1 INTRODUCTION

Periodontitis is a chronic inflammatory disease that is mainly caused by pathogenic bacterial infection. Periodontal pathogenic bacteria and their metabolites trigger inflammation in periodontal tissue, resulting in the destruction of periodontal-supporting tissue and even tooth loss.1, 2 Pathogenic bacteria deliver microbial virulence factors such as microbial peptides, and bacterial antigens directly act on host cells,1, 3 and host-derived enzymes and cytokines further participate in tissue destruction.

GroEL, part of the heat shock protein (HSP) family, plays a key part in protein folding, intracellular protein trafficking, and responding to denatured proteins and is found in almost all prokaryotes and eukaryotes.4, 5 Moreover, GroEL is an important molecule associated with bacterial infectious diseases and autoimmune diseases.4, 5 There are increased anti-HSP60 levels in the serum of cardiovascular patients with severe periodontal infection,6 and the antibodies against Porphyromonas gingivalis GroEL in the serum are markedly decreased by periodontal treatment.7 Several previous studies reported that GroEL acts on host cells, which can cause changes in their biological behavior. For example, GroEL upregulated the secretion of cytokines in human periodontal ligament (PDL) cells, human gingival fibroblasts and osteoblasts, potentially via NF-κB activation.8-10 Our previous study also illustrated that GroEL can stimulate osteoblasts to boost gelatinase secretion.11 However, the potential mechanisms of the GroEL-host interaction are not completely clear.

PDLSCs, which consist in PDL tissue, possess characteristics of mesenchymal stem cells and have good potential for proliferation and multidirectional differentiation.12 Previous study proved that PDLSC transplantation contributes to periodontal tissue repair in immunocompromised rats,13 and PDLSCs can repair alveolar bone defects in periodontitis,14, 15 indicating that PDLSCs possess the osteogenic potential of repairing and regenerating impaired periodontal bone tissue. Moreover, some studies have reported that bacterial virulence factors such as lipopolysaccharide (LPS) inhibit PDLSC osteogenesis,16-18 although it is still controversial.19-21 Cheng M et al showed that LPS could inhibit osteogenic capacity of PDLSCs,18 whereas Xing Y et al. reported that E. coli-derived LPS promoted osteogenic differentiation of PDLSCs by Wnt/β-catenin-induced TAZ elevation.21 As another important virulence factor, GroEL, has been proved to increase the cytokines levels in PDLCs.8 Local injection of bacterial GroEL induced absorption of the skull in mice and alveolar bone in rats.8, 9 E. coli GroEL showed significant osteolytic activity.22 These results strongly inferred the contribution of GroEL to bone homeostasis. PDLSCs serve as seed cells for periodontal bone regeneration, but the effect of GroEL on the differentiation potential of PDLSCs is not clear. Therefore, it is of importance to understand the differentiation changes of PDLSCs when exposed to bacterial virulence factor GroEL and its inner regulation mechanism.

In this study, we aimed to explore the effect of GroEL on the osteogenic and adipogenic differentiation of hPDLSCs and the potential mechanisms, which might be helpful for us to better understand the effect of bacterial virulence factors on the biological behavior of oral cells and potential effect of GroEL on tissue regeneration.

2 MATERIALS AND METHODS 2.1 Cell culture

The procedures were approved by Human Research Ethics Committee of west China hospital of stomatology (WCHSIRB-D-2020-048, Sichuan University, Chengdu, China) and all patients signed informed consent form. PDL tissues were acquired from extracted human premolars because of orthodontic treatment (aged 11 to 24 years). hPDLSCs were isolated according to the previous study.23 Briefly, PDL tissues in the middle 1/3 of the root were gently scratched and cut up into 1 mm3 sections. Next, the sections were digested with 3 mg/mL collagenase I* for 30 minutes at 37°C.Then PDL sections were transferred into α-MEM medium complement with 1% penicillin–streptomycin† solution and 10% fetal bovine serum (FBS)† in an atmosphere of 5% CO2 at 37°C. The medium was changed after 5 days. PDLSCs at passage 3∼5 were used in this study.

2.2 Colony-forming assay

To assay colony-formation efficiency, hPDLSCs (1 × 103 cells) were distributed onto 10 cm2 cell culture dishes for 10 days and the medium were renewed every 3 days. The cultures were fixed with 4% paraformaldehyde (PFA) for 15 minutes, washed 3 times with PBS, and dyed with crystal violet‡ . The collection > 50 cells was counted as a colony.

