In vitro vitamin K2 and 1α,25-dihydroxyvitamin D3 combination enhances osteoblasts anabolism of diabetic mice

European Journal of Pharmacology 767 (2015) 30–40

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

Endocrine pharmacology

In vitro vitamin K2 and 1α,25-dihydroxyvitamin D3 combination enhances osteoblasts anabolism of diabetic mice Christina C.W. Poon a, Rachel W.S. Li a,b, Sai Wang Seto g, Siu Kai Kong d, Ho Pui Ho e, Maggie P.M. Hoi f, Simon M.Y. Lee f, Sai Ming Ngai d, Shun Wan Chan c, George P.H. Leung b,n, Yiu Wa Kwan a,nn a

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Department of Pharmacology and Pharmacy, Faculty of Medicine, The University of Hong Kong, Hong Kong c State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong d School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Hong Kong e Department of Electronic Engineering, Faculty of Engineering, The Chinese University of Hong Kong, Hong Kong f Institute of Chinese Medical Sciences, The University of Macau, Macau, China g National Institute of Complementary Medicine, School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia b

art ic l e i nf o

a b s t r a c t

Article history: Received 24 June 2015 Received in revised form 25 September 2015 Accepted 29 September 2015 Available online 8 October 2015

In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K2 (10 nM) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (þ / þ) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K2 (10 nM) and 1,25(OH)2D3 (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K2 and 1,25(OH)2D3 co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K2 plus 1,25 (OH)2D3 treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K2 plus D3 combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis. & 2015 Elsevier B.V. All rights reserved.

Keywords: Type 2 diabetes mellitus Osteoporosis Osteoblasts Vitamin K2 1,25(OH)2D3

1. Introduction Diabetes mellitus (DM) is a growing global epidemic affecting largely the elderly population (Yaturu, 2009). DM patients suffer increased morbidity and mortality not only because of increased complication in cardiovascular diseases, but also an increased fracture related to osteoporosis (Leidig-Bruckner and Ziegler, 2001)—a skeletal disorder of bone loss and fractures (Merlotti et al., 2010; Wongdee and Charoenphandhu, 2011; Hamann et al., 2012). Diabetic patients have a higher risk of developing n

Corresponding author. Fax: þ852 2817 0859. Correspondence to: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong , Room 207A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Hong Kong. Fax: þ852 2603 5139. E-mail addresses: [email protected] (G.P.H. Leung), [email protected] (Y.W. Kwan). nn

http://dx.doi.org/10.1016/j.ejphar.2015.09.048 0014-2999/& 2015 Elsevier B.V. All rights reserved.

spontaneous hip fractures by falls (Leidig-Bruckner and Ziegler, 2001; de Paula et al., 2010). More than 90% diabetes cases are Type 2 diabetes mellitus (T2DM) (Sivitz and Yorek, 2010) and T2DMassociated osteoporosis is a complicated phenomenon. Vitamin D is a fat-soluble vitamin, through a serial of biochemical conversions to form 25-hydroxyvitamin D [25(OH)D], which is the major circulating form of vitamin D. 25(OH)D is further converted to 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] which is the biologically-active form of vitamin D (Holick, 2006). 1,25 (OH)2D3 is responsible for regulating the calcium (Ca2 þ ) and phosphorus homeostasis (Lips and van Schoor, 2011). 1,25(OH)2D3 promotes bone remodelling via the vitamin D receptor which enhances osteoblasts differentiation and activities (Misof et al., 2003). Low serum 25(OH)D in T2DM patients caused reduced Ca2 þ absorption and bone formation (Busse et al., 2013). However, recent clinical studies reported that Ca2 þ with vitamin D supplements prescribed for patients with osteoporosis only marginally

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improved hip’s bone mineral density (BMD) but failed to reduce the overall risk of fractures (Jackson et al., 2006; Eriksen et al., 2013). Decreased osteoblast number and functions are associated with high serum undercarboxylated osteocalcin (ucOC) levels in T2DM patients which is a reflection of low serum vitamin K level (Wang et al., 2014). Vitamin K is an essential cofactor of γ-glutamyl carboxylase which converts the glutamyl (Glu) residues to γ-carboxyglutamic acid (Gla) residues (γ-carboxylation of osteocalcin) for bone mineralisation (Price et al., 1976; Vermeer et al., 1995; Ducy et al., 1996; Miyake et al., 2001; Iwamoto et al., 2003). Thus, patients with low serum vitamin K level are prone to osteoporotic fractures especially in elderly women with diabetes (Szulc et al., 1993; Weber, 1997). Surprisingly, the beneficial effects of vitamin K supplementation in treating osteoporosis could not confirmed in a randomized clinical trial (Ahmadieh and Arabi, 2011). In ovariectomized (OVX) rats, vitamin K2 and vitamin D3 supplementation prevented bone loss by stimulating osteocalcin production (Hara et al., 1994; Matsunaga et al., 1999). Vitamins K2 and D3 co-administration has synergistic effects on reducing bone loss in OVX rats, but no obvious effect was detected when these vitamins were given alone (Shiraishi et al., 2002). Besides, vitamin K2 is more effective in increasing bone mineralisation of osteoporotic patients with high serum 25(OH)D levels (Miyake et al., 2001). In this study, in vitro anabolic effects and the underlying mechanisms involved of vitamin K2 and 1,25(OH)2D3 co-administration on osteoblasts harvested from the iliac crests (anatomically equivalent to the hips of humans) of db/db mice (mice which exhibit phenotypes of human T2DM) were examined.

