Inhibition of PTGS1 promotes osteogenic differentiation of adipose-derived stem cells by suppressing NF-kB signaling
Tissue inflammation is an important problem in the field of human adipose-derived stem cell (ASC)-based therapeutic bone regeneration. Many studies indicate that inflammatory cytokines are disadvantageous for osteogenic differentiation and bone formation. Therefore, overcoming inflammation would be greatly beneficial in promoting ASC-mediated bone regeneration. The present study aims to investigate the potential anti-inflammatory role of Prostaglandin G/H synthase 1 (PTGS1) during the osteogenic differentiation of ASCs.
We performed TNFα treatment to investigate the response of PTGS1 to inflammation. Loss- and gain-of-function experiments were applied to investigate the function of PTGS1 in the osteogenic differentiation of ASCs ex vivo and in vivo. Western blot and confocal analyses were used to determine the molecular mechanism of PTGS1-regulated osteogenic differentiation.
Our work demonstrates that PTGS1 expression is significantly increased upon inflammatory cytokine treatment. Both ex vivo and in vivo studies indicate that PTGS1 is required for the osteogenic differentiation of ASCs. Mechanistically, we show that PTGS1 regulates osteogenesis of ASCs via modulating the NF-κB signaling pathway.
Collectively, this work confirms that the PTGS1-NF-κB signaling pathway is a novel molecular target for ASC-mediated regenerative medicine.
KeywordsPTGS1 NF-κB Osteogenic differentiation ASCs
Alizarin red staining
Adipose-derived stem cells
Hematoxylin and eosin
Mesenchymal stem cells
Nuclear factor kappa B
Prostaglandin G/H synthase 1
Runt-related transcription factor 2
Tumor necrosis factor alpha
Traditional treatments for bone augmentation using autologous or allogeneic bone graft, bone substitute material, or guided bone regeneration technology often show limited clinical benefit . In addition, most therapeutic bone defects or causes of bone loss, such as periodontitis, traumatic bone injury, or insufficient bone mass around implants, are usually accompanied by inflammation. The above methods fail to control the inflammatory microenvironment. Current advances in bone tissue engineering hold significant future for regenerating bone tissue. Considering their capabilities of self-renewal, proliferation, multiple differentiation, and abundant availability, human adipose-derived stem cells (ASCs) are an ideal origin of adult mesenchymal stem cells (MSCs) for bone tissue regeneration [2, 3, 4]. Therefore, our focus herein is to determine how to vigorously activate the osteogenesis capability and inflammatory regulation potential of ASCs and to identify key parameters of a stem cell-based approach for bone tissue regeneration.
Prostaglandin G/H synthase 1 (PTGS1) is an enzyme of prostaglandin synthesis that is of great physiological significance. The promoter region of PTGS1 is GC-rich and has multiple transcription factor binding sites, but lacks functional TATA and CAAT sequences. As an inherent housekeeping enzyme, PTGS1 is mainly expressed in the stomach, kidney, and platelets, and its fundamental functions include regulating the resistance of peripheral vasculature, maintenance of renal blood flow, protection of gastric mucosa, and modulation of platelet aggregation. PTGS1 is also perceived as acting critical roles on pathophysiological progress of inflammation, arthritic disease, and cancer [5, 6, 7, 8, 9, 10]. Clinical studies have shown that as a specific inhibitor of PTGS1, low-dose aspirin can effectively exert antipyretic, analgesic, and anti-inflammatory functions; these are reported to prevent or treat numerous associated diseases . Importantly, it has been reported that application of PTGS1 inhibitors or silencing of PTGS1 expression greatly attenuates the inflammatory response induced by LPS via negatively governing the nuclear factor kappa B (NF-κB) signaling pathway [12, 13]. In addition, previous studies have indicated PTGS1 is involved in the differentiation of macrophages and monocytes . Another PTGS1 inhibitor, BGJb, has been found to inhibit bone resorption . However, the role of PTGS1 in the osteogenic differentiation of ASCs and its potential role in the regulation of inflammation have not been reported.
