Fitoterapia 98 (2014) 59–65
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Kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells Mi-Bo Kim a, Youngwoo Song b, Jae-Kwan Hwang a,b,⁎ a b
Department of Biomaterials Science and Engineering, Yonsei University, Seoul 120-749, Republic of Korea Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 19 May 2014 Accepted in revised form 13 July 2014 Available online 23 July 2014 Keywords: Kirenol Osteoblast differentiation BMP Wnt/β-catenin
a b s t r a c t Kirenol has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, anti-adipogenic, and anti-arthritic activities; however, its effect on osteoblast differentiation has not yet been reported. The aim of the present study was to evaluate the effect of kirenol on osteoblast differentiation through activation of the bone morphogenetic protein (BMP) and Wnt/β-catenin signaling pathways in MC3T3-E1 cells. Kirenol markedly promoted alkaline phosphatase (ALP) activity and mineralization. Kirenol not only increased the expression of osteoblast differentiation markers, such as ALP, type I collagen (ColA1), and osteopontin (OPN), but also increased the expression of osteoprotegerin/receptor activator of nuclear factor kappa B ligand (OPG/RANKL) ratio. The effects of kirenol on osteoblast differentiation were accompanied by stimulating the expression of the BMP and Wnt/β-catenin signaling pathways, including BMP2, runt-related transcription factor 2 (Runx2), osterix (Osx), low density lipoprotein receptor related protein 5 (LRP5), disheveled 2 (DVL2), β-catenin, cyclin D1 (CCND1), and phosphorylated glycogen synthase kinase 3β (GSK3β). In addition, kirenol up-regulated the expression of β-catenin, CCND1, ALP, and ColA1 which were down-regulated by siRNA knockdown of β-catenin. Overall, these results demonstrate that kirenol is capable of promoting osteoblast differentiation in MC3T3-E1 cells through activation of the BMP and Wnt/β-catenin signaling pathways, suggesting that it is a potential candidate target for treating or preventing osteoporosis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis is a systemic bone disease characterized by a reduction in bone mass, bone quality, and microarchitectural deterioration, leading to low bone strength and high risk of fractures [1,2]. Bone metabolism is controlled by the mutual interaction between osteoblasts and osteoclasts. Osteoblasts induce bone formation by producing new bone, while osteoclasts induce bone resorption by breaking down bone [3]. Osteoblasts stimulate bone production by regulating the proliferation and differentiation of osteoblast precursors [4]. Osteoblast differentiation is regulated by the action of key ⁎ Corresponding author at: Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Republic of Korea. Tel.: +82 2 2123 5881; fax: +82 2 362 7265. E-mail address: [email protected] (J.-K. Hwang).
http://dx.doi.org/10.1016/j.fitote.2014.07.013 0367-326X/© 2014 Elsevier B.V. All rights reserved.
transcription factors, including runt-related transcription factor 2 (Runx2) and osterix (Osx), accompanied by the increased expression of bone matrix proteins, such as alkaline phosphatase (ALP), type I collagen (ColA1), and osteopontin (OPN). These osteoblast differentiation factors stimulate mineralization and lead to bone formation [5]. The bone morphogenetic protein (BMP) and winglesstype MMTV integration site (Wnt)/β-catenin signaling pathways cooperatively control bone formation and osteoblast differentiation [6]. Among the BMP family members, BMP2 is an important autocrine and paracrine growth factor and modulates mesenchymal precursor cells to differentiate into mature osteoblasts [6]. In addition, BMP2 regulates osteoblast differentiation by stimulating osteoblast-specific transcription factors, such as Runx2 and Osx, which are downstream regulators of the BMP signaling pathway [7].
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Another key pathway contributing to osteoblast differentiation is the Wnt/β-catenin signaling pathway, which positively modulates bone mass by enhancing pre-osteoblast replication, stimulating osteoblastogenesis, and inhibiting osteoblast and osteocyte apoptosis [4,8]. Kirenol (Fig. 1) is a natural diterpenoid compound isolated from Herba Siegesbeckia, including S. orientalis, S. glabrescens, and S. pubescens [9]. It was reported that dried Herba Siegesbeckia contained 0.482–1.302 mg/g of kirenol [10]. Kirenol has been reported to possess anti-inflammatory, immunoregulatory, anti-oxidant, wound healing, anti-arthritic, and anti-photoaging activities [9–12]; however its effect on osteoblast differentiation has not yet been investigated. Previously, we reported that kirenol exerted anti-adipogenic effect through activation of the Wnt/β-catenin signaling pathway [13]. It is known that the Wnt/β-catenin signaling pathway stimulates the expression of osteoblast differentiation markers and master osteogenic factor BMP2 [6]. The present study reports the effect of kirenol on osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells. 2. Materials and methods 2.1. Chemical reagents Kirenol was purchased from Institute for Korea Traditional Medical Industry (Daegu, Korea). Alpha modification of Eagle's minimum essential medium (α-MEM) was purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). βGlycerophosphate, L-ascorbic acid, 3-[4,5-dimethylthiazol-2yl]-2, 5-diphenyltetrazolium bromide (MTT), and Alizarin red S were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against phosphorylated glycogen synthase kinase 3β (GSK3β) and α-tubulin were purchased from Cell Signaling Technology (Beverly, MA, USA). An antibody against β-catenin was purchased from BD Biosiences (Franklin Lakes, NJ, USA). βCatenin small interfering RNA (siRNA) and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Lipofectamine RNAiMAX transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA). 2.2. Cell culture and differentiation MC3T3-E1 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were
Fig. 1. Chemical structure of kirenol.
maintained in α-MEM with antibiotics (100 units/mL penicillin A and 100 μg/mL streptomycin) and 10% FBS in an atmosphere of 5% CO2 at 37 °C. MC3T3-E1 cells were induced to differentiate in maintenance medium with 10 mM β-glycerophosphate and 50 μg/mL L-ascorbic acid. To examine the effect of kirenol on osteoblast differentiation, the cells were cultured with the differentiation medium in the presence of various concentrations (10–40 μM) of kirenol.
