Inhibition of PP2A Enhances the Osteogenic Differentiation of Human Aortic Valvular Interstitial Cells via ERK and p38 MAPK Pathways
Fei Xie a, #; Fei Li a, #; Rui Li; Zongtao Liu; Jiawei Shi; Chao Zhang a, *; Nianguo Dong a, *
Abstract
Aims: To investigate the role of PP2A in calcified aortic valve disease (CAVD). Materials and Methods: The expressions of PP2A subunits were detected by real-time polymerase chain reaction (RT-PCR) and western blot in aortic valves from patients with CAVD and normal controls, the activities of PP2A were analyzed by commercial assay kit at the same time. Aortic valve calcification of mice was evaluated through histological and echocardiographic analysis. ApoE−/− mice and ApoE−/− mice injected intraperitoneally with PP2A inhibitor LB100 were fed a high-cholesterol diet for 24 weeks. Immunofluorescent staining was used to locate the cell-type in which PP2A activity was decreased, the PP2A activity of valvular interstitial cells (VICs) treated with osteogenic induction medium was assessed by western blot and commercial assay kit. After changing the activity of VICs through pharmacologic and genetic intervention, the osteoblast differentiation and mineralization were assessed by western blot and Alizarin Red staining. Finally, the mechanism was clarified by using several specific inhibitors.
Key findings: PP2A activity was decreased both in calcified aortic valves and human VICs under osteogenic induction. The PP2A inhibitor LB100 aggravated the aortic valve calcification of mice. Furthermore, PPP2CA overexpression inhibited osteogenic differentiation of VICs, whereas PPP2CA knockdown promoted the process. Further study revealed that the ERK/p38 MAPKs signaling pathways mediated the osteogenic differentiation of VICs induced by PP2A inactivation.
Significance: This study demonstrated that PP2A plays an important role in CAVD pathophysiology, PP2A activation may provide a novel strategy for the pharmacological treatment of CAVD.
Keywords: PP2A, Osteogenic differentiation, ERK/p38 MAPKs
1. Introduction
Calcific aortic valve disease (CAVD) is the most prevalent heart valve disease in developed countries, with increasing morbidity due to the acceleration of population aging [1]. Previously, CAVD was considered a passive and degenerative valve disorder. However, recent studies have found that it involves a variety of complex and active pathological processes, including endothelial injury, inflammation, matrix remodeling, angiogenesis, and osteogenesis [2-4]. As the thickening and calcification of the valve leaflets progress, end-stage calcific aortic stenosis is formed, which requires surgical or interventional aortic valve replacement [1]. Unfortunately, valve replacement presents high risks of surgical complications in elderly patients. Mechanical heart valves last for a long time, but they require anticoagulants, while bioprosthetic heart valves face recalculation and patients may even need reoperation [5]. Thus, deep investigation of the pathophysiology of CAVD is required to develop novel therapeutic strategies for slowing or even reversing the progress of the disease [6]. Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase that counter-balances kinase-mediated phosphorylation in various cellular processes. It belongs to the phosphoprotein phosphatase (PPP) family and consists of a catalytic subunit PP2A-C (PPP2CA, PPP2CB), a scaffolding subunit PP2A-A (PPP2R1A, PPP2R1B), and a regulatory subunit PP2A-B (B/PR55, B′/PR61, B″/PR72, and B‴ subunits) [7]. PP2A plays a critical role in cellular signaling, cell cycle, cell metabolism, apoptosis, and stress responses [8-10]. Previous studies have confirmed that PP2A is a tumor suppressor in human malignancies, and allosteric inactivation of PP2A contributes to the development of cancer [11-14]. PP2A activity also affects the pathogenesis in diabetes and Alzheimer’s disease [15, 16]. In the heart, reversible protein phosphorylation is pivotal to a myriad of cardiac physiological and pathophysiological processes. Dysregulation of PP2A has been associated with a variety of cardiovascular diseases including arrhythmia, ischemia-reperfusion injury and heart failure [17, 18]. Terentyev D et al. found translational inhibition of PP2A regulatory subunit B56α promotes arrhythmogenic sarcoplasmic reticulum Ca2+ release [19]. Other reports showed that pretreating hearts with PP2A inhibitors reduces the myocardial infarct size in ischemia-reperfusion injury [20, 21]. Several genetically modified mice models also pinpointed an important role for PP2A in heart failure. Transgenic mice expressing a mutant A subunit of protein phosphatase 2A in muscle tissue develops dilated cardiomyopathy [22]. Furthermore, both cardiomyocyte-specific deletion as well as cardiomyocyte-specific overexpression of PPP2CA gene causes cardiomyocyte hypertrophy and fibrosis [23, 24], indicating that PP2A activity needs to be precisely regulated to prevent cardiac dysfunction. Many critical cellular molecules, such as protein kinase B (PKB/Akt), glycogen synthase kinase-3β (GSK-3β), mitogen-activated protein kinases (ERK/p38 MAPKs), and nuclear transcription factor-κB (NF-κB) are dephosphorylated by PP2A [25-28], which also participate in the development and progression of CAVD [29-33]. Consequently, the purpose of this study is to investigate the role and underlying molecular mechanisms of PP2A in aortic valve calcification, which may provide a new approach for the effective treatment of CAVD.
