ORIGINAL RESEARCH ARTICLE

Journal of

Bradykinin Regulates Osteoblast Differentiation by Akt/ERK/NFkB Signaling Axis SWATI SRIVASTAVA, KIRTI SHARMA, NARENDER KUMAR,

AND

Cellular Physiology

PARTHA ROY*

Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India Bradykinin (BK), a well known mediator of pain and inflammation, is also known to be involved in the process of bone resorption. The present study therefore evaluated the role of BK in osteoblast lineage commitment. Our data showed that BK inhibits the migration of bone marrow mesenchymal stem cells, but does not affect their viability. Moreover, BK also inhibits osteoblastic differentiation by significantly downregulating the levels of mRNAs for osteopontin, runX2, col24, osterix, osteocalcin genes and bone mineralization (P < 0.05). Further, BK was found to elicit the BK receptors (BDKR1 and BDKR2) mediated activation of ERK1/2 and Akt pathways, which finally led to the activation of NFkB. BK also promoted the osteoclast differentiation of bone marrow derived preosteoclast cells by upregulating the expression of c-fos, NFATC1, TRAP, clcn7, cathK, and OSCAR genes and increasing TRAP activity through NFkB pathway. In conclusion, our data suggest that BK decreases the differentiation of osteoblasts with concomitant increase in osteoclast formation and thus provides new insight into the mechanism of action of BK in modulating bone resorption. J. Cell. Physiol. 229: 2088–2105, 2014. © 2014 Wiley Periodicals, Inc.

Chronic inflammatory conditions such as rheumatoid arthritis and periodontal diseases are often associated with local loss of bone tissue, mainly due to activation of osteoclastic bone resorption. BK has also been implicated as a potential mediator of bone resorption, due to its presence in the close vicinity of chronic inflammatory processes (Lerner et al., 1987; Selwyn et al., 1989). BK is a potent vasoactive nonapeptide formed by the proteolytic cleavage of kininogen by plasma kallikrein (Regoli and Barabe, 1980). Kininogens are the key inflammatory proteins, secreted in plasma mainly responsible for blood coagulation (Scott et al., 1984). BK exerts their biological activities through two main bradykinin receptor subtypes, named as bradykinin receptor 1 (BDKR1) and bradykinin receptor 2 (BDKR2) (Regoli et al., 1977). Although, BDKR1 is not expressed at significant levels in normal tissues, its synthesis can be induced during inflammation. On the other hand, BDKR2 is constitutively expressed in different types of cells including smooth muscle cells, certain neurons, fibroblasts, epithelial cells of lung and osteoblast cells (Marceau et al., 1998). Presence of both BDKR1 and BDKR2 in osteoblast cells signifies the involvement of BK stimulated pathways in the bone metabolism. Although, there are no reports published yet showing the direct involvement of BK in the differentiation of osteoblasts and osteoclasts (Yamamura et al., 2006), but it is already known that they stimulate the biosynthesis of prostaglandin (Warren et al., 1987). Prostaglandin E2 (PGE2) has been shown, both in vitro and in vivo, to affect bones by stimulating osteoblastic differentiation and bone formation (Keila et al., 2001; Yoshida et al., 2002) and simultaneously increasing osteoclast differentiation and bone resorption (Lader and Flanagan, 1998). Some reports even suggest that BK can stimulate bone resorption in neonatal mouse calvaria (Gustafson and Lerner, 1984) and hence can be categorized as osteoclastogenic agent. In another report, it was shown that stimulation of osteoblast cell line with BK increased the expression of NFkB, an important transcription factor, known to be involved in osteoclastogenesis (Brechter and Lerner, 2007). However, in contrast, Kakoki et al. (2010) have recently shown that the lack of both BDKR1 and BDKR2 results in a severe reduction in bone mineral density in diabetic © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .

and non-diabetic mice. Further, another important cellular defect in arthritis is the degeneration of cartilage tissues. BDKR1 and BDKR2 are also known to be expressed in human chondrocytes, indicating that both bone and cartilage are responsive to BK (Meini et al., 2011). However, direct effect of BK on chondrocyte formation is not yet fully studied. Based on the existing information, it could be presumed that the exact action of BK on the differentiation of osteoblasts is not completely understood. The possible reason for this lack of information could be the fact that the exact physiology of BK at the cellular level is not well defined atleast in case of BK responsive cells such as osteoblasts, osteoclasts, and chondrocytes. The bone marrow is recognized as the ultimate source for different types of cells, and the microenvironment in the bone marrow highly dictates the fate of the cells as they differentiate

Current address of Kirti Sharma is DKFZ—German Cancer Research Center, Heidelberg, Germany. Contract grant sponsor: Department of Biotechnology, Government of India; Contract grant number: DBT-JRF/08-09/459. Contract grant sponsor: Ministry of Human Resource and Development; Contract grant number: MHR03-412-Fig ‘B’. Contract grant sponsor: Uttarakhand State Council for Science and Technology; Contract grant number: UCS&T/R&D/LS-10/11-12/4224. *Correspondence to: Partha Roy, Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667 Uttarakhand India. E-mail: [email protected] Manuscript Received: 12 September 2013 Manuscript Accepted: 9 May 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 13 May 2014. DOI: 10.1002/jcp.24668

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from its stem cells. The homeostasis of bone is highly regulated by the bone marrow stem cells (BMSCs), which subsequently differentiate into osteoblasts and osteoclasts, the cells responsible for bone remodeling (Korkalainen et al., 2009). Consequently, impaired functions of BMSCs can give rise to dramatic consequences in bone homeostasis. Therefore, the objective of this study was to elucidate the role of BK in modulating various processes involved in bone metabolism, such as formation of osteoblasts and osteoclasts from BMSCs. To explore the signaling pathways underlying BK induced events, we analyzed the involvement of Akt, ERK1/2 and NFkB, since numerous reports indicated their roles in the responses elicited by BK in various cell types (Blaukat et al., 2000; Xie et al., 2000). Our data showed that BK is capable of modulating the multilineage commitment of BMSCs by inhibiting osteoblast and chondrocyte differentiation, and promoting the osteoclast formation through the activation of Akt and ERK1/2 mediated NFkB signaling cascades. Further, BK mediated action was found to be mainly regulated by its receptors and hence the antagonists for BK may prove to be useful as therapeutic agents in the management of bone related disorders. Materials and Methods Materials Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and streptomycin–penicillin antibiotic solutions were procured from Gibco-BRL (Inchinnan, UK). Dimethyl sulfoxide (DMSO) and 3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide (MTT) were purchased from HiMedia (Mumbai, India). NFkB luciferase reporter vectors were purchased from BD Biosciences (Clontech, UK), while polyfect transfection reagent was bought from Qiagen (Alameda, CA). bgal reporter plasmid was procured from Promega (Madison, WI). Antibodies were purchased from Santa Cruz (Santa Cruz, CA). BK, HOE157, HOE158, and all other chemicals used were of cell culture grade and were purchased from Sigma Aldrich (St. Louis, MO) and Himedia. The cell lines used in present study that is, MG63, a human osteosarcoma cell line and RAW 264.7, a mouse macrophage cell line, were procured from National Center for Cell Sciences, Pune, India. Animals Male albino mice (Mus musculus) (about 1 month old) were purchased from National Institute of Pharmaceutical Education and Research (NIPER), Chandigarh, India, and were in healthy condition at the time of purchase. They were housed in a wellventilated animal house with 12:12 h light-dark cycle, and were acclimatized for about 10 days. The animal handling and experimentation were in accordance with the guidelines of Institutional Ethical Committee (Regn. no. 563/02/a/CPCSEA). BMSCs isolation BMSCs were isolated from bone marrow of femurs and tibias of male albino mice. The collection of BMSCs was carried out according to the method as described earlier by Nadri et al. (2007). Briefly, the tibias and femurs were harvested, and the medullar channels were flushed using a syringe with 26-gauge needle containing 5 ml culture medium (DMEM containing 10% FBS). The cells were then collected and washed twice with 10 ml of PBS (without Ca2þ and Mg2þ) by centrifugation at 1,000 rpm for 5 min and suspended in 5 ml of growth medium. Finally, 5  106 cells/ml was placed on 6 cm petridish. The non-adherent bone marrow hematopoietic cells (BMHCs) were collected after 24 h and used as osteoclast precursors. At the same time the adherent cells were maintained for 15 days with change of medium on every 5th consecutive day. These cells were then characterized for their JOURNAL OF CELLULAR PHYSIOLOGY

