidulin inhibits osteoclast formation in two different cell culture systems

0

15

-

-

30 60

15

30

60

Time (min)

-

10

10

10

Hispidulin (μ μM)

-

Phospho-ERK We used two types of cells for osteoclastic differentiation to evaluate the effect of hispidulin on osteoclastogenesis. RAW 264.7 cells were induced with RANKL to differentiate into osteoclasts, while BMMs were induced with RANKL and M-CSF for 6 days. Hispidulin added to both cultures significantly inhibited the formation of TRAP-positive multinucleated cells (MNC), as shown in Fig. 2A for BMMs and Fig. 2B for Raw 264.7 cells. Notably, MNCs in cultures treated with hispidulin were smaller than control cells. The addition of hispidulin into both cell cultures showed dosedependent inhibition of osteoclast formation as measured by the number of TRAP-positive MNCs (Fig. 2C and D). In order to find out that the inhibition of osteoclast is not due to unspecific cytotoxicity of hispidulin, we performed a cell viability assay up to 10 mM of hispidulin treatment. There was no observable cell loss at the concentrations tested in this study (Fig. 2E; data not shown for BMMs).

ERK Phospho-JNK JNK1 Phospho-p38 p38 β-actin 0

15

30

60

15

30

60

-

-

-

-

10

10 10

Time (min) Hispidulin (μM)

3.2. Hispidulin reduced the level of RANKL-induced osteoclast activity and the expression of osteoclastic genes in RAW 264.7 and increased ALP activity in MC3T3E1 cells

Phospho-p65

To further evaluate the osteoclastic changes, we examined the effects of hispidulin on the mRNA expression levels of osteoclast specific genes using semi-quantitative RT-PCR. Osteoclast differentiation is associated with up-regulation of specific genes such as TRAP, RANK, and cathepsin K in response to RANKL (Lubberts et al., 2002). Hispidulin significantly reduced the expressions of the osteoclast-specific genes in a concentrationdependent manner (Fig. 3A), as shown in the quantitative measurements (Fig. 3B), although it did not affect the expression of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Furthermore hispidulin inhibited RANKL induced osteoclast enzyme activities in a concentration dependent manner (Fig. 3C). These data show consistency with the inhibitory effects on osteoclast formation. On the other hand, we tried to find out the effect of hispidulin in osteoblast differentiation in MC3T3 E1 cells. MC3T3 E1 cells supplemented with or without βglycerolphosphate and ascorbic acid with the treatment of 1 and 10 mM of hispidulin were incubated for 0, 7, 14 and 21 days. Then equal amounts of protein were taken to measure the ALP activity at 405 nm. Among all, hispidulin treated cells exhibited greatest differentiation. Cell treated with β-glycerolphosphate and ascorbic acid also showed a significant differentiation in compare with control (Fig. 3D). These results implied that hispidulin could not only inhibit the osteoclast formation but also stimulate osteoblast differentiation.

Phospho-IkB

3.3. Hispidulin inhibits RANKL-induced activation of MAPK and NF-κB in RAW 264.7 cells To explore pathways by which hispidulin regulates osteoclast differentiation and function, the effects of hispidulin on RANKLinduced MAPK and NF-κB activation were examined in RAW 264.7 cells. The 3 families of MAPKs, ERK, JNK, and p38, were all activated as indicated by their phosphorylation within 30 min of treatment with RANKL. However, hispidulin significantly inhibited the phosphorylation of JNK and p38, though it did not have much of an effect on ERK activation (Fig. 4A). NF-κB is one of the key transcription factors activated by the osteoclast differentiation factor RANKL (Vaira et al., 2008), and its activation is suppressed via dephosphorylation of inhibitory κB (IκB) and p65, as shown in

p65

IkB β-actin Fig. 4. Suppression of RANKL-induced MAPKs and NF-κB activation by hispidulin in RANKL-induced osteoclastogenesis in RAW 264.7 cells. RAW 264.7 cells were serum-starved for 16 h, pretreated with 10 μM hispidulin for 30 min, and stimulated with RANKL (200 ng/ml) for the indicated time. (A) Cell extracts were analyzed by immunoblot analysis with specific antibodies for phospho-ERK, phospho-JNK, phospho-p38 and ERK, JNK, and p38, respectively. An equal amount of protein was loaded in each lane, as demonstrated by the level of β-actin. (B) Cell extracts were analyzed by immunoblot analysis with specific antibodies for phospo-p65, phospo-IκB, p65 and IκB. An equal amount of protein was loaded in each lane as demonstrated by the level of β-actin.