2.3. Cell viability Cell viability was determined using the MTT assay. MC3T3E1 cells were treated with various concentrations (10–60 μM) of kirenol for 24 h, and MTT solution (0.5 mg/mL) was added. After 3 h of incubation at 37 °C for MTT-formazan formation, the supernatant was removed, and dimethyl sulfoxide (DMSO) was added. Absorbance at 540 nm was determined spectrophotometrically by using a VERSAmax tunable microplate reader (Molecular Devices, Inc., Sunnyvale, CA, USA). Cell viability revealed no significant toxicity at various concentrations of kirenol: 10, 20, and 40 μM for 24 h (data not shown). Further studies were carried out at kirenol concentrations under 40 μM.
2.4. Quantitative assay of ALP activity ALP activity was determined with a SensoLyte p-nitrophenyl phosphate (p-NPP) ALP activity assay kit (AnaSpec, Inc., Fremont, CA, USA) according to the manufacturer's protocol. Three days following osteoblast differentiation induction, the cells were washed twice using phosphate-buffered saline (PBS) and lysed with 50 μL of lysis buffer containing 0.2% Triton X-100. The lysate was centrifuged at 2500 ×g for 15 min at 4 °C. The supernatant was collected for the measurement of ALP activity using p-NPP as a phosphatase substrate and alkaline phosphatase as a standard. The ALP activity in the cell was measured at 405 nm with a VERSAmax tunable microplate reader (Molecular Devices, Inc.). Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The data are expressed as a ratio of ALP activity per ng of protein.
2.5. Quantification of mineralization Mineralization in osteoblast cultures was determined by staining with Alizarin red S solution. Cells were incubated in the differentiation medium for 14 days in the absence or presence of kirenol. During the differentiation period, the medium was changed every 3 days. After differentiation induction for 14 days, the cells were washed with PBS and fixed with 70% ethanol for 1 h, and rinsed with distilled water. The cells were stained with Alizarin red S solution (40 mM, pH 4.2) for 10 min at room temperature. Excess stain was removed by washing five times with distilled water. Quantification was carried out by extracting Alizarin red Sstained mineral deposits with 0.1 N NaOH, and OD was measured at 540 nm with a VERSAmax tunable microplate reader (Molecular Devices, Inc.).
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2.6. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from MC3T3-E1cells using Trizol reagent (Invitrogen). The cDNA was converted in a 20 μL reaction containing 2 μg of total RNA, oligo (dT), and Reverse Transcription Premix (ELPIS-Biotech, Daejeon, Korea). PCR amplification of the cDNA products (3 μL) was performed with PCR premix (ELPIS-Biotech) and the following primer pairs (Bioneer, Daejeon, Korea): ALP forward 5′-CCA TGG TAG ATT ACG CTC ACA-3′ and ALP reverse 5′-ATG GAG GAT TCC AGA TAC AGG-3′ (433 bp); ColA1 forward 5′-ACT CAG CCG TCT GTG CCT CA-3′ and ColA1 reverse 5′-GGA GGC CTC GGT GGA CAT TA-3′ (183 bp); OPN forward 5′-GAC CAC ATG GAC GAC GAT G-3′ and OPN reverse 5′-TGG AAC TTG CTT GAC TAT CGA3′ (499 bp); osteoprotegerin (OPG) forward 5′-AAA GCA CCC TGT ATA AAA CA-3′ and OPG reverse 5′-CCG TTT TAT CCT CTC TAC ACT C-3′ (258 bp); receptor activator of nuclear factor kappa B ligand (RANKL) forward 5′-CGC TCT GTT CCT GTA CTT TCG AGC G-3′ and RANKL reverse 5′-TCG TGC TCC CTC CTT TCA TCA GGT T-3′ (586 bp); BMP2 forward 5′-CCA AGA CAC AGT TCC CTA CA-3′ and BMP2 reverse 5′-CAC GGC TTC TAG TTG ATG GA-3′ (531 bp); Runx2 forward 5′-GCC GGG AAT GAT GAG AAC TA-3′ and Runx2 reverse 5′-TGG GGA GGA TTT GTG AAG AC-3′ (155 bp); Osx forward 5′-CTT CCC AAT CCT ATT TGC CGT TT-3′ and Osx reverse 5′-CGG CCA GGT TAC TAA CAC CAA TCT-3′ (184 bp); low density lipoprotein receptor related protein 5 (LRP5) forward 5′-AAG GGT GCT GTG TAC TGG AC-3′ and LRP5 reverse 5′-AGA AGA GAA CCT TAC GGG ACG-3′ (220 bp); disheveled 2 (DVL2) forward 5′-GCT TCC ACA TGG CCA TGG GC-3′ and DVL2 reverse 5′-TGG CAC TGC TGG TGA GAG TCA CAG-3′ (195 bp); β-catenin forward 5′-GCC AAG TGG GTG GTA TAG AG-3′ and β-catenin reverse 5′-CTG GGT ATC CTG ATG TGC-3′ (329 bp); cyclin D1 (CCND1) forward 5′-AAA ATC GTG GCC ACC TGG AT-3′ and CCND1 reverse 5′-CAT CCG CCT CTG GCA TTT TG-3′ (346 bp); β-actin forward 5′-CCA CAC CTT CTA CAA TGA GC-3′ and β-actin reverse 5′-TGA GGT AGT CAG TCA GGT C-3′ (308 bp). All primers were denatured at 94 °C for 5 min prior to performing PCR amplification. Amplification consisted of 15–38 cycles as follows: denaturing at 94 °C for 30 s, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min, followed by a final 5 min extension phase at 72 °C. PCR was performed using a Gene Amp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). PCR products were electrophoresed by 1.5% agarose gel electrophoresis and visualized using a G:BOX EF imaging system and the Gene Snap program (Syngene, Cambridge, UK). β-Actin was used as an internal control.
