Curcumin suppresses increased bone resorption by inhibiting osteoclastogenesis in rats with streptozotocin-induced diabetes

European Journal of Pharmacology 621 (2009) 1–9

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Molecular and Cellular Pharmacology

Curcumin suppresses increased bone resorption by inhibiting osteoclastogenesis in rats with streptozotocin-induced diabetes Mamiko Hie, Mariko Yamazaki, Ikuyo Tsukamoto ⁎ Department of Food Science and Nutrition, Nara Women's University, Nara 630, Japan

a r t i c l e

i n f o

Article history: Received 11 June 2009 Accepted 11 August 2009 Available online 21 August 2009 Keywords: Curcumin Diabetes mellitus Osteoclastogenesis AP-1 (activator protein-1) c-fos c-jun

a b s t r a c t Curcumin is a potent inhibitor of the transcription factor activator protein-1 which plays an essential role in osteoclastogenesis. However, the effects of curcumin on bone metabolism have not been clarified in vivo. We reported herein the inhibitory effects of curcumin on the stimulated osteoclastic activity in insulin-dependent diabetes mellitus using rats with streptozotocin-induced diabetes. A dietary supplement of curcumin reversed the increase in levels of activity and mRNA of tartrate-resistant acid phosphatase (TRAP) and cathepsin K to control values. A histochemical analysis showed that the increase in TRAP-positive cells in the distal femur of the diabetic rats was reduced to the control level by the supplement. These results suggested that curcumin reduced diabetes-stimulated bone resorptive activity and the number of osteoclasts. When bone marrow cells were cultured with macrophage colony stimulating factor and receptor activator NFκB ligand (RANKL), the increased activity to form TRAP-positive multinucleated cells and the increased levels of mRNA and protein of c-fos and c-jun in the cultured cells from diabetic rats decreased to control levels in the curcumin-supplemented rats. Similarly, the increased expression of c-fos and c-jun in the distal femur of the diabetic rats was significantly reduced by the supplement. These results suggested that curcumin suppressed the increased bone resorptive activity through the prevention of osteoclastogenesis associated with inhibition of the expression of c-fos and c-jun in the diabetic rats. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Insulin-dependent diabetes mellitus is associated with decreased bone mass and osteoporosis (Krakauer et al., 1995; Tuominen et al., 1999; Kemink et al., 2000). Streptozotocin-induced diabetes in rats is a wellrecognized model of insulin-dependent diabetes mellitus (Szkudelski, 2001). The effects of insulin-dependent diabetes mellitus have generally been attributed to insulin deficiency and to an impairment in osteoblastic function since insulin has a stimulatory effect on osteoblasts. However, increases in markers of osteoclastic function such as the excretion of calcium, hydroxyproline, and deoxypyridinoline in urine were reported in subjects with insulin-dependent diabetes mellitus (Selby et al., 1995; Bjorgaas et al., 1999). In fact, we have reported increases in the expression of cathepsin K and tartrate-resistant acid phosphatase (TRAP) at the early stages of streptozotocin-induced diabetes in rats (Hie et al., 2007). The increase in osteoclastic activity associated with the decreased osteoblastic activity leads to a diabetic osteopenia later on. The suppression of the increased bone resorptive activity at the early stages of diabetes would be useful for preventing, or at least delaying, the later loss of bone mass.

⁎ Corresponding author. Tel./fax: +81 742 20 3452. E-mail address: [email protected] (I. Tsukamoto). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.08.025

Bone resorption is carried out by hematopoietically derived osteoclasts (Udagawa et al., 1990; Kurihara et al., 1990). Their number and activity is determined by cell lineage allocation, the proliferation and differentiation of osteoclast precursors and the resorptive efficacy of mature osteoclasts (Harada and Rodan, 2003). Osteoclastic differentiation which requires macrophage colony-stimulating factor (M-CSF) and receptor for activation of NF-κB ligand (RANKL) is a multi-step process that eventually leads to the expression of TRAP, multinucleation, and bone-resorbing activity (Boyle et al., 2003; Asagiri and Takayanagi, 2007). The binding of M-CSF to c-Fms stimulates the expression of RANK in the hematopoietic osteoclast precursor cells. The binding of RANKL to its receptor RANK activates NF-κB and activator protein-1(AP-1) in osteoclast precursors and induces osteoclastic differentiation (Asagiri and Takayanagi, 2007). From a clinical point of view, the RANKL signaling pathway has promise as a strategy for suppressing excessive osteoclastic formation. Curcumin (diferuloyl methane), a polyphenolic phytochemical, is a primary component of the dietary spice tumeric. It has been used for centuries in indigenous medicine for the treatment of a variety of inflammatory conditions and other diseases (Ammon and Wahl, 1991). Curcumin has been demonstrated to be a powerful inhibitor of AP-1 (Hanazawa et al., 1993; Bierhaus et al., 1997; Chen and Tan, 1998; Squires et al., 2003). The transcription factor AP-1, a dimeric complex composed of Fos and Jun, is activated by RANKL and induces the expression of osteoclast-specific genes. c-Fos is crucial for AP-1

