Alendronate promotes the gene expression of extracellular matrix mediated by SP-1/SOX-9

Abstract

Background and purpose:

Osteoarthritis (OA) is a disease with significant degenerative changes of articular cartilage, which is reported to be closely related to the integrity of chondrocytes extracellular matrix (ECM). Alendronate belongs to the family of bisphosphonates with promising cartilage repair function. In the present study, the effects of Alendronate on the gene expression of chondrocytes ECM and the potential mechanism will be investigated to explore the potential therapeutic property of Alendronate on OA.

Methods:

Human SW1353 chondrocytes were stimulated with 1 and 2 μM Alendronate for 12 h. The gene expression of Col2α1, COL9α2, and Acan in the treated chondrocytes was determined by qRT-PCR. QRT-PCR and western blot analysis were used to evaluate the expression level of SOX-9 in the treated chondrocytes. The expression level of SP-1 was checked by qRT-PCR and immunostaining. SiRNA against SP-1 was transfected into chondrocytes to knockdown the expression of SP-1. The levels of p-ERK1/2 and total ERK1/2 were examined using western blot analysis. TNF-α was used to induce an OA-like in vitro model in the chondrocytes for therapeutic evaluations.

Results:

Treatment with Alendronate increased the levels of ECM related genes (Col2α1, COL9α2, and Acan) in a dose-dependent manner through increasing the expression of SOX-9, a central regulator of ECM genes. Additionally, our findings demonstrate that the effects of Alendronate in the expression of SOX-9 are mediated by SP-1 as silencing of SP-1 abolished these effects. Notably, Alendronate increased the phosphorylation of ERK1/2 and inhibition of ERK1/2 using its specific inhibitor U0126 blocked the expression of SP-1. Finally, we found that treatment with Alendronate could rescue TNF-α-induced reduction of Col2α1, COL9α2, Acan and SOX-9.

Conclusion:

Our data indicated that Alendronate might promote the gene expression of extracellular matrix through SOX-9 mediated by the ERK1/2/SP1 signaling pathway.

Keywords

Alendronate chondrocytes extracellular matrix SOX-9 osteoarthritis

Introduction

Osteoarthritis (OA) is a common disease induced by degenerative changes of articular cartilage, which is mainly clinically characterized with joint pain and dysfunction.¹ According to the survey data of America, the morbidity of OA in the population with age over 75 detected by radiation is approximately 80% and OA has become the second reason contributing to the disablement for males over 50 years old.² The pathological mechanism underlying OA is currently unknown. Recently, more and more reports claim that cartilage deficiency induced by the imbalance between synthesis and metabolism in the extracellular matrix (ECM) of chondrocytes plays an important role in the development and processing of OA.³ Generally, OA is pathologically characterized with the apoptosis of chondrocytes and the degenerative degradation of ECM, which is the site where chondrocytes exert physiological functions and the vector by which chondrocytes uptake the nutrition and deliver the signals. The normal function of cartilage tissues is maintained by the metabolic balance of ECM. The ECM of chondrocytes are mainly consisting of collagens and proteoglycans. The ability of a cartilage to withstand stress and tension is directly depending on collagenous fibers. Type II collagen, which occupies approximately 90%–95% in total collagens and encoded mainly by Col2α1, is found to be expressed in almost all kinds of articular cartilages and control the expansional and water absorption characteristics of proteoglycans to engraft the cartilage with tension.^4,5 Matyas et al. reported that the collagen encoded genes, such as Col2α1 and COL9α2, were significantly upregulated with compensatory extend in the early stage of animal OA model. However, as the development of the disease and the increasing of collagenase and polyproteoglycolase, the network structure of collagen fiber would be destroyed, companied by collagen fibrinolysis and loss, which contribute to the decreased protective property of collagens on chondrocytes and cartilage tissues.⁶ Therefore, it is of great significance to maintain the structure of chondrocytes ECM for the treatment of OA. SOX-9 is recently discovered as a pivotal transcription factor in developing cartilage, which is indispensable to enhance the chondrocytes survival, maintain the expression of cartilage markers, such as Col2α1, COL9α2, and Acan,^7,8 the expression of which could be regulated by SP-1, an important transcription factor discovered recently.⁹ SOX-9 might become a promising molecular target for the clinical treatment of OA.