trilineage differentiation potential (Srivastava et al., 2013) and termed as bone marrow mesenchymal stem cells (BMMSCs). The isolated BMMSCs were then used for further osteoblast differentiation studies. MTT assay MTT assay was carried out as described previously (Mosmann, 1983). Briefly, cells were seeded on 96-well plates at low density (1  103 cells/cm2) and grown in culture medium along with BK at 1, 10, and 100 nM concentrations. After various time periods of incubation, the medium was replaced with fresh medium without any test chemicals. MTT was added to each well at a final concentration of 0.5 mg/ml, followed by incubation for 4 h to form formazan. The MTT containing medium was then aspirated, and 200 ml of DMSO and 25 ml of Sorensen glycine buffer (0.1 M glycine and 0.1 M NaCl, pH 10.5) were added to it to solubilize the water insoluble formazan. Absorbance was measured at 570 nm in an ELISA plate reader (Oasys, Austria). Scratch assay The motility of BMMSCs (passage 4) was analyzed by in vitro scratch assay. For this the cells were first seeded in a 12-well plate in culture medium. When the cells achieved desired confluency, a scratch was made on the monolayer over the total diameter of each well using a sterile pipette tip, and subsequently the cells were allowed to grow for another 12 h in the culture medium with 0, 1, 10, and 100 nM BK. Migration of the cells into the scratch area was then documented by phase contrast microscopy (Axiovert 25, Jena, Germany) and quantified by using ImageJ 1.43 software (NIH, USA). Osteoblast differentiation For evaluating the impact of BK and its antagonists on osteoblast differentiation, the BMMSCs (Passage 5) and human osteoblast-like cell line, MG-63 (Passage 62) were seeded on to six-well plates, and cultured in osteoblast differentiation media (DMEM containing 10% FBS, 50 mM ascorbic acid, 10 nM dexamethasone and 10 mM b-glycerophosphate). The cells were then treated with BK (1– 100 nM) in the presence/absence of 100 nM each of HOE157 (BDKR1 antagonist) and HOE158 (BDKR2 antagonist). The cells were then maintained in this differentiation media for further 10 days. Alizarin red staining After complete osteoblast differentiation, mineralization of extracellular matrix (ECM) was analyzed using alizarin red staining. For staining, the medium was removed, and the cells were washed with phosphate-buffered saline (PBS). The cells were then fixed in 10% formaldehyde solution and incubated at 4°C for 30 min. Then the formaldehyde solution was discarded, and the cells were washed with distilled water to remove excess formaldehyde. Subsequently, the alizarin red staining solution was added and incubated at room temperature for 20 min. Finally, the excess stain was removed, and the cells were washed with PBS and observed using an inverted microscope (Axiovert 25, Zeiss, Jena, Germany). von Kossa staining This staining is generally performed to observe the extent of phosphate deposition in the ECM after mineralization. We performed the staining according to the method described earlier (Meloan and Puchtler, 1985). The staining method involved addition of 5% solution of silver nitrate to each well of the plate containing differentiated cells, which was then exposed in sunlight for 30 min or until calcium turned black. After removing the excess

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stain, the cells were washed with distilled water, and finally incubated in 5% sodium thiosulfate solution for an additional 2 min. The cells were again washed and observed under microscope as stated above. Osteoclast differentiation and tartrate-resistant acid phosphatase (TRAP) staining For testing the role of BK in osteoclast differentiation, BMHCs (passage 3) were seeded at a density of 1  106 cells/well in 24-well plates in the presence of human recombinant soluble RANKL (50 ng/ml) for 7 days. Murine RAW264.7 cells (passage 53) were grown in DMEM supplemented with 10% FBS and 1% penicillin/ streptomycin. For differentiation of osteoclasts, RAW264.7 cells (2  104, in 24-well plate) were cultured in the presence of RANKL (50 ng/ml) for 5 days. The culture medium was replaced every 3 days. After completion of differentiation, the cells were fixed and stained for TRAP activity. The TRAP positive multinucleated cells containing two or more nuclei were considered to be osteoclastlike cells, and were counted under the inverted microscope (Axiovert 25, Zeiss, Jena, Germany).

luciferase reporter plasmids namely; SRE, NFkB, CRE, and AP-1 luciferase vectors, activation of which indicate the involvement of MAPK, NFkB, cAMP and AP-1 mediated signaling. In order to determine signaling pathways, BMMSCs were seeded in the 24well plate at a concentration of 4  104 cells/well and incubated for 24 h before transfection. The cells were then transiently transfected with various luciferase reporter vectors each containing a specific cis-acting DNA sequence (enhancer element) upstream of the luciferase gene, using polyfect transfection reagent, according to manufacturer’s instructions (Qiagen, Alameda, CA). Briefly, the cells were co-transfected with 200 ng/ well of reporter plasmids and 0.6 ng/well of b-gal as internal control. The cells were then exposed to test samples in charcoalstripped medium for 12 h. On completion of treatment the cells were lysed with lysis buffer (0.6 M NaCl, 0.1 M EDTA, 0.2 M MgSO4, 0.2 M DTT, 0.1% Triton X-100, 0.08 M Tricine), and the luminescence was measured using luciferin as substrate. Each experiment was performed in triplicates and the results varied by less than 10%. The value of luciferase for each lysate was normalized to the b-gal activity. Western blotting and immunofluorescence staining

Measurement of matrix metalloproteinase activities Matrix metalloproteinase (MMP) activities were determined by zymography assay as described earlier (Hu and Beeton, 2010). After the culture period (24 h in serum-free culture medium in the presence or absence of BK), MMP activities were detected in culture supernatants of BMMSCs. The equal volumes of supernatant were loaded on electrophoresis gel which was composed of 10% polyacrylamide gel containing 0.2% gelatin. After electrophoresis, the gels were washed twice for 30 min each in 2.5% (v/v) Triton X-100 at room temperature and then incubated in substrate reaction buffer (50 mM Tris-HCl, 5 mM CaCl2, 0.02% (w/v) NaN3, pH 8.0) for 16 h at 37 °C. The gels were then stained with Coomassie Blue R250 in 10% acetic acid and 30% methanol for 1–2 h and destained briefly in the same solution without dye. Proteolytic activities were detected by clear bands indicating the lysis of the substrate. Finally the gel was photographed and band density was measured using ImageJ 1.43 software (NIH, USA). Chondrocyte differentiation and staining