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interaction with its specific transcriptional partner, which is required in osteoclastogenesis. Mice expressing an inactivated c-fos or deficient in c-fos manifested arrested osteoclastic development and osteopetrosis (Wang et al., 1992; Grigoriadis et al., 1994). These findings suggest a potential role for curcumin in treatment for preventing bone resorptive activity. In the present study, we examined the effects of a dietary supplement of curcumin on the stimulated osteoclastic activity and demonstrated that curcumin inhibited bone resorptive activity with the suppression of osteoclastogenesis in rats with streptozotocininduced diabetes.

2. Materials and methods 2.1. Animals and study design Ten-week-old female rats of the Wistar/ST strain were purchased from Japan SLC (Shizuoka, Japan) and housed individually in a temperature-controlled room with a 12-h light cycle. After fasting overnight, sixteen rats were treated with streptozotocin (45 mg/kg body weight in 0.05 M citrate buffer, pH 4.5, i.p.), a pancreatic beta-cell cytotoxin, to render them diabetic. Eight control animals received the same volume of the streptozotocin diluent. The diagnosis of diabetes was based on glycosuria and hyperglycemia (blood glucose (nonfasting) N300mg/dl). All rats subjected to the streptozotocin-injection were rendered diabetic and no animals died during the experimental period. Four days following the injection of streptozotocin, eight diabetic rats were fed a standard diet (AIN 76A) with 0.5% curcumin (Curcumin), while the other eight diabetic rats (Diabetes) or the control rats (Control) were given a standard diet during the experimental period (14 days). The rats had free access to both food and water which were provided fresh daily. The estimated amount of curcumin taken in per individual was about 120 mg/day. At 14 days after the injection of streptozotocin or the diluent, blood and femoral and tibial bone were collected under anaesthesia with sodium pentobarbital (25 mg/kg) after overnight access to feed (non-fasting). Urine was collected over 24 h to determine the level of deoxypyridinoline. The sera were used to determine the concentrations of glucose and osteocalcin. After the removal of muscle and tendons, the femoral bone was used for the biochemical analysis, the histological analysis, or the preparation of the bone marrow cells. The bone marrow cells were used for the assay of osteoclastic differentiation, the frequency analysis of clonogenic osteoclast precursors, and the in vitro colony formation assay. Animal experiments were performed in accordance with protocols approved by the Animal Care Research Committee of Nara Women's University.

2.2. Preparation of bone extract For the biochemical analysis of bone, the femoral bone was sectioned into quarters as described previously (Goto and Tsukamoto, 2003). The quarter from the aspect of the knee of the femur (distal femur) was homogenized with 10 volumes of 10 mM triethanolamine buffer, pH 7.5, and stirred for 1.5 h at 4 °C. After centrifugation, an aliquot of the bone extracts was used for determining the activities of ALP, TRAP, and cathepsin K and for the Western blot analysis. The insoluble pellets were hydrolyzed with 6 N HCl at 105 °C for 24 h and analyzed for calcium and hydroxyproline. 2.3. Analytical methods The glucose concentration in serum and urine was assayed by the mutarotase–glucose oxidase method using commercial kits (Wako Diagnostic, Osaka, Japan).