Bone remodeling is a constant homeostasis that is frequently disturbed by pro-inflammatory cytokines which could curb bone formation and lead to bone loss [16, 17]. NF-κB is a core transcription factor that governs osteogenesis and inflammatory response in MSCs. Significant evidence has accumulated implying the strong potential of NF-κB as a therapeutic target for treating inflammation-associated bone remodeling [18, 19]. In this study, we aimed to evaluate the role of PTGS1 in the osteogenic differentiation and inflammatory regulation of human ASCs. Our results demonstrate that deletion of PTGS1 greatly promotes the osteogenesis of ASCs ex vivo and in vivo and depletion of PTGS1 possesses potential anti-inflammatory function via repressing NF-κB pathway, suggesting the potential utility of PTGS1 in ASC-based bone tissue engineering.
Cell cultures and osteogenic induction
Primary human ASCs from three donors (Batch number 2249, 11537, and 19382) were purchased from the ScienCell Research Laboratories (Carlsbad, CA, USA; catalogue number 7510). ASCs were cultured in a humidified incubator at 37 °C under 5% CO2 in the DMEM alpha modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 2 mmol/L glutamine, and 100 μg/mL streptomycin (Invitrogen). For TNFα (R&D Systems, Minneapolis, MN, USA) treatment, ASCs were synchronized by starvation for 24 h in culture medium without fetal bovine serum, then changed to ordinary medium with TNFα treatment.
For osteogenic differentiation induction, cells were cultured in osteogenic induction medium consisting of DMEM alpha modified Eagle’s medium with 10% (v/v) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM β-glycerophosphate, 0.2 mM l-ascorbic acid, and 100 nM dexamethasone.
Recombinant lentivirus targeting PTGS1 was purchased from the GenePharma company. The study was performed as described previously [20, 21, 22]. For viral infection, ASCs were cultured overnight, infected with lentivirus with 4 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA) for 8 h, and then cultured with an ordinary medium. After 96 h, 1 mg/mL puromycin (Sigma-Aldrich) was added into the medium to select the infected cells. The shRNA sequences were as follows: NC, TTCTCCGAACGTGTCACGT; PTGS1sh1, GATCCAGAACAGTGGCTCG; and PTGS1sh2, AAGTGCCATCCAAACTCTATCTT.
For gene overexpression, a lentivirus expressing PTGS1 was purchased from the Cyagen company and used for ASC infection.
ALP activity and ALP staining
For alkaline phosphatase (ALP) activity, ASCs were cultured in osteogenic induction medium. After 5 days, 5 μL protein lysate was extracted and then tested following an ALP activity kit (Sigma-Aldrich). Signal was normalized by protein concentration.
After 1-week osteogenic induction, cells were fixed in 4% paraformaldehyde for 15 min. Then cells were stained with a BCIP/NBT staining kit (CWBIO, Beijing, China) at room temperature for 10 min.
Alizarin red staining and quantification
After osteogenic induction for 2 weeks, cells were fixed in 95% ethanol and then incubated with 2% Alizarin red buffer (Sigma-Aldrich). To exam the level of calcium, the coloration was destained and the absorbance of calcium was measured on a multiplate reader at 562 nm. The final concentration of calcium was normalized to protein concentration of each plate.