2. Materials and Methods
2.1. Human aortic valves collection
This study complied with the Declaration of Helsinki and was approved by an ethics committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. All of the patients recruited from the department of cardiovascular surgery provided informed consent and agreed to participate in the trial. Calcific aortic valves (AVs) were obtained from patients undergoing valve replacement due to severe aortic stenosis. Normal AVs were collected from age-matched patients with acute Stanford type A aortic dissection or dilated cardiomyopathy. All of the valves were divided into three parts according to the purpose of the experiment. For detection of PP2A activity via protein and mRNA expression level, the tissues were kept frozen in liquid nitrogen. For immunofluorescence staining and Alizarin Red staining, the tissues were fixed in 4% paraformaldehyde. For cell culture and treatment, the tissues were put in sterile phosphate buffered saline on ice, transported to the lab, and digested by collagenase immediately. All of the valves were tricuspid.
2.2. Animal model
ApoE−/− (C57BL/6 background) mice were purchased from Beijing HFK Bioscience Co. Ltd., housed under the standard light-dark cycle (12 h/12 h), and allowed ad libitum access to water and diet. The animal protocol was reviewed and approved by The Institutional Animal Research Committee of Tongji Medical College. Aortic valve calcification was induced in male mice at age 6–8 weeks by a 24-week protocol as described previously [34]. After initial quarantine, mice were assigned into three groups: ApoE−/− mice receiving standard rodent diet (control group); ApoE−/− mice receiving 0.25% high-cholesterol diet injected intraperitoneally with PP2A inhibitor LB100 (Selleck Chemicals, 1.0 mg/kg) every other day (LB100 + high-cholesterol diet group); and ApoE−/− mice receiving 0.25% high-cholesterol diet injected intraperitoneally with equivalent saline (saline + high-cholesterol diet group). At the end of the experiment, mice were anesthetized and sacrificed. The heart and ascending aorta were collected and fixed in 4% paraformaldehyde.
2.3. Echocardiography
Mice were narcotized under 2.5% isoflurane, and transthoracic echocardiography was performed by an experienced operator blinded to the assignments. The images were acquired by using an 18–38 MHz phased-array probe (MS400) connected to a Vevo 1100 Imaging System. The transvalvular velocity was evaluated through continuous wave Doppler by parasternal long axis view.
2.4. Isolation, culture and treatment of VICs
Human VICs were isolated from normal valves by the collagenase I digestion method as previously described [35]. Briefly, aortic valve leaflets were digested in DMEM (Dulbecco’s Modified Eagle Medium; Gibco, USA) containing 1.0 mg/ml collagenase I at 37°C for 30 min. After vortexing to remove endothelial cells, the leaflets were further digested with a fresh solution of 1.0 mg/ml collagenase medium for 4–6 h at 37°C. The suspension was spun at 300 g for 10 min to precipitate cells. Cells were re-suspended and cultured in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco, USA), and 10% fetal bovine serum (FBS; Gibco, Australia) in an incubator with 5% CO2 at 37°C. VICs from passages 3 through 5 were selected for further studies and plated at the concentration of 1.0 × 105/ml. Osteogenic induction medium (OST) containing 2% FBS, 10 mmol of β-glycerophosphate, 100 nmol of dexamethasone, and 50 mg/ml ascorbic acid (all three reagents from Sigma-Aldrich, St Louis, Mo) was used to provoke calcification. The cells were starved with DMEM containing only 2% FBS overnight and treated with either PP2A inhibitor LB100 or agonist FTY720 (all purchased from Selleck Chemicals, USA) at appropriate concentrations 4 h before the addition of osteogenic induction medium.
