Open Access

Pinoresinol promotes MC3T3‑E1 cell proliferation and differentiation via the cyclic AMP/protein kinase A signaling pathway

  • Authors:
    • Xin Jiang
    • Wenjing Chen
    • Fuguo Shen
    • Wenlong Xiao
    • Hongliang Guo
    • Hang Su
    • Jiang Xiu
    • Wencai Sun
  • View Affiliations

  • Published online on: July 3, 2019     https://doi.org/10.3892/mmr.2019.10468
  • Pages: 2143-2150
  • Copyright: © Jiang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Estradiol (E2) is a first‑line drug for osteoporosis (OP) treatment via promotion of osteoblastic proliferation and differentiation. However, a long‑term use of E2 would produce side effects thus, it is imperative to discover safer and more effective drugs. Pinoresinol (PINO) has a similar chemical structure to E2. The present study aimed to investigate whether PINO could promote osteoblastic proliferation and differentiation and the potential mechanisms. After treatment with 0.1 µg/l PINO for 2 days, MC3T3‑E1 cell migration was assessed by wound healing assay. Estrogen (E2) treatment served as a positive control. RT‑qPCR and western blotting were used for mRNA and protein expression analyses. Alkaline phosphatase (ALP) activity assay and Alizarin red staining were performed to investigate the calcification and mineralization, and the cyclic AMP (cAMP) level was detected by enzyme‑linked immunosorbent assay (ELISA). H89, an inhibitor of protein kinase A (PKA), was introduced to verify the role of cAMP/PKA in the effect of PINO on MC3T3‑E1 cells. Cell viability was the highest under 48 h of 0.1 µg/l PINO treatment. After treatment with PINO, a significant increase was observed in the migration rate and the expression of collagen type I (Col‑I), ALP, osteopontin (OPN), runt‑related transcription factor 2 (Runx2) and bone morphogenetic protein‑2 (BMP‑2) (P<0.01). The ALP activity and Alizarin red size in PINO and E2 groups were notably increased. The increased cAMP, PKA and phosphorylated cAMP response element‑binding protein (CREB) levels were also observed in the PINO group. Furthermore, H89 co‑treatment abolished the positive effects of PINO on cell viability and migration. PINO had similar effects to E2 on the osteoblastic proliferation and differentiation, and these positive effects may be attributed to the regulation of the cAMP/PKA signaling pathway.

Introduction

Osteoporosis (OP), which is a type of body-wide metabolic osteopathy, is characterized by osteopenia, skeletal fragility, and microarchitectural deterioration. The main cause of OP is the imbalance between bone formation and resorption (1). The chronic pain, malformation and pathological fracture induced by OP severely affect the quality of life of the elderly, postmenopausal or estrogen-deficient women, and it even leads to death (2,3). With the increase of the aging population, OP has gradually attracted the attention of many countries and society. It has been reported that the balance of bone metabolism is maintained by osteoclasts and osteoblasts, and the weakness of osteoblast proliferation is one of the most important pathogenesis of OP (4,5). Therefore, enhancing the proliferation and differentiation of osteoblasts is vital to develop OP treatment strategies. To date, the main treatments of OP were based on drug therapies, including calcitonin, and bisphosphonates which functioned as bone resorption inhibitors by blocking the function of osteoclasts (6). Estrogens could directly regulate the expression of bone formation-related genes by binding to estrogen receptor (ER) in osteoblasts (4,7). However, a long-term use of these agents has been associated with severe side effects (8,9). For instance, a long term use of bisphosphonates in clinical practice could lead to gastrointestinal intolerance, osteonecrosis of the jaw, atypical femur fractures, oesophageal cancer, atrial fibrillation and chronic musculoskeletal pain (10). In addition, although estrogen (E2) therapy was believed to be the most effective treatment for postmenopausal osteoporosis (PMOP) via the notch signaling pathway (11), recent research has indicated that the prolonged use of E2 could increase the risk of endometrial and breast cancer (12,13). Hence, it is necessary to identify safer and more effective natural compounds for OP treatment.

Bone formation contains a complex series of events including osteoblastic differentiation. In the process of osteoblastic differentiation, bone morphogenetic protein-2 (BMP-2), a member of the BMP subfamily, is responsible for the production of bone-specific matrix proteins (14). BMP-2 participates in osteoblastic differentiation by activating osteoblast-associated transcriptional factors such as Osterix, runt-related transcription factor 2 (Runx2) and osteocalcin (15,16). Notably, numerous studies have indicated that protein kinase A (PKA) plays a crucial role in osteoblast differentiation and could regulate several osteoblast-specific transcriptional factors (17,18). However, the functional role of PKA still requires further exploration.