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tubulin (1:1000 dilution) at 4 °C. After washing three times in Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (1:5000 dilution; Bethyl Laboratories, Inc., Montgomery, TX). Proteins were detected with the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Little Chalfont, UK) and visualized with the G:BOX EF imaging system and the Gene Snap program (Syngene). 2.8. β-Catenin knockdown by siRNA transfection β-Catenin knockdown by siRNA transfection was performed as previously described [14], with a slight modification. MC3T3-E1 cells were cultured in serum-free medium for 1 h and transfected with 60 nM of β-catenin siRNA or control siRNA using the Lipofectamine RNAiMAX transfection reagent. After 9 h, the transfected cells were differentiated according to the differentiation protocol. After 3 days, total RNA was extracted for RT-PCR. 2.9. Statistical analysis All experiments were repeated at least three times, and each experiment was performed in triplicate. Results are presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). Group differences were assessed by one-way analysis of variance (ANOVA) followed by Scheffe's test. P values b 0.05 were considered statistically significant. 3. Results 3.1. Kirenol stimulates cell differentiation and mineralization in MC3T3-E1 cells ALP is an early phenotypic marker and essential enzyme for osteoblast differentiation [15]. Kirenol treatment significantly increased ALP activity in a dose-dependent manner (Fig. 2A). The maximal effect was observed at 40 μM of kirenol, which increased ALP activity by 49%, as compared to the control. Mineralized bone nodule formation is an osteoblast maturation marker [2]. At 40 μM of kirenol the mineralization level was significantly increased by 26%, as compared to the control (Fig. 2B). Thus, kirenol effectively stimulated markers of osteoblast maturation, which resulted in enhanced osteoblast differentiation in MC3T3-E1 cells.
2.7. Western blot analysis MC3T3-E1 cells were lysed using RIPA lysis buffer (ElpisBiotech) with a protease inhibitor cocktail (Sigma–Aldrich). The lysate protein concentrations were determined using the BioRad protein assay kit (Bio-Rad Laboratories). Equal amounts of protein (35–50 μg) in each sample were separated by 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Whatman GmBH, Dassel, Germany). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature and then incubated overnight with primary antibodies against β-catenin, phosphorylated GSK3β, and α-
3.2. Kirenol promotes the expression of osteoblast differentiation markers Pre-osteoblastic cells produce extracellular matrix proteins, including ALP, ColA1, and OPN, which induce deposition and mineralization of osteoblasts [15]. The mRNA expression of osteoblast differentiation markers, such as ALP, ColA1, and OPN, was significantly increased by kirenol treatment in a dose-dependent manner (Fig. 2C). The levels of ALP, ColA1, and OPN mRNA expression were markedly increased at 40 μM of kirenol, by 67.8, 40.0, and 67.7%, respectively, as compared to the control. These results suggest that kirenol
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Fig. 2. Effects of kirenol treatment on osteoblast differentiation in MC3T3-E1cells. Cells were cultured with the differentiation medium in the various concentrations of kirenol for 3 days (ALP activity) or 14 days (mineralization). (A) ALP activity was measured using a commercial ALP assay kit. (B) Mineralization was assessed using Alizarin red S staining. (C) The mRNA expression of osteoblast differentiation factors, such as ALP, ColA1, and OPN was detected by RT-PCR. β-Actin was used as internal control. The results are expressed as the mean ± SD (% control) of three independent experiments. ⁎P b 0.05 and ⁎⁎P b 0.01 (control vs. sample-treated cells).
promotes mineralization and bone formation by increasing markers of osteoblast differentiation. 3.3. Kirenol increases the expression of OPG/RANKL ratio and the BMP signaling pathway components The expression of OPG/RANKL ratio is an informative marker for evaluation of effects on bone resorption and bone remodeling [16]. Kirenol treatment increased the mRNA expression of OPG and simultaneously decreased the mRNA expression of RANKL (Fig. 3A). Consequently, kirenol treatment increases the expression of OPG/RANKL ratio, which can result in inhibition of osteoclastogenesis. The BMP signaling pathway promotes the differentiation of mesenchymal cells into an osteoblastic lineage, as suggested by its ability to stimulate the expression of osteoblast differentiation markers [15]. Kirenol treatment significantly activated the mRNA expression of genes in the BMP signaling pathway-related regulators, including BMP2, Runx2, and Osx (Fig. 3B). Taken together, kirenol treatment effectively increased the expression of OPG/RANKL ratio and regulated the expression of the BMP signaling pathway components, leading to stimulation of osteoblast differentiation and prevention of osteoclastogenesis. 3.4. Kirenol activates the expression of the Wnt/β-catenin signaling pathway components The Wnt/β-catenin signaling pathway serves as an endogenous modulator of osteoblast differentiation and bone formation
[17]. Kirenol treatment significantly up-regulated the mRNA expression of genes in the Wnt/β-catenin signaling pathwayrelated regulators, including LRP5, DVL2, β-catenin, and CCND1 (Fig. 4A). In addition, kirenol efficiently activated β-catenin but inactivated GSK3β by increasing its phosphorylation (Fig. 4B). These results indicate that kirenol stimulates the expression of the Wnt/β-catenin signaling pathway components, leading to enhanced expression of osteoblast differentiation markers. 3.5. Kirenol regulates osteoblast differentiation through the Wnt/ β-catenin signaling pathway To clarify the function of β-catenin in kirenol-induced stimulation of osteoblast differentiation, β-catenin knockdown by siRNA was performed in MC3T3-E1 cells with or without kirenol treatment. The mRNA expression of β-catenin and its target gene CCND1 were markedly reduced by β-catenin knockdown, as compared to control siRNA, in both the presence and absence of kirenol. Furthermore, kirenol treatment increased the mRNA expression of osteoblast differentiation markers, ALP and ColA1, which were decreased by β-catenin knockdown. These results show that the Wnt/β-catenin signaling pathway is involved in kirenol-enhanced osteoblast differentiation. 4. Discussion The Wnt/β-catenin signaling pathway stimulates the expression of osteoblast differentiation markers and mineralization, while it inhibits adipogenesis by down-regulating
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Fig. 3. Effects of kirenol treatment on the OPG/RANKL ratio and the BMP signaling pathway in MC3T3-E1 cells. Cells were cultured with the differentiation medium in the various concentrations of kirenol for 3 days. (A) The mRNA expression of OPG/RANKL ratio, such as OPG and RANKL was detected by RT-PCR. (B) The mRNA expression of the BMP signaling pathway related markers, such as BMP2, Runx2, and Osx was detected by RT-PCR. β-Actin was used as an internal control. The results are expressed as the mean ± SD (% control) of three independent experiments. ⁎P b 0.05 and ⁎⁎P b 0.01 (control vs. sample-treated cells).