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puncture. Blood samples collected were allowed to clot at room temperature for 2 h, followed by centrifugation (4000 g) for 10 min, and serum was carefully pipetted and stored at  80 °C for subsequent biochemical analysis within 24 h Serum levels of 25 (OH)D, 1,25(OH)2D3 and ucOC of lean mice and db/db mice were determined by competitive enzyme immunoassay techniques using mouse-selective 25(OH)D, 1,25(OH)2D3 and ucOC ELISA kits respectively, and the absorbance was measured at 450 nm. 2.4. Bone histological determination The iliac crests freshly dissected from lean mice and db/db mice were fixed with 10% formalin, followed by de-calcification using 9% acetic acid and 4% mollifex solution. The de-calcified specimens were embedded in paraffin prior to the preparation of 5 μm sections and haematoxylin and eosin (H and E) staining for histological examinations. 2.5. Cell culture and vitamins treatment Under sterile conditions, the primary osteoblasts were isolated from iliac crests of individual mouse according to the protocols as described in previous study (Sheng et al., 2004) with enzymatic washing procedures. Cell pellet of primary osteoblasts were then resuspended and cultured with complete DMEM (supplemented with 10% FBS and 1% Penicillin–Streptomycin solution) at 37 °C in a humidified atmosphere of 5% CO2. For determination of osteoblastic activity and mineralisation, primary osteoblasts were grown with additional supplements of 10 mM β-glycerophosphate and 50 mg/ml of ascorbic acid. The medium was changed every 48 hours.

2. Materials and methods

2.6. Alizarin red S staining for mineralisation

2.1. Materials and drugs

Osteoblasts were seeded in 24-well plates (22  104 cells per well) after cultured for 7, 14 and 21 days with drug treatments. The cells were then fixed in 70% ethanol and stained with 40 mM Alizarin Red S (AR-S) (Sigma, USA). The stained matrix was photographed using a digital camera (Nikon, Japan). AR-S stain was released from the cell matrix by incubation in 10% (wt/vol) cetylpyridinium chloride for 15 min. The amount of dye released was quantified by spectrophotometry at 562 nm and the calcium ion concentration of each sample was normalised to its protein concentration.

Dulbecco's Modified Eagle Medium (DMEM) (low glucose: 5.56 mM), foetal bovine serum (FBS), HRP Chemilluminescence Substrate Reagents were purchased from Invitrogen (USA). Penicillin–Streptomycin solution was purchased from Thermo Scientific (USA). Reagents and molecular weight standards for Western blotting were purchased from Bio-Rad (USA). Mouse 25 hydroxyvitamin D3 (25OH-VD3) ELISA kit, mouse 1,25 hydroxyvitamin D3 (1,25OH-VD3) ELISA kit and mouse undercarboxylated osteocalcin (ucOC) ELISA kit were purchased from BlueGene Biotech (Shanghai, PR of China). ALP reagents were purchased from Stanbio Laboratory (USA). Menaquinone-4 (vitamin K2) was purchased from Santa Cruz Biotechnology (USA). 1α,25-dihydroxyvitamin D3 (vitamin D3) was purchased from Tocris Bioscience (UK). Other chemicals were purchased from Sigma-Aldrich (USA). 2.2. Animals C57BL/KsJ (female; 4–6 months old) lean ( þ/ þ) and obese/ diabetic (db/db) (leptin receptor-deficient) mice were used. The experimental procedures were approved by the Animals Experimentation Ethics Committee of the Chinese University of Hong Kong (Ref. no. 04/054/MIS). Animals were housed individually and maintained under controlled temperature (25 72 °C) with a 12-h light/dark cycle. They were fed standard chow and water ad libitum. 2.3. Serum collection and the measurements of 25(OH)D, 1,25 (OH)2D3 and ucOC Blood samples of anaesthetised mouse were collected via heart

2.7. Alkaline phosphatase (ALP) activities Osteoblasts were seeded in 24-well plates (22  104 cells per well) after cultured for 7, 14 and 21 days drug treatments. Cells were washed twice with 50 mM Tris–HCl (pH 7.3) before they were kept in 0.05% Triton X-100 lysis buffer overnight at  20 °C. The ALP activity of cell lysates was determined by measuring the absorbance of p-nitrophenol (yellow) catalyserd by ALP at 405 nm (Bio-Rad microplate reader). The ALP activity of each sample was normalised to its protein concentration and expressed as units per milligram of protein. 2.8. Immunocytochemistry Osteoblasts after drug treatment (7, 14 and 21 days) were seeded on glass coverslips (cell density: 18  104 cells per well) and cultured overnight in complete DMEM medium. Then, cells were fixed with paraformaldehyde (4% vol/vol) and permeabilized with Triton X-100 (0.3% vol/vol). After blocking with BSA (0.5% vol/ vol), cells were incubated with primary antibodies – osteocalcin (Santa Cruz Biotechnology, USA; 1:250) or V-ATPase A1 (Santa