Serum osteocalcin levels were measured using a rat osteocalcin enzyme immunoassay kit (Rat EIA kit; Biomedical Technologies, Stoughton, MA). ALP and TRAP activities were determined using 10 mM pnitrophenyl phosphate as the substrate in 0.1 M diethanolamine buffer (pH 9.8) and in 50 mM acetate buffer (pH 5.5) containing 20 mM sodium tartrate, respectively, for 30 min at 25 °C, as reported (Hie et al., 2007). One unit of activity is defined as the release of 1 nmol of p-nitrophenol per minute. The activity of cathepsin K was determined by a fluorogenic assay as described previously (Hie et al., 2007) and expressed as pmol of liberated 7-amino-4-methylcoumarin per minute. The amounts of Ca were determined by an o-cresolphthalein complexone (OCPC) method using commercial kits (Wako Diagnostic, Osaka, Japan). The level of hydroxyproline was measured by the method of Bergman and Loxley (1970). The concentration of deoxypyridinoline in urine was determined by an enzyme-linked immunoabsorbent assay using a Pyrilinks Dassay kit (Metra Biosystems, PaloAlto, CA). 2.4. Western blot analysis The protein concentrations of the bone extract or cell lysate were measured using the BCA protein assay kit. Equal amounts of the bone extract or cell lysate were electrophoresed in SDS-polyacrylamide gels and transferred to membranes. The membranes were blocked in 10 mM Tris–HCl buffer, pH 7.2, containing 0.15 M NaCl, 0.05% Tween 20, and 10% nonfat powdered milk overnight and incubated with a specific antibody to c-Fos or c-Jun (Santa Cruze Biotechhnology). After incubation with a secondary antibody conjugated to horseradish peroxidase, immunoreactive proteins were detected with the enhanced chemiluminescense system (ECL; Amersham). The chemiluminescent signals were quantified by a densitometer. The equal loading of protein samples was confirmed by staining of the gel with Coomassie brilliant blue and the reprobing of membranes with actin antibody. For reprobing, membranes were stripped with STRIP reagent (Nacalai Tesque Ltd., Japan) according to the manufacturer's protocol and reprobed with the corresponding antibodies. The molecular sizes of the developed proteins were determined by comparison with pre-stained markers (New England Biolabs). 2.5. Quantitative real-time PCR Total RNA from the distal femur or the cultured cells was prepared using a commercial kit (“NucleoSpin RNA II kit”, Macherey-Nagel, France). In the case of the distal femur, the bone marrow cells were washed out and RNA extraction was carried out after homogenization in the presence of 0.1 M EDTA. The total RNA was reverse transcribed by a first-strand cDNA synthesis kit (Toyobo, Tokyo, Japan). Real-time PCR was performed using the cDNA, or total RNA for the negative control, with SYBR-Green Real-time PCR Master Mix plus (Toyobo, Tokyo, Japan) and specific primers (ALP: 5′-GCACAACATC-3′AAGGACATCG-3′ and 5′TGGCCTTCTCATCCAGTTCA-3′; osteocalcin: 5′-AATAGACTCCGGCGCTACCT-3′ and 5′-GAGCTCACACACCTCCCTGT; TRAP: 5′-CAGCCCTTATTACCGTTTGC-3′ and 5′-GAATTGCCACACAGCATCAC-3′; cathepsin K: 5′TGTCTGAGAACTATGGCTGTGG-3′ and 5′-ATACGGGTAAGCGTCTTCAGAG-3′; RANKL: 5′-AGCGCAGATGGATCCTAACA-3′ and 5′TCGAGTCCTGCAAACCTGTA-3′; osteoprotegerin: 5′-TGTTCTGGTGGACAGTTTGC-3′ and 5′-GCTGGAAAGTTTGCTCTTGC-3′; c-fms: 5′GTGGCTGTGAAGATGCTCAA-3′ and 5′-GCAGCAGTATTCGGTGATGA-3′; RANK: 5′-TAGCCATCCGTTGATTGGA-3′ and 5′-ATATGCCTGCATCCCCTGAA-3′; c-fos: 5′-TCAAAGGGTTCAGCCTTCAG-3′ and 5′-CTTCACCCTGCCTCTTCTCA-3′; c-jun: 5′-TGAAGCAGAGCATGACCTTG-3′ and 5′TAGTGGTGATGTGCCCATTG-3′; actin: 5′-AGCCATGTACGTAGCCATCCA3′ and 5′-TCTCCGGAGTCCATCACAATG-3′) using a Light Cycler real-time PCR detection system (Toyobo, Tokyo, Japan). The amplification

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program consisted of 1 cycle for 1 min at 95 °C followed by 45 cycles of 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. Melting curve and gel analyses were used to verify specific products of appropriate size. Levels of gene expression were shown relative to an internal standard (actin).

cultured in 1 ml of MEM containing 1.2% methylcellulose, 30% FCS, 1% BSA and M-CSF (10 ng/ml) for 7 days, and the colonies were counted.

2.6. Histochemical analysis of distal femur

All statistical analyses were performed with a one-way analysis of variance with pairwise comparison by the Bonferroni method using the Microsoft Excel data analysis program. Values are expressed as the mean± S.E.M. Values of P b 0.05 were considered statistically significant.