2.5. Alizarin Red staining
For mineralization experiments of VICs, cells were seeded on 24-well plates. Once the cells reached 70%–80% confluence, they were incubated with indicated interventions in osteogenic induction medium for 21 days. The medium was changed every 3 days. Alizarin Red staining was performed to detect calcium deposits as described previously [36]. Briefly, cell monolayers were washed three times with phosphate-buffered saline and then fixed for 15 min in 4% paraformaldehyde. After incubation with 0.2% Alizarin Red solution (pH: 4.2) for 30 min, excessive dye was removed by washing with distilled water. For analysis of calcium deposits in the aortic valve tissue of humans or mice, Alizarin Red staining was performed according to the manufacturer’s instructions. To quantify the staining, the percentage of Alizarin Red-positive staining area was analyzed by Image-Pro Plus software.
2.6. Gene knockdown
To achieve PPP2CA knockdown, cultured VICs at 70%–80% confluence were transfected with specific or control siRNA using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s recommendations. The PPP2CA siRNA was purchased from Thermo Fisher Scientific and consisted of three distinct RNA sequences (si-PPP2CA #1 F: 5’-UCA AGA GCC UCU GCG AGA AGG CUA A-3’ R: 5’-UUA GCC UUC UCG CAG AGG CUC UUG A-3’ HSS108358; si-PPP2CA #2 F: 5’-GAG AGC AGA CAG AUC ACA CAA GUU U-3’ R: 5’-AAA CUU GUG UGA UCU GUC UGC UCU C-3’ HSS108359; and si-PPP2CA #3 F: 5’-GCC AUG ACC GGA AUG UAG UAA CGA U-3’ R: 5’-AUC GUU ACU ACA UUC CGGUCA UGG C-3’ HSS108360). The medium was changed 8 h after treatment with siRNA. Cells were harvested after 24 h for polymerase chain reaction (PCR) and 72 h for western blot analysis to validate the knockdown efficiency, or stimulated with osteogenic induction medium for 72 h to determine the effect of PPP2CA knockdown on the regulation of Runx2 and ALP.
2.7. Gene overexpression
To overexpress PPP2CA, VICs were infected with adenovirus expressing PPP2CA or carrying empty vector (Vigene Bioscience, China) according to the manufacturer’s instructions. Three days later, cells were harvested for validation of overexpression efficiency, and cellular levels of Runx2 and ALP were examined. Ad-PPP2CA-FH expression was confirmed by western blot analysis with an antibody against the His Tag.
2.8. PP2A phosphatase activity assay
A PP2A immunoprecipitation phosphatase assay kit (Millipore, Germany) was used to measure the PP2A activity of human aortic valves and VICs. Tissues or cells were lysed using a lysis buffer (20 mM imidazole-HCL, 2 mM EDTA, 2 mM EGTA, 1 mM benzamidine, pH 7.0 with 10 μg/ml aprotinin, and 1 mM PMSF). The extracts were sonicated for 10 s and centrifuged at 2000 g for 5 min. Next, 200 μg of total protein extracts were incubated with 4 μg anti-PP2A-C subunit antibody 18 h at 4°C with gentle rocking. Then 40 μl Protein A agarose slurry was added and rocked for 2 h at 4°C. Beads were washed three times with 700 μl TBS and once with 500 μl Ser/Thr assay buffer. The beads were then incubated with 60 μl diluted phosphopeptide and 20 μl Ser/Thr assay buffer at 30°C for 10 min in a constant temperature shaker. The beads were centrifuged briefly, and the samples were analyzed in a colorimetric assay using malachite green at an absorbance of 650 nm.
2.9. SDS–PAGE and western blot analysis
Total protein was extracted from VICs using commercial RIPA buffers (Thermo Fisher, USA) supplemented with protease and phosphatase inhibitors (Thermo Fisher, USA) according to the manufacturer’s instructions. Protein samples were separated by SurePAGE™ precast polyacrylamide gels with a gradient between 4%–20% (GenScript, Nanjing, China) and blotted onto 0.22-μm polyvinylidene difluoride membranes (Millipore, Germany) by eBlot L1 Fast Wet Transfer System (GenScript, Nanjing, China). The membranes were incubated in 5% skimmed milk in a TBS-T solution for 1 h at room temperature to block nonspecific binding and then incubated with appropriate primary antibodies overnight at 4°C on the shaker. Specific binding was detected by incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and an enhanced chemiluminescence (ECL) system (Thermo, USA). Band density was analyzed by using ImageJ software (NIH, USA). The primary antibodies are listed in Table 1.