2.3 Osteogenic differentiation

The hPDLSCs were seeded onto six-well (2.5 × 105 cells/well, for Western blot and real-time PCR), 24-well (6 × 104 cells/well, for alizarin red staining) or 48-well (4 × 104 cells/well, for alkaline phosphatase [ALP] staining) plates and cultured in 2, 0.8, or 0.5 mL 10% FBS α-MEM medium, separately. After achieving 80 to 90% confluence, hPDLSCs were induced with osteogenic differentiation medium [10% FBS α-MEM with 50 μg/mL ascorbic acid§ , 0.02μM dexamethasone§ and 8 mM β-glycerol phosphate§] with or without GroEL§ (recombinant chaperonin 60 from E. coli) (0.5, 1, 5, and 10 μg/mL). For signal pathway inhibition experiments, hPDLSCs were pretreated with 5 μM ST2825∥ (a myeloid differentiation factor 88 [MyD88] inhibitor), 5 μM BAY-117085¶ or 10 μM SP600125# for 1 hour and then added with or without GroEL (10 μg/mL). The cell samples were harvested at 3 days (for real-time PCR and Western blot), 4 days (for ALP staining), 7 days (for real-time PCR, Western blot and ALP staining), 10 days (for alizarin red staining), 14 days (for Western blot and alizarin red staining), and 21 days (for alizarin red staining) after induction.

2.4 Adipogenic differentiation

The hPDLSCs were seeded onto six-well (2.5 × 105 cells/well, for Western blot) or 24-well (8 × 104 cells/well, for oil red staining) plates and separately cultured in 2 or 0.8 mL 10% FBS α-MEM medium. After achieving 100% confluence, cells were induced in adipogenic differentiation medium [10% FBS α-MEM with 5 μM indomethacin§, 10 μg/mL bovine insulin¶, 2.5 × 10−2 μM dexamethasone and 50 μM 3-isobutyl-1-methylxanthine¶] with or without GroEL (1 and 10 μg/mL). For signal pathway inhibition experiments, hPDLSCs were pretreated with 5 μM ST2825, 5 μM BAY117085, or 10 μM SP600125 for 1 hour and then added with or without GroEL (10 μg/mL). After stimulation for 21 and 30 days, cells were collected for Western blot and oil red staining.

2.5 staining

BCIP/NBT ALP Color Development Kit** was used for ALP staining. Briefly, the cells were rinsed with PBS and fixed in 4% PFA for 15 minutes. Next, the cells were stained with BCIP/NBT dye solution for 15 to 30 min at room temperature in dark, followed by rinsing with ddH2O. The images of cells were observed with the light microscope†† .

2.6 Alizarin red staining

The degree of extracellular matrix calcification was measured by Alizarin Red staining. In short, after fixation with 4% PFA for 15 minutes, the cells were stained with 1% Alizarin Red S solution‡‡ for 30 to 60 min, followed by removing the remaining Alizarin Red solution. Then pictures of calcium nodus were obtained with the light microscope∥∥.

2.7 Oil red staining

The presence of lipid was detected by oil red O staining. After fixation with 4% PFA, the cells were flushed with ddH2O and then stained with oil red O solution§§ for 1 hour. After irrigation by ddH2O, the images of lipid were taken by the light microscope∥∥ .

2.8 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from hPDLSCs was extracted with a RNApure total RNA fast isolation Kit¶¶ . The RNA samples were quantitated with the Nanodrop spectrophotometer## . Then, RNA was reverse transcripted into cDNA with the cDNA synthesis kit. Quantitative RT-PCR reactions were done on the iCycler*** conforming to the protocols recommended by the manufacturer. The expression of osteogenic related genes and toll-like receptor (TLR) genes were calculated using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control by a ΔΔCt method. The sequences of specific primers are listed in Table S1.

2.9 Western blot analysis

After washed three times with PBS, the cells were lysed in radio immunoprecipitation assay lysis buffer††† and then added 1% phenylmethanesμlfonyl fluoride‡‡‡ immediately. The protein concentration of the samples was detected by BCA protein concentration determination kit§§§ . The protein samples were then heated at 100°C for 5 minutes. Proteins were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were then transferred from the gels to polyvinylidene difluoride (PVDF) membranes∥∥∥ . After blocking with 5% non-fat dry milk solution for 1 hour, the membranes were incubated at 4°C for 12 hours with 1:1000 primary antibody: mouse anti-β-actin¶¶¶ ; rabbit anti-NF-κB### ; mouse anti-NF-κB p65**** ; rabbit anti-JNK****; rabbit anti-Phospho-JNK****; rabbit anti-p38****; rabbit anti-p38-MAPK(Phospho-Thr180/Tyr182)****; rabbit anti-ERK1+ERK2###; rabbit anti-ERK1 (phospho T202) + ERK2 (phospho T185)###; rabbit anti-Collagen I###; rabbit anti-Runx2###; rabbit anti-Sp7/Osterix****; rabbit anti-ALP###; rabbit anti-C/EBP****; rabbit anti-PPAR****. The secondary antibodies were incubated at room temperature for 2 hours. The bands on the PVDF membrane were displayed by a Western Blotting Luminol Reagent Kit¶¶¶. Image J software was used for quantitative analysis of protein bands.