Inflammatory factors are reported to be the main inducer to the degradation of ECM in chondrocytes. Tumor necrosis factor-α (TNF-α) has been proved to inhibit the synthesis of cartilage matrix and induce the production of matrix metalloproteinase (MMPs) to trigger the degradation of cartilage matrix.^10,11 The results of immunohistochemistry indicate that the destroy of osteoarthritis cartilage is closely correlated to the production of TNF-α in local tissues.¹² TNF-α exerts an important role in the development of osteoarthritis cartilage degeneration by targeting and binding with the specific receptors, TNFRs.¹³ In the present study, TNF-α was used to induce an OA-like in vitro model in the chondrocytes for therapeutic evaluations.

Alendronate, the structure of which is shown in Figure 1, is the third generation of bisphosphonates approved by Food and Drug Administration (FDA) for the treatment of osteoporosis.¹⁴ Similar to other bisphosphonates widely applied in clinic, Alendronate inhibits the bone resorption indirectly by inducing the apoptosis of osteoclasts, promoting the production of osteoblasts and maintaining the bone balance.^15,16 Notably, protective effects of Alendronate against inflammatory factors stimulated chondrocytes have been recently reported.^17,18 In the present study, molecular mechanism underlying the protective effects of Alendronate on chondrocytes will be investigated by exploring the impact of Alendronate on the chondrocytes ECM. The findings will provide fundamental basis for the clinical treatment of OA with Alendronate.

Figure 1.

Molecular structure of Alendronate.

Materials and methods

Cell culture and treatment

Human SW1353 chondrocytes were purchased from the Type Culture Collection of the Chinese Academy of Sciences (TCCCAS, Shanghai, China) and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO₂ for subsequent experiments. To determine the effects of Alendronate in gene expression of ECM, cells were stimulated with 1, 2 μM Alendronate for 12 h.

Real-time PCR assay

A TRIzol solution (Thermo Fisher, Massachusetts, USA) was used to extract total RNA from the treated chondrocytes. The cDNAs were transcribed from RNA using a SuperScript III (Takara, Tokyo, Japan) reagent. A SYBR green quantitative PCR kit (Takara, Tokyo, Japan) was used to determine the relative expression of detected proteins, with GADPH taken as a negative control. The ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems) was applied in the present study for qRT-PCR. Levels of relative expression were calculated and quantified with the 2^−ΔΔCt method.

Transfection of small-interfering RNA (siRNA)

To knockdown the expression of SOX-9, the chondrocytes were transfected with a siRNA against the mRNA of SOX-9 (Genscript, Nanjing, China). The siRNA was co-transfected with a lipofectamine® 3000 transfection reagent (Takara, Tokyo, Japan) according to the instructions of the manufacturer.

Immunofluorescence staining

The treated chondrocytes were washed with PBS solution and incubated with the primary antibodies against SP-1, followed by incubation with appropriate TRITC-conjugated secondary antibodies. Nuclei were mounted with 4′,6-diamidino-2-phenylindole (DAPI). Negative control staining was performed by omitting the primary antibody. The pictures of the stained cells were taken by a fluorescence microscope.

Western blotting assay

The chondrocytes were lysed with a RIPA lysis buffer containing the protease and phosphatase inhibitors, followed by centrifugation at 14,000 rpm for 20 min at 4°C. The total concentration of proteins was detected using a BCA assay kit (Thermo Fisher Scientific, USA). Approximately 25 µg of protein for each sample was separated by a SDS-PAGE, followed by transferring to a PVDF membranes (Millipore, Massachusetts, USA). The membranes were incubated with 5% non-fat milk for 90 min to remove the non-specific proteins binding sites, followed by incubation with rabbit anti-SOX-9, ERK1/2, or p-ERK1/2 (1:1,000, Abcam, Massachusetts, USA) antibody at 4°C overnight. Following washing with 0.1% TBST, the membranes were incubated with secondary antibodies and the immuno-reactive bands were visualized using an ECL kit (Beyotime, Shanghai, China). The specific bands were analyzed using the software Image J.