major adipogenesis transcription factors [17]. Our previous study demonstrated that kirenol inhibited adipogenesis through activation of the Wnt/β-catenin signaling pathway [13]. The Wnt/β-catenin signaling pathway activates the expression of the master osteogenic factor BMP2 in osteoblasts, suggesting that the BMP signaling pathway is controlled by functional cross talk between the Wnt/β-catenin signaling pathway [6]. Accordingly, it is conceivable that kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways. The process of osteoblast differentiation occurs in two stages. In the early stage, ALP is a phenotypic marker and essential enzyme, which produces and up-regulates specific osteoblast differentiation genes, including ColA1 and OPN. In the terminal stage, the extracellular matrix gradually becomes mineralized by calcium deposition [15,18]. Kirenol effectively promotes ALP activity and the expression of osteoblast differentiation markers, leading to enhance the osteoblast mineralization (Fig. 2). These results demonstrate that kirenol enhances the osteoblast differentiation process from the early to the terminal stage, upregulating maturation and differentiation in MC3T3-E1 cells. Osteoclastogenesis is affected by cytokines like RANKL and OPG, synthesized by osteoblasts. RANKL plays a critical role in stimulation of osteoclast differentiation through binding to its receptor RANK [16]. The interaction of RANKL-RANK modulates osteoclastogenesis, which is involved in the formation and survival of osteoclasts. The secretion of OPG by osteoblasts prevents RANKL-RANK interaction and RANK activation, resulting in inhibition of bone resorption [2,4]. Therefore, the ratio of OPG/RANKL is a determinative and evaluative
factor for bone resorption and bone remodeling [16]. Kirenol increased the expression of OPG/RANKL ratio in MC3T3-E1 cells (Fig. 3A), suggesting that kirenol suppressed osteoclastogenesis by regulating the balance of OPG/RANKL. The BMP signaling pathway induces bone formation and bone remodeling by stimulating the differentiation of osteoblast [8]. Kirenol activated the expression of BMP signaling pathway components, such as BMP2, Runx2, and Osx (Fig. 3B). Runx2 and Osx are essential transcription factors for osteoblast differentiation and have been reported to be induced by BMP2. These two transcription factors regulate the expression of the osteoblastrelated genes, including ALP, ColA1, and OPN [19]. Thus, kirenol promotes osteoblast differentiation through activation of the BMP signaling pathway, increasing the expression of the downstream regulators, Runx2 and Osx. The Wnt/β-catenin signaling pathway modulates differentiation, proliferation, and mineralization in osteoblastogenesis [8]. The Wnt ligands bind to frizzled receptors and the co-receptors LRP5 and LRP6, leading to activation of the Wnt/β-catenin signaling pathway [1]. Kirenol significantly increased the mRNA expression of LRP5 in differentiated MC3T3-E1cells (Fig. 4A). LRP5 controls bone formation and bone mass within the bone microenvironment, mostly at the level of osteocytes [20]. Kirenol continually activated DVL2 and disrupted the β-catenin destruction complex by inactivating GSK3β, allowing the stabilization and nuclear translocation of β-catenin. CCND1 is a β-catenin target gene that is activated by kirenol treatment (Figs. 4A and B). β-Catenin increases the expression of OPG in osteoblast differentiation, which indirectly represses osteoclast differentiation by inhibiting bone resorption and bone remodeling [20]. The
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Fig. 4. Effect of kirenol treatment on the Wnt/β-catenin signaling pathway in MC3T3-E1 cells. Cells were cultured with the differentiation medium in the various concentrations of kirenol for 3 days. (A) The mRNA expression of the Wnt/β-catenin signaling pathway related markers, such as LRP5, DVL2, β-catenin, and CCND1 was detected by RT-PCR. (B) The protein levels of the β-catenin and p-GSK3β were evaluated by Western blotting. β-Catenin knockdown by siRNA was performed in MC3T3E1 cells with or without kirenol treatment. (C) The mRNA expression of β-catenin, CCND1, ALP, and ColA1 was detected by RT-PCR. β-Actin and α-tubulin were used as internal controls. The results are expressed as the mean ± SD (% control) of three independent experiments. ⁎P b 0.05 and ⁎⁎P b 0.01 (control vs. sample-treated cells).
essential transcription factor of Runx2 integrates the BMP and Wnt/β-catenin signaling pathways in the regulation of osteoblast differentiation [7]. The Wnt/β-catenin signaling pathway synergies with the BMP signaling pathway to stimulate osteoblast differentiation and bone formation [4]. Therefore, kirenol affects both osteoblast differentiation and osteoclastogenesis through cooperative interactions with the BMP and Wnt/β-catenin signaling pathways. The Wnt/β-catenin signaling pathway can induce osteoblast differentiation genes, including ALP, ColA1, and OPN [21]. β-Catenin plays an important role in regulating bone fracture regeneration [4]. β-Catenin knockdown by siRNA transfection decreased the expression of ALP and ColA1 (Fig. 4C), suggesting that kirenol stimulated ALP and ColA1 through β-catenin up-regulation. These results support the conclusion that effect of kirenol on osteoblast differentiation is regulated through activating components of the Wnt/β-catenin signaling pathway. It was reported that the absorption, distribution and excretion of kirenol were rapid after oral administration in male SD rats. The pharmacokinetic data suggest that kirenol can be absorbed quickly to play a pharmacological effect, and does not easily accumulate in the body to cause toxicity [22].