To analyze the protein expression profiles of cells in response to BK, after completion of the respective treatments, the cell lysates were prepared in cell lysis buffer [20 mM Tris (pH 7.2), 5 mM EGTA, 5 mM EDTA, 0.4% (w/v) sodium dodecyl sulfate (SDS), and 1 protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO)]. The protein concentrations were quantified using a BCA protein estimation kit (Sigma Aldrich, St. Louis, MO), according to the manufacturer’s instruction. The total protein samples (50 mg) were then separated on 10% polyacrylamide gels, followed by immunoblot analysis as per our previously reported protocol (Srivastava et al., 2012). Briefly, the proteins were transferred onto a nylon membrane and blocked with Tris-buffered saline with Tween-20 [TBST; 20 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 0.05% Tween-20] containing 5% skim milk powder. The blots were then washed with TBST and incubated with diluted primary antibody (1:500) at 4°C overnight. The details of the antibodies used in this study are provided in Table 1. Subsequently, the membranes were washed and incubated with the anti-rabbit secondary antibody (1:10,000) conjugated to horseradish peroxidase (HRP). Finally, the blots were developed using ECL detection kit (GE Healthcare, Little Chalfont, UK) as per manufacturer’s instructions. The developed blots were then subjected to densitometric analysis using b-actin as an internal control.

For Chondrogenesis both monolayer and pellet cultures were used. For monolayer culture, BMMSCs (Passage 6) were incubated in chondrogenic medium containing DMEM, supplemented with 1 mM sodium pyruvate, 20 ng/ml proline, 107 M dexamethasone, 50 mM ascorbate, ITSþ supplement (mixture of insulin, transferring and selenium) (catalog # 41400-045, Gibco-BRL) and TGF-b3. For pellet culture, a suspension of 250,000 BMMSCs in chondrogenic differentiation medium was added to 15-ml conical tubes and centrifuged at 300g for 5 min to form a pellet at the bottom of each tube. The tubes were then incubated with loosened tops at 37°C and 5% CO2. The culture medium was changed two times per week for up to 3 weeks. On completion of the treatment period the fixed monolayer cells and pellet sections were stained with 0.1% alcian blue. The periodic acid schiffs (PAS) staining was performed to stain the glycogen fibers in ECM. For PAS staining the cells were first fixed in 4% glutaraldehyde and then 0.5% periodic acid solution was added to it followed by incubation for 10 min. Finally the cells were washed in PBS and incubated in Schiff’s solution for 3 min and observed under light microscope.

Total RNA was extracted from the treated cells according to the previously described method (Chomczynski and Sacchi, 1987). Briefly, the RNA was quantified and equal amounts of it were reverse transcribed with the help of the RT-PCR kit from Bangalore Genei (Bangalore, India), according to the manufacturer’s instructions. PCR was then performed using cDNA as the template for 25–30 cycles (denaturation at 94°C for 60 sec, annealing for 30 sec at various temperatures and extension at 72°C for 60 sec). The primer sequences and the size of the amplified products are listed in Table 2. The PCR products were separated on 1.5% agarose gel and visualized using a gel documentation system (BioRad, Hercules, CA).

Transactivation assay

Statistical analysis

The signal transduction pathway was determined using pathway profiling luciferase system (BD Biosciences). Details of the various reporter plasmids used in this study has been reported in our earlier publication (Srivastava et al., 2014). The kit contains four

Values are expressed as the mean  SE. The statistical significance was evaluated by one-way ANOVA at 5% level of significance. Dunnett’s test was used to compare the statistical significance between various group means to a control mean. Further, Tukey–

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RT-PCR analysis

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TABLE 1. Details of antibodies used for Western blot and immuno-fluorescence analysis Primary antibody Akt1/2 pAkt1/2/3 ERK pERK pIkB pNFkB osteopontin pJNK p-P38 b- actin

Cat # Santacruze

Phosphorylated residue

SC-1619 SC-7985 SC-154 SC-7383 SC-101713 SC-101749 SC-20788 SC-6254 SC-17852 SC-130656

— Akt1 ¼ Ser 473, Akt2 ¼ Ser 474, Akt3 ¼ Ser 472 — Tyr 204 Ser 32/36 of IkB-a Ser 276 of NFkB p65 — Thr 183 and Tyr 185 of JNK1, JNK2, and JNK3 Thr 180 and Tyr 182 of p38a, p38b, p38g and p38d —

Kramer test was used to compare the means of all groups with each other. The statistical package used was GraphPad Prism 5.04 (GraphPad Software, San Diego, CA). Results BK inhibits migration without affecting the viability of BMMSCs

In order to analyze the effect of BK on cellular functions of BMMSCs, first we determined the effect of BK on the viability and migration of these cells. As shown by MTT assay, BK treatment did not alter the viability of BMMSCs even after its prolonged exposure to the cells (Fig. 1A). When tested for its in vitro migration activity, the cells grown in vehicle treated medium displayed higher migration capacity, since after 12 h of incubation they completely colonized within the scratched area (Fig. 1B). However, during the similar time period, the cells treated with varying doses of BK demonstrated a regressive pattern of cell migration with its increasing doses.

BK inhibits osteogenic differentiation of BMMSCs

To analyze whether BK affects osteoblast differentiation of BMMSCs, the cells were induced to differentiate by culturing in osteoblast differentiation medium for 10 days in the presence of various doses of BK. After the differentiation period, deposited calcium phosphate nodules and characteristics of matrix mineralization, were detected by alizarin red and von Kossa staining, where the former and latter combines with calcium and phosphate ions, respectively, thus indicating ECM. As shown in Figure 2, BK showed decreased mineralization in dose dependent manner as depicted by both alizarin red and von Kossa staining. At 1 nM of BK the mineralization reduced by almost 50% (P < 0.05) as compared to control, which further reduced to almost 10% of mineralization at 100 nM of BK treatment. To confirm that the decrease in mineralization at 100 nM of BK was not due to any cellular toxicity or reduction in the number of cells, acridine orange (AO) and ethidium bromide (EB) counter staining and total protein

TABLE 2. List of primers and product size (in bp) used for RT-PCR Sequence (50 –30 )

Gene Osteopontin Osteoprotegerin Osteocalcin Runx2 BDKR1 BDKR2 cFos OSCAR NAFTC1 RANKL Col2A1 Aggrecan b-actin Osterix Col24 Clcn7 CathK TRAP

Fwd: TCTCCTTGCGCCACAGAATG Rev: TCCTTAGACTCACCGTCTTT Fwd: TGCTCCTGGCACCTACCTA Rev: ACTCCTGCTTCACGGACTG Fwd: TCTGACAAAGCCTTCATGTCC Rev: AAATAGTGATACCGTAGATGCG Fwd: CCTGACTCTGCACCAAGTC Rev: GAGGTGGCAGTGTCATCATC Fwd: GCCAGCAGAACCCGGTGTGG Rev: GGCTACCCTGTCCTCCGGCT Fwd: ACGGTGCGCATCTTGCAGGT Rev: ACCAGTGGGTTGAGGCCGCT Fwd: GTCGACCTAGGGAGGACCTT Rev: AGGCCTTGACTCACATGCTC Fwd: AGGCAGGGTCAACCTTTTCC Rev: CCCAGTCTGTCTTGCGGTAG Fwd: CAGGACCCGGAGTTCGACTT Rev: CTTCGGGGAAAACCCTCCTC Fwd: GGAAGCGTACCTACAGACTA Rev: AGTACGTCGCATCTTGATCC Fwd: CAGGATGCCCGAAAATTAGGG Rev: ACCACGATCACCTCTGGGT Fwd: CACGCTACACCCTGGACTTTG Rev: CCATCTCCTCAGCGAAGCAGT Fwd: TCACCCACACTGTGCCCCATCTACGA Rev: CAGCGGAACCGCTCATTG-CCAATGG Fwd: CCTCTGCGGGACTCAACAAC Rev: TGCCTGGACCTGGTGAGATG Fwd: CGCTGTGACTCTCCCAAACT Rev: CGAGGGCCCGTACATAACTC Fwd: CGAGATGCCTATCCACGCTT Rev: TGGAGGGTCAGGGAATAGGG Fwd: GACACCCAGTGGGAGCTATG Rev: TCCGTTCTGCTGCACGTATT Fwd: GGTAATGGCTGAGGCAGGAT Rev: CACAAGCCGCCCAATCTTTC