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Cruz Biotechnology, USA; 1:250) overnight at 4 °C. The primary antibodies were detected using either Texas Red-conjugated antirabbit antibody or Texas Red-conjugated anti-mouse antibody (1:250; Invitrogen, USA) for 1 h and/or post-stained with Alexa Fluor 488 phalloidin (5 U/ml) (for labelling F-actin cytoskeleton) (Invitrogen, USA) for 30 min at room temperature. Osteoblasts were rinsed before mounting on glass slides. Cells were scanned (X, Y and Z panes; step size: 1 μm) using the confocal laser scanning microscope (Olympus FV1000-ZCD, Japan) for z-stacking to establish 3D images of individual cells examined. The overall intensities of the fluorescent signals (per cell) were collected and quantified using the computer software Olympus FV1000 ver.1.7 (Japan). 2.9. Measurement of intracellular calcium levels with 1,25(OH)2D3 (10 nM) challenge Osteoblasts were seeded on glass-bottom confocal culture dishes with complete low glucose DMEM medium overnight at 37 °C in a humidified atmosphere of 5% CO2 incubator. Cells were then incubated with fluo-4 AM fluorescent probe (Invitrogen, USA) (2 μM in 0.05% DMSO) for 1 h in buffer (in mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 1, glucose 10, HEPES 10 (pH 7.2) at 37 °C in a 95% air and 5% CO2 atmosphere. After 1 h incubation, osteoblasts were washed with dye-free buffer before commencing the experiments. 1,25(OH)2D3 (10 nM) was added to selected osteoblasts and the fluorescence emission (fluo-4 AM: 494–506 nm) was guided through an oil-immersion objective (60  , numerical aperture 1.45) and recorded (every 10 s for 25 min) using the confocal laser scanning microscope (Olympus FV1000-ZCD, Japan). The intensities of the fluorescent signals captured (equivalent to total intracellular calcium levels) were quantified using the computer software (Olympus FV1000), and plotted versus time using GraphPad Prism 5 (San Diego, USA). 2.10. Western immunoblots Osteoblasts were collected at 7, 14 and 21 days after drug treatments, and disrupted in lysis buffer (in mM): Trizma base 50, NaCl 100, EDTA 5, sodium pyrophosphate 67, sodium orthovanadate 0.5 and Triton X-100 1% (vol/vol) (pH 7.5). Cell lysates collected were centrifuged for 10 min (20,000 g), and the supernatants were stored at  20 °C. Protein concentrations were measured using Bicinchoninic Acid (BCA) assay. Proteins (20 μg per lane) were separated on 10% SDS-polyacrylamide gels and electro-transferred onto nitrocellulose membranes. Primary antibodies of calcium-sensing (CaS) receptor (1:500; Santa Cruz Biotechnology, USA), vitamin D receptor (1:500; Santa Cruz Biotechnology, USA), pregnane X receptor (1:500; Santa Cruz Biotechnology, USA), MEK1/2 (1:1000; Cell Signalling Technology, USA), phospho-MEK1/2 (Ser217/221) (1:1000; Cell Signalling Technology, USA), p44/42 MAPK (Erk1/2) (1:1000; Cell Signalling Technology, USA), phospho-p44/42 MAPK (Erk1/2) (Thr202/ Tyr204) (1:1000; Cell Signalling Technology, USA), F-actin (1:1000; Abcam, USA), anti-Osteocalcin (1:500; Millipore, USA), bone morphogenetic protein 2 (BMP-2) (1:500; Santa Cruz Biotechnology, USA), anti-runt-related transcription factor 2 (antiRunx2) (1:500; Abcam, USA), anti-Dlx5 (1:1000; Abcam, USA), osterix (OSX) (1:500; Santa Cruz Biotechnology, USA), ATF4 (1:1000; Cell Signalling Technology, USA), Smad1/5/8 (1:500; Santa Cruz Biotechnology, USA), phospho-Smad1 (Ser463/465)/ Smad5(Ser463/465)/Smad8(Ser426/428) (1: 1000; Cell Signalling Technology, USA) and V-ATPase A1 (1:500; Santa Cruz Biotechnology, USA) were used for the determination of protein expressions. GAPDH (1:2000) (Millipore, USA) was used as control for equal protein loading. The membranes were washed and

immunoblotted with anti-mouse IgG/anti-rabbit IgG (1:1000; BioRad, USA) or anti-goat IgG (1:1000; Invitrogen, USA). Labelled protein bands were visualised using HRP Chemiluminescence Substrate Reagents (Invitrogen, USA). The chemiluminescence intensity of each band was quantified by densitometry using Scion Image Programme (Version 1.63, Scion Image, USA). 2.11. Wound healing assay After 7, 14 and 21 days of drug treatments, osteoblasts of lean mice and db/db mice and the time-matched drug-free controls were seeded (25  104/well) into a 12-well plate and starved for 24 h. The cell layer was scratched by a 10 μl-pipette tip and one straight vertical wound was introduced in each well. Immediately after this procedure, culture medium was removed and photos of each well were taken. Cells were then incubated in DMEM medium (0.5% FBS) and allowed to migrate (37 °C, 48 h). Photos were taken after 48 h, and the area covered by cells that had migrated into the wound (i.e. wound closure) was estimated and analysed using ImageJ programme (USA). 2.12. Determination of cell surface morphology using scanning electron microscopy (SEM) After 7, 14 and 21 days treatments, osteoblasts of lean mice and db/db mice, and the time-matched drug-free controls were seeded (8  104 cell/coverslip) and cultured on sterile coverslips. After 24 h, samples were washed with 0.1 M pH 7.2 Sorensen's phosphate buffer (PB), fixed with 2.5% glutaraldehyde for 30 min and post-fixed with 2% osmium tetroxide for 10 min. They were then dehydrated in a series of ethanol solutions (70%, 75%, 85%, 95% and 100%). Samples mounted onto the round SEM stubs, critical pointdried and ion sputter-coated with a thin layer of gold before the surface morphology of adhered osteoblasts was examined and compared by scanning electron microscope (Hitachi SU8010, Japan). 2.13. Statistical analysis All quantitative data were analysed using GraphPad Prism 5 (USA) and presented as means 7S.E.M. Comparisons between groups were performed using either unpaired Student's t-test, one-way ANOVA followed by Dunnett's multiple comparison test or two-way ANOVA followed by Bonferroni post-tests, where appropriate. Po 0.05 was considered significant.