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Kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells Mi-Bo Kim a, Youngwoo Song b, Jae-Kwan Hwang a,b,⁎ a b
Department of Biomaterials Science and Engineering, Yonsei University, Seoul 120-749, Republic of Korea Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 19 May 2014 Accepted in revised form 13 July 2014 Available online 23 July 2014 Keywords: Kirenol Osteoblast differentiation BMP Wnt/β-catenin
a b s t r a c t Kirenol has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, anti-adipogenic, and anti-arthritic activities; however, its effect on osteoblast differentiation has not yet been reported. The aim of the present study was to evaluate the effect of kirenol on osteoblast differentiation through activation of the bone morphogenetic protein (BMP) and Wnt/β-catenin signaling pathways in MC3T3-E1 cells. Kirenol markedly promoted alkaline phosphatase (ALP) activity and mineralization. Kirenol not only increased the expression of osteoblast differentiation markers, such as ALP, type I collagen (ColA1), and osteopontin (OPN), but also increased the expression of osteoprotegerin/receptor activator of nuclear factor kappa B ligand (OPG/RANKL) ratio. The effects of kirenol on osteoblast differentiation were accompanied by stimulating the expression of the BMP and Wnt/β-catenin signaling pathways, including BMP2, runt-related transcription factor 2 (Runx2), osterix (Osx), low density lipoprotein receptor related protein 5 (LRP5), disheveled 2 (DVL2), β-catenin, cyclin D1 (CCND1), and phosphorylated glycogen synthase kinase 3β (GSK3β). In addition, kirenol up-regulated the expression of β-catenin, CCND1, ALP, and ColA1 which were down-regulated by siRNA knockdown of β-catenin. Overall, these results demonstrate that kirenol is capable of promoting osteoblast differentiation in MC3T3-E1 cells through activation of the BMP and Wnt/β-catenin signaling pathways, suggesting that it is a potential candidate target for treating or preventing osteoporosis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis is a systemic bone disease characterized by a reduction in bone mass, bone quality, and microarchitectural deterioration, leading to low bone strength and high risk of fractures [1,2]. Bone metabolism is controlled by the mutual interaction between osteoblasts and osteoclasts. Osteoblasts induce bone formation by producing new bone, while osteoclasts induce bone resorption by breaking down bone [3]. Osteoblasts stimulate bone production by regulating the proliferation and differentiation of osteoblast precursors [4]. Osteoblast differentiation is regulated by the action of key ⁎ Corresponding author at: Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Republic of Korea. Tel.: +82 2 2123 5881; fax: +82 2 362 7265. E-mail address: [email protected] (J.-K. Hwang).
http://dx.doi.org/10.1016/j.fitote.2014.07.013 0367-326X/© 2014 Elsevier B.V. All rights reserved.
transcription factors, including runt-related transcription factor 2 (Runx2) and osterix (Osx), accompanied by the increased expression of bone matrix proteins, such as alkaline phosphatase (ALP), type I collagen (ColA1), and osteopontin (OPN). These osteoblast differentiation factors stimulate mineralization and lead to bone formation [5]. The bone morphogenetic protein (BMP) and winglesstype MMTV integration site (Wnt)/β-catenin signaling pathways cooperatively control bone formation and osteoblast differentiation [6]. Among the BMP family members, BMP2 is an important autocrine and paracrine growth factor and modulates mesenchymal precursor cells to differentiate into mature osteoblasts [6]. In addition, BMP2 regulates osteoblast differentiation by stimulating osteoblast-specific transcription factors, such as Runx2 and Osx, which are downstream regulators of the BMP signaling pathway [7].
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Another key pathway contributing to osteoblast differentiation is the Wnt/β-catenin signaling pathway, which positively modulates bone mass by enhancing pre-osteoblast replication, stimulating osteoblastogenesis, and inhibiting osteoblast and osteocyte apoptosis [4,8]. Kirenol (Fig. 1) is a natural diterpenoid compound isolated from Herba Siegesbeckia, including S. orientalis, S. glabrescens, and S. pubescens [9]. It was reported that dried Herba Siegesbeckia contained 0.482–1.302 mg/g of kirenol [10]. Kirenol has been reported to possess anti-inflammatory, immunoregulatory, anti-oxidant, wound healing, anti-arthritic, and anti-photoaging activities [9–12]; however its effect on osteoblast differentiation has not yet been investigated. Previously, we reported that kirenol exerted anti-adipogenic effect through activation of the Wnt/β-catenin signaling pathway [13]. It is known that the Wnt/β-catenin signaling pathway stimulates the expression of osteoblast differentiation markers and master osteogenic factor BMP2 [6]. The present study reports the effect of kirenol on osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells. 2. Materials and methods 2.1. Chemical reagents Kirenol was purchased from Institute for Korea Traditional Medical Industry (Daegu, Korea). Alpha modification of Eagle's minimum essential medium (α-MEM) was purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). βGlycerophosphate, L-ascorbic acid, 3-[4,5-dimethylthiazol-2yl]-2, 5-diphenyltetrazolium bromide (MTT), and Alizarin red S were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against phosphorylated glycogen synthase kinase 3β (GSK3β) and α-tubulin were purchased from Cell Signaling Technology (Beverly, MA, USA). An antibody against β-catenin was purchased from BD Biosiences (Franklin Lakes, NJ, USA). βCatenin small interfering RNA (siRNA) and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Lipofectamine RNAiMAX transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA). 2.2. Cell culture and differentiation MC3T3-E1 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were
Fig. 1. Chemical structure of kirenol.
maintained in α-MEM with antibiotics (100 units/mL penicillin A and 100 μg/mL streptomycin) and 10% FBS in an atmosphere of 5% CO2 at 37 °C. MC3T3-E1 cells were induced to differentiate in maintenance medium with 10 mM β-glycerophosphate and 50 μg/mL L-ascorbic acid. To examine the effect of kirenol on osteoblast differentiation, the cells were cultured with the differentiation medium in the presence of various concentrations (10–40 μM) of kirenol.