2.10. Immunofluorescence staining
Cryosections (5 µm) from normal and calcified human aortic valves were prepared, and immunofluorescence staining was applied as previously described [37]. After permeabilization, 10% goat serum was applied for 30 min at room temperature to block the nonspecific binding sites of proteins; then, the sections were first incubated with primary antibodies against Y307 (1:50 dilution) and Vimentin (1:200 dilution) at 4°C overnight, and then subsequently incubated with a secondary antibody Alexa Fluor 568 (imaged on red channel) or Alexa Fluor 488 (imaged on green channel) for 2 h at room temperature. Finally, DAPI was used for nucleus counterstaining (imaged on blue channel). Thus, dual immunofluorescence staining was accomplished to co-localize Vimentin and Y307. Microscopy was performed with an Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany).
2.11. Real-time polymerase chain reaction analysis
Total RNAs were isolated from aortic valve tissue or VICs by using Trizol reagent (Thermo Fisher, USA) and then reversely transcribed into cDNA. Real-time polymerase chain reaction (RT-PCR) was performed with SYBR Green (Takara, Japan) using specific primers that are listed in Table 2 and GAPDH as the control. Quantitative RT-PCR was performed on a StepOne Plus thermal cycler (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. All of the data were analyzed by the 2‐ΔΔct method, and each sample was analyzed in triplicate.
2.12. Statistical analysis
The data are presented as mean ± standard error and represent data from at least three independent experiments. Parameters were evaluated via either Student’s t-test for comparison between two groups or ANOVA followed by Bonferroni’s multiple comparison test for comparison among three or more groups. P < 0.05 was considered to be statistically significant. 3. Results 3.1. The activity of PP2A was decreased in calcified human aortic valves In order to evaluate the activity of PP2A in both normal and calcified human aortic valves (Valve donor demographics in Table S1), we analyzed the protein levels of PP2A subunits and the phosphorylation levels of catalytic subunits (Y307) through immunoblotting. The results showed that there was no statistical difference among the PP2A subunits’ expression, while the phosphorylation of catalytic subunits (Y307) significantly increased in calcified human aortic valves (Figure 1A). Then RT-PCR was used to determine the mRNA expression levels of PP2A subunit isoforms; we found that PPP2R1A, PPP2R5B, and PPP2R5D decreased in calcified human aortic valves, whereas PPP2R3C increased (Figure 1B). Finally, we used a PP2A immunoprecipitation phosphatase assay kit to test the activity directly. The results showed that PP2A activity decreased markedly in calcified human aortic valves compared to that in normal valves (Figure 1C). These results suggested that PP2A participated in calcific aortic valve disease (CAVD), playing an important role in osteogenesis transition and aortic valve calcification. 3.2. PP2A inhibitor LB100 aggravated high-cholesterol-diet-induced AV calcification in ApoE−/− mice LB100 is a hydrophilic small-molecule inhibitor of the PP2A-C subunit with a significantly more favorable toxicity profile compared to Okadaic Acid, a traditional PP2A inhibitor. A phase I clinical trial using LB100 for the treatment of adult solid tumors was completed recently [38]. In this section, we describe how we used LB100 to suppress the activity of PP2A and investigated its effect on aortic valve calcification in vivo. Transthoracic echocardiography demonstrated that compared with mice that received a standard rodent diet, ApoE−/− mice that received a high-cholesterol diet for 24 weeks had increased peak transvalvular velocities, suggesting the existence of aortic valve stenosis. In addition, a significant increase in transvalvular velocity was observed in ApoE−/− mice injected intraperitoneally with LB100 under a high-cholesterol diet (Figure 2A-B). No significant difference of left ventricular function was observed among groups (Table S2). We further evaluated the morphology of valve leaflet and the degrees of calcification via HE and Alizarin Red staining. Compared with ApoE−/− mice on a high-cholesterol diet, LB100-treated ApoE−/− mice exhibited thickened AV leaflets. At the same time, we found significant calcium deposits in AV leaflets of LB100-treated ApoE−/− mice by Alizarin Red staining (Figure 2A, 2C). Therefore, inhibition of PP2A aggravated high-cholesterol-diet-induced AV calcification in ApoE−/− mice. 3.3. PP2A activity of VICs