Statistical analysis

The software SPSS 19.0 was used for the data analysis. All data were presented as mean ± standard deviation (S.D.). For comparisons of three or more groups, analysis of variance (ANOVA) analysis was carried out following by the Bonferroni post-hoc test. P < 0.05 was considered as statistically significant.

Results

The expression of Col2α1, COL9α2, and Acan in human SW1353 chondrocytes was promoted by Alendronate

To evaluate the effects of Alendronate on collagen proteins, the chondrocytes were stimulated with 1, 2 μM Alendronate for 12 h. As shown in Figure 2, we found that the gene expression of Col2α1, COL9α2, and Acan in the chondrocytes was significantly upregulated by the introduction of Alendronate in a dose-dependent manner, indicating a potential protective effect of Alendronate in maintaining the integrity of ECM in chondrocytes.

Figure 2.

Alendronate increased the gene expression of Col2α1, COL9α2, and Acan in human SW1353 chondrocytes. Cells were stimulated with 1 and 2 μM Alendronate for 12 h. The gene expressions of (a) Col2α1, (b) COL9α2, and (c) Acan were measured (*P < 0.05, **P < 0.01, ***P < 0.005 vs. untreated control group).

Alendronate increased the expression of SOX-9 and SP-1 in human SW1353 chondrocytes

SOX-9 is a main transcriptional factor governing the expressions of Col2α1, COL9α2, and Acan. We further investigated the impact of Alendronate on the expression of SOX-9 to explore the potential mechanism. As shown in Figure 3, the expression of SOX-9 in chondrocytes was significantly elevated by the administration of Alendronate. To verify whether the regulatory effect of Alendronate against SOX-9 was related to SP-1, which is a transcriptional factor that mediates the expression of SOX-9. As shown in Figure 4, we found that SP-1 was greatly upregulated in chondrocytes by the administration of Alendronate, indicating a possible correlation between Alendronate and SP-1/SOX-9 signaling.

Figure 3.

Alendronate increased the expression of SOX-9 in human SW1353 chondrocytes. Cells were stimulated with 1 and 2 μM Alendronate for 12 h. (a) Gene of SOX-9; (b) protein of SOX-9 (**P < 0.01, ***P < 0.005 vs. untreated control group).

Figure 4.

Alendronate upregulated the expression of SP-1 in human SW1353 chondrocytes. Cells were stimulated with 1 and 2 μM Alendronate for 12 h. (a) mRNA of SP-1; (b) protein of SP-1 as measured by immunostaining (**P < 0.01, ***P < 0.005 vs. untreated control group).

The effects of Alendronate on SOX-9 is mediated by SP-1

To further verify the correlation between Alendronate and the SP-1/SOX-9 signaling, human SW1353 chondrocytes were transfected with SP-1 siRNA, followed by stimulation with 2 μM Alendronate for 12 h. As shown in Figure 5(a), SP-1 was significantly down-regulated in the SP-1 siRNA group, indicating that the SP-1 knockdown chondrocytes were successfully established. Figure 5(b) and (c); showed the expression level of SOX-9 in the Alendronate treated normal chondrocytes or SP-1 knocked down chondrocytes. We found that the expression level of SOX-9 was significantly promoted by the introduction of Alendronate, which was greatly suppressed by knocking down the expression level of SP-1, indicating that the effects of Alendronate in SOX-9 expression are mediated by SP-1.

Figure 5.

The effects of Alendronate in SOX-9 is mediated by SP-1. Cells were transfected with SP-1 siRNA, followed by stimulation with 2 μM Alendronate for 12 h. (a) Successful knockdown of SP-1 was measured by western blot analysis; (b and c) the gene and protein expression of SOX-9 were measured (***P < 0.005 vs. vehicle group; ^##P < 0.01 vs. Alendronate group).