2.3. Cell viability Cell viability was determined using the MTT assay. MC3T3E1 cells were treated with various concentrations (10–60 μM) of kirenol for 24 h, and MTT solution (0.5 mg/mL) was added. After 3 h of incubation at 37 °C for MTT-formazan formation, the supernatant was removed, and dimethyl sulfoxide (DMSO) was added. Absorbance at 540 nm was determined spectrophotometrically by using a VERSAmax tunable microplate reader (Molecular Devices, Inc., Sunnyvale, CA, USA). Cell viability revealed no significant toxicity at various concentrations of kirenol: 10, 20, and 40 μM for 24 h (data not shown). Further studies were carried out at kirenol concentrations under 40 μM.
2.4. Quantitative assay of ALP activity ALP activity was determined with a SensoLyte p-nitrophenyl phosphate (p-NPP) ALP activity assay kit (AnaSpec, Inc., Fremont, CA, USA) according to the manufacturer's protocol. Three days following osteoblast differentiation induction, the cells were washed twice using phosphate-buffered saline (PBS) and lysed with 50 μL of lysis buffer containing 0.2% Triton X-100. The lysate was centrifuged at 2500 ×g for 15 min at 4 °C. The supernatant was collected for the measurement of ALP activity using p-NPP as a phosphatase substrate and alkaline phosphatase as a standard. The ALP activity in the cell was measured at 405 nm with a VERSAmax tunable microplate reader (Molecular Devices, Inc.). Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The data are expressed as a ratio of ALP activity per ng of protein.
2.5. Quantification of mineralization Mineralization in osteoblast cultures was determined by staining with Alizarin red S solution. Cells were incubated in the differentiation medium for 14 days in the absence or presence of kirenol. During the differentiation period, the medium was changed every 3 days. After differentiation induction for 14 days, the cells were washed with PBS and fixed with 70% ethanol for 1 h, and rinsed with distilled water. The cells were stained with Alizarin red S solution (40 mM, pH 4.2) for 10 min at room temperature. Excess stain was removed by washing five times with distilled water. Quantification was carried out by extracting Alizarin red Sstained mineral deposits with 0.1 N NaOH, and OD was measured at 540 nm with a VERSAmax tunable microplate reader (Molecular Devices, Inc.).
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2.6. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from MC3T3-E1cells using Trizol reagent (Invitrogen). The cDNA was converted in a 20 μL reaction containing 2 μg of total RNA, oligo (dT), and Reverse Transcription Premix (ELPIS-Biotech, Daejeon, Korea). PCR amplification of the cDNA products (3 μL) was performed with PCR premix (ELPIS-Biotech) and the following primer pairs (Bioneer, Daejeon, Korea): ALP forward 5′-CCA TGG TAG ATT ACG CTC ACA-3′ and ALP reverse 5′-ATG GAG GAT TCC AGA TAC AGG-3′ (433 bp); ColA1 forward 5′-ACT CAG CCG TCT GTG CCT CA-3′ and ColA1 reverse 5′-GGA GGC CTC GGT GGA CAT TA-3′ (183 bp); OPN forward 5′-GAC CAC ATG GAC GAC GAT G-3′ and OPN reverse 5′-TGG AAC TTG CTT GAC TAT CGA3′ (499 bp); osteoprotegerin (OPG) forward 5′-AAA GCA CCC TGT ATA AAA CA-3′ and OPG reverse 5′-CCG TTT TAT CCT CTC TAC ACT C-3′ (258 bp); receptor activator of nuclear factor kappa B ligand (RANKL) forward 5′-CGC TCT GTT CCT GTA CTT TCG AGC G-3′ and RANKL reverse 5′-TCG TGC TCC CTC CTT TCA TCA GGT T-3′ (586 bp); BMP2 forward 5′-CCA AGA CAC AGT TCC CTA CA-3′ and BMP2 reverse 5′-CAC GGC TTC TAG TTG ATG GA-3′ (531 bp); Runx2 forward 5′-GCC GGG AAT GAT GAG AAC TA-3′ and Runx2 reverse 5′-TGG GGA GGA TTT GTG AAG AC-3′ (155 bp); Osx forward 5′-CTT CCC AAT CCT ATT TGC CGT TT-3′ and Osx reverse 5′-CGG CCA GGT TAC TAA CAC CAA TCT-3′ (184 bp); low density lipoprotein receptor related protein 5 (LRP5) forward 5′-AAG GGT GCT GTG TAC TGG AC-3′ and LRP5 reverse 5′-AGA AGA GAA CCT TAC GGG ACG-3′ (220 bp); disheveled 2 (DVL2) forward 5′-GCT TCC ACA TGG CCA TGG GC-3′ and DVL2 reverse 5′-TGG CAC TGC TGG TGA GAG TCA CAG-3′ (195 bp); β-catenin forward 5′-GCC AAG TGG GTG GTA TAG AG-3′ and β-catenin reverse 5′-CTG GGT ATC CTG ATG TGC-3′ (329 bp); cyclin D1 (CCND1) forward 5′-AAA ATC GTG GCC ACC TGG AT-3′ and CCND1 reverse 5′-CAT CCG CCT CTG GCA TTT TG-3′ (346 bp); β-actin forward 5′-CCA CAC CTT CTA CAA TGA GC-3′ and β-actin reverse 5′-TGA GGT AGT CAG TCA GGT C-3′ (308 bp). All primers were denatured at 94 °C for 5 min prior to performing PCR amplification. Amplification consisted of 15–38 cycles as follows: denaturing at 94 °C for 30 s, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min, followed by a final 5 min extension phase at 72 °C. PCR was performed using a Gene Amp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). PCR products were electrophoresed by 1.5% agarose gel electrophoresis and visualized using a G:BOX EF imaging system and the Gene Snap program (Syngene, Cambridge, UK). β-Actin was used as an internal control.