Pinoresinol (PINO), a high-value plant-derived lignan, is the third most abundant phenolic compound in virgin olive oil (VOO) (19). PINO was demonstrated to be able to notably inhibit the proliferative ability of human leukemia cells (20), and attenuate colon cancer progression by inducing tumor cell cycle arrest and apoptosis (21). Furthermore, a previous study indicated that the phenolic compounds of VOO could promote osteoblast proliferation, however, the signaling pathways involved in this treatment effect of VOO are still unclear (22). PINO is a phytoestrogen and has a chemical structure that is similar to that of E2, therefore, it was speculated that PINO could have similar functional effects as estrogen on OP. The aim of the present study was to investigate the effects of PINO on osteoblastic proliferation and differentiation and the underlying mechanism.

Materials and methods

Cell culture and viability detection

The mouse osteoblast MC3T3-E1 cell line (cat. no. CRL-2595; ATCC) was cultured in α-Minimum Essential Medium (α-MEM, cat. no. A1049001; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; cat. no. 30-2020; ATCC), 1% penicillin and 1% streptomycin at 37°C in the presence of 5% CO2. The medium was refreshed every 3 days. The morphology of MC3T3-E1 cells was observed using an inverted microscope (Nikon Eclipse TC 100; Nikon Corporation).

MC3T3-E1 cell viability was examined using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc.). Briefly, MC3T3-E1 cells were embedded in 96-well plates at a cell density of 4×103 cells/well for 24 h at 37°C with 5% CO2. Then, the cells were treated with 0, 10−7, 10−6, 10−5, 10−4, 10−3, 10−2, 0.1, 1 and 100 µg/l PINO (CAS no. 487-36-5; Sigma-Aldrich; Merck KGaA) concentrations. The treatment of 0.01 µmol/l E2 served as a positive control. After 24 and 48 h of incubation, 10 µl CCK-8 reagent was added to each well and cultured for another 1 h. Absorbance at 450 nm was examined using a microplate reader (Bio-Rad Laboratories, Inc.).

Cell migration assay

The logarithmic phase of MC3T3-E1 cells was trypsinized and collected by centrifugation at 3,000 × g for 5 min. The cells were randomly divided into five groups and incubated in 96-well plates (3×104 cells/well) at 37°C with 5% CO2, including a control group (untreated cells), PINO group (cells were treated with 0.1 µg/l PINO for 2 days) and E2 group. The treated MC3T3-E1 cells were plated in 6-cm culture dishes and maintained in the presence of 5% CO2 at 37°C. After cell confluence reached >90%, a cell-free line was created by a 200 µl sterile pipette tip. At 48 h after the scratch, wound healing images were captured on a light microscope and the migration rates were calculated based on the changes of the width of wound closure.

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA from MC3T3-E1 cells was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.). Total extract (1 µg) was reversely-transcribed by iScript™ reverse transcription (Bio-Rad Laboratories, Inc.). RT-qPCR was performed using the SYBR Green real-time PCR Master mix (Toyobo Co., Ltd.) on an ABI PRISM 7000 Sequence 10 Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Reaction mixture (20 µl) consisted of each primer (10 µM), 10 µl SYBR fluorescent dye, 2 µl cDNA and RNase-Free dH2O. The basic protocol used for the RT-PCR analysis was an initial denaturation at 95°C for 10 min, followed by 45 cycles at 95°C for 15 sec, 56°C for 30 sec and elongation at 72°C for 2 min. The mRNA expression levels were normalized against that of GAPDH, and the levels of the transcripts were quantified using the 2−ΔΔCq method (23). All primers are listed in Table I.

Table I.

Primers for reverse transcription-quantitative PCR.

Table I.

Primers for reverse transcription-quantitative PCR.

Gene namePrimer sequences
Col-IF: 5′-GAGCGGAGTACTGGATCG-3′
R: 5′-GCTTCTTTTCCTTGGGGTT-3′
ALPF: 5′-GATCATTCCCACGTTTTCACATT-3′
R: 5′-TTCACCGTCCACCACCTTGT-3′
OPNF: 5′- GCCACAAGTTTCACAGCCAC-3′
R: 5′-AAAATGCAGTGGCCGTTTGC-3′
Runx2F: 5′-AGCGGCAGAATGGATGAGTC-3′
R: 5′-ACCAGACAACACCTTTGACG-3
BMP-2F: 5′-TGAGCAAAGTGCTTGCACAC-3′
R: 5′-AGCCCCCTGGAAGGGATTAT-3′
GAPDHF: 5′-AGGTCGGTGTGAACGGATTTG-3′
R: 5′-GGGGTCGTTGATGGCAACA-3

[i] Col-I, collagen type I; ALP, alkaline phosphatase; OPN, osteopontin; Runx2, runt-related transcription factor 2; BMP-2, bone morphogenetic protein-2; F, forward; R, reverse.