3. Results 3.1. Histological examinations Cross-sections of the iliac crest of obese/diabetic (db/db) mice appeared porous and filled up with marrow adipose tissues whereas a denser structure was observed in lean (þ /þ ) mice (Fig. 1A). 3.2. Serum levels of 25(OH)D, 1,25(OH)2D3 and ucOC Serum levels of 25(OH)D and 1,25(OH)2D3 were significantly lower in db/db mice compared with lean mice (Table 1). In contrast, a significant higher serum ucOC level (i.e. a lower vitamin K2 level) was detected in db/db mice compared with lean mice (Table 1). 3.3. Measurements of cytosolic calcium changes in response to 1,25

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Fig. 1. Evaluation of physiological differences between lean (þ / þ ) and obese/diabetic (db/db) mice. (A) Micrographs of representative sections of the iliac crests using haematoxylin and eosin (H and E) staining. Magnification: 200  . Scale bar: 15 μm. (B) Evaluation of intracellular cytosolic Ca2 þ concentration ([Ca2 þ ]i) changes in osteoblasts in response to 1,25(OH)2D3 (10 nM) challenge. [Ca2 þ ]i levels were measured using fluo-4 AM fluorescent probe by confocal laser scanning microscope (magnification: 600  ). Data are expressed as means7 S.E.M. (n¼ 13 for each strain of mice).

Table 1 Determinations of serum levels of 25(OH)D, 1,25(OH)2D3 and ucOC in lean ( þ/ þ ) and obese/diabetic (db/db) mice. Results are expressed as means 7 S.E.M. (n ¼3). Serum Level

þ /þ Mice

db/db mice

25(OH)D (ng/ml) 1,25(OH)2D3 (pg/ml) ucOC (ng/ml)

19.617 0.4869 80.50 7 2.446 5.3747 0.3467

13.65 7 1.014a 54.377 8.320b 37.96 7 1.107c

a

P o0.01. b Po 0.05. c Po 0.001.

(OH)2D3 A relatively lower basal cytosolic Ca2 þ ([Ca2 þ ]i) was observed in single osteoblasts of db/db mice compared with lean mice (Fig. 1E). Acute 1,25(OH)2D3 (10 nM) application induced a rapid, transient increase in [Ca2 þ ]i that reached the maximum at ∼140 s in osteoblasts of db/db mice whereas a sustained, greater magnitude of increase in [Ca2 þ ]i was detected in osteoblasts of lean mice (Fig. 1B). In our preliminary studies, vitamin K2 (1 and 10 nM) and 1,25 (OH)2D3 (1 and 10 nM) when applied alone, and a combination of

vitamin K2 (1 nM) plus 1,25(OH)2D3 (1 nM) all failed to alter the bone anabolic biomarkers of osteoblasts of both strains of mice. Thus, we decided to concentrate our efforts in evaluating the effects of a combination of vitamin K2 (10 nM) plus 1,25(OH)2D3 (10 nM) (i.e. combined vitamins) on osteoblasts of lean mice and db/db mice. 3.4. Effects of vitamin K2 plus 1,25(OH)2D3 and/or warfarin on osteoblastogenesis As shown in Fig. 2A, more Ca2 þ deposits were consistently detected in control (i.e. drug-free) osteoblasts of lean mice compared with db/db mice (Supplementary Fig. 1A). Combined vitamins significantly increased Ca2 þ deposits in a time-dependent manner in osteoblasts of lean mice and db/db mice, albeit a smaller magnitude of increase was observed in db/db mice (Supplementary Fig. 1B). Warfarin (20 mM, a vitamin K epoxide reductase inhibitor) pre-treatment eradicated the effects of combined vitamins on Ca2 þ deposits of osteoblasts of both strains of mice (Supplementary Fig. 1C). Similar to Ca2 þ deposits, a higher ALP activity was consistently detected in osteoblasts of lean mice compared with db/db mice (Fig. 2B). Combined vitamins treatment resulted in a time-

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Ca2+ deposits ( M / mg protein)

** #

20000

*

^^^

10000

0

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+/+ db/db

^^^

***

***

Osteocalcin (intensity per cell)

5000

** ##

30000

Vitamin K 2 + 1,25(OH) 2 D3

###

4000

2000

^^^

**

^

1000

Control

# Vitamin K2 + 1,25(OH) 2 D3

7 days

7 days

14 days

14 days 21 days

Warfarin

Warfarin

ALP activity (Unit / L / mg protein)

Warfarin

***

**

###

*** 0

Control

db/db

^^^

Fig. 3. Evaluation of osteocalcin levels in osteoblasts of lean (þ / þ) and obese/ diabetic (db/db) mice after vitamin K2 (10 nM) plus 1,25(OH)2D3 (10 nM), with or without warfarin (20 μM), were quantified by total fluorescent intensity measurements. Data are expressed as means 7 S.E.M. (n¼3 for each group). **Po 0.01 and # Po 0.05, ##P o0.01, ###Po 0.001 vs. ( þ /þ ) control; ^P o0.05, ^Po 0.01, ^Po 0.001 vs. db/db control.