Femoral bone was fixed in 4% paraformaldehyde, decalcified in 10% EDTA, and then embedded in paraffin. Sections (4 µm) were stained for TRAP activity using the leukocyte acid phosphatase kit (sigma 387-A) as described (Goto and Tsukamoto, 2003). The TRAP stained cells were measured on the metaphysis of the distal femur at ×200 magnification and shown as the number of osteoclasts (more than 3 nuclei) per field. Fifteen to 20 fields were counted for the metaphysis of the distal femur. 2.7. Osteoclastic differentiation of bone marrow cells Bone marrow cells were prepared by removing femurs and tibia from rats and flushing the bone marrow cavity with MEM containing penicillin (100 U/ml) and streptomycin (100 μg/ml). The bone marrow cells (2 × 105 cells/well of a 96-well plate) were cultured in MEM containing 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), 1% nonessential amino acid (NEAA), 1% sodium pyruvate, M-CSF (20 ng/ml) and RANKL (10 ng/ml). Cultures were maintained with a change of medium every 3 days. After 5 days, cells were used for TRAP staining and the assessment of cell viability. For the determination of mRNA and protein levels, the cells were harvested at 24 h and used for quantitative realtime PCR or Western blotting. 2.8. TRAP staining and viability of cultured cells Cells were fixed with 10% formaldehyde in phosphate-buffered saline for 10 min, treated with ethanol–acetone (50:50, v/v) for 1 min, and subjected to TRAP staining using the leukocyte acid phosphatase kit (Sigma 387-A). The TRAP-positive multinucleated cells (MNCs) (3 or more nuclei/cell) were counted manually by light microscopy. Cell viability was assessed by colorimetric assay using WST-8 (2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)2H-tetrazolium monosodium salt) (Cell Counting Kit-8: Dojin Ltd., Japan) according to the manufacturer's instructions. 2.9. Frequency analysis of clonogenic osteoclast precursors in bone marrow cells The frequency of clonogenic osteoclast precursors in bone marrow cells from the rats was assessed based on a limiting dilution assay (Sato et al., 2001) with some modifications. Briefly, the bone marrow cells were seeded into 96-well plates at 25, 50, 100, or 200 cells per well and cultured for 5 days in MEM containing 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), 1% non-essential amino acid (NEAA), 1% sodium pyruvate, M-CSF (5 ng/ml) and RANKL (5 ng/ml). The wells containing TRAP-positive MNCs were counted as osteoclast-positive after the TRAP staining. Plates with appropriate numbers of osteoclastpositive wells (6–20 of the 96 wells) from each experimental group were selected, and 1 / frequency of osteoclast precursors was calculated according to the formula; 1 / frequency=N / {ln[T/(T −P)]}, where N is the number of cells seeded in a well (25, 50, 100, or 200 cells), T is the number of wells per group, and P is the number of osteoclast-positive wells. 2.10. In vitro colony formation assay The numbers of colony formation unit-macrophage (CFU-M) in bone marrow cells from the rats were determined as described (Hayashi et al., 1997). Briefly, bone marrow cells (1 × 104) were

2.11. Statistical analysis

3. Results 3.1. Streptozotocin-induced diabetes and effects of curcumin on glucose levels in urine and serum, food intake, body weight, and bone length and weight The injection of streptozotocin increased glucose levels in urine before the curcumin supplement was given (at 4 days after the Streptozotocin-injection) (Table 1). The diabetic status induced by streptozotocin persisted throughout the study period as evidenced by hyperglycemia, high water intake, high food intake and polyuria. The curcumin supplement did not affect the serum glucose levels and food intakes of diabetic rats. Body weight (final) and bone weights of the femur, tibia and distal femur were significantly lower in the diabetic group than the control. No effect of curcumin on body weight, bone length or tibia weight was observed. The weights of the femur and distal femur of the curcuminsupplemented rats were not significantly different from the control value. 3.2. Effects of the dietary supplement of curcumin on the activities of ALP, TRAP, and cathepsin K, and the amounts of hydroxyproline and Ca in the distal femurs, serum osteocalcin level, and urinary deoxypyridinoline level The ALP activity in the distal femur and serum osteocalcin level in the diabetic group significantly decreased to about 55% and 15% of the control value, respectively (Table 2). The curcumin supplement did not affect these levels. The activities of TRAP and cathepsin K in the diabetic group significantly increased to about 1.5- and 2.4-fold the control level, respectively. These activities were significantly reduced to the control level by the supplement (Table 2). The levels of hydroxyproline and Ca were significantly lower in the diabetic rats