Western blotting

The treated MC3T3-E1 cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentrations were quantified by BCA Protein Assay reagent (Pierce; Thermo Fisher Scientific, Inc.). Total protein (20 µg) was run on 12% sodium dodecyl sulfate-polyacrylamide gel and electro-transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.), which were then blocked with 5% non-fat dry milk in Tris-buffered saline, followed by the incubation with the primary antibodies anti-collagen type I (Col-I; 1:5,000; 130 kDa; cat. no. ab34710), anti-ALP (1:500; 39 kDa, cat. no. ab83259), anti-osteopontin (OPN; 1:1,000; 66 kDa, cat. no. ab8448), anti-Runx2 (1:1,000; 57 kDa; cat. no. ab23981), and anti-BMP-2 (1:1,000; 45 kDa; cat. no. ab14933; all from Abcam), anti-PKA (1:1,000; 42 kDa; cat. no. 4782), anti-p-cAMP response element-binding protein (CREB; 1:1,000; 43 kDa; cat. no. 9198) and anti-CREB (1:1,000; 43 kDa; cat. no. 9197; all from Cell Signaling Technology (CST)) overnight at 4°C. Following the primary antibodies, the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibodies (cat. no. ab205718; Abcam) at 4°C for 1 h. Protein bands were detected with an enhanced chemiluminescence detection system (EMD Millipore), and GAPDH (1:1,000; 36 kDa; cat. no. ab8245; Abcam) was used as the internal control.

Alkaline phosphatase (ALP) activity assay

The cells from all the groups were embedded into 24-well plates and maintained in 5% CO2 at 37°C for 2 days. The cells were washed 2 times with phosphate-buffered saline (PBS) and lysed in 0.2% Triton X-100. Then, cell lysates were subjected to a 5-min centrifugation at 10,000 × g. The supernatant was mixed with p-nitrophenyl phosphate (pNPP) (Sigma-Aldrich; Merck KGaA) and maintained at 37°C for 15 min. NaOH (2 mol/l) was used for terminating the reaction. ALP activity at 405 nm was examined under a microplate reader.

Mineralization detection

Alizarin red staining was used to determine the mineralization level of MC3T3-E1 cells. Firstly, Alizarin red S could selectively bind to calcium and produce a dark red stain. Each group of cells (2×105) was respectively cultured in 24-well plates for 14 days and the medium was changed every 3 days. After wiping off the medium, the cells were then stained with 40 mM Alizarin red S (pH 4.2; Sigma-Aldrich; Merck KGaA) for 15 min with gentle agitation, and the images of calcified matrices were captured by light microscope.

Enzyme-linked immunosorbent assay (ELISA)

All treated MC3T3-E1 cells were resuspended by cell extraction buffer (Thermo Fisher Scientific, Inc.) containing 1 mM PMSF and protease inhibitor cocktail. Total cAMP levels in the supernatant of each group of cells were assessed by commercially available cAMP ELISA kits (cat. no. STA-501; Cell Biolabs, Inc.). In this assay, these plates were coated overnight with specific antibodies to cAMP (cat. no. ab134901; 1:1,000; Abcam), and the following morning, the plates were washed and blocked for 2 h. The prepared standard liquid and diluted supernatant (supernatant: diluent, 1:4) were added to an ELISA plate. After being washed by PBS, the streptavidin-horseradish peroxidase-conjugated (HRP) antibody-labeled secondary cAMP (cat. no. ab205719; Abcam) was added to each well at room temperature for 1 h of incubation. After an additional washing step, tetramethylbenzidine substrate solution (Clinical Science Products, Inc.) was added to produce a substrate solution. The enzymatic activities were then quantified by detecting the optical density at 450 nm with a microplate reader.

Pathway inhibition

To further explore whether the cAMP/PKA signaling pathway participated in the functional effects of PINO on osteoblast proliferation and differentiation, H89, an effective inhibitor of PKA, was used for co-treatment with PINO. The effects on the MC3T3-E1 cells were assessed by CCK-8 kit and wound healing assay.