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Annealing temp. (°C)/ Cycle

Product size (bp)

55/30

398

55/35

158

197/35

57

55/30

234

69/30

499

65/30

263

60/35

209

63.7/35

381

64/35

620

57/30

207

62/32

131

60/32

200

58/22

300

57/35

320

64/35

343

63/35

493

63.5/35

513

63/35

475

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Fig. 2. Dose dependent effect of BK on osteoblast differentiation of BMMSCs after 10 days of differentiation as depicted by optical images of alizarin red and von Kossa stained cells. Images shown here are representative of three individual experiments. Histogram showing the quantification of alizarin red in stained cells. The data are mean  SE, n ¼ 3. a and b indicate significant levels differences at P < 0.05 with respect to control and a, respectively. Optical density of control (vehicle treated) was considered as 100%.

Fig. 1. A: Dose and time dependent effects of BK on the viability of BMMSCs as analyzed by MTT assay. The data are mean  SE, n ¼ 3. B: Effect of BK on migration of BMMSCs analyzed by scratch assay. Image shown here is representative of three individual experiments. Histogram in the lower panel of (B) indicates the quantification of scratched area using ImageJ 1.43 software (NIH, USA). a indicates significantly different from control at P < 0.05.

concentration of cells were also determined (Supplementary Fig. S1). The results showed that cells were completely viable through out the differentiation process at every tested concentrations of BK. Further, the time dependent effect of BK on osteoblast differentiation was also analyzed. Two osteoblast specific marker genes, osteopontin (early marker) and osteocalcin (late marker), were analyzed and shown in Figure 3A. As shown here, the expression of osteopontin was minimum at day 1 and gradually increased at day 7 of differentiation. On the other hand, the expression of osteocalcin was evident only after 4 JOURNAL OF CELLULAR PHYSIOLOGY

days of differentiation. Interestingly, the decrease in the expressions of both the genes was found to be dose dependent with maximum decrease at 100 nM of BK. However, the mineralization was detected only after 5 days of differentiation which was even restricted to vehicle treated cells indicating inhibitory effects of BK (Fig. 3B). At day 9, although there was increase in mineralization at all the doses including control, the level decreased with increasing doses of BK (Fig. 3B). BK inhibits osteogenic differentiation of BMMSCs through its cognate receptors BDKR1 and BDKR2

To check if the action of BK on mineralization was through its cognate receptors in the cell, they were also treated with BK receptor antagonists in the presence or absence of BK. As shown in Figure 4, there was a noticeable decrease in the mineralization in BK induced cells as compared to the control cells (Fig. 4A,B). The cells subjected to BK showed only 8% of alizarin red staining, as compared to the control (100%;

BRADYKININ ACTION ON OSTEOBLAST

in the expressions of osteocalcin and col24 which are the late osteoblast marker genes. Interestingly, similar to mineralization data, the reduced expression levels of most of these marker genes reverted back almost to 50–60% of the levels of vehicle treated cells when the cells were co-treated with corresponding BK receptor antagonists, that is, BDKR1 (lane 3) and BDKR2 (lane 4). But, here also the BK induced inhibition was more efficiently rescued by HOE157 than that of HOE158, which is more evident in expression patterns of col24 and osteocalcin. When we checked for the expression patterns of BDKR1 and BDKR2 during osteoblast differentiation, a tremendous increment in the expression levels of BDKR1 (5.5fold; P < 0.05) and BDKR2 (4-fold; P < 0.05) was observed in BK induced cells, which further suggests the involvement of BDKR1 and BDKR2 in BK induced effect on osteoblast differentiation. Further, our data also showed that the expression of BDKR1 was significantly down-regulated in the presence of HOE157, and reached to the level of vehicle treated cells, whereas the expression of BDKR2 was less affected in this case. Similarly, in the presence of HOE158, the expression pattern of BDKR2 was inhibited to a greater extent than that of BDKR1. This data showed that the response of antagonists were specific to its cognate receptors. BK activates ERK/Akt/NFkB signaling in BMMSCs

Fig. 3. Dose dependent effect of BK on osteoblast differentiation of BMMSCs after various time intervals of differentiation process as depicted by (A) Transcriptional analysis for the expression of two osteogenic marker genes: osteopontin (an early marker), osteocalcin (a late marker) as determined by RT-PCR and (B) mineralization assay as determined by alizarin red staining.

P < 0.05; Fig. 4C). Interestingly, the BK induced inhibition of mineralization was rescued and reached to 60% and 45% (P < 0.05) of the control, when the cells were co-treated with HOE157 (antagonist for BDKR1) or HOE158 (antagonist for BDKR2) respectively, albeit to a significantly greater extent in presence of former antagonist than latter. However, when both the antagonists were co-treated with BK, the cells did not exhibit any significant (P < 0.05) effect on osteoblast differentiation, thus indicating the involvement of both the receptors in BK induced effect on osteoblast differentiation. During this entire period of differentiation the cells were found to be completely viable and equally dense, confirming the fact that the above mentioned effects were not due to cytotoxicity (Supplementary Fig. S2). To further validate the obtained data, similar experiment was repeated on MG63 human osteosarcoma cell line, which is preosteoblastic by nature and has already been reported to express both BDKR1 and BDKR2 (Brechter and Lerner, 2002). The patterns of mineralization by BK in the presence and absence of its receptor antagonists in MG63 cell line were almost the same as in BMMSCs (Supplementary Fig. S3). The effect of BK on osteoblast differentiation was further confirmed by analyzing the expression patterns of some prominent osteoblast marker genes such as osteopontin, osterix, runx2, osteocalcin and col24. As shown in Figure 5, almost 50% (P < 0.05) down-regulation was found in the expressions of osteopontin, Runx2 and osterix in BK induced cells (lane 2). Interestingly, this down-regulation was found to be almost 95% JOURNAL OF CELLULAR PHYSIOLOGY