Table 1 Effects of curcumin on glucose levels in serum and urine, food intake, body weight, and length and weight of femur and tibia. Control Glucose level In urine (mg/24 h urine) At 4 day (before the supplement) At 14 day (final) In serum (mg/dl) Food intake (g/day) Body weight (g) Start (before fasting) Before the injection Final Bone length Femur (mm) Tibia Bone weight Femur Tibia (g) Distal femur

Diabetes

Curcumin

1.1 ± 0.4

8441.5 ± 446.6a

8655.7 ± 599.8a

1.1 ± 0.06 145.2 ± 11.1 14.4 ± 0.5

13059.8 ± 538.1a 465.3 ± 16.6a 24.6 ± 0.6a

13347.9 ± 349.4a 425.4 ± 18.9a 23.9 ± 0.6a

198.8 ± 3.1 183.4 ± 2.6 185.5 ± 2.5a 32.5 ± 0.2 36.2 ± 0.2 0.607 ± 0.010a 0.482 ± 0.005a 0.220 ± 0.004a

199.7 ± 2.6 181.4 ± 2.4 184.2 ± 2.7a 32.4 ± 0.1 36.2 ± 0.2 0.622 ± 0.011 0.488 ± 0.012a 0.226 ± 0.007

192.2 ± 3.4 182.1 ± 1.8 226.5 ± 2.5 32.8 ± 0.2 36.6 ± 0.2 0.655 ± 0.010 0.518 ± 0.009 0.245 ± 0.004

Streptozotocin (45 mg/kg body weight in 0.05 M citrate buffer) (diabetes) or the buffer (control) was intraperitoneally injected into rats fasted overnight. The supplementation with curcumin was started at 4 days after the streptozotocin-injection. At 2 weeks after the injection, blood (non-fasting) and femoral and tibial bone were collected under anesthesia. Values are the mean± S.E.M. for 8 rats. a Significantly different from the control value (P b 0.05).

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Table 2 Effects of curcumin on bone biochemical markers.

Distal femur ALP activity (U/g) TRAP activity (U/g) Cathepsin K activity (U/g) Ca (mg/g) Hydroxyproline (µmol/g) Serum osteocalcin (ng/ml) Urinary deoxypyridinoline (nmol/24 h urine)

Control

Diabetes

Curcumin

8.51 ± 0.41 0.358 ± 0.011 533.3 ± 30.2 117.5 ± 3.0 111.9 ± 1.9 13.69 ± 1.40 8.46 ± 0.23

4.67 ± 0.65a 0.545 ± 0.047a 1303.3 ± 133.1a 93.3 ± 3.9a 95.0 ± 5.0a 2.09 ± 0.35a 13.17 ± 0.58a

4.23 ± 0.22a 0.241 ± 0.026b 694.7 ± 26.2b 109.7 ± 4.6 100.7 ± 2.1 1.81 ± 0.06a 10.94 ± 0.22a,b

At 2 weeks after the streptozotocin-injection, blood and femoral bone were collected under anesthesia. The femoral bone was processed for the determination of the activities of ALP, TRAP, and cathepsin K and the amounts of Ca and Hyp as described in Materials and methods. The sera and urine were used to determine the osteocalcin concentration and the amount of deoxypyridinoline, respectively, as described in Materials and methods. Values are the mean ± S.E.M. for 8 rats. a Significantly different from the control value (P b 0.05). b Significantly different from diabetes (P b 0.05).

than the control. The levels of hydroxyproline and Ca in the curcuminsupplemented rats were not significantly different from the control values, but these levels were not significantly higher than those in the diabetic rats. The urinary deoxypyridinoline level, which significantly increased to 1.6-fold the control value in the diabetic rats, was significantly reduced by the curcumin supplement (Table 2).

3.3. Effects of the dietary supplement of curcumin on the mRNA levels of ALP, osteocalcin, TRAP, and cathepsin K in the distal femurs The expression levels of ALP, osteocalcin, TRAP, and cathepsin K relative to the internal control, actin, in the distal femur are shown in Fig. 1. The mRNA levels of ALP and osteocalcin in the diabetic group significantly decreased to about 30% and 25% of the control level, respectively. These decreases were not restored by the supplementation.