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tubulin (1:1000 dilution) at 4 °C. After washing three times in Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (1:5000 dilution; Bethyl Laboratories, Inc., Montgomery, TX). Proteins were detected with the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Little Chalfont, UK) and visualized with the G:BOX EF imaging system and the Gene Snap program (Syngene). 2.8. β-Catenin knockdown by siRNA transfection β-Catenin knockdown by siRNA transfection was performed as previously described [14], with a slight modification. MC3T3-E1 cells were cultured in serum-free medium for 1 h and transfected with 60 nM of β-catenin siRNA or control siRNA using the Lipofectamine RNAiMAX transfection reagent. After 9 h, the transfected cells were differentiated according to the differentiation protocol. After 3 days, total RNA was extracted for RT-PCR. 2.9. Statistical analysis All experiments were repeated at least three times, and each experiment was performed in triplicate. Results are presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). Group differences were assessed by one-way analysis of variance (ANOVA) followed by Scheffe's test. P values b 0.05 were considered statistically significant. 3. Results 3.1. Kirenol stimulates cell differentiation and mineralization in MC3T3-E1 cells ALP is an early phenotypic marker and essential enzyme for osteoblast differentiation [15]. Kirenol treatment significantly increased ALP activity in a dose-dependent manner (Fig. 2A). The maximal effect was observed at 40 μM of kirenol, which increased ALP activity by 49%, as compared to the control. Mineralized bone nodule formation is an osteoblast maturation marker [2]. At 40 μM of kirenol the mineralization level was significantly increased by 26%, as compared to the control (Fig. 2B). Thus, kirenol effectively stimulated markers of osteoblast maturation, which resulted in enhanced osteoblast differentiation in MC3T3-E1 cells.
2.7. Western blot analysis MC3T3-E1 cells were lysed using RIPA lysis buffer (ElpisBiotech) with a protease inhibitor cocktail (Sigma–Aldrich). The lysate protein concentrations were determined using the BioRad protein assay kit (Bio-Rad Laboratories). Equal amounts of protein (35–50 μg) in each sample were separated by 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Whatman GmBH, Dassel, Germany). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature and then incubated overnight with primary antibodies against β-catenin, phosphorylated GSK3β, and α-
3.2. Kirenol promotes the expression of osteoblast differentiation markers Pre-osteoblastic cells produce extracellular matrix proteins, including ALP, ColA1, and OPN, which induce deposition and mineralization of osteoblasts [15]. The mRNA expression of osteoblast differentiation markers, such as ALP, ColA1, and OPN, was significantly increased by kirenol treatment in a dose-dependent manner (Fig. 2C). The levels of ALP, ColA1, and OPN mRNA expression were markedly increased at 40 μM of kirenol, by 67.8, 40.0, and 67.7%, respectively, as compared to the control. These results suggest that kirenol
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Fig. 2. Effects of kirenol treatment on osteoblast differentiation in MC3T3-E1cells. Cells were cultured with the differentiation medium in the various concentrations of kirenol for 3 days (ALP activity) or 14 days (mineralization). (A) ALP activity was measured using a commercial ALP assay kit. (B) Mineralization was assessed using Alizarin red S staining. (C) The mRNA expression of osteoblast differentiation factors, such as ALP, ColA1, and OPN was detected by RT-PCR. β-Actin was used as internal control. The results are expressed as the mean ± SD (% control) of three independent experiments. ⁎P b 0.05 and ⁎⁎P b 0.01 (control vs. sample-treated cells).
promotes mineralization and bone formation by increasing markers of osteoblast differentiation. 3.3. Kirenol increases the expression of OPG/RANKL ratio and the BMP signaling pathway components The expression of OPG/RANKL ratio is an informative marker for evaluation of effects on bone resorption and bone remodeling [16]. Kirenol treatment increased the mRNA expression of OPG and simultaneously decreased the mRNA expression of RANKL (Fig. 3A). Consequently, kirenol treatment increases the expression of OPG/RANKL ratio, which can result in inhibition of osteoclastogenesis. The BMP signaling pathway promotes the differentiation of mesenchymal cells into an osteoblastic lineage, as suggested by its ability to stimulate the expression of osteoblast differentiation markers [15]. Kirenol treatment significantly activated the mRNA expression of genes in the BMP signaling pathway-related regulators, including BMP2, Runx2, and Osx (Fig. 3B). Taken together, kirenol treatment effectively increased the expression of OPG/RANKL ratio and regulated the expression of the BMP signaling pathway components, leading to stimulation of osteoblast differentiation and prevention of osteoclastogenesis. 3.4. Kirenol activates the expression of the Wnt/β-catenin signaling pathway components The Wnt/β-catenin signaling pathway serves as an endogenous modulator of osteoblast differentiation and bone formation
[17]. Kirenol treatment significantly up-regulated the mRNA expression of genes in the Wnt/β-catenin signaling pathwayrelated regulators, including LRP5, DVL2, β-catenin, and CCND1 (Fig. 4A). In addition, kirenol efficiently activated β-catenin but inactivated GSK3β by increasing its phosphorylation (Fig. 4B). These results indicate that kirenol stimulates the expression of the Wnt/β-catenin signaling pathway components, leading to enhanced expression of osteoblast differentiation markers. 3.5. Kirenol regulates osteoblast differentiation through the Wnt/ β-catenin signaling pathway To clarify the function of β-catenin in kirenol-induced stimulation of osteoblast differentiation, β-catenin knockdown by siRNA was performed in MC3T3-E1 cells with or without kirenol treatment. The mRNA expression of β-catenin and its target gene CCND1 were markedly reduced by β-catenin knockdown, as compared to control siRNA, in both the presence and absence of kirenol. Furthermore, kirenol treatment increased the mRNA expression of osteoblast differentiation markers, ALP and ColA1, which were decreased by β-catenin knockdown. These results show that the Wnt/β-catenin signaling pathway is involved in kirenol-enhanced osteoblast differentiation. 4. Discussion The Wnt/β-catenin signaling pathway stimulates the expression of osteoblast differentiation markers and mineralization, while it inhibits adipogenesis by down-regulating
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Fig. 3. Effects of kirenol treatment on the OPG/RANKL ratio and the BMP signaling pathway in MC3T3-E1 cells. Cells were cultured with the differentiation medium in the various concentrations of kirenol for 3 days. (A) The mRNA expression of OPG/RANKL ratio, such as OPG and RANKL was detected by RT-PCR. (B) The mRNA expression of the BMP signaling pathway related markers, such as BMP2, Runx2, and Osx was detected by RT-PCR. β-Actin was used as an internal control. The results are expressed as the mean ± SD (% control) of three independent experiments. ⁎P b 0.05 and ⁎⁎P b 0.01 (control vs. sample-treated cells).