Next, we analyzed the signaling events triggered by BK in BMMSCs. For this, the effect of BK on transcriptional activity of some selected signaling molecules viz. MAPK, NFkB, CRE, and AP1 were determined. As shown in Figure 6A, BK had no effects on the activation of CRE-luc and AP1-luc reporter plasmids. On the other hand, it stimulated the activation of SRE-luc and NFkB-luc reporter plasmids by about 3.1- and 4fold, respectively, (P < 0.05) at 100 nM concentrations. This data suggested that BK activated only MAPK and NFkB signaling pathways, atleast in this cell. To further explore the involvement of MAPK/NFkB signaling pathways in BK mediated action, the cells were incubated with different concentrations of BK, and the cell lysates were analyzed for the expressions of phosphorylated MAPK (i.e., pERK, pP38, and pJNK), IkB, and NFkB. As shown in Figure 6B, there was a significant increase in the phosphorylation of ERK, which was almost 2.2-fold over the control (P < 0.05) at 100 nM of BK treatment. On the other hand, no significant changes were observed in the phosphorylation levels of P38 and JNK as compared to that of control even at the highest concentration tested (100 nM). Further, as shown in Figure 6B, there was also a dosedependent increase in the levels of phosphorylated IkB and NFkB proteins by about 3- and 1.6-fold, respectively (P < 0.05), thus indicating the involvement of these pro-inflammatory proteins in its action. Another important signaling molecule which is known to be regulated by BK is Akt. As shown in Figure 6B, the phosphorylation of Akt at Ser473 residue increased by about 3-fold in response to 100 nM of BK treatment (P < 0.05), thus confirming the involvement of ERK, NFkB, and Akt signaling in BK mediated actions. Further, osteopontin level decreased significantly in a dose-dependent manner which was almost 40% of the control level (P < 0.05) at 100 nM of BK treatment. ERK/Akt/NFkB signaling pathway mediates BK-induced inhibition of osteoblast differentiation

In the next series of experiments, attempts were made to analyze the possible pathway(s) which might be involved in the differentiation of osteoblasts. To establish the direct involvement of ERK, Akt and NFkB in osteoblast differentiation, BMMSCs were initially treated with specific

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Fig. 4. Effect of BK and its receptor antagonists (100 nM each) on osteoblast differentiation of BMMSCs as shown by optical images of (A) alizarin red and (B) von Kossa stained cells. Images shown here are representative of three individual experiments. C: Histogram showing the quantification of alizarin red in stained cells. The data are mean  SE, n ¼ 3. a, b, c, and d indicate significant levels differences at P < 0.05 with respect to control, a, b, and c, respectively. Optical density of control (vehicle treated) was considered as 100%.

inhibitors for ERK (PD98059, Sigma catalogue # P215; inhibits the phosphorylation of ERK1 and ERK2), Akt (Akt1/2 kinase inhibitor, Sigma cat # A6730; inhibits the phosphorylation of Akt1 and Akt2) and NFkB (parthenolide, Sigma catalogue # P0667; inhibits IKKa and IKKb, the upstream regulator of NFkB), prior to their exposure to 100 nM of BK and subsequent induction for osteogenic differentiation. As expected, the osteopontin expression was significantly downregulated by almost 60% (P < 0.05) in presence of BK in all the treatments (Fig. 7). However, interestingly, the BK induced inhibition of osteopontin was rescued by pretreatment of cells with specific inhibitors for ERK (Fig. 7A), Akt (Fig. 7B), and NFkB (Fig. 7C). The expression levels of pERK, pAkt (Ser473), JOURNAL OF CELLULAR PHYSIOLOGY

pIkB, and pNFkB were significantly reduced (P < 0.05) when BK stimulated cells were pretreated with their respective inhibitors, thus indicating the specificity of their actions (Fig. 7A–C). In conclusion, the level of osteopontin may be considered to be tightly regulated by a dynamic balance between BK-activated ERK and PD98059 mediated inhibition of ERK atleast in this cell (BMMSC). The same may be attributed to AKT and NFkB signaling pathways as well. BDKR1 and BDKR2 act through ERK and Akt pathways

Next, we checked the probable mode of regulation of these signaling pathways by BK receptors, namely BDKR1 and

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BK mediated NFkB activation is dependent on ERK and Akt activation

Once it was established that BK activates ERK/Akt/NFkB signaling pathways, it was important to elucidate the exact mode of action of BK in this signaling cascade since there are several reports of complex cross talks between these signaling pathways (Armstrong et al., 2006). To determine whether NFkB activation in BK induced osteoblast differentiation is dependent on ERK/Akt activation, the cells were treated with BK with or without pre-treatment of specific inhibitors, and the activation of pNFkB was then analyzed. As shown in Figure 8B, the inhibition of Akt and ERK by their specific inhibitors, significantly suppressed the BK induced phosphorylation of NFkB (lane 2 as compared to lanes 4 and 6, respectively) (P < 0.05), suggesting direct regulation of the inflammatory pathway by ERK/Akt atleast in response to BK. BK induces osteoclast differentiation of BMHCs by up-regulating the marker genes for osteoclast formation

Fig. 5. Transcriptional analysis for the expression of osteogenic marker genes after 10 days of differentiation of BMMSCs in presence of BK (100 nM), and/or its respective receptor antagonists (100 nM) as determined by RT-PCR. Histogram in the lower part shows the mean  SE, of arbitrary pixel intensities of three individual RT-PCRs. a, b, and c indicate significant levels differences at P < 0.05 with respect to control, a and b, respectively.

BDKR2. As shown in Figure 8A, BK caused almost 90% inhibition in the expression of osteopontin, which was reversed by both BDKR1 and BDKR2 antagonists (lane 3 and 4, respectively) to about 50%, as that of the control cells (P < 0.05). The inhibition was found to be only 10% when the BK induced cells were treated with both HOE157 and HOE158 together (lane 5). When checked for the phosphorylation patterns of Akt, we found that BK mediated increased level of pAkt (2.2-fold) was dropped almost to the level of vehicle treated cells, in presence of HOE158 (lane 4). On the other hand, in presence of HOE157, though the BK induced pAkt level dropped close to 1.3-fold, still it did not achieve the basal level of phosphorylation. Interestingly, the data on the phosphorylation patterns of ERK (pERK) was just the reverse, where HOE157 treatment reduced the BK induced pERK expression from 2.5- to 1.2-fold (P < 0.05) which was found to be 1.6-fold in case of HOE158 treated cells. However, unlike pAKT and pERK expression patterns, BK induced expressions of pIkB and pNFkB were suppressed in the presence of both HOE157 and HOE158, albeit to a slightly greater extent in case of former than the latter. From this data, it could be conceived that both BDKR1 and BDKR2 acts through Akt and ERK pathways, where the former (BDKR1) is marginally more efficient in regulating ERK pathway while the latter (BDKR2) favors Akt pathway. However, further detailed analyses are needed to confirm this fact. JOURNAL OF CELLULAR PHYSIOLOGY

In the next phase of study we checked the role, if any, for BK in the osteoclastogenesis process using BMHCs. For this analysis, BMHCs were cultured in osteoclast differentiation medium and the expressions of transcription factors, which may directly or indirectly regulate osteoclast differentiation namely, cathK, clcn7, TRAP, cFos, NAFTC1 and OSCAR, were analyzed. As shown in Figure 9A there was a dose dependent increase in expressions of clcn7, TRAP and OSCAR genes. But the genes such as cFos, cathK, and NFATC1 showed significant increase in their expression only at higher concentrations, which suggests that 100 nM BK is more efficient in inducing osteoclast differentiation. In addition to the expressions of osteoclast marker genes, next we investigated the effects of BK on MMP activities. As shown in Figure 9B, only 100 nM of BK induced a significant increase in MMP-9 activity. Interestingly, unlike MMP-9, a dosedependent enhancing effect of BK was observed on MMP-2 activity. Our results hence clearly showed a differential effect of BK on the proteases activities of MMP-9 and MMP-2. BK regulates osteoclast differentiation of BMHCs through its BDKR1 and BDKR2