The mRNA levels of TRAP and cathepsin K in the diabetic rats increased to 2.7- and 2.6-fold the control level, respectively. The increased levels were reduced to the control value by the curcumin supplement. 3.4. Histochemical TRAP staining As shown in Fig. 2, the number of TRAP-positive cells increased in the diabetic rats compared with the control. The number of osteoclasts was 36± 8, 74± 12, and 43 ± 7 cells per field (means± S.D) in the control, diabetic, and curcumin-supplemented diabetic rats, respectively. The curcumin supplement significantly decreased the number of osteoclasts in the diabetic rats. 3.5. Effects of the dietary supplement of curcumin on osteoclastogenesis of bone marrow cells Bone marrow cells obtained from the control, diabetic or curcuminsupplemented diabetic rats were cultured in the presence of M-CSF and RANKL, and their osteoclastogenic potential was assessed by TRAP staining. As shown in Fig. 3A, the activity to form TRAP-positive MNCs was greater in bone marrow cells from the diabetic rats than control or curcumin-supplemented rats. The number of TRAP-positive MNCs in the culture of the bone marrow cells from the diabetic rats was about 2.4-fold the control value (Fig. 3B). In the curcumin-supplemented diabetic rats, the number of TRAP-positive MNCs decreased to the control level. The viability of the cultured cells from the control, diabetic and supplemented diabetic rats was similar (Fig. 3C). To examine the inhibitory mechanism involved in osteoclastogenesis of the bone marrow cells from the curcumin-supplemented rats, the expression of factors essential for osteoclastogenesis, c-fms, RANK, c-fos and c-jun, was determined (Fig. 3D). The mRNA levels of c-fms and RANK were similar among the three groups. The mRNA levels of c-fos and c-jun in the cultured cells from the diabetic rats were about 2.5- and 2.2-fold the control level, respectively. These higher levels decreased to the control level in the curcumin-supplemented rats. The protein levels of c-fos and c-jun in the diabetic rats were about 2.3- and 2-fold the

Fig. 1. Effects of the dietary supplement of curcumin on the mRNA levels of ALP, osteocalcin, TRAP and cathepsin K in the distal femurs. Total RNA was extracted from distal femurs of rats at 2 weeks after the streptozotocin-injection. The mRNA levels of ALP, osteocalcin, TRAP and cathepsin K were determined by real-time RT-PCR and are expressed relative to an internal standard, actin. Values represent the mean ± S.E.M. for 4 independent experiments. ⁎Significantly different from the control value (P b 0.05). # Significantly different from diabetes (P b 0.05).

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precursors to the control level. A colony assay showed that the number of colony forming cells by M-CSF (CFU-M) was similar among the three groups (Fig. 4B). 3.7. Effects of the dietary supplement of curcumin on the mRNA levels of RANKL, osteoprotegerin, c-fms, RANK, c-fos and c-jun and the protein levels of c-Fos and c-Jun in distal femur The expression levels of the factors necessary for osteoclastogenesis were determined in the distal femoral bone (Fig. 5). The mRNA levels of RANKL and osteoprotegerin in diabetic rats were similar to the control level and were not affected by curcumin (Fig. 5A). The mRNA levels of c-fms, RANK, c-fos, and c-jun in the diabetic rats were about 1.5-, 10-, 4.6-, and 1.9-fold the control level, respectively. The curcumin supplement did not affect the expression of c-fms or RANK. However, the c-fos mRNA levels in the curcumin-supplemented rats returned to the control level, while c-jun mRNA levels were significantly decreased but still higher than the control value. The protein levels of c-Fos and c-Jun in the diabetic rats were about 3.6- and 3.4-fold the control level, respectively. The elevated levels of c-Fos protein were significantly reduced to the control level by the curcumin supplement. The level of c-Jun protein also decreased but was still significantly higher than the control level in the curcuminsupplemented rats. 4. Discussion

Fig. 2. TRAP staining of the distal femur in control (A), the diabetic (B), and curcuminsupplemented diabetic (C) rats. Paraformaldehyde-fixed, decalcified, and paraffinembedded femur obtained from control (A), diabetic (B), and curcumin-supplemented diabetic (C) rats, was processed for TRAP staining. TRAP-positive cells appeared red. Hematoxylin counterstaining. × 200 magnification. The results presented here are typical of four separate experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

control value, respectively (Fig. 3E). The higher levels returned to the control value in the curcumin-supplemented rats.

3.6. Effects of the dietary supplement of curcumin on the pool of osteoclast precursors and the CFU-M in bone marrow Based on the frequency analysis, the population of osteoclast precursor cells of bone marrow (frequency) was 0.28%, 0.66%, and 0.38% in the control, diabetic, and curcumin-supplemented diabetic rats, respectively, as shown in Fig. 4A. The curcumin supplement significantly reduced the diabetes-increased population of osteoclast