was decreased by osteogenic induction medium To further investigate the cell type in which PP2A activity was decreased, we used immunofluorescence staining to co-localize the Y307 and cell biomarkers. Dual immunofluorescence staining and Alizarin Red staining demonstrated that Y307 was co-localized with Vimentin, especially in calcified aortic valves (Figure 3A). The PP2A activity of VICs from calcified aortic valves was significantly lower compared to that from normal aortic valves (Figure 3B, Valve donor demographics in Table S1). We next detected PP2A activity of VICs in response to osteogenic induction medium. After incubating VICs with OST for several days (0, 1, 3, 5, and 7 d), we evaluated the PP2A activity of VICs through western blot and PP2A activity assay. The immunoblotting results showed that Y307 phosphorylation was enhanced from the third day, whereas the expression of PP2A subunits did not change (Figure 3C). PP2A activity assay further confirmed that the PP2A activity of VICs was decreased by OST (Figure 3D). These results demonstrated that the PP2A activity of VICs was decreased, with enhanced phosphorylation of Y307 after osteogenic differentiation. 3.4. PP2A activity regulated the osteoblastic differentiation and mineralization of VICs Since we found that the PP2A activity decreased in calcified human aortic valves and the osteogenic differentiation of VICs suppressed the activity of PP2A, we changed the PP2A activity of VICs to study the effects on the pro-osteogenic factors’ expression and calcium deposits. FTY720, as a sphingosine analogue, has been confirmed to activate PP2A through preventing the binding of SET (I2PP2A) to PP2A [39]. Pre-treatment with FTY720 in VICs significantly blocked the OST-induced expression of RUNX2 and ALP (Figure 4A), as well as the calcium deposits shown through Alizarin Red staining (Figure 4B). To the contrary, the PP2A inhibitor LB100 promoted the osteoblastic differentiation and calcification of VICs (Figure 4C-D). To further clarify the osteoblast differentiation affected by PP2A activity, we conducted an experiment using siRNA interference and adenovirus overexpressing the gene PPP2CA (catalytic Cα subunit of PP2A). Three siRNA sequences (si-PPP2CA #1, si-PPP2CA #2, and si-PPP2CA #3) were used for silencing PPP2CA. The PCR results in Figure S1A show that si-PPP2CA #2 had the strongest silencing efficacy, and the protein expression of PPP2CA was significantly decreased (Figure S1B). Administration of si-PPP2CA #2 also aggravated the effect of OST-induced production of RUNX2 and ALP by VICs and calcium deposits (Figure 4G-H). However, overexpression of PPP2CA using adenovirus (Figure S1C) inhibited the upregulation of RUNX2 and ALP and also alleviated the mineralization of VICs (Figure 4E-F). Taken together, these results elucidated that the OST-induced osteogenic differentiation of VICs was regulated by the activity of PP2A. 3.5. PP2A regulated the osteoblastic differentiation of VICs through ERK/p38 MAPKs signaling pathways We treated VICs with OST for several durations ranging from 0–6 h (0, 10min, 30 min, 1 h, 3 h, and 6 h) and detected the changes of signaling pathway phosphorylation through immunoblotting. As shown in Figure S2, both ERK1/2 and p38 were activated immediately in 10 min and then gradually decreased over time, and p-Akt (Ser473) reached the peak at 3 h. Meanwhile, the phosphorylation of GSK-3β and p65 did not show obvious changes as osteogenesis proceeded. The results show that ERK/p38 MAPKs and Akt took part in OST-induced osteoblastic differentiation of VICs. We further used siRNA and adenovirus to verify the role of PP2A in signaling pathways of osteoblast differentiation. Interestingly, overexpressing PPP2CA attenuated the phosphorylation levels of ERK/p38 MAPKs and Akt at each point in time (Figure 5A), while siRNA interference aggravated the phosphorylation (Figure 5B). We then explored which signaling pathways regulating osteoblastic differentiation were affected by PP2A. After being transfected with scramble siRNA or si-PPP2CA #2, VICs were treated with SB203580 (p38 inhibitor), PD184352 (MEK1/2 inhibitor), or MK2206 (Akt1/2/3 inhibitor) for 4 h before OST treatment. Immunoblotting showed that the ERK/p38 MAPKs inhibitors markedly blocked the deteriorating effect of PP2A-mediated RUNX2 and ALP expression in osteoblastic differentiation and calcium deposition of VICs, but no statistical difference was found in the Akt inhibitor group (Figure 5C-F). These results revealed that PP2A promotes calcification of VICs through activating ERK/p38 MAPKs signaling pathways. 