previous studies (Granholm et al., 2007; Vaira et al., 2008). The RANKL-induced phosphorylation of IκB and the phosphorylation of p65 were significantly suppressed by hispidulin (Fig. 4B). These results suggest that the anti-osteoclastogenic effect of hispidulin was mainly caused by disrupting the signaling of NF-κB, JNK and p38 rather than ERK signaling. 3.4. Hispidulin inhibits RANKL-induced NFATc1 activation in RAW 264.7 cells NFATc1, a transcription factor, plays a role in regulating the expressions of osteoclast-specific downstream target genes including TRAP. RANKL induces NFATc1 expression during osteoclastogenesis at both the transcriptional and post-translational levels (Kim et al., 2011). The NFATc1 transcriptional level is dependent on both the NF-κB and the c-Fos pathways (Takayanagi et al., 2002). We investigated whether hispidulin reduced the activation of NFATc1 in the osteoclastogenesis of RAW 264.7 cells. It seems that the NFATc1 mRNA level is increased by RANKL treatment in RAW 264.7 cells, as shown by RT-PCR. Treatment with 10 μM of hispidulin had a significant influence on the inhibition of the NFATc1 mRNA level 12 h after hispidulin treatment (Fig. 5A). We also examined NFATc1 transcriptional activity using a luciferase

M. Nepal et al. / European Journal of Pharmacology 715 (2013) 96–104

Time (h) Hispidulin (μ μM)

0 -

3 -

6 -

12 -

3 10

6 12 10 10

Time (h) Hispidulin (μM)

NFATc1

0 -

101

3 -

6 -

12 -

3 10

6 10

12 10

c-Fos

10 9 8 7 6 5 4 3 2 1 0

Time (h) Hispidulin (μM)

Relative luciferase Activity (%)

GAPDH # *

0

3

6

12

3

6

-

-

-

-

10

10

12 10

#

500 450 400 350 300 250 200 150 100 50 0

**

-

+

+

RANKL

-

-

10

Hispidulin (μM)

Fig. 5. Suppression of RANKL-induced NFATc1 activation by hispidulin in RANKLinduced osteoclastogenesis in RAW 264.7 cells. (A) RAW 264.7 cells were cultured with 10 μM of hispidulin or vehicle in the presence of RANKL (50 ng/ml) for 6 days. For the NFATc1 mRNA expression levels, RT-PCR was performed using total RNA, and the results were compared with GAPDH levels. The lower histogram represents the mRNA expression fold-change compared with that of GAPDH. (B) RAW 264.7 cells were transfected with NFATc1-dependent luciferase reporter constructs and then treated with RANKL (200 ng/ml) in the absence or presence of 10 μM hispidulin. Cells were extracted, and relative luciferase activity was obtained by normalizing the firefly luciferase activity against the β-galactosidase activity. The results are expressed as mean 7 S.E.M. n ¼5 per group. nPo 0.05 and nnPo 0.01 versus vehicle-treated cells (#), respectively.

reporter plasmid-construct harboring the NFATc1 binding site. RANKL caused a 4.5-fold increase in NFATc1 transcriptional activity. However, 10 μM of hispidulin significantly suppressed RANKLinduced NFATc1 transcriptional activity (Fig. 5B). These results suggest that hispidulin might suppress NFATc1 by inhibiting its transcriptional activity without affecting the mRNA level in RANKL-stimulated RAW 264.7 cells. 3.5. Hispidulin inhibits RANKL-induced c-Fos expression in RAW 264.7 cells c-Fos, one of the signaling molecules involved in the induction of NFATc1 can bind to its promoter region and regulate NFATc1 expression (Giannoudis et al., 2007; Takayanagi, 2007). To further elucidate pathways by which hispidulin regulates osteoclast differentiation and function, we examined the effect of hispidulin on RANKL-induced c-Fos expression using RT-PCR following the hispidulin treatments in RAW 264.7 cells. The RANKL-induced increase in c-Fos mRNA level was significantly attenuated by hispidulin (Fig. 6). In this study, hispidulin suppressed not only

mRNA level (fold change)

mRNA l evel (fold ch ange)

GAPDH

8 7 6 5 4 3 2 1 0

#

**

Time (h)

0

3

6

12

3

6

12

Hispidulin (μM)

-

-

-

-

10

10

10

Fig. 6. Suppression of RANKL-induced c-Fos expression by hispidulin in RANKLinduced osteoclastogenesis in RAW 264.7 cells. RAW 264.7 cells were serumstarved for 16 h, pretreated with 10 μM hispidulin for 30 min, and stimulated with RANKL (200 ng/ml) for the indicated time. (A) The mRNA expression of c-Fos was determined by RT-PCR and compared with that of GAPDH. (B) The histogram represents the levels of the mRNA expression fold-change compared with that of GAPDH. The results are expressed as mean7 S.E.M. n¼ 5 per group. nnPo 0.01 versus vehicle-treated cells (#).