The effects of Alendronate in SP-1 expression were mediated by ERK1/2

It has been reported that activation of ERK1/2 plays an important role in regulating the expression of SP-1. To further explore the mechanism underlying the regulatory effect of Alendronate in SP-1, the activation state of ERK1/2 signal pathway was evaluated. As shown in Figure 6(a), the level of p-ERK1/2 in chondrocytes was significantly elevated by treatment with Alendronate, with the level of total ERK1/2 nearly unchanged. To verify the involvement of the ERK1/2 signaling pathway in the regulatory effect of Alendronate in SP-1 in chondrocytes, the ERK1/2 signal pathway inhibitor, U0126, was introduced into the incubation system. Cells were stimulated with 2 μM Alendronate in the presence or absence of U0126 (10 μM) for 24 h. As shown in Figure 6(b) and (c), SP-1 was significantly upregulated by the stimulation of Alendronate, which was greatly suppressed by the introduction of U0126, indicating a possible correlation between the regulatory effect of Alendronate on the expression level of SP-1 and the activation of ERK1/2 signal pathway.

Figure 6.

The effects of Alendronate in SP-1 expression is mediated by ERK1/2. (a) Cells were stimulated with 1 and 2 μM Alendronate for 2 h. Phosphorylated and total levels of ERK1/2 were measured; (b and c) cells were stimulated with 2 μM Alendronate in the presence or absence of U0126 (10 μM) for 24 h, mRNA and protein levels of SP1 were measured (***P < 0.005 vs. vehicle group; ^##P < 0.01 vs. Alendronate group).

Alendronate ameliorated TNF-α-induced reduction of gene expression of extracellular matrix and SOX-9

To claim the potential therapeutic property of Alendronate against OA, the in vitro inflammatory model on chondrocytes was established by stimulation with TNF-α. Briefly, human SW1353 chondrocytes were stimulated with 10 ng/mL TNF-α in the presence or absence of 2 μM Alendronate for 12 h. As shown in Figure 7(a) to (c), the expression of Col2α1, COL9α2, and Acan in the chondrocytes was significantly down-regulated by the stimulation of TNF-α, which was greatly rescued by the administration of Alendronate. In addition, as shown in Figure 7(d) and (e), the suppressed expression level of SOX-9 induced by TNF-α was significantly reversed by the introduction of Alendronate. These data indicate that Alendronate impacted the ECM balance and SOX-9 expression in the chondrocytes not only under physiological state, but also under pathological state.

Figure 7.

Alendronate ameliorated TNF-α-induced reduction of gene expression of extracellular matrix and SOX-9. Human SW1353 chondrocytes were stimulated with 10 ng/mL TNF-α in the presence or absence of 2 μM Alendronate for 12 h. (a) Gene level of Col2α1; (b) gene level of COL9α2; (c) gene level of Acan; (d) mRNA of SOX-9; (e) protein of SOX-9 (***P < 0.005 vs. vehicle group; ^##P < 0.01 vs. TNF-α group).