4. Discussion In this study, we demonstrated that PP2A activity is a crucial regulator in osteoblast differentiation and aortic valve calcification due to its ability to control the expressions of RUNX2 and ALP through the ERK/p38 MAPKs signaling pathway. First, we detected the PP2A activity from calcified and normal aortic valves by immunoblotting, RT-PCR, and PP2A phosphatase activity assay. We found that PP2A activity was obviously decreased in calcified aortic valves, accompanied by the up-regulation of PP2A phosphorylation (Y307). The pathological accumulation of Y307 in calcified aortic valves was accompanied by increased ALP and RUNX2 expression, suggesting that the post-translational modification of PP2A at the Y307 site might be an indicator in the pathogenesis of CAVD. Previous reports have shown that the PP2A holoenzyme can quickly respond to specific environmental cues by the rapid transformations of B subunits [40]. Beg M et al. found that PPP2R5B is a negative regulator of Akt phosphorylation contributing partly to the chronic hyperinsulinemia induced insulin resistance in adipocytes [41], and PPP2R5D was also reported to dephosphorylate Akt at the positions Thr-308 and Ser-473 in tau phosphorylation homeostasis [42]. In our study, we observed both PPP2R5B and PPP2R5D expression were decreased in calcified aortic valves with low PP2A activity. All of these may provide an insight to explain our findings that inhibition of PP2A activity enhanced the phosphorylation of Akt in VICs under OST stimulation. What’s more, the gene expression of PPP2R5B has been reported to be strongly correlated with femur bone mineral density in rat [43]. Our results further verified the correlation between PPP2R5B and osteogenic differentiation. Since current studies have demonstrated that PPP2R2C (B55γ) is induced by glucocorticoid receptor (GR) in human primary Bone Marrow-Mesenchymal Stem Cells (BM-MSCs) during differentiation to osteoblasts [44], the expression changes of PPP2R1A, PPP2R3C and PPP2R5D discovered in our study also provided a new essential factor in how PP2A affects the osteoblast differentiation of CAVD. Our further findings demonstrate that LB100, a water-soluble inhibitor of PP2A, also aggravated the aortic valve calcification of ApoE−/− mice in vivo. These results implied that PP2A might be a promising therapeutic strategy for CAVD. The aortic valve tissue architecture is synthesized and maintained by the resident VICs, which play a vital role in the pathophysiology of aortic valve calcification [45-47]. In the present study, we observed that Y307 phosphorylation was common in VICs of calcified aortic valves. We next isolated primary VICs and discovered that PP2A activity decreased in VICs from calcified aortic valves. The OST induced osteoblast differentiation, reduced the PP2A activity of VICs, and increased Y307 phosphorylation. It has been reported that PP2A hyperphosphorylation (Y307) is associated with Alzheimer’s disease, specifically the neurofibrillary pathology of the disease, and colorectal cancer [48, 49]. These findings in our study also demonstrate that PP2A phosphorylation (Y307) is the key factor for osteoblast differentiation of VICs in vitro. We further showed that the osteoblast differentiation and mineralization of VICs were exacerbated after PP2A inactivation, while PP2A activation attenuated the progression. These results implied that alteration of PP2A activity is involved in osteoblast differentiation through the expression of ALP and RUNX2, which are early-stage osteogenesis-specific markers for osteoblast differentiation and bone formation. In a previous study, Okamura et al. proved that PPP2CA ablation induced osteoblast differentiation and was accompanied by increases in ALP activity and the expression of bone-related genes, including Osterix, Bsp, and OCN [50]. To the contrary, the abilities of osteoblast differentiation and mineralization were suppressed by PPP2CA overexpression in MC3T3-E1 cells [51], a kind of mouse pre-osteoblast cell line. In our study, we used primary VICs to induce osteogenic differentiation and obtained similar results. These reports and our results suggest that PP2A activity plays an important role in cellular osteoblast differentiation. Mitogen-activated protein kinases (MAPKs), some of the oldest and conserved serine/threonine kinases, transmit extracellular signals to a wide range of stimuli [52]. Recent reports have shown that MAPKs are vital signal transducers in osteoblast differentiation and bone formation [53, 54]. MAPKs are composed of ERK, p38, and JNK, three