2.10 Immunofluorescence and confocal laser scanning microscopy (CLSM)

The operating procedures of CLSM were done as previously described.24 Briefly, hPDLSCs were plated onto Glass Bottom Dish and cultured with 10% FBS α-MEM medium with or without GroEL (10 μg/mL) for 10 hours. For signal pathway inhibition experiments, hPDLSCs were pretreated with 5 μM ST2825 for 1 hour and then added with GroEL (10 μg/mL) for 10 hours. After rinsing with PBS, the cells were fixed with 4% PFA for 15 minutes. Following irrigation with PBS, the cells were permeabilized with 0.5% Triton X-100. The samples were blocked for 1 hour with 5% bull serum albumin. Next, all samples were incubated at 4°C overnight with 1:200 primary antibody: NF-κB(acetyl K310)###; total NF-κB****; Phospho-JNK****. A fluorescein-conjugated anti-rabbit antibody AlexaFluor 647### and a fluorescein-conjugated anti-mouse antibody AlexaFluor 647### were incubated for 2 hours at room temperature. The cytoskeletons and nuclei were respectively stained with FITC-phalloidin†††† and DAPI‡‡‡‡ . The fluorescent staining images were captured by CLSM§§§§ .

2.11 Statistical analysis

All data are obtained from at least three different donors. The results are averaged by three independent experiments and meet the minimum biological repetition to make sure data reproducibility (n = 3), presented as the mean ± SEM. We used one-way ANOVA to confirm whether differences existed among groups in the data. GraphPad Prism 8 software was used to test the normal distribution and perform one-way ANOVA analysis. P < 0.05 (α = 0.05) is a significant difference.

3 RESULTS 3.1 Characteristics of isolated PDLSCs

Primary cells from PDL tissues typically grew around the tissue mass and multiplied after 5 to 7 days of culture (the red rectangle indicates the scope, and the black arrow shows the hPDL, Figure S1A). The hPDLSCs exhibited fusiform shapes and were arranged spirally (Figure S1B). The formation of single colonies confirmed the high colony-formation efficiency of hPDLSCs (Figure S1C). Multidirectional differentiation potential of hPDLSCs was confirmed by Alizarin Red staining (Figure S1D), ALP staining (Figure S1E) and Oil Red O staining (Figure S1F). Immunofluorescence staining showed that the STRO-1 was positive in harvested PDLSCs (Figure S1G). These results indicate that we successfully harvested hPDLSCs.

3.2 GroEL influences the osteogenic capacity of hPDLSCs

To explore the effects of GroEL on the osteogenic capacity of hPDLSCs, osteogenic-induced hPDLSCs were stimulated with 0, 0.5, 1, 5, and 10 μg/mL GroEL. ALP staining and Alizarin Red staining showed that GroEL treatment significantly reduced ALP expression on days 4 and 7 and suppressed the formation of calcified nodules on days 10 and 21 in a dose-dependent manner (Figure 1A, B). The q-PCR results revealed that the mRNA expression of the osteogenic transcription factors ALP, Collagen I, and Runx2 was significantly decreased in GroEL treatment groups at days 3 and 7 in comparison with the control group (Figure 1C). Further, Western blotting verified that GroEL treatment reduced the expression of proteins (ALP, Collagen I, Osx, and Runx2) at days 3, 7, and 14 of osteogenic induction (Figure 1D, E). These results indicated that GroEL impaired the osteogenic capacity of hPDLSCs. To rule out the possibility that GroEL impaired osteogenic capacity of hPDLSCs through attenuating cell viability, we used CCK-8 assay to measure hPDLSCs viability under different concentrations of GroEL, GroEL/ST2825, GroEL/BAY-117085 and GroEL/SP 600125, and did not find significant difference (Figure S2).

The impact of GroEL on the osteogenic capacity of hPDLSCs. (A-B) Osteogenic-induced hPDLSCs were stimulated with different concentrations of GroEL. ALP staining on days 4 and 7 (A) showing reduced osteogenesis in hPDLSCs treated with GroEL. Alizarin Red staining on days 10 and 21 (B) showing fewer mineralized nodules in hPDLSCs treated with GroEL than in the control group. (C) Representative q-PCR results showing the mRNA expression of osteogenic transcription factors (ALP, Collagen I, and Runx2) in hPDLSCs with or without 10 μg/mL GroEL treatment, which were detected on 3rd and 7th day after osteogenic induction. (D) Representative western blots showing the protein expression of osteogenic transcription factors, including ALP, Collagen I, Osx, and Runx2, in hPDLSCs stimulated with 0, 1, and 10 μg/mL GroEL, which were detected on days 3, 7, and 14 of osteogenic induction. (E) Histograms showing the quantitative analysis of ALP, collagen I, Osx, and Runx