Real-time reverse transcriptase-polymerase chain reaction
Total cellular RNA was extracted from ASCs using Trizol reagent (Invitrogen). We synthesized cDNA from 1 μg RNA aliquots. Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was performed with Power SYBR Green PCR Master Mix (Roche, Basel, Switzerland) and tested by a 7500 Real-Time PCR Detection System (Applied Biosystems, Thermo Fisher, MA, USA). GAPDH was used as the internal control. The primer sequences were as follows: GAPDH (forward) 5′-CGGACCAATACGACCAAATCCG-3′ and (reverse) 5′-AGCCACATCGCTCAGACACC-3′; PTGS1 (forward) 5′-CAATGCCACCTTCATCCGAG-3′ and (reverse) 5′-GATAAGGTTGGAGCGCACTG-3′; ALP (forward) 5′-GACCTCCTCGGAAGACACTC-3′ and (reverse) 5′-TGAAGGGCTTCTTGTCTGTG-3′; OCN (forward) 5′-AGCAAAGGTGCAGCCTTTGT-3′ and (reverse) 5′-GCGCCTGGGTCTCTTCACT-3′; BSP (forward) 5′-CAGGCCACGATATTATCTTTACA-3′ and (reverse) 5′-CTCCTCTTCTTCCTCCTCCTC-3′; RUNX2 (forward) 5′-TCTTAGAACAAATTCTGCCCTTT-3′ and (reverse) 5′-TGCTTTGGTCTTGAAATCACA-3′; OSX (forward) 5′-CCTCCTCAGCTCACCTTCTC-3′ and (reverse) 5′-GTTGGGAGCCCAAATAGAAA-3′; IL6 (forward) 5′-CGCAACAACTCATCTCATTCTGCG-3′ and (reverse) 5′-CATGCTACATTTGCCGAAGAGC-3′; IL8 (forward) 5′-CGGATAAAGGGCCAAGAGAATATCCG-3′ and (reverse) 5′-TCACATTCTAGCAAACCCATTCAA-3′; and SELE (forward) 5′-AGCTTCCCATGGAACACAAC-3′ and (reverse) 5′-CTGGGCTCCCATTAGTTCAA-3′. Relative gene expression is calculated by using the 2−ΔΔCt method.
Nuclear and cytoplasmic extraction
Cells were suspended and swollen in buffer A (10 mM HEPES, 0.1 mM EDTA, 1 mM DTT, 0.1 mM EGTA, 10 mM KCl, 0.15% NP-40, and 1:100 proteinase inhibitor cocktail) on ice for 10 min. After centrifugation, the supernatant of the sample was collected as the cytoplasmic extract. The rest of pellet was washed with PBS and then resuspended in buffer B (20 mM HEPES, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 400 mM NaCl, 0.5% NP-40, and 1:100 proteinase inhibitor cocktail) at 4 °C for 25 min. After centrifugation, the supernatant was collected and used as the nuclear extract.
Western blot experiment
ASCs were lysed in the RIPA buffer (10 mM Tris-HCl, 1% sodium dodecyl sulfate, 50 mM sodium fluoride, 50 mM β-glycerophosphate, 1 mM EDTA, 1% NP-40, and 1:100 proteinase inhibitor cocktail). Thirty-microgram protein of each sample was used for analysis as described previously [20, 21, 22]. Primary antibodies against PTGS1, GAPDH, p-IκBα (ser32/ser36), p-p65 (Ser536), p65, tubulin, PCAF (Cell Signaling Technology), and IκBα (Abcam) were diluted 1:1500 and then incubated with the membrane overnight at 4 °C. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology) were diluted 1:10,000 and incubated with the membrane for 1 h, and the membrane was visualized. Next, band intensity was quantified by using the Image J software, and target band signal was normalized to the corresponding internal control (GAPDH, PCAF, or tubulin).
Firstly, cells that grew on glass coverslips were fixed and then permeabilized with 0.25% Triton X-100 for 10 min, blocked with 0.8% BSA for 1 h at room temperature. Next, cells were incubated with the primary antibody against p65 (Cell Signaling Technology) overnight at 4 °C and treated with appropriate secondary antibody. The nucleus was counterstained with DAPI. The experiment was performed as described previously [20, 21].