well-studied families. In particular, ERK and p38 MAPKs can phosphorylate RUNX2, which is the master regulator of early osteoblast differentiation [55, 56], while treatment with a JNK inhibitor blocks late-stage osteoblast differentiation in vitro. At the end of the study, we explored the underlying mechanisms of how PP2A participates in osteoblast differentiation of VICs through detecting the relevant signal pathways by immunoblotting. We found ERK and p38 MAPKs were activated immediately and Akt followed about 3 h later. Moreover, the PPP2CA genetic level gain- and loss-of function studies confirmed PP2A activity was strongly interlinked with the phosphorylation level of these three signal pathways. Further, we used SB203580 (p38 inhibitor), PD184352 (MEK1/2 inhibitor), and MK2206 (Akt1/2/3 inhibitor) to check the precise signal pathways involved in PP2A-mediated osteoblast differentiation of VICs. The results indicated that ERK/p38 MAPKs inhibitors can reverse the acceleration effect of osteoblast differentiation and mineralization induced by PP2A inactivation. Xu et al. demonstrated that the inhibition of the MAPK/ERK pathway can dramatically reduce VIC calcification [57]. Our study verified this conclusion and further discovered that the inhibition of the p38 MAPK pathway also alleviates calcification in VICs. There are some studies explaining the possible mechanisms of PP2A in osteoblast differentiation. Bengtsson et al. found that PP2A dephosphorylates Smads, facilitating their nuclear translocation, and thus amplifies BMP-Smad signaling, which is an important pathway for skeletal development and bone formation [58, 59]. Huang et al. reported that the inhibition of PP2A activity may partly rescue osteoblastic apoptosis under oxidative stress conditions [60]. To the best of our knowledge, our study is the first to confirm that OST-induced VIC calcification is mediated by PP2A through the ERK/p38 MAPKs signaling pathway, and it may indicate a new molecular mechanism in osteoblast differentiation of VICs. Limitations In our study we used LB100 to inhibit PP2A activity in ApoE-/- mice and came to the conclusion that PP2A inhibition aggravated the aortic valve calcification of ApoE-/- mice. In vitro studies, we also found PP2A activation alleviated the osteogenic differentiation of VICs. However, further studies involving activating PP2A activity in ApoE-/- mice are necessary to verify if PP2A activation prevents the aortic valve calcification of ApoE-/- mice in vivo, and this may be more helpful for PP2A-target drug development in CAVD. Furthermore, although VICs are the most prevalent cell type in aortic valves and play a crucial role in aortic valve calcification, multiple cell types like endothelial cells and macrophages also participate in the pathological process of CAVD [61]. We cannot rule out that PP2A activity of other cell types contribute to increased aortic valve calcification in our model. What’s more, since administration of pharmacological substances faces off-target effects, deeper analyses using genetically modified mice models with cell-specific inhibition and activation of PP2A are also needed to complete our conclusions. We adopted both pharmacological and genetic approaches to assess the role of PP2A activity in osteoblastic differentiation of VICs. Additionally, we identified that PP2A regulates RUNX2 and ALP expression by involving ERK and p38 MAPK Pathways. However, the exact molecular mechanism by which ERK and p38 MAPK Pathways affects RUNX2 and ALP in VICs remains to be determined. On the other hand, we have only investigated the relationship between PP2A activity and osteogenic transition of VICs in the change of signaling pathway phosphorylation. Current studies also indicating the relationship about PP2A and matrix metalloproteinases (MMPs) activity on the regulating effect of PP2A in tumor invasion [62, 63]. As extensive remodeling of the extracellular matrix in valve leaflets is one of the histopathologic characteristics in CAVD, further studies will be needed to discover whether PP2A affects the regulation of MMPs activity in the pathophysiology of CAVD. Conclusion In summary, the present findings demonstrate that OST-induced VIC osteoblast differentiation is closely associated with the inactivation of PP2A. The inhibition of PP2A activity promotes osteoblast differentiation and mineralization of VICs through the ERK/p38 MAPKs signaling pathway (Figure 6). These findings may contribute to the development of a new LB-100 approach to clinical therapy for CAVD.
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