Statistical analysis

Data are expressed as the mean ± SD, and statistical analysis among three and more groups was performed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A P-value <0.05 was considered to indicate a statistically significant difference.

Results

PINO treatment enhances cell viability and migratory capacity of MC3T3-E1 cells

The morphology of MC3T3-E1 cells was observed under a light microscope, and as revealed in Fig. 1A, the MC3T3-E1 cells were cuboidal or fusiform. After treatment with various concentrations of PINO, it was revealed (Fig. 1B) that low concentrations of PINO had a positive effect on MC3T3-E1 cell viability, and PINO treatment at 0.1 µg/l for 48 h had the most significant effect on cell viability. Hence, PINO at a concentration of 0.1 µg/l was selected for the following experiments. To further investigate the functional effects of PINO on the MC3T3-E1 cells, the migratory capacity was assessed by wound healing assay. As revealed in Fig. 1C, E2 was set as the positive control, and the migration rate in the E2 group was greater than that in the control group (P<0.01). PINO treatment had similar effects to E2 on MC3T3-E1 cells and it significantly enhanced the migratory ability. Collectively, the results revealed that PINO had similar effects to E2 on osteoblastic viability and migration.

PINO treatment promotes the expression of differentiation-associated factors in MC3T3-E1 cells

For the assessment of osteoblastic differentiation, the expression levels of several osteoblast-specific genes (Col-I, ALP, OPN, Runx2, and BMP-2) were detected in MC3T3-E1 cells. As one of the first-line drugs for OP treatment, E2 significantly increased the mRNA and protein levels of these osteoblastic differentiation-related genes (P<0.01, Fig. 2). In addition, the present results revealed that PINO had similar effects to E2. Although the effects of PINO on inducing Col-I, ALP, OPN, Runx2 and BMP-2 expression were not as strong as those of E2, the mRNA and protein levels of these genes were also significantly upregulated under PINO treatment. Therefore, the results indicated that PINO had the ability to promote osteoblastic differentiation.

PINO stimulates ALP activity and mineralization in MC3T3-E1 cells

ALP activity and mineralization were also detected to further determine MC3T3-E1 cell differentiation. After incubation with α-MEM in the presence of PINO and E2, pNPP was used as a substrate for ALP activity detection. In comparison to the control group, the ALP activity in the E2 group was the highest and the PINO group also had a significant increase (P<0.05), as revealed in Fig. 3A. After Alizarin red staining, the red region in the PINO and E2 groups were denser than in the control group (Fig. 3B), indicating that both PINO and E2 contributed to the mineralization of MC3T3-E1 cells. Collectively, our findings indicated that PINO treatment could promote the differentiation of osteoblastic MC3T3-E1 cells.

The cAMP/PKA signaling pathway participates in the promotive effects of PINO on osteoblastic differentiation

As aforementioned, the cAMP/PKA signaling pathway was involved in the regulation of multiple osteoblast-specific transcriptional factors. The protein levels of cAMP, PKA, phosphorylated and unphosphorylated CREB were assessed to further study the molecular mechanism of the effect of PINO on MC3T3-E1 cells. As revealed in Fig. 4A, a significantly increased cAMP level was revealed in the PINO and E2 groups, compared to the control group (P<0.01), and the cAMP level in the PINO group was slightly lower than that in the E2 group. Similarly, the protein level of PKA was also notably upregulated under the treatment of PINO or E2 (P<0.01). PINO had limited effect on unphosphorylated CREB expression, however, the phosphorylation levels of CREB were markedly increased (P<0.01, Fig. 4B). Moreover, the effects of PINO on cAMP/PKA signals were not as strong as E2. Collectively, the promoting effect of PINO on osteoblastic differentiation may partially rely on the activation of cAMP/PKA signals.

H89 co-treatment reverses the positive effects of PINO on the viability and migration of MC3T3-E1 cells

The H89 solution was used as an inhibitor of the cAMP/PKA signaling pathway to further verify the role of cAMP/PKA in the functional effects of PINO on MC3T3-E1 cells. In comparison to the control, H89 treatment alone significantly decreased cell viability (P<0.01, Fig. 5A). When cells were co-treated with H89, the positive effects of PINO on MC3T3-E1 cell viability were also abolished (P<0.01). In addition, H89 treatment alone could significantly decrease the migration rate by less than half of the control group. Furthermore, H89 co-treatment could also significantly limit the capacity of PINO in enhancing the migration of MC3T3-E1 cells (P<0.01, Fig. 5B and C). Therefore, the present results indicated that the functional effects of PINO on osteoblastic differentiation and proliferation may be attributed to the activation of cAMP/PKA signals.