major adipogenesis transcription factors [17]. Our previous study demonstrated that kirenol inhibited adipogenesis through activation of the Wnt/β-catenin signaling pathway [13]. The Wnt/β-catenin signaling pathway activates the expression of the master osteogenic factor BMP2 in osteoblasts, suggesting that the BMP signaling pathway is controlled by functional cross talk between the Wnt/β-catenin signaling pathway [6]. Accordingly, it is conceivable that kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways. The process of osteoblast differentiation occurs in two stages. In the early stage, ALP is a phenotypic marker and essential enzyme, which produces and up-regulates specific osteoblast differentiation genes, including ColA1 and OPN. In the terminal stage, the extracellular matrix gradually becomes mineralized by calcium deposition [15,18]. Kirenol effectively promotes ALP activity and the expression of osteoblast differentiation markers, leading to enhance the osteoblast mineralization (Fig. 2). These results demonstrate that kirenol enhances the osteoblast differentiation process from the early to the terminal stage, upregulating maturation and differentiation in MC3T3-E1 cells. Osteoclastogenesis is affected by cytokines like RANKL and OPG, synthesized by osteoblasts. RANKL plays a critical role in stimulation of osteoclast differentiation through binding to its receptor RANK [16]. The interaction of RANKL-RANK modulates osteoclastogenesis, which is involved in the formation and survival of osteoclasts. The secretion of OPG by osteoblasts prevents RANKL-RANK interaction and RANK activation, resulting in inhibition of bone resorption [2,4]. Therefore, the ratio of OPG/RANKL is a determinative and evaluative
factor for bone resorption and bone remodeling [16]. Kirenol increased the expression of OPG/RANKL ratio in MC3T3-E1 cells (Fig. 3A), suggesting that kirenol suppressed osteoclastogenesis by regulating the balance of OPG/RANKL. The BMP signaling pathway induces bone formation and bone remodeling by stimulating the differentiation of osteoblast [8]. Kirenol activated the expression of BMP signaling pathway components, such as BMP2, Runx2, and Osx (Fig. 3B). Runx2 and Osx are essential transcription factors for osteoblast differentiation and have been reported to be induced by BMP2. These two transcription factors regulate the expression of the osteoblastrelated genes, including ALP, ColA1, and OPN [19]. Thus, kirenol promotes osteoblast differentiation through activation of the BMP signaling pathway, increasing the expression of the downstream regulators, Runx2 and Osx. The Wnt/β-catenin signaling pathway modulates differentiation, proliferation, and mineralization in osteoblastogenesis [8]. The Wnt ligands bind to frizzled receptors and the co-receptors LRP5 and LRP6, leading to activation of the Wnt/β-catenin signaling pathway [1]. Kirenol significantly increased the mRNA expression of LRP5 in differentiated MC3T3-E1cells (Fig. 4A). LRP5 controls bone formation and bone mass within the bone microenvironment, mostly at the level of osteocytes [20]. Kirenol continually activated DVL2 and disrupted the β-catenin destruction complex by inactivating GSK3β, allowing the stabilization and nuclear translocation of β-catenin. CCND1 is a β-catenin target gene that is activated by kirenol treatment (Figs. 4A and B). β-Catenin increases the expression of OPG in osteoblast differentiation, which indirectly represses osteoclast differentiation by inhibiting bone resorption and bone remodeling [20]. The
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Fig. 4. Effect of kirenol treatment on the Wnt/β-catenin signaling pathway in MC3T3-E1 cells. Cells were cultured with the differentiation medium in the various concentrations of kirenol for 3 days. (A) The mRNA expression of the Wnt/β-catenin signaling pathway related markers, such as LRP5, DVL2, β-catenin, and CCND1 was detected by RT-PCR. (B) The protein levels of the β-catenin and p-GSK3β were evaluated by Western blotting. β-Catenin knockdown by siRNA was performed in MC3T3E1 cells with or without kirenol treatment. (C) The mRNA expression of β-catenin, CCND1, ALP, and ColA1 was detected by RT-PCR. β-Actin and α-tubulin were used as internal controls. The results are expressed as the mean ± SD (% control) of three independent experiments. ⁎P b 0.05 and ⁎⁎P b 0.01 (control vs. sample-treated cells).
essential transcription factor of Runx2 integrates the BMP and Wnt/β-catenin signaling pathways in the regulation of osteoblast differentiation [7]. The Wnt/β-catenin signaling pathway synergies with the BMP signaling pathway to stimulate osteoblast differentiation and bone formation [4]. Therefore, kirenol affects both osteoblast differentiation and osteoclastogenesis through cooperative interactions with the BMP and Wnt/β-catenin signaling pathways. The Wnt/β-catenin signaling pathway can induce osteoblast differentiation genes, including ALP, ColA1, and OPN [21]. β-Catenin plays an important role in regulating bone fracture regeneration [4]. β-Catenin knockdown by siRNA transfection decreased the expression of ALP and ColA1 (Fig. 4C), suggesting that kirenol stimulated ALP and ColA1 through β-catenin up-regulation. These results support the conclusion that effect of kirenol on osteoblast differentiation is regulated through activating components of the Wnt/β-catenin signaling pathway. It was reported that the absorption, distribution and excretion of kirenol were rapid after oral administration in male SD rats. The pharmacokinetic data suggest that kirenol can be absorbed quickly to play a pharmacological effect, and does not easily accumulate in the body to cause toxicity [22].
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