^^^

###

1000

Warfarin

+/+

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**

21 days

Warfarin

Warfarin

2000

db/db

^^^

#

3000

0 Control

+/+

^^^ ***

**

**

Vitamin K 2 + 1,25(OH) 2 D3 7 days 14 days 21 days

Warfarin Warfarin

Warfarin Fig. 2. Determination of bone formation biomarkers in osteoblasts. Effects of vitamin K2 (10 nM) plus 1,25(OH)2D3 (10 nM), with or without warfarin (20 μM) on (A) calcium deposits formation and (B) ALP activities in osteoblasts of lean ( þ/ þ ) and db/db mice. Data are expressed as means 7 S.E.M. (n ¼3–6) * Po 0.05, ** Po 0.01, ***Po 0.001 and #Po 0.05, ##Po 0.01, ###P o0.001 vs. ( þ/ þ ) control; ^ Po 0.001 vs. db/db control.

dependent (7 and 14 days) increase of ALP activities in osteoblasts of lean mice and no further increase at 21 days. On the other hand, combined vitamins resulted in a time-dependent increase in ALP activities of osteoblasts of db/db mice, albeit a lesser magnitude of increase compared with that was detected in osteoblasts of lean mice (Fig. 2B). Warfarin abolished the effects of combined vitamins on ALP activities of osteoblasts of both strains of mice (Fig. 2B). In controls, a significantly higher level of osteocalcin was detected in osteoblasts of lean mice compared with db/db mice (Fig. 3). Combined vitamins treatment resulted in a time-dependent, warfarin-sensitive, increase of osteocalcin expression in osteoblasts of lean and db/db mice with a greater magnitude of increase was detected in db/db mice (Fig. 3 and Supplementary Fig. 2). 3.5. Effects of vitamins K2 plus 1,25(OH)2D3, with and without warfarin, on bone formation and differentiation biomarkers expression As shown in Fig. 4 and Supplemental Fig. 3, a lower protein expression of bone formation and differentiation markers: ATF4, OSX, Runx2, Dlx5 and osteocalcin was consistently detected in the control groups of osteoblasts of db/db mice compared with lean

Fig. 4. Determination of protein expression of bone formation transcription factors in osteoblasts. (A–B) Effects of vitamin K2 (10 nM) plus 1,25(OH)2D3 (10 nM), with or without warfarin (20 μM), on protein expressions of the bone formation transcription factors (ATF4, OSX, Runx2, Dlx5 and osteocalcin) in osteoblasts of lean (þ / þ ) and obese/diabetic (db/db) mice after indicated periods of treatment.

mice. In db/db mice, 14 and 21 days incubation with combined vitamins resulted in a significant increase of ATF4 expression whereas a moderate increase in ATF4 expression was observed in

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lean mice. In db/db mice and lean mice, 7 days incubation with combined vitamins resulted in an increase of OSX expression, and no further increase was observed with a longer incubation period. Fourteen days incubation resulted in a significant increase of Runx2 expression, and no further significant increase of Runx2 expression was observed after 21 days treatment. In osteoblasts of db/db mice, combined vitamins treatment resulted in a time-dependent increase in Dlx5 expression. On the other hand, a moderate increase in Dlx5 expression was observed only after 21 days treatments in osteoblasts of lean mice. In db/db mice, 7 days incubation with combined vitamins significantly increased osteocalcin expression and no further increase was detected with longer periods of incubation. In lean mice, 7 and 14 days treatment caused a marginal change of osteocalcin expression, and a significant increase was detected at 21 days. Co-incubation with warfarin eradicated the effects of combined vitamins on ATF4, OSX, Runx2, Dlx5 and osteocalcin expression of osteoblasts of both strain of mice (Fig. 4B and Supplementary Fig. 3). At 21 days, warfarin inhibited these biomarkers moderately (ATF4) or markedly (OSX, Runx2, Dlx5 and osteocalcin) of osteoblasts of lean mice. 3.6. Effects of vitamin K2 plus 1,25(OH)2D3, with and without warfarin, on osteoblast migration The migration rate, as determined using wound healing assay, of osteoblasts of db/db mice was  52% slower compared with lean mice in controls before combined vitamins treatment (Fig. 5). Combined vitamins treatment significantly increased the migration rate of osteoblasts in a time-dependent (14 and 21 days) fashion in both strains of mice, and a completed wound closure (i.e. 100%) was achieved at 21 days. Warfarin abolished, in a timedependent manner, the enhancement effects of combined vitamins treatment (Fig. 5 and Supplementary Fig. 4). 3.7. Effects of vitamin K2 plus 1,25(OH)2D3, with and without warfarin, on cell surface morphology of osteoblasts In controls, osteoblasts of lean mice exhibited cobblestone-like morphology and its surface was densely covered with microvilli

Wound closure (%)

150

100

+/+

**

**

** ##

50

0

###

Control

db/db

^^^ ###

# ^^^

* ###

^

^^

Vitamin K2 + 1,25(OH) 2 D3 7 days 14 days 21 days

Warfarin Warfarin

Warfarin Fig. 5. Determination of migration of osteoblasts. Effects of vitamin K2 (10 nM) plus 1,25(OH)2D3 (10 nM), with or without warfarin (20 μM), on migration of osteoblasts of lean ( þ/ þ ) and obese/diabetic (db/db) mice using wound-healing assay. Wound closure was expressed as percentage of the width of the initial wound area and data are expressed as means 7 S.E.M. (n ¼6 for each group). *P o0.05, **Po 0.01, ***P o0.001; #Po 0.05, ##Po 0.01, ###Po 0.001 vs. ( þ /þ ) control; ^Po 0.05, ^ Po 0.01, ^Po 0.001 vs. db/db control.