Analyses of bone formation in vivo
This study was approved by the Institutional Animal Care and Use Committee of the Peking University Health Science Center (LA2014233), and all related experiments were performed in accordance with the Institutional Animal Guidelines. In vivo study was performed as described previously [20, 21]. The third passage of ASCs that transfected with target gene was cultured in a proliferation medium before implantation. For each implantation site, 2 × 106 cells were mixed with 40-mg synthograft (Bicon) at 37 °C for 1 h, then the mixture pellet was acquired and implanted on the dorsal subcutaneous site of homozygous nude mice (6-week-old BALB/c, n = 10). After 8 weeks, the mice were euthanized by CO2 asphyxiation. Next, specimens were harvested and then fixed, followed by decalcification in 10% EDTA buffer (pH 7.4) for 1.5 weeks, then dehydrated, and embedded. Tissue section (thickness 5 μm) was cut and then stained with HE, Masson’s trichrome, and immunohistochemical staining (primary antibody against OCN (Abcam)). To quantify the bone-like tissue, 15 images from each sample were randomly collected, and the area of new bone formation versus total area was calculated by the SPOT 4.0 software (Diagnostic Instruments, Sterling Heights, MI, USA).
All statistical calculations were performed by using SPSS10 statistical software. Comparisons between two groups were analyzed by independent two-tailed Student’s t tests, and comparisons between more than two groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Data presented are derived from three ASC strains. All data are showed as the mean ± standard deviation (SD) of three to ten experiments per group. P value < 0.05 was considered statistically significant.
PTGS1 expression is stimulated by TNFα treatment
PTGS1 is required for the osteogenic differentiation of ASCs in vitro
In order to avoid off-target effect, an additional sequence targeting PTGS1 was used. After transfection, results indicated that the efficiency of infection was greater than 80% (Additional file 1: Figure S1A-C). Consistently, PTGS1 knockdown promoted osteogenic differentiation as revealed by ALP activity, ALP staining, Alizarin red staining, and quantitative calcium analysis (Additional file 1: Figure S1D-G). Moreover, real-time RT-qPCR results showed upregulation of related genes (ALP, OCN, BSP, RUNX2, and OSX) (Additional file 1: Figure S1H-L) in PTGS1sh cells. Taken together, these data demonstrate that PTGS1 plays a critical function in the osteogenic differentiation of ASCs.
PTGS1 influences osteogenesis of ASCs in vivo
Knockdown of PTGS1 inhibits the translocation of p65 from the cytoplasm to the nucleus
PTGS1 has been implicated in numerous pathophysiological processes including arthritic disease, cancer, pain, and inflammation, but its role in osteogenic differentiation and inflammatory regulation in MSCs remains fairly vague. Significant evidence has accumulated revealing that inhibition of PTGS1 yields an anti-inflammatory effect. Previous research showed that genetic deletion or pharmacological inhibition of PTGS1 could reduce the LPS-induced inflammatory response, potentially acting through the NF-κB pathway. Ptgs1-deficient mice showed a decreased level of arachidonic acid-induced inflammation [12, 23].
In this study, we found that PTGS1 was activated upon TNFα treatment in ASCs. Moreover, deletion of PTGS1 substantially attenuated the expressions of IL6, IL8, and SELE. These results indicate that inhibition of PTGS1 may have the potential to reduce inflammation in ASCs. Previously, the role of PTGS1 in osteogenesis has rarely been documented. A related study reported that the PTGS1 inhibitor BGJb can rescue bone resorption induced by IL1 . Herein, we demonstrated knockdown of PTGS1 significantly enhanced the osteogenic differentiation of ASCs, and overexpression of PTGS1 inhibited osteogenesis capacity both in vitro and in vivo. This is consistent with the above report, which indicated PTGS1 exerts a negative regulatory role in bone formation. Research about bone forming related to blocking inflammation is a meaningful part for bone tissue regeneration, which was closely coordinated by cell microenvironment. Bone injury model with inflammation is needed to carry out in our future research.