c-Fos induction, but also NFATc1 up-regulation by RANKL. Results suggest that the down-regulation of c-Fos by hispidulin may be, at least in part, a causal factor of suppression of NFATc1 expression by RANKL. 3.6. Hispidulin reduces in vivo LPS-induced bone resorption We next aimed to examine the potential suppressive effects of hispidulin on bone lysis in vivo. For this experiment, we employed an animal model for endotoxin-induced bone destruction. Mice were challenged with LPS and treated with hispidulin. LPS-injected mice displayed profound decreases in trabecular and cortical bone densities of femurs and tibias. Micro CT revealed that LPS-induced bone loss was clearly reduced in the femurs of hispidulin-treated LPS-challenged mice (Fig. 7A). In order to find out the more precise effect of hispidulin in bone microstructure, the radiographic results of bone resorption were further analyzed to assess the trabecular bone microstructure of the femurs. The calculation of the microstructural indices of trabecular bone density (BV/TV) revealed the amount of trabecular bone volume per unit of total bone mass. The BV/TV was reduced by LPS induction, and this reduction was observed to a lesser extent in the hispidulin-treated group (Fig. 7B). The LPS-induced decrease in trabecular thickness (Tb.Th.) was also attenuated by hispidulin (Fig. 7C). Histology with H&E staining showed that osteoclast formation and bone loss induced by LPS were largely inhibited in the tibia of hispidulin-treated mice (Fig. 7D). Lower magnified picture revealed that the LPS-induced bone loss was higher in the tibia of mice while treatment with the hispidulin showed efficient improvements in bone loss. These results suggested that hispidulin have beneficial effect in bone loss. We further examined the osteoclast activity in the perichondrium of tibia of mice. Interestingly, numerous osteoclast cells were detected in the perichondrium of LPS challenged groups (Fig. 7D, upper panel).

M. Nepal et al. / European Journal of Pharmacology 715 (2013) 96–104

Vehicle

LPS E

E

60

E G

G

G M

LPS + His

BV/TV (%)

102

M

M

50 #

40 30 20 10 0

V

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Fig. 7. The inhibitory effect of hispidulin on LPS-induced bone absorption. Six-week-old ICR mice were intraperiotoneally injected at 2 time points with LPS (5 mg/kg body weight) or vehicle, while hispidulin (25 μg/kg body weight) was administered orally on every second day from day −1 to day 7 (for a total of 5 times), as shown in Fig. 1 (n¼ 5). (A) Radiographs of the longitudinal and transverse section of the proximal femurs were obtained with a μCT apparatus. (B) The histogram represents the femur's bone volume/tissue volume (BV/TV), which was compared to the control group (#) for the microstructural indices of trabecular bone density. (C) The trabecular thickness (Tb.Th.) was also measured by Skyscan1076 μCT scanner software. (D) Histological analysis of mouse tibia for bone morphology and osteoclast detection was performed using H&E staining. (E) Percentage of eroded bone surface (BS) near the growth plate of the femur was analyzed using the histomorphometric results. The results are expressed as mean 7 S.E.M. n ¼10 per group. *nPo 0.05 and nnPo 0.01 versus control group (#).

The osteoclasts were not detected in control and hispidulintreated groups (Fig. 7D, lower panel). Furthermore, the percentage of eroded bone surface (BS) near the growth plate of the tibia was significantly increased in the LPS-treated mice, and this increase was blocked in hispidulin-treated mice (Fig. 7E). Taken together, these results strongly suggest that hispidulin inhibits osteoclast differentiation at the cellular level and also inhibits in vivo LPS-induced bone loss.

4. Discussion During recent years, an advanced understanding of the genetic and biological mechanisms involved in bone resorption has revealed new therapeutic targets for antiresorptive treatments. Antiresorptive agents work by inhibiting the activity of osteoclasts and, therefore, reducing bone resorption. Currently available antiresorptive agents include bisphosphonates, selective estrogen-receptor modulators, calcitonin and estrogen (Deal, 2009). However, many recent studies have reported that some of those drugs are associated with severe