This study clearly demonstrated that the dietary supplement of curcumin suppressed the increased bone resorptive activity in rats with streptozotocin-induced diabetes. Consistent with previous findings at 1 week after the injection of streptozotocin (Hie et al., 2007), the TRAP and cathepsin K activity in the distal femoral bone increased at 2 weeks. In diabetic rats, the elevated level of cathepsin K activity stimulated degradation of the bone matrix, resulting in a decrease in the amount of hydroxyproline in the distal femur and an increase in the urinary excretion of deoxypyridinoline, which is a product of the degradation of collagen. These results are consistent with reports that the excretion of hydroxyproline was increased in subjects with Type 1 diabetes (Bjorgaas et al., 1999) and in rats with streptozotocin-induced diabetes (Hie et al., 2007). The increase in cathepsin K activity was associated with the up-regulation of mRNA expression. The activity and mRNA levels of TRAP, another marker of osteoclastic activity, also significantly increased in the distal femur of diabetic rats. The curcumin supplement restored the levels of activity and mRNA of cathepsin K and TRAP to the control value. The urinary deoxypyridinoline level was also decreased by curcumin. Further, the histochemical analysis showed that the increased number of osteoclasts in the distal femur of the diabetic rats was recovered to the control level in the curcumin-supplemented rats. However, the decreased levels of expression of ALP and osteocalcin in diabetic rats were not affected by the supplement in diabetic rats. A previous study on rat calvarial osteoblastic cells also reported that curcumin did not affect markers of osteoblastic differentiation such as the activity of ALP and the level of expression of osteocalcin mRNA (Notoya et al., 2006). Although the inhibition of mineralization by curcumin was observed in the cultured osteoblasts (Notoya et al., 2006), the calcium content of the distal femur was not decreased by the curcuminsupplement in the diabetic rats, but rather partially recovered to levels which were not significantly different from the control value. These results suggested that curcumin affected the activity and number of osteoclasts rather than osteoblasts in bone of diabetic rats. When bone marrow cells were cultured with M-CSF and RANKL, the number of TRAP-positive MNCs in the cultured cells of the diabetic rats was about 2.4-fold the control value. The formation of TRAPpositive MNCs in curcumin-supplemented diabetic rats was reduced to the control level. The osteoclastogenesis-supporting compounds

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Fig. 4. Effects of the dietary supplement of curcumin on the population of osteoclast precursors and CFU-M in bone marrow cells. A frequency analysis of clonogenic osteoclast precursors in bone marrow cells (A) and the in vitro colony formation assay (B) were performed as described in Materials and methods. Data represent the mean ± S.E.M. for 6 independent experiments. ⁎Significantly different from the control value (P b 0.05). #Significantly different from diabetes (P b 0.05).

M-CSF and RANKL were present in excess in the cultures and, therefore, were not rate-limiting. Therefore, these results suggested that the number of osteoclast progenitors which respond to M-CSF and RANKL and differentiate into osteoclasts in bone marrow increased in the diabetic rats and decreased in the curcuminsupplemented diabetic rats. The frequency analysis also showed an increase in osteoclast precursors in bone marrow of diabetic rats and the reduction caused by the curcumin-supplement. However, a significant difference in CFU-M was not observed among the control, diabetic and curcumin-supplemented diabetic rats. In bone marrow, the phenotype of the progenitor for the osteoclast was comparable with that of CFU-M (Sudo et al., 1995; Hayashi et al., 1997; Arai et al., 1999). These results suggested that the difference in activity for osteoclastic formation of bone marrow cells was due to the potential of the precursors to differentiate into osteoclasts in response to RANKL. In fact, the levels of c-fms and RANK, the receptors of M-CSF and RANKL, respectively, in the bone marrow cell cultures were similar among the three groups. On the other hand, the mRNA levels of c-fos and c-jun of the cultured cells were higher in the diabetic rats than the control rats. The increased levels were reduced to the control levels in the curcumin-supplemented diabetic rats. Consistent with the mRNA levels, the increased protein levels of c-Fos and c-Jun in diabetic rats were also reduced to the control level by the curcuminsupplement. c-Fos and c-Jun are components of the transcription factor AP-1, a member of the osteoclast-specific transcriptional complex needed for the efficient expression of osteoclast-specific genes (Asagiri and Takayanagi, 2007). The expression of AP-1 (c-fos and c-jun) is required in the RANK–RANKL signaling pathway for the differentiation into osteoclasts (Grigoriadis et al., 1994; Asagiri and Takayanagi, 2007). These results suggested that the suppression of osteoclastic formation in the bone marrow cells of the curcuminsupplemented rats was due to the inhibition of c-fos and c-jun expression. The precursors of osteoclasts are hematopoietic cells of the monocyte/macrophage lineage originating in the bone marrow. The differentiation into osteoclasts, however, occurs on the bone surface in vivo (Hayashi et al., 1997; Manolagas, 2000). Therefore, the expression of RANKL, osteoprotegerin, c-fms, RANK, c-fos and c-jun was examined in the bone. RANKL plays a critical role in the differentiation of osteoclast precursors into mature osteoclasts. Osteoclastic differentiation is principally stimulated by an increase