The next objective was to determine the role of BDKR1 and BDKR2 in BK mediated osteoclast differentiation. As 100 nM of BK was found to increase osteoclast differentiation maximally, this concentration was subsequently used unless otherwise stated. BMHCs were induced for osteoclast differentiation, with or without BK and its antagonists, and after 7 days they were subjected to TRAP staining. The TRAP-positive multinucleated cells were counted as osteoclast-like cells. As shown in Figure 10, multinucleated osteoclast (indicated with arrow) was maximum (25  4) in case of BK induced cells. The numbers of TRAP positive cells were reduced to 10  2 and 5  1, in case of HOE158 and HOE157 treated cells, respectively (P < 0.05). Interestingly, when the cells were treated with both HOE157 and HOE158, the multinucleated osteoclasts almost disappeared; indicating the involvement of both BDKR1 and BDKR2 in BK mediated osteoclast differentiation. Similar pattern of the results were obtained after calcium film resorption assay (Supplementary Fig. S4). Finally, RAW 264.7 cells were used as a positive model for analyzing the osteoclast differentiation, since it is a macrophage cell line with the capacity to form osteoclast-like cells. Our data showed that the pattern of osteoclast formation was almost similar to that of BMHSCs, confirming the specificity of action of BK regardless the type of cell line (Supplementary Fig. S5). A

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Fig. 6. A: Dose dependent effect of BK on promoter activities of various signaling pathways as determined by transactivation assay in BMMSCs. The cells were transfected with reporter vectors followed by treatment with BK after 24 h of transfection. After incubating the cells for another 24 h the cell lysates were collected and luciferase activity was determined. The data are mean  SE, n ¼ 3. a, b, and c indicate significant levels differences at P < 0.05 with respect to control, a and b, respectively. B: Dose dependent effect of BK on the expressions of various signaling molecules in differentiated BMMSCs as determined by Western blot analysis. Histograms in the right panel show the mean  SE, of arbitrary pixel intensities of three individual immunoblots. a and b indicate significant levels differences at P < 0.05 with respect to their corresponding controls and a, respectively.

similar pattern of inhibition of BK induced osteoclast specific genes were observed when the cells were co-treated with BK and its receptor antagonists (BDKR1 and BDKR2) indicating a direct involvement of these receptors in its action (data not shown). On order to get a deeper insight into the effects of BK on osteoclast differentiation, time dependent changes in the gene expression profiles of some of the prominent osteoclast markers were also analyzed. As shown in Figure 11, both vehicle and BK treated cells induced the expression of clcn7 and TRAP in a time dependent manner which is obviously more pronounced in case of BK treated cells. This data further confirmed the role of BK in inducing osteoclast differentiation.

(OPG) (Lacey et al., 1998). Decrease in OPG/RANKL ratio indicates the increased migration and differentiation of osteoclasts at inflammatory site. To check the effect of BK on OPG/RANKL ratio, BMMSCs were differentiated towards osteoblast lineage in presence or absence of BK, HOE157, and HOE158. On completion of differentiation, the levels of expression of mRNA for OPG and RANKL were analyzed and demonstrated in Figure 12A. BK induced the OPG/RANKL ratio to decrease by about 90% (P < 0.05) as compared to the vehicle treated cells. As expected, this ratio was significantly elevated almost to control level in presence of both the antagonists together (Fig. 12A, lane 5). BK stimulates osteoclast formation by NFkB pathway

Regulation of osteoprotegerin/RANKL mRNA ratio in response to BK

Osteoblasts regulate the recruitment and activity of osteoclasts through expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin JOURNAL OF CELLULAR PHYSIOLOGY

Since it is known that, stimulation of RANKL leads to the activation of NFkB, we next assessed the effect of BK on RANKL-induced NFkB activation. For this, RAW 264.7 cells were transfected with NFkB-luc reporter vector, and then stimulated with RANKL and BK. As shown in Figure 12C,

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Fig. 7. Role of BK on various signaling molecules: Effect of 20 mM of (A) ERK inhibitor, that is, PD98059; (B) Akt inhibitor, that is, Akt1/2 kinase inhibitor; (C) NFkB inhibitor, that is, parthenolide on BK (100 nM) mediated expression of various signaling proteins. Histograms in the right part of each figures show the mean  SE of arbitrary pixel intensities of three individual immunoblots. a, b, and c indicate significant levels differences at P < 0.05 with respect to their corresponding control, a and b, respectively, in each figures.

RANKL induced NFkB transcriptional activation, which was considered as 1-fold (P < 0.05), was significantly enhanced to almost 4.5-fold (P < 0.05) after BK induction. Involvement of NFkB signaling during BK induced osteoclast differentiation was also confirmed by Western blot analysis. BK significantly enhanced the phosphorylation of IkB and NFkB by 4- and 3.5fold, respectively (P < 0.05) (Fig. 12B). However, the effect of BK was almost completely abolished when the cells were JOURNAL OF CELLULAR PHYSIOLOGY

treated with both HOE157 and HOE158 as observed in both transactivation (Fig. 12C) and immunoblot analysis (Fig. 12B). BK inhibits chondrocyte differentiation of BMMSCs

Since osteoarthritis is also characterized by a progressive degeneration of the articular cartilage, we finally checked if BK also affects the chondrocyte differentiation of BMMSCs both in

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Fig. 8. A: Western blot analysis of osteoblast differentiated BMMSCs for the expression of various signaling proteins in response to 100 nM of BK and its receptor antagonists. B: Effects of 20 mM of PD98059 and Akt1/2 kinase inhibitor on the expression of NFkB. Histograms in each figures shows the mean  SE of arbitrary pixel intensities of three individual immunoblots. a, b, c, d, e, and f indicate significant levels differences at P < 0.05 with respect to their corresponding vehicle treated control, a, b, c, d, and e, respectively, for each individual figures.

monolayer and pellet culture. For this assay, the cells were differentiated towards chondrocyte lineage in presence or absence of BK, HOE157, and HOE158. The data were represented by the patterns of PAS and alcian blue staining, which indicates the deposition of acidic and neutral mucin, respectively, specifically in chondrocytes. In monolayer culture, as shown in Figure 13A, BK substantially decreased the formation of both acidic and neutral mucin, while deposition of both kind of mucins were found to be increased when BK JOURNAL OF CELLULAR PHYSIOLOGY

induced cells were treated separately with HOE157 and HOE158. The staining of cells was found to be almost equivalent to vehicle treated cells in presence of HOE157 and HOE158 together. In order to reconfirm the monolayer data as obtained above (Fig. 13A) in the next phase we tested the pattern of alcian blue staining in pellet culture. As shown in Figure 13B, alcian blue staining pattern of pellet culture is similar to the monolayer culture, where BK induced inhibition was significantly rescued by both the receptor inhibitors. The

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Fig. 9. Dose dependent effect of BK on osteoclast differentiation. A: Transcriptional analysis for the expression of some osteoclast marker genes after 7 days of differentiation of BMHCs as determined by RT-PCR. B: Detection of MMPs by gelatin zymography as depicted by scanned image of the gelatin gel. Histograms in the right panel of each figures show the mean  SE of arbitrary pixel intensities of band products for three individual experiments. a, b, and c indicate significant levels differences at P < 0.05 with respect to their corresponding vehicle treated control, a and b, respectively, for each individual figures.