Discussion

It is recognized that osteoblasts occupy a fundamental place in bone formation and remodeling, therefore, enhancing osteoblastic proliferation and differentiation can radically improve OP treatment (24,25). E2, one of the first-line drugs for OP treatment, could effectively enhance osteoblastic proliferation and differentiation, however, the side effects of a long-term use of E2 make it imperative to identify safer and more effective drugs. In the present study, E2 treatment was set as the positive control to assess whether PINO treatment had the ability to promote osteoblastic proliferation and differentiation in MC3T3-E1 cells. Although a previous study revealed that PINO did not affect cell proliferation of osteoblasts (22), the result was not in conflict with our study due to the differences in the concentration of PINO. The present results indicated that only PINO at a concentration of 1×10−2 µg/l could induce a slight upregulation of MC3T3-E1 cell viability, and promote cell migration. In addition, PINO had a positive effect on osteoblastic differentiation by inducing the expression of multiple differentiation-associated genes. Moreover, the involvement of cAMP/PKA signaling pathway played a vital role in the molecular mechanism of the effect of PINO on osteoblastic proliferation and differentiation. Although the positive effects of PINO on osteoblastic proliferation and differentiation were not as strong as those of E2, the present results indicated that PINO has the potential to replace E2 in terms of OP treatment.

Osteoblastic differentiation is a complex process mediated by a variety of regulators such as Col-I, ALP, OPN, Runx2, and BMP-2. In the present study, when treating with PINO, a significant increase of these differentiation-associated genes was revealed in MC3T3-E1 cells. The Col-I protein is a fundamental component of the bone extracellular matrix and it is responsible for connecting the cell surface integrins with other extracellular matrix proteins (26). ALP can hydrolyze organic phosphate and pyrophosphate, subsequently promoting bone mineral formation and mineralization (27). In addition, ALP and Col-I both are recognized as the earliest biomarkers of osteoblastic differentiation (28,29). A previous study indicated that the treatment of fermented red ginseng extract (FRGE) could enhance the ALP level and Col-I expression, ultimately improving bone formation and MC3T3-E1 cell differentiation (30). Therefore, the increased ALP activity, mineralization and Col-I in the present results indicated that PINO treatment could promote osteoblastic MC3T3-E1 cell differentiation. OPN is a type of acidic protein secreted by osteoblasts and plays a crucial role in biomineralization and bone remodeling (31). As another specific marker of bone formation, increased OPN expression in the present study also indicated that PINO treatment contributed to the improvement of osteoblastic differentiation. It is recognized that BMPs can induce undifferentiated mesenchymal cells to gather and differentiate to osteoblasts and chondrocytes, finally promoting osteogenic activity (32). In addition, BMP-2 expression induced the activated downstream transcriptional factors (Runx2 and Osterix) to bind to the promoter regions of ALP and OPN (33). Aucubin, an iridoid glucoside separated from multiple Chinese herbs, was reported to inhibit titanium particle-induced osteoblast apoptosis and promote bone formation by activating the BMP-2/Runx2 pathway (34). Collectively, the promoting effect of PINO treatment on osteoblastic differentiation and osteogenesis may rely on the regulation of BMP-2/Runx2 pathway.

In the cAMP/PKA signaling pathway, PKA functions as a second messenger to mediate the various functions of cAMP in mammalian cells. A previous study demonstrated that PKA played an essential role in the process of the promotion of osteoblastic differentiation (35). In addition, another study demonstrated that cAMP could promote drosophila mothers against decapentaplegic protein (SMAD)-mediated BMP-2/Runx2 signaling via the PKA pathway (36). Hence, the potential relationship between the cAMP/PKA and BMP-2/Runx2 signaling pathways was surmised based on this research. Recent research revealed that linarin (LIN), a natural flavonoid compound in Flos Chrysanthemi Indici (FCI), could enhance the expression of BMP-2/Runx2 signals via the activation of the cAMP/PKA signaling pathway. However, when the cells were treated with H89, the positive effects of LIN were effectively abrogated (37). This research was basically consistent with the present results. After co-treatment with a PKA inhibitor (H89), the increased cAMP, PKA and phosphorylated CREB expression levels in the PINO group were nearly decreased to the control levels (data not shown). Furthermore, PINO was also reported to be able to inhibit cell viability and promote apoptosis of human leukemia cell line (HL60) and colorectal cancer (CRC) cell lines through the upregulation of the CDK inhibitor p21Waf1/Cip1 or ATM-p53 cascade (20,21). However, there are many differences in the gene expression, metabolism and other cellular morphological and phenotypic profiles between HL60 or CRC cells and MC3T3-E1 cells due to cell heterogeneity. This does not signify that PINO could also upregulate the p21 and ATM-p53 cascade, and subsequently, inhibit cell proliferation and differentiation in MC3T3-E1 cells. In the present study, the data demonstrated a promoting effect of PINO on osteoblast proliferation and differentiation. Collectively, the present study indicated that PINO-induced osteoblastic differentiation was largely attributed to the regulation of the cAMP/PKA signaling pathway.