in the biological availability of RANKL assessed by the ratio of RANKL to its decoy receptor, osteoprotegerin (Suda et al., 1999; Riggs, 2000; Hofbauer et al., 2000). In bone tissues, osteoblasts express two functionally conflicting factors, RANKL and osteoprotegerin, and the RANKL/osteoprotegerin ratio decreases with osteoblastic differentiation (Hofbauer and Heufelder, 2000). The mRNA levels of RANKL and osteoprotegerin in the curcumin-supplemented diabetic rats were not significantly different from those in the diabetic rats. These results suggested that curcumin did not affect the expression of osteoclastogenesis-supporting factors of osteoblasts, although a contribution by other cells including chondrocytes to the expression was not excluded. Regardless of the cell types which produced RANKL and osteoprotegerin, the levels of RANKL available in bone were not affected by curcumin in the diabetic rats. It seemed that the suppression of the increased number of osteoclasts in the diabetic rats by curcumin was not due to the change in the RANKL level. The mRNA levels of c-fms, RANK, c-fos and c-jun significantly increased in the diabetic rats. The levels of c-fms and RANK, which are expressed by osteoclast precursors and osteoclasts in bone, were not affected by curcumin. As observed in the bone marrow cell cultures, however, the mRNA and protein levels of c-fos and c-jun were reduced by the supplement. AP-1 is essential for osteoclastogenesis. In addition, it regulates the expression of osteoblast-specific genes including the ALP and osteocalcin genes in osteoblasts (McCabe et al., 1996) and plays an important role in chondrocytic differentiation (Thomas et al., 2000). In our experiments, however, no significant changes in the expression of ALP and osteocalcin were caused by the curcuminsupplement as described above. Effects of curcumin on chondrocytes were not found in the histological analysis either. These results suggested that the decrease in the expression of c-fos and c-jun by curcumin occurred in the precursors of osteoclasts rather than osteoblasts or chondrocytes in the bone. Taken together with the results of bone marrow cultures, curcumin appeared to suppress osteoclastogenesis by inhibiting the expression of c-fos and c-jun but not the expression of c-fms or RANK in the diabetic rats, although curcumin inhibited osteoclastogenesis through inhibition of NF-κB in RAW 264.7 cells (Bharti et al., 2004). The present study showed for the first time that curcumin suppressed osteoclastogenesis in association with the inhibition of c-fos and c-jun expression in vivo. Curcumin may have a potential clinical application in bone diseases by virtue of its suppressive effects on osteoclasts. However, further study is

Fig. 3. Effects of the dietary supplement of curcumin on osteoclastogenesis of bone marrow cells from the control, diabetic, and curcumin-supplemented rats. The bone marrow cells obtained from the control, diabetic, and curcumin-supplemented rats were cultured with M-CSF and RANKL for 5 days (A–C) or 24 h (D, E) as described in Materials and methods. (A) The cells were fixed and stained for TRAP. (B) The TRAP-positive MNCs were counted. (C) Cell viability was determined using WST-8 as described in Materials and methods. (D) The mRNA levels of c-fms, RANK, c-fos, and c-jun mRNA were expressed relative to an internal standard, actin. (E) Western blot analysis for c-Fos and c-Jun. The protein levels of c-Fos and c-Jun were quantified by densitometry and represented graphically. Data represent mean ± S.E.M. for 4 independent experiments. ⁎Significantly different from the control value (P b 0.05). # Significantly different from diabetes (P b 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Effects of the dietary supplement of curcumin on the mRNA levels of RANKL, osteoprotegerin, c-fms, RANK, c-fos and c-jun and the protein levels of c-Fos and c-Jun in the distal femur. (A) The mRNA levels of RANKL, osteoprotegerin, c-fms, RANK, c-fos and c-jun. Total RNA was extracted from distal femurs at 2 weeks after the streptozotocin-injection. The mRNA levels were determined by real-time RT-PCR and are expressed relative to an internal standard, actin. (B) Western blot analysis for c-Fos and c-Jun. The protein levels of c-Fos and c-Jun were quantified by densitometry and represented graphically. Values represent the mean ± S.E.M. for 4 independent experiments. ⁎Significantly different from the control value (P b 0.05). # Significantly different from diabetes (P b 0.05).

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