staining data thus indicated a significant inhibition of chondrocytic differentiation of BMMSCs, in response to BK. Further, RT-PCR analysis showed a tremendous downregulation of Col2a1 and aggrecan expressions, the crucial markers of chondrocytic differentiated cells, which was rescued by HOE157 and HOE158 (Fig. 13C). In parallel to that, BDKR1 and BDKR2 expressions were also found to be upregulated, during BK induced chondrocyte differentiation (Fig. 13C). Discussion

Although BK, a potent mediator of pain, is known to regulate diverse biological activities in many somatic tissues, its functional role in cellular differentiation is still poorly studied. This study was designed to demonstrate the effects of BK on differentiation of various cells involved in regulation of bone metabolism, such as osteoblasts and osteoclasts. It was based on two facts: firstly, BK is reportedly present in the synovial fluid of patients with rheumatoid arthritis (Lerner et al., 1987), and secondly, its receptors are present in the bone cells (Brechter and Lerner, 2002). Therefore, a cross talk between them was expected. We first determined the potential involvement of BK in regulating osteoblast differentiation. The inhibitory effect of BK JOURNAL OF CELLULAR PHYSIOLOGY

on osteoblast differentiation, associated with the attenuated expression of proteins and genes involved in osteoblast differentiation, is a novel regulatory role of BK, which to the best of our knowledge has not been reported in the literature. Although there is no study on direct action of BK on osteoblast differentiation, but it has been reported to stimulate expression of a-smooth muscle actin of human mesenchymal stem cells (Kim et al., 2008), indicating role of BK in inducing myocyte differentiation. Yamaguchi et al. (1991) reported that cells of the osteoblast lineage has a close relationship with those of the muscular lineage, and the development of two cell lineages may be mutually regulated by same factor(s). Several factors, for example, BMP-2, Smuf-1 and IL-7 have been reported to inhibit myogenic differentiation with concomitant stimulation of osteoblast differentiation (Yamaguchi et al., 1991; Ying et al., 2003; Kocic et al., 2012). Based on these existing reports we hypothesized that, since BK is known to stimulate myocyte formation, it is most likely to have inhibitory effect on osteoblast differentiation. It is known that during bone injury or inflammation, osteogenic cell migration from the bone marrow compartment to the injury site is regulated by factors released during inflammation and injury (Karpa et al., 2005). The ubiquitous expression of BK at the sites of bone inflammation and expression of receptors for BK by mesenchymal stem cells

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Fig. 10. Effect of BK and its receptor antagonists (100 nM each) on BMHCs after 7 days of osteoclast differentiation as shown by optical images of TRAP stained cells. Histogram shows the approximate numbers of osteoclasts/ well. The data are mean  SE, n ¼ 3. a, b, c, and d indicate significant levels differences at P < 0.05 with respect to control, a, b, and c, respectively.

prompted us to assume that BK may have a direct effect on stem cell migration. Our data showed that BK did not alter BMMSC’s viability, but significantly inhibited the migration of BMMSCs. Based on these data, it could be predicted that inflammation-induced bone resorption may be linked to decreased differentiation and migration of preosteoblast towards inflammatory site, which collectively results in higher rate of bone resorption than that of formation. BK, a potent vasoactive kinin peptide of kallikrein - kinin system exerts several physiological effects on various cells (Kondo and Togari, 2004; Suzuki et al., 2011). For example, in inflammatory condition, BK promotes the migration of blood cells to the target tissue (Dray and Perkins, 1993), vasoconstriction by stimulation of smooth muscle cells and promoting release of several endothelium-derived hyperpolarizing factors (Regoli and Barabe, 1980). These varied effects are mediated by the activation of two G proteincoupled BK receptors, BDKR1 and BDKR2. Different types of functions of BK indicate that efficiency of these two receptors varies with different species and pathological conditions (LeebLundberg et al., 2005). In the present study, we demonstrated that the negative effect of BK on osteoblast differentiation was JOURNAL OF CELLULAR PHYSIOLOGY

reduced more efficiently by BDKR1 antagonist (HOE157) as compared to BDKR2 antagonist (HOE158). Similar observation was also found during osteoclast differentiation, as depicted by TRAP staining and osteoclast marker gene analysis. Based on these results, we predict that although both the receptors are involved in BK mediated action, but the major inhibition of osteoblast differentiation in presence of BK might be mediated through BDKR1 than BDKR2. Thus, as soon as the cells are induced with BDKR1 antagonist HOE157, the effect of BK is neutralized to a greater extent. On the other hand, since the response of BK through BDKR2 was minimal, the inhibitory effect could not be reversed completely even in the presence of BDKR2 antagonist, which was mostly lesser than that of HOE157. The above finding can be better explained on the basis of several lines of facts/evidences. Firstly, BDKR1 is constitutively internalized without any agonist treatment, likely due to its basal constitutive activity. Once activated with ligand, BDKR1 is resistant to desensitization and internalization. On the other hand, BDKR2 undergoes rapid desensitization and internalization in response to its ligands (Prado et al., 2002). Leeb-Lundberg et al. (2001, 2005) have shown that BDKR1 has higher basal constitutive activity in hydrolyzing

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Fig. 11. Effect of 100 nM of BK treatment on the transcription of osteoclast specific marker genes at various time intervals of osteoclast differentiation process as determined by RT-PCR.

phosphoinositide even in the absence of its agonist, which is almost equivalent to that generated by BDKR2 after agonist treatment. In line with these findings, we demonstrated that BDKR2 mediated signaling is transient, whereas BDKR1 induced signaling is sustained and prolonged. Hence, BK

mediated response might be more effective through BDKR1 as compared to BDKR2, which is suppressed significantly in presence of BDKR1 antagonist as compared to that of BDKR2 antagonist. However, further in depth studies are needed to conclusively validate this activity. In several studies BK has been shown to activate ERK, Akt and NFkB (Blaukat et al., 2000; Xie et al., 2000; Uchida et al., 2012), thereby regulating the expression of a wide array of inducible genes. In this study, we demonstrated that all the three pathways, that is, ERK1/2, Akt and NFkB, were activated by BK in BMMSCs in a dose-dependent manner which was concomitant with dose-dependent reduction of osteoblast differentiation. By using the Akt, ERK and NFkB specific pharmacological inhibitors we further confirmed that all the three pathways were simultaneously involved in response to BK in inhibiting the differentiation of osteoblast from BMMSCs. Although the direct role of this triad Akt/ERK/NFkB signaling in the process of osteoblast differentiation has not yet been evaluated, yet, this cascade is known to regulate various cellular processes. According to some recent reports, in the event of both up and down regulation of these signaling pathways by some

Fig. 12. A: Determination of OPG/RANKL ratio in osteoblast differentiated BMMSCs in response to BK and its receptor antagonists. B: Immunoblot analysis of osteoclast differentiated BMHCs for the expression of various signaling proteins in response to test chemicals or their combinations. The histogram in the right panel of each figure shows the mean  SE of arbitrary pixel intensities of band products for three individual experiments. a, b, and c indicate significant levels differences at P < 0.05 with respect to corresponding control, a and b, respectively, for each figure. C: Effect of BK on RANKL induced NFkB-luciferase activity as determined by transactivation assay. The data are mean  SE, n ¼ 3. a, b, and c indicate significant levels of differences at P < 0.05 with respect to control, a and b, respectively. The value of only RANKL treated group was considered to be 1.