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along and embedded in large ruffles. In contrast, osteoblasts of db/ db mice were bigger in size and flat, and the surface was only scarcely covered with shorter microvilli, smaller ruffles and abundant fenestrations (Fig. 6A). In db/db mice, 7 and 14 days combined vitamins treatment resulted in the “re-appearance” of microvilli with some large blebs along the edges of large membrane ruffles on cell surface. A higher density of longer microvilli appeared on the surface at 21 days (Fig. 6B). Co-incubation with warfarin for 7 days resulted in a disappearance of microvilli and membrane ruffles, and abundant fenestrations appeared on the surface. At 14 days, no microvilli and membrane ruffles could be found, and more fenestrations appeared at 21 days (Fig. 6C). In osteoblasts of lean mice, irrespective of the duration of combined vitamins treatment, more membrane ruffles were appeared on the cell surface with no change in the cobblestone-like morphology (Fig. 6B). Co-incubation with warfarin for 7 days treatment resulted in a disappearance of microvilli and membrane ruffles with no apparent change in the cobblestone-like morphology (Fig. 6C). Moreover, fenestrations started to appear on the surface of osteoblasts. At 14 days, no microvilli and membrane ruffles was found, and abundant fenestrations were found at 21 days with warfarin. More importantly, in lean mice 14 and 21 days treated with warfarin resulted in a complete morphological change of osteoblasts from the cobblestone-like into flat shapes which were similar to the morphology of osteoblasts of db/db mice. 3.8. Effects of vitamins K2 plus 1,25(OH)2D3, with and without warfarin, on protein expression of F-actin cytoskeleton and V-ATPase In controls, a significantly higher level of F-actin cytoskeleton (illustrated as green fluorescent signal) and V-ATPase subunit A1 (shown as red fluorescent signal) was consistently observed in osteoblasts of lean mice compared with db/db mice. In osteoblasts of db/db mice, 7 days of combined vitamins treatment resulted in a marked increase in F-actin cytoskeleton. Interestingly, the levels of F-actin cytoskeleton measured were higher than that was observed in osteoblasts of lean mice after 7 days treatment which was slightly increased compared with drug-free controls (Fig. 7A and Supplementary Figs. 5A and 6). In both strains of mice, 14 days treatment resulted in a further increase in F-actin cytoskeleton, and no further increase was observed at 21 days (Supplementary Figs. 5A and 6). The enhancement effects of combined vitamins treatment on F-actin cytoskeleton levels was abolished by warfarin with a greater inhibition occurred in db/db mice (Fig. 7C and Supplementary Figs. 5A and 6). In controls, V-ATPase subunit A1 was mainly concentrated around the nucleus region of osteoblasts with a lower level in db/ db mice compared with lean mice (Fig. 7). In both lean mice and db/db mice, 14 days treatment resulted in a significant increase in V-ATPase subunit A1 levels and no further increase was observed with a longer duration of incubation (Fig. 7B and Supplementary Fig. 5B). Warfarin eradicated the enhancement effects of combined vitamins treatment on V-ATPase subunit A1 levels, with a greater inhibition on osteoblasts of db/db mice (Fig. 7C and Supplementary Figs. 5B and 6). 3.9. Effects of vitamins K2 plus 1,25(OH)2D3, with and without warfarin, on protein expression of calcium-sensing (CaS) receptor, pregnane X receptor and vitamin D receptor Protein expression of CaS receptor in both lean mice and db/db mice was markedly increased, in a warfarin-sensitive manner, after 7 days combined vitamins treatment and no further increase was observed with a longer duration (i.e. 14 and 21 days) (Fig. 8

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Fig. 6. Comparison of the surface morphologies of osteoblasts. (A–C) Cell surface morphology determinations of osteoblasts of lean ( þ /þ ) and obese/diabetic (db/db) mice before and after vitamin K2 (10 nM) plus 1,25(OH)2D3 (10 nM), with or without warfarin (20 μM). Representative high magnification images were captured by scanning electron microscopy (magnification 4000  for overview of cells; scale bar: 10 μm and magnification 35,000  for detailed surface structures; scale bar: 1 μm) (n¼3).

and Supplementary Fig. 7). Expression of pregnane X receptor of osteoblasts of db/db mice was increased after 14 days treatment and no further increase was observed at 21 days. In osteoblasts of lean mice, an increase in pregnane X receptor expression was observed after 21 days treatment (Fig. 8A and Supplementary Fig. 7). An increase in pregnane X receptor expression by combined vitamins treatments was eradicated by warfarin (Fig. 8B and Supplementary Fig. 7). Protein expression of vitamin D receptor of osteoblasts of db/db mice was lower compared with lean mice. A significant increase in vitamin D receptor expression was observed after 21 days

combined vitamins treatment in both strains of mice (Fig. 8A and Supplementary Fig. 7), and the enhancement effect was eradicated by warfarin (Fig. 8B and Supplementary Fig. 7). 3.10. Effects of vitamin K2 plus 1,25(OH)2D3, with and without warfarin, on BMP2-Smad and MAPK signalling pathways In controls, a lower protein expression of bone morphogenetic protein 2 (BMP-2) was detected in osteoblasts of db/db mice compared with lean mice. Combined vitamins treatment for 7 days resulted in an increase in BMP-2 protein expression in

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Fig. 7. Determination of cellular location and expressions of F-actin cytoskeleton and V-ATPase A1 in osteoblasts. (A–C) Representative images of cellular localisation of F-actin cytoskeleton and V-ATPase A1 in osteoblasts of lean (þ / þ) and obese/diabetic (db/db) mice before and after vitamin K2 (10 nM) plus 1,25(OH)2D3 (10 nM), with or without warfarin (20 μM). Images were captured and visualised using confocal laser scanning microscope (magnification: 600  ) and the total intensities were estimated using fluorescent Alexa Fluor 488 Phalloidin labelled F-actin cytoskeleton (shown as green colour) and Texas Red-conjugated antibodies labelled with V-ATPase A1 (shown as red colour). The representative images shown were captured at the mid-plane of cells. Scale bar: 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