We found that PTGS1 expression is highly induced in response to TNFα treatment and that the inflammatory cytokine could inhibit osteogenesis and bone formation by downregulating two core transcription factors for osteogenic differentiation: RUNX2 and OSX [24, 25]. TNFα is the key stimulating factor for activating the classical NF-κB pathway; for this reason, we hypothesized that targeting the NF-κB pathway may be a novel mechanism through which PTGS1 regulates the osteogenic differentiation of MSCs. For NF-κB family, the p50-p65 heterodimers remain segregated in the cytoplasm through interaction with IκBa. After stimulation by TNFα, the classical pathway is triggered as follows: IKK complexes are phosphorylated by the IκB kinase, IκBa protein undergoes phosphorylation and ubiquitin-dependent degradation, and the p50-p65 dimers are released from IκBa protein, translocating to the nucleus. In addition, the nuclear p50-p65 dimer can also combine with IκBa protein to form a strong nuclear export signal. The heterodimer is crucial for the expression of genes encoding pro-inflammatory mediators and anti-osteogenesis regulators [26, 27].
Mechanistically, we found that knockdown of PTGS1 substantially repressed the expressions of NF-κB target genes (IL6, IL8, and SELE). Furthermore, deletion of PTGS1 reduced the phosphorylation and degradation levels of IκBa protein and the phosphorylation of p65. Conversely, overexpression of PTGS1 could induce the phosphorylation and degradation levels of IκBa protein and the phosphorylation of p65. These results reveal that inhibition of PTGS1 exerts pro-osteogenic function in ASCs by regulating the classical NF-κB pathway. Furthermore, we also found that PTGS1 gene silencing can prevent nuclear translocation of p65, while its overexpression resulted in accelerated nuclear entrance of p65, as verified by confocal microscopy and Western blot analyses of nuclear and cytoplasmic cell fractions. The new link between PTGS1 and NF-κB suggests their collaboration in regulating the osteogenic differentiation and anti-inflammation of ASCs; future research is needed to further define the underlying mechanism.
In summary, our study demonstrates that knockdown of PTGS1 promotes the osteogenic differentiation of ASCs both in vitro and in vivo by targeting p65 cytoplasmic/nuclear translocation. More importantly, silencing of PTGS1 may provide a potential means for modulating the inflammatory microenvironment during bone remodeling. Our findings demonstrate that PTGS1 can be a potential target for treating inflammation-associated bone defects and is a key factor to consider in stem cell-based approaches for bone tissue regeneration.
Our results first demonstrated that inhibition of PTGS1 promoted the osteogenic differentiation of ASCs through repressing NF-κB pathway by targeting p65 cytoplasmic/nuclear translocation. Furthermore, knockdown of PTGS1 may provide a potential means for regulating inflammatory microenvironment in the process of bone remodeling. Collectively, PTGS1-NF-κB signaling pathway is a novel molecular target for ASC-mediated regenerative medicine.
This study was supported by grants from the National Natural Science Foundation of China (No. 81500822 to P.Z., No. 81570953 to Y.Z.), the Project for Culturing Leading Talents in Scientific and Technological Innovation of Beijing (Z171100001117169 to Y.Z.), the PKU School of Stomatology for Talented Young Investigators (PKUSS20140109 to P.Z.), the Beijing Nova Program (Z181100006218037).
Availability of data and materials
The authors confirm that all data underlying the findings are fully available.
YW was responsible for the collection and/or assembly of data, data analysis and interpretation, and manuscript writing; YL and MZ were responsible for the collection and/or assembly of data; LL and XZ were responsible for the provision of study material; PZ was responsible for the conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and financial support; YZ was responsible for the conception and design, financial support, and manuscript writing. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Peking University Health Science Center (approval no. LA2014233). All surgeries were performed under anesthesia, and all efforts were made to minimize animal suffering.
Consent for publication
The authors declare that they have no competing interests.
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