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Fig. 13. Effect of various combinations of treatments of 100 nM of each BK, HOE157, and HOE158 on chondrocyte differentiation of BMMSCs after 20 days of differentiation, as determined by (A) PAS and alcian blue stained cells in monolayer culture and (B) Alcian blue stained cells in pellet culture. C: Transcriptional analysis for the expression of chondrocyte marker genes as determined by RT-PCR. Histogram in the lower panel shows the mean  SE, of arbitrary pixel intensities of three individual RT-PCRs. a, b, c, and d indicate significant levels differences at P < 0.05 with respect to their corresponding vehicle treated control, a, b, and c, respectively.

external factors/agents, leads to the inhibition of osteoblast differentiation, indicating the requirement of a threshold level of these signaling molecules (Matsushita et al., 2009; McGonnell et al. 2012). In bone metastasis, which is a common complication of breast cancer, Akt/ERK/NFkB cascade was reported to be responsible for increased breast cancer cell migration which in turn aggravated the metastatic JOURNAL OF CELLULAR PHYSIOLOGY

condition (Wei et al., 2008). Further, it was also reported that Akt/ERK/NFkB cascade is crucial for the maintenance of pluripotency in human embryonic stem cells and components of this cascade were decreased upon differentiation (Armstrong et al., 2006). The interplay between extracellular signals and transcriptional regulation is a crucial nexus of control for cell

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lineage determination. Based on this notion, we attempted to demonstrate if the activation of transcription factor, NFkB, can be regulated by extracellular signals like Akt and ERK. We observed that both ERK and Akt positively mediated the activation of NFkB signaling. Several studies have already shown that ERK mediates increase in IKKa/b and NFkB activities in various cell types (Rangaswami et al., 2006; Wei et al., 2008). So far the Akt and NFkB crosstalk is concerned, studies suggest that other than classical NFkB activation pathway, phosphoinositide 3-kinases (PI3K) and its downstream kinase (Akt) are involved in NFkB activation (Ozes et al., 1999). In this study, the authors demonstrated the presence of Akt signaling in parallel with classical NFkB pathways that converge at the point of IKK activation. Our results illustrated that, inhibition of either Akt or ERK did not abolish IkB activation completely, suggesting the presence of other classical NFkB signaling pathways, in addition to Akt and ERK signaling for BK-induced NFkB activation. Similarly, Xie et al. (2000) has also reported that Akt is a component of the BK signaling pathway that leads to NFkB activation where knocking down of Akt failed to completely abolish

the BK-induced luciferase activity. Thus, it is possible that BK simultaneously modulates ERK, Akt and NFkB pathways, and each pathway would act in concert, either independently or in combination, in order to downregulate osteoblast differentiation, and enhance bone resorption. One of the interesting finding of our study is BDKR1 and BDKR2 dependent activation of ERK and Akt signaling, respectively, during osteoblast differentiation process. It has been shown that GPCRs can activate ERK pathway, which is necessary for the control of proliferation in different cellular systems (Gutkind, 1998). As BDKR1 and BDKR2 belong to the class of GPCRs, both are reported to activate ERK signaling, but their differential action on Akt and ERK signaling can be justified by several lines of evidences. First, under inflammatory conditions, BK stimulates the production of nitric oxide (NO) by up-regulating nitric oxide synthase expression such as iNOS and eNOS (Ghalayini, 2004). Second, further investigation revealed that iNOS-mediated NO output is dependent on BDKR1 mediated ERK activation, while eNOS-mediated NO production is dependent on BDKR2 mediated Akt activation (Kuhr et al., 2010). This could be the

Fig. 14. Schematic diagram showing the possible mode of action of BK during inflammatory condition. BK is a direct ligand for BDKR2 which is also converted to corresponding agonist of BDKR1 by removal of the C-terminal Arg by membrane bound kininase. Both the BDKR1 and BDKR2 can couple to activate NFkB signaling through Akt and/or ERK1/2 pathways. Increased NFkB activity is responsible for BK-induced dampening of osteoblast and chondrocyte differentiation with subsequent activation of osteoclast formation. Circles beside various signalling molecules indicate their phosphorylated states.

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SRIVASTAVA ET AL. appropriate explanation of our finding, where we demonstrated that BDKR1 activates ERK more efficiently as compared to BDKR2, while BDKR2 activates Akt signaling to a marginally greater extent than BDKR1 in BK mediated osteoblast differentiation. However, further studies are needed to conclusively draw a line of evidence for this differential action of BDKR1 and BDKR2. Matrix metalloproteinases (MMPs) are capable of processing certain components of bone tissue and they are expressed by both osteoclasts and osteoblasts. Since MMPs are expressed by bone cells, they are most likely to have a role in influencing the strength and toughness of bone. The protein MMP-9 which is expressed mainly by osteoclasts is involved in osteoclast recruitment to ossification centers during development. On the other hand, MMP-2 which is expressed mainly by osteoblasts weakens bone and this effect is associated with a decrease in mineralization density of the bone (Nyman et al., 2011). Above stated facts are in accordance to our finding in BK induced differential role in osteoblast and osteoclast differentiation. While it could inhibit the osteoblast differentiation even at the lowest dose (1 nM) as tested by us, it could significantly increase osteoclast only at highest dose of BK (100 nM), suggesting its biased activity towards osteoblast differentiation. A similar trend was observed in the expressions of MMP-2 and MMP-9 which are linked to the actions of osteoblasts and osteoclast, respectively. Bone is formed through two distinct phases: (1) endochondral ossification, in which a cartilage model is replaced by bone, and (2) intramembranous ossification, in which bones are shaped directly from condensations of mesenchymal cells without a cartilage intermediate (Soltanoff et al., 2009). In view of this concept, we observed that along with reducing intramembranous ossifications, BK also dampened the endochondral ossification by directly inhibiting chondrocyte differentiation of mesenchymal stem cells in our study. In conclusion, our results demonstrated that BK elicits multidirectional actions by activating different signaling cascades, which can finally stimulate bone resorption in bone inflammatory diseases (Fig. 14). However, the exact crosstalk between both the receptors (BDKR1 and BDKR2) and downstream signaling pathways (ERK and Akt) as proposed in our hypothesis needs further validation to conclusively prove their specificity for a particular pathway. To the best of our knowledge there are no other reports on the regulation of osteoblastic differentiation of mesenchymal stem cells by BK, although there are some reports on the presence of this molecule in various bone related disorders. Overall, stimulation of BK receptors can have both protective and detrimental effects in a variety of disease states. Therefore, it is difficult to predict the exact cross talks between various signaling pathways in response to BK due to their complex interactions; however, our study provides sufficient evidence for its probable mode of action in bone remodeling process. Hence, based on these results, further detailed studies using in vitro and in vivo animal models are warranted prior to the development of potent receptor specific agonists and/or antagonists for BK. This may prove to be useful as therapeutic agents in cure and management of bone related disorders in future. Acknowledgments

Authors would like to thank the technical support team of the Institute Instrumentation Centre, Indian Institute of Technology Roorkee, India. Author would also like to acknowledge the professional editing service; “Reseapro Scientific Services (P) Ltd.” India, for English editing of this manuscript. JOURNAL OF CELLULAR PHYSIOLOGY

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Supporting Information

Additional supporting information may be found in the online version of this article at the publisher's web-site.

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NFκB signaling axis.

Bradykinin (BK), a well known mediator of pain and inflammation, is also known to be involved in the process of bone resorption. The present study the...
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