osteoblasts of db/db mice, and no further increase in BMP-2 expression was detected after a longer duration (Fig. 8A and Supplementary Fig. 7). In contrast to db/db mice, BMP-2 expression in osteoblasts of lean mice was not altered by combined vitamins treatment. BMP-2 expression in osteoblasts of both strains of mice was sensitive to warfarin and BMP-2 expression was markedly suppressed to levels below the drug-free controls after warfarin treatment (Fig. 8B and Supplementary Fig. 7). In controls, a lower protein expression of p-Smad1/5/8 / Smad1/5/8 was detected in osteoblasts of db/db mice compared with lean mice (Fig. 8A and Supplementary Fig. 7). Similar to BMP2, 7 days treatment resulted in an increase in p-Smad1/5/8/Smad1/ 5/8 protein expression in osteoblasts of db/db mice with no further increase after a longer duration of treatment (14 and 21 days) (Fig. 8A and Supplementary Fig. 7). In contrast, p-Smad1/5/8/ Smad1/5/8 protein expression of lean mice was not altered by combined vitamins treatment. Warfarin abolished the effects of combined vitamins treatment on p-Smad1/5/8/Smad1/5/8 protein expression, and 21 days warfarin treatment resulted in a marked suppression of p-Smad1/5/8/Smad1/5/8 protein expression to a level below the drug-free controls (Fig. 8B and Supplementary Fig. 7). In controls, a similar protein expression of p-MEK1/2 / MEK1/2

was observed in osteoblasts of both strains of mice. 21 days of combined vitamins treatment resulted in a warfarin-sensitive increase in protein expression of p-MEK1/2/MEK1/2 of osteoblasts of both strains of mice, and no apparent effects were observed with a shorter duration (i.e. 7 and 14 days) (Fig. 8A and Supplementary Fig. 7). In controls, a lower protein expression of p-Erk1/2/Erk1/2 was detected in osteoblasts of db/db mice compared with lean mice. Combined vitamins treatment for 14 days resulted in an increase in p-Erk1/2/Erk1/2 protein expression in osteoblasts of db/db mice with no further increase after a longer duration (i.e. 21 days) (Fig. 8A and Supplementary Fig. 7), and the enhancement effects was eradicated by warfarin (Fig. 8B and Supplementary Fig. 7). In contrast, p-Erk1/2/Erk1/2 protein expression in osteoblasts of lean mice was not altered by combined vitamins treatment (Fig. 8A and Supplementary Fig. 7).

4. Discussion Patients with T2DM have higher fracture risks especially at the hip (Leidig-Bruckner and Ziegler, 2001). However, development of new treatments for obese/diabetes-associated osteoporosis has

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Fig. 8. Determination of protein expressions of CaS receptor, pregnane X receptor, vitamin D receptor and bone formation biomarkers in osteoblasts. (A – B) Effects of vitamin K2 (10 nM) plus 1,25(OH)2D3 (10 nM), with or without warfarin (20 μM), on protein expressions of CaS receptor, pregnane X receptor, vitamin D receptor, BMP2, p-Smad1/5/8, Smad1/5/8, p-MEK1/2, MEK1/2, p-Erk1/2 and Erk1/2 of osteoblasts of lean ( þ/ þ ) and obese/diabetic (db/db) mice after indicated periods of treatment.

been hampered by a poor understanding of the role(s) of leptin receptor (Lepr) and its cellular signalling cascades in osteoblasts – bone-building cells. Leptin secretion from adipocytes is up-regulated with increased adipose tissues, and leptin receptors are present in the hypothalamus and the skeleton which are important peripheral sites of action of leptin. In leptin-receptor deficient db/db mice, leptin receptor was demonstrated as a negative modulator of bone mechanosensitivity (Kapur et al., 2010) whereas the bone mass and strength are reduced in the absence of leptin signalling (Williams et al., 2011). Similar to the typical porous structures commonly observed in osteoporotic patients, a highly porous structure filled with large numbers of marrow adipose tissue was found in the iliac crest of db/db mice in contrast to a denser structure in lean mice (Bouxsein et al., 2009). Hyperglycaemia promotes differentiation of mesenchymal stromal cells into adipocytes instead of osteoblasts (Pittenger et al., 1999, 2000; Botolin et al., 2005; Botolin and McCabe, 2007; Wang et al., 2014) which further deteriorates bone quality. Moreover, large adipocytes of marrow adipose tissues