There were some limitations in our study. The duration of PINO treatment was too short to completely reflect the effects of PINO on the growth and differentiation of osteoblasts. Moreover, the effects of PINO on the ER pathway require further investigation.

In summary, the present study revealed that PINO had a similar function to E2 in OP treatment. PINO induced osteoblast differentiation and mineralization by regulating BMP-2/Runx2 signaling via activation of the cAMP/PKA pathway. Collectively, PINO may be considered as an alternative to E2 in OP treatment.

Acknowledgements

Not applicable.

Funding

The present study was supported by The Qiqihar Science and Technology Plan Project (grant no. SFZD-2015037).

Availability of data and materials

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Authors' contributions

XJ and WC substantially contributed to the conception and design of the study. FS, JX, HG, WS, HS and WX performed the data acquisition, data analysis and interpretation. XJ and WC drafted the study and critically revised it for important intellectual content. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Tang D, Ju C, Liu Y, Xu F, Wang Z and Wang D: Therapeutic effect of icariin combined with stem cells on postmenopausal osteoporosis in rats. J Bone Miner Metab. 36:180–188. 2018. View Article : Google Scholar : PubMed/NCBI

2 

Krege JH and Wan X: Teriparatide and the risk of nonvertebral fractures in women with postmenopausal osteoporosis. Bone. 50:161–164. 2012. View Article : Google Scholar : PubMed/NCBI

3 

Hannafon F and Cadogan MP: Recognition and treatment of postmenopausal osteoporosis. J Gerontol Nurs. 40:10–14. 2014. View Article : Google Scholar : PubMed/NCBI

4 

Ming LG, Lv X, Ma XN, Ge BF, Zhen P, Song P, Zhou J, Ma HP, Xian CJ and Chen KM: The prenyl group contributes to activities of phytoestrogen 8-prenynaringenin in enhancing bone formation and inhibiting bone resorption in vitro. Endocrinology. 154:1202–1214. 2013. View Article : Google Scholar : PubMed/NCBI

5 

Ke K, Li Q, Yang X, Xie Z, Wang Y, Shi J, Chi L, Xu W, Hu L and Shi H: Asperosaponin VI promotes bone marrow stromal cell osteogenic differentiation through the PI3K/AKT signaling pathway in an osteoporosis model. Sci Rep. 6:352332016. View Article : Google Scholar : PubMed/NCBI

6 

Antebi B, Pelled G and Gazit D: Stem cell therapy for osteoporosis. Curr Osteoporos Rep. 12:41–47. 2014. View Article : Google Scholar : PubMed/NCBI

7 

Vico L and Vanacker JM: Sex hormones and their receptors in bone homeostasis: Insights from genetically modified mouse models. Osteoporos Int. 21:365–372. 2010. View Article : Google Scholar : PubMed/NCBI

8 

Reginster JY, Pelousse F and Bruyere O: Safety concerns with the long-term management of osteoporosis. Expert Opin Drug Saf. 12:507–522. 2013. View Article : Google Scholar : PubMed/NCBI

9 

Cosman F: Long-term treatment strategies for postmenopausal osteoporosis. Curr Opin Rheumatol. 30:420–426. 2018. View Article : Google Scholar : PubMed/NCBI

10 

Lewiecki EM: Safety of long-term bisphosphonate therapy for the management of osteoporosis. Drugs. 71:791–814. 2011. View Article : Google Scholar : PubMed/NCBI

11 

Fan JZ, Yang L, Meng GL, Lin YS, Wei BY, Fan J, Hu HM, Liu YW, Chen S, Zhang JK, et al: Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway. Mol Cell Biochem. 392:85–93. 2014. View Article : Google Scholar : PubMed/NCBI