risk factors including breast cancer, endometritis, thromboembolism, hypercalcemia, osteonecrosis of the jaw and atrial fibrillation (O'Regan and Gradishar, 2001; Rejnmark and Mosekilde, 2011). Accordingly, the safety of new therapeutic treatments must be highly considered. It is also desirable that new therapeutic compounds not be deposited within bone, and that they have a prolonged presence in plasma in order to prevent the anabolic activity. Hispidulin is a naturally occurring flavonoid commonly found in several Artemisia and Salvia species, which have been traditionally and widely used as medicinal plants (He et al., 2011; Yang et al., 2010). It has been reported that hispidulin has antifungal, antiproliferative, antioxidant and antithrombic properties (Dabaghi-Barbosa et al., 2005). Hispidulin has also been shown to be a partial positive allosteric modulator at the benzodiazepine receptor (Kavvadias et al., 2004). Recent studies showed that hispidulin could potentiate the ability to modulate tumor growth and tumor angiogenesis (He et al., 2011). For instance, it was shown to prevent pancreatic cancer growth in xenograft mice and to induce apoptosis in human ovarian cancer cells and human glioblastoma multiform cells (Kavvadias et al., 2004; Lin et al., 2010; Yang et al., 2010). However, scant

M. Nepal et al. / European Journal of Pharmacology 715 (2013) 96–104

information is available on the actions of hispidulin in bone health. In this study, we investigated the functional roles of hispidulin in osteoclastogenesis and bone resorption for the first time. Understanding the cellular and molecular mechanisms by which this natural compound inhibits osteoclasts might provide invaluable information for the treatment of osteolysis. RANK recruits the adapter molecule, TNF receptor-associated factor 6 (TRAF6), which activates NF-κB and MAPKs, including JNK and p38 in RANKL-induced osteoclastogenesis (Ikeda et al., 2008; Matsumoto et al., 2000). In the canonical NF-κB pathway, ligation of RANK activates the inhibitor of the IκB kinase (IKK) complex, which phosphorylates NF-κB-associated IκBα, leading to its ubiquitination and proteosomal degradation (Viatour et al., 2005). These events release into the cytosol NF-κB dimers containing p65 (RelA) and c-Rel, allowing them to translocate into the nucleus where they enhance transcription of target genes (Lee and Kim, 2003). In addition to NF-κB, RANKL-induced activation of c-Fos is required for the initial expression of the key transcription factor, NFATc1 (Takayanagi et al., 2002; Viatour et al., 2005). NFATc1 is an NFAT family member which is activated by the Ca2+/calmodulin regulated phosphatase, calcineurin (Ang et al., 2007). In osteoclast precursors, calcium signaling activates the existing NFATc1, and an AP-1 complex containing c-Fos may cooperate with NFATc1 to trigger NFATc1 amplification (Lee et al., 2000). Upon activation, NFATc1 proteins are dephosphorylated by calcineurin and then translocate from the cytoplasm into the nucleus, where they direct transcription of osteoclast-specific genes such as TRAP, cathepsin K and MMP9 at the terminal differentiation of osteoclasts (Sundaram et al., 2007; Teitelbaum, 2000). Our study showed that, during RANKL-induced osteoclastogenesis, hispidulin inhibited the activation of JNK and p38 and also decreased the transcriptional activity of NFATc1. Hispidulin also reduced NF-κB activity as well as phosphorylation of IκB and p65, which are markedly induced by RANKL. Hispidulin dampened RANKL-triggered osteoclast differentiation, diminishing the TRAP, cathepsin K and MMP-9 expressions in murine monocyte/macrophage cell lines. These results imply that hispidulin has the potential to inhibit osteoclast differentiation by attenuating the downstream signaling cascades associated with RANKL, and that these phenomena did not result from cytotoxic effects. Our study also showed that hispidulin reduced bone loss in an animal model for endotoxin-induced bone destruction. We employed an animal model of LPS-injected mice which displayed profound decreases in trabecular and cortical bone area densities in femurs. Bone loss induced by LPS was reduced by hispidulin treatment in the femurs of LPS-injected mice as shown by radiographic results as well as histological analysis. The results suggest that hispidulin suppresses bone loss via pre-osteoclast fusion and osteoclast activity, while LPS induces the production of inflammatory factors, supporting the survival of mature osteoclasts and stimulating osteoclastic bone resorption. In summary, our findings clearly show that hispidulin has an antiosteoclastogenic potential by reducing the in vitro RANKL induction of NF-κB, JNK, and p38 and NFATc1 in osteoclast precursors. Moreover, hispidulin also prevented in vivo LPSinduced bone destruction in RANKL-stimulated RAW 264.7 cells as well as in BMMs. Thus, our findings strongly indicate that hispidulin deserves new evaluation as a potential treatment option in various bone diseases associated with excessive osteoclast formation and bone destruction.

Acknowledgments This work was supported by the grant from Ministry of Knowledge Economy, Republic of Korea (R0000949).

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