Discussion

SOX-9 is an important gene related to early embryonic development and involved in multiple process of early embryonic development such as gender determination and cartilage formation.¹⁹ In 1997, Lefebvre et al.²⁰ reported a parallel expression correlation between SOX-9 and Col2α1 in the processing of cartilage formation. Col2α1 is a specific proliferous gene for type II collagen in the chondrocytes and encodes type II collagen to maintain the intergrity of ECM.²¹ In addition, in the normal cartilage tissues, relatively high expression level of SOX-9 is observed and on the contrary, SOX-9 is lowly expressed in the hypertrophic chondrocytes.²⁰ Tew et al. reported that the expression level of SOX-9 is significantly elevated in human articular chondrocytes incubated in the hypertonic cultural medium, accompanied by promoted expression level of Col2α1 in the subsequent subculturing generations.²² Bell et al. further explained that SOX-9 regulated the expression level of Col2α1 by directly binding with the specific promoter of Col2α1. ²³ In addition, in the skeletal anomalies induced by SOX-9 mutated CD syndrome, abnormal regulation of SOX-9 to Col2α1 might be involved in the pathological mechanism. In the present study, we found that ECM balance in the chondrocytes was protected by the introduction of Alendronate, accompanied by the elevated expression of SOX-9, indicating that Alendronate might maintain the function of chondrocytes ECM by promoting the expression of SOX-9. In the processing of OA, inflammatory factors released by lymphocytes are the main promoter for the degradation and injury on cartilage tissues by interfering the regular function of chondrocytes and destroying the balance of chondrocytes ECM.^24,25 In the present study, TNF-α was used to induce an OA-like in vitro model for the evaluation of Alendronate. ECM was significantly destroyed by the stimulation of TNF-α, which was verified by the decreased expression level of collagen biomarkers, including Col2α1, COL9α2, and Acan. By introducing Alendronate for the treatment, the impairment of ECM was greatly rescued, indicating a promising protective effect of Alendronate on chondrocytes ECM when the chondrocytes were injured by inflammations. Meanwhile, the suppressed expression level of SOX-9 induced by TNF-α was significantly reversed by the introduction of Alendronate, which further indicated that the protective effect of Alendronate against chondrocytes ECM was related to the regulatory effect of Alendronate on the expression level of SOX-9. However, further verification will be made in our future work to confirm the correlation between the biofunction of Alendronate on chondrocytes ECM and the regulatory effect of Alendronate on SOX-9, such as introducing the inhibitor of SOX-9. In addition, the pharmacodynamics study of Alendronate on OA will be investigated by treating the animal OA model with Alendronate to provide more direct verifications for the potential application of Alendronate on the clinical OA treatments.

SP-1, an important nuclear transcription factor that mediates the multiple mammalian gene expression, which is widely reported to affect the growth and metastasis of tumors by regulating the expressions of tumor-related genes.^26
–28 Recently, SP-1 is reported to regulate OA related proteins to impact the processing of OA. Wang et al. reported that TGF-β1 promoted chondrocyte proliferation by regulating SP-1 through MSC-exosomes derived miR-135b and induced cartilage repair.²⁹ Xue et al.³⁰ reported that by suppressing the expression level of lncRNA NKILA, the proliferation was inhibited and the apoptosis was promoted in the chondrocytes by regulating the miR-145/SP1/NF-κB signaling in human osteoarthritis. In addition, SP-1 is one of the substrates of ERK1/2 signaling pathway, and could be activated by phosphorylation of ERK1/2.³¹ In the present study, to explore the molecular mechanism underlying the regulatory effects of Alendronate on SOX-9, we further checked the impact of Alendronate on the expression level of SP-1 and found that the SP-1 was also greatly upregulated by Alendronate. By knocking down the expression level of SP-1, the regulatory effect of Alendronate against SOX-9 expression was significantly reversed, indicating that Alendronate might promote the expression of SOX-9 by upregulating its transcriptional factor. In addition, we found that the ERK1/2 signal pathway was greatly activated by Alendronate and the regulatory effect of Alendronate on the expression level of SP-1 was significantly reversed by blocking ERK1/2 signaling pathway using U0126, indicating that the expression of SP-1 was regulated by Alendronate through activating the ERK1/2 signaling pathway. In our future work, the direct target of Alendronate on regulating the ERK1/2 signal pathway will be explored to better understand the regulatory effect of Alendronate in the ERK1/2/SP-1/SOX-9 axis and thereafter the therapeutic effect of Alendronate on OA. Schematic drawing of the underlying mechanism is shown in Figure 8.

Figure 8.

Schematic drawing of the effects of Alendronate in the gene expression of extracellular matrix.

Taken together, our data indicated that Alendronate might promote the gene expression of extracellular matrix by increasing SOX-9.

Footnotes

Author contributions

LW and BM contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was supported by the Program of Linyi Science and Technology Innovation Development (Grant No. 201919058).

ORCID iD

H Zhu

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