12 

Burkman RT: Hormone replacement therapy. Current controversies. Minerva Ginecol. 55:107–116. 2003.PubMed/NCBI

13 

Komm BS, Morgenstern D, A Yamamoto L and Jenkins SN: The safety and tolerability profile of therapies for the prevention and treatment of osteoporosis in postmenopausal women. Expert Rev Clin Pharmacol. 8:769–784. 2015. View Article : Google Scholar : PubMed/NCBI

14 

Canalis E, Economides AN and Gazzerro E: Bone morphogenetic proteins, their antagonists and the skeleton. Endocr Rev. 24:218–235. 2003. View Article : Google Scholar : PubMed/NCBI

15 

Phimphilai M, Zhao Z, Boules H, Roca H and Franceschi RT: BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype. J Bone Miner Res. 21:637–646. 2006. View Article : Google Scholar : PubMed/NCBI

16 

Yang B, Lin X, Tan J, She X, Liu Y and Kuang H: Root bark of Sambucus Williamsii Hance promotes rat femoral fracture healing by the BMP-2/Runx2 signaling pathway. J Ethnopharmacol. 191:107–114. 2016. View Article : Google Scholar : PubMed/NCBI

17 

He S, Choi YH, Choi JK, Yeo CY, Chun C and Lee KY: Protein kinase A regulates the osteogenic activity of Osterix. J Cell Biochem. 115:1808–1815. 2014. View Article : Google Scholar : PubMed/NCBI

18 

Li H, Jeong HM, Choi YH, Kim JH, Choi JK, Yeo CY, Jeong HG, Jeong TC, Chun C and Lee KY: Protein kinase a phosphorylates Dlx3 and regulates the function of Dlx3 during osteoblast differentiation. J Cell Biochem. 115:2004–2011. 2014.PubMed/NCBI

19 

Owen RW, Mier W, Giacosa A, Hull WE, Spiegelhalder B and Bartsch H: Identification of lignans as major components in the phenolic fraction of olive oil. Clin Chem. 46:976–988. 2000.PubMed/NCBI

20 

Sepporta MV, Mazza T, Morozzi G and Fabiani R: Pinoresinol inhibits proliferation and induces differentiation on human HL60 leukemia cells. Nutr Cancer. 65:1208–1218. 2013. View Article : Google Scholar : PubMed/NCBI

21 

Fini L, Hotchkiss E, Fogliano V, Graziani G, Romano M, De Vol EB, Qin H, Selgrad M, Boland CR and Ricciardiello L: Chemopreventive properties of pinoresinol-rich olive oil involve a selective activation of the ATM-p53 cascade in colon cancer cell lines. Carcinogenesis. 29:139–146. 2008. View Article : Google Scholar : PubMed/NCBI

22 

García-Martínez O, De Luna-Bertos E, Ramos-Torrecillas J, Ruiz C, Milia E, Lorenzo ML, Jimenez B, Sánchez-Ortiz A and Rivas A: Phenolic compounds in extra virgin olive oil stimulate human osteoblastic cell proliferation. PLoS One. 11:e01500452016. View Article : Google Scholar : PubMed/NCBI

23 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

24 

Luo Z, Liu M, Sun L and Rui F: Icariin recovers the osteogenic differentiation and bone formation of bone marrow stromal cells from a rat model of estrogen deficiency-induced osteoporosis. Mol Med Rep. 12:382–388. 2015. View Article : Google Scholar : PubMed/NCBI

25 

Zhu C, Bao N, Chen S and Zhao J: Dioscin enhances osteoblastic cell differentiation and proliferation by inhibiting cell autophagy via the ASPP2/NF-κβ pathway. Mol Med Rep. 16:4922–4926. 2017. View Article : Google Scholar : PubMed/NCBI

26 

Li B, Hu RY, Sun L, Luo R, Lu KH and Tian XB: Potential role of andrographolide in the proliferation of osteoblasts mediated by the ERK signaling pathway. Biomed Pharmacother. 83:1335–1344. 2016. View Article : Google Scholar : PubMed/NCBI

27 

Ma XN, Ma CX, Shi WG, Zhou J, Ma HP, Gao YH, Xian CJ and Chen KM: Primary cilium is required for the stimulating effect of icaritin on osteogenic differentiation and mineralization of osteoblasts in vitro. J Endocrinol Invest. 40:357–366. 2017. View Article : Google Scholar : PubMed/NCBI

28 

Shao X, Cao X, Song G, Zhao Y and Shi B: Metformin rescues the MG63 osteoblasts against the effect of high glucose on proliferation. J Diabetes Res. 2014:4539402014. View Article : Google Scholar : PubMed/NCBI