released adipokines and inflammatory cytokines which suppress bone formation and promote bone resorption (Rosen and Bouxsein, 2006; Rosen and Klibanski, 2009). To establish the importance of vitamin K and vitamin D in the development of osteoporosis in db/db mice, serum ucOC, 25(OH)D and 1,25(OH)2D3 levels were measured. Our results illustrate that db/db mice have low serum levels of both vitamins K and D3 which are probably responsible for the development of osteoporotic features of the iliac crests. Surface morphologies of osteoblasts reveal a marked difference in terms of cell size and the structural features of lean mice and db/ db mice. The unique surface morphological features in osteoblasts of db/db mice i.e. distinct fenestrations revealed for the first time highlight the importance of fenestrations in the overall weakening of bones in patients with DM/obesity. Microvilli are actin filaments organised in parallel bundles and cross-linked to each other which are important in cell migration (Bartles, 2000; Majstoravich et al., 2004). Integrin cytoplasmic domains at the focal adhesion sites linking actin cytoskeleton to the extracellular matrix on the outer surface of osteoblasts for the attachment to the bone matrix (Pavalko et al., 1991). Our results for the first time illustrate that surface of osteoblasts of db/db mice is devoid of microvilli and ruffles, and combined vitamins treatment resulted in the re-appearance of long microvilli and ruffles which is similar to the surface features found in lean mice. Besides, osteoblasts are bonebuilding cells which are attracted and subsequently crawled along to where bone cracks exist for repairing. The physiological functions of surface microvilli and ruffles in osteoblasts are unknown at present. It is tempting to speculate that surface microvilli and ruffles found in osteoblasts are important for migration (Bartles, 2000; Majstoravich et al., 2004). Thus, osteoblasts of db/db mice which are devoid of surface microvilli and ruffles before combined vitamins treatment are incapable of/less efficient in bone repairing i.e. a greater risk of fracture. In addition to surface microvilli and ruffles, F-actin cytoskeleton is important in cells migration. Our results demonstrate the poor migration of osteoblasts of db/db mice before combined vitamins treatment is probably related to a lower F-actin cytoskeleton expression and the lack of surface microvilli and ruffles. Besides, combined vitamins treatment markedly improved the sluggish migration and the restoration of suppressed F-actin cytoskeleton of osteoblasts db/db mice. The deficiency of serum vitamin D3 and vitamin K levels detected in db/db mice leads to a decreased osteocalcin level and mineralisation in osteoblasts. Our observations are consistent with a previous study which reported a reduced osteocalcin level in db/ db mice is associated with reduced bone formation rates and decreased bone strength (Williams et al., 2011). ALP is produced by osteoblasts mainly during bone formation stage for hard tissues calcification and extracellular matrix mineralisation (Cerovic et al., 2007), and a decreased ALP activity as illustrated in osteoblasts of db/db mice is associated with poor bone structures. During bone remodelling, extracellular CaS receptor of osteoblasts plays an important role in sensing the changes of Ca2 þ influx which is important for bone mineralisation (Uchida et al., 2010). 1,25 (OH)2D3 is a major hormone involved in Ca2 þ homoeostasis by regulating osteocalcin synthesis, osteoblastic proliferation and differentiation via Ca2 þ signals (Uchida et al., 2010; Rosso et al., 2012) which in turn modifies the functions of protein kinases including MAPKs (Norman et al., 2004; Pardo et al., 2006; Menegaz et al., 2010; Rosso et al., 2012). In addition, 1,25(OH)2D3 activated the membrane-associated vitamin D receptor in osteoblasts to increase [Ca2 þ ]i via Ca2 þ channels (Zanello and Norman, 2006). Thus, a reduction in serum 25(OH)D and 1,25(OH)2D3 levels, and a decrease in CaS receptor and vitamin D receptor expression in osteoblasts of db/db mice are associated with a reduction in

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[Ca2 þ ]i (thus a lower level of Ca2 þ deposits) plus a curtailment of [Ca2 þ ]i-induced signalling cascades inside osteoblasts are anticipated-weaker bone structures. Dlx5 is a homeobox protein which is necessary for induction of Runx2, OSX and osteocalcin expression, and inactive Dlx5 resulted in a decreased proliferation and differentiation (Holleville et al., 2007). Besides, Runx2 is important for promoting osteoblasts maturation from mesenchymal stem cells and it interacts with ATF4 to modulate osteocalcin expression (Ziros et al., 2008). Mice with inactive Runx2 and ATF4 have no functional osteoblasts and no proper bone development (Yang et al., 2004). BMP-2 plays an important role in osteoblasts differentiation through activation of downstream molecules such as Smad1, Smad5 and Smad8, in addition to Erk1/2 in MAPK pathways, which serve as positive regulators of osteoblasts differentiation via the activation of Runx2 and OSX (Matsui et al., 2014). Moreover, patients with long-term diabetes exhibit poor wound healing and altered peripheral blood flow circulation due to abnormal angiogenic mechanisms which are regulated by vacuolar type H þ -ATPase (V-H þ -ATPase) (Rojas et al., 2004). Inhibition of V-H þ -ATPase suppressed cell migration and a lower activity of V-H þ -ATPase was observed in cells harvested from diabetic animals (Rojas et al., 2004). Reduction of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4, OSX, ALP activity), V-H þ -ATPase, p-Smad1/5/8/Smad1/5/8 and pERK1/2/ERK1/2 expressions are probably related to the poor quality/porous structure of the iliac crests of db/db mice compared with lean mice. 1,25(OH)2D3 induces extracellular matrix mineralisation and promotes Ca2 þ resorption for calcification (Koshihara and Hoshi, 1997). Interestingly, 1,25(OH)2D3 stimulated vitamin K2-dependent epoxidase (γ-glutamyl carboxylase) activity for osteocalcin production in cultured human osteoblasts (Miyake et al., 2001). Thus, 1,25(OH)2D3 is necessary for γ-carboxylation of osteocalcin induced by vitamin K2 suggesting that there is an important interplay between 1,25(OH)2D3 and vitamin K2 in bone formation. Vitamin K2 and 1,25(OH)2D3, when applied alone, provided no significant improvement in bone anabolism of OVX rats (Hara et al., 1994). These results are consistent with our observations in which these two vitamins when applied alone failed to alter the expression of all bone anabolic biomarkers measured.

5. Conclusion Our in vitro study demonstrates that combined vitamin K2 plus 1,25(OH)2D3 treatment could be a potential treatment for DMassociated osteoporosis. It remains to determine the clinical efficacy of using this combined vitamins strategy in treating osteoporosis in diabetic patients. Future studies are needed by analysing the isoboles to investigate the nature of pharmacological interactions (additive or synergistic) (Tallarida, 2002, 2006) between vitamin K2 and 1,25(OH)2D3.

Conflicts of interest No potential conflicts of interest relevant to this article were reported.

Acknowledgements The work described in this paper was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project no. CUHK467613) and the

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Health and Medical Research Funds (Food and Health Bureau, Department of Health, Hong Kong SAR) (Project reference number: HMRF: 10110371; HMRF reference number: 10110371). Provision of Ph.D. studentship to Miss Christina Chui Wa Poon by the School of Biomedical Sciences (Faculty of Medicine, The Chinese University of Hong Kong) is acknowledged. Dr. S.W. Seto is a recipient of fellowship from the National Heart Foundation, Australia (PF12B6825).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2015.09. 048.

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