29 

Fusaro M, Crepaldi G, Maggi S, D'Angelo A, Calo L, Miozzo D, Fornasieri A and Gallieni M: Bleeding, vertebral fractures and vascular calcifications in patients treated with warfarin: Hope for lower risks with alternative therapies. Curr Vasc Pharmacol. 9:763–769. 2011. View Article : Google Scholar : PubMed/NCBI

30 

Siddiqi MZ, Siddiqi MH, Kim YJ, Jin Y, Huq MA and Yang DC: Effect of fermented red ginseng extract enriched in ginsenoside Rg3 on the differentiation and mineralization of preosteoblastic MC3T3-E1 cells. J Med Food. 18:542–548. 2015. View Article : Google Scholar : PubMed/NCBI

31 

Choi ST, Kim JH, Kang EJ, Lee SW, Park MC, Park YB and Lee SK: Osteopontin might be involved in bone remodelling rather than in inflammation in ankylosing spondylitis. Rheumatology (Oxford). 47:1775–1779. 2008. View Article : Google Scholar : PubMed/NCBI

32 

Lee SH, Park Y, Song M, Srikanth S, Kim S, Kang MK, Gwack Y, Park NH, Kim RH and Shin KH: Orai1 mediates osteogenic differentiation via BMP signaling pathway in bone marrow mesenchymal stem cells. Biochem Biophys Res Commun. 473:1309–1314. 2016. View Article : Google Scholar : PubMed/NCBI

33 

Nishimura R, Hata K, Matsubara T, Wakabayashi M and Yoneda T: Regulation of bone and cartilage development by network between BMP signalling and transcription factors. J Biochem. 151:247–254. 2012. View Article : Google Scholar : PubMed/NCBI

34 

Zhu Z, Xie Q, Huang Y, Zhang S and Chen Y: Aucubin suppresses Titanium particles-mediated apoptosis of MC3T3-E1 cells and facilitates osteogenesis by affecting the BMP2/Smads/RunX2 signaling pathway. Mol Med Rep. 18:2561–2570. 2018.PubMed/NCBI

35 

Lo KW, Kan HM, Ashe KM and Laurencin CT: The small molecule PKA-specific cyclic AMP analogue as an inducer of osteoblast-like cells differentiation and mineralization. J Tissue Eng Regen Med. 6:40–48. 2012. View Article : Google Scholar : PubMed/NCBI

36 

Ohta Y, Nakagawa K, Imai Y, Katagiri T, Koike T and Takaoka K: Cyclic AMP enhances Smad-mediated BMP signaling through PKA-CREB pathway. J Bone Miner Metab. 26:478–484. 2008. View Article : Google Scholar : PubMed/NCBI

37 

Li J, Hao L, Wu J, Zhang J and Su J: Linarin promotes osteogenic differentiation by activating the BMP-2/RUNX2 pathway via protein kinase A signaling. Int J Mol Med. 37:901–910. 2016. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

September-2019
Volume 20 Issue 3

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Jiang X, Chen W, Shen F, Xiao W, Guo H, Su H, Xiu J and Sun W: Pinoresinol promotes MC3T3‑E1 cell proliferation and differentiation via the cyclic AMP/protein kinase A signaling pathway. Mol Med Rep 20: 2143-2150, 2019
APA
Jiang, X., Chen, W., Shen, F., Xiao, W., Guo, H., Su, H. ... Sun, W. (2019). Pinoresinol promotes MC3T3‑E1 cell proliferation and differentiation via the cyclic AMP/protein kinase A signaling pathway. Molecular Medicine Reports, 20, 2143-2150. https://doi.org/10.3892/mmr.2019.10468
MLA
Jiang, X., Chen, W., Shen, F., Xiao, W., Guo, H., Su, H., Xiu, J., Sun, W."Pinoresinol promotes MC3T3‑E1 cell proliferation and differentiation via the cyclic AMP/protein kinase A signaling pathway". Molecular Medicine Reports 20.3 (2019): 2143-2150.
Chicago
Jiang, X., Chen, W., Shen, F., Xiao, W., Guo, H., Su, H., Xiu, J., Sun, W."Pinoresinol promotes MC3T3‑E1 cell proliferation and differentiation via the cyclic AMP/protein kinase A signaling pathway". Molecular Medicine Reports 20, no. 3 (2019): 2143-2150. https://doi.org/10.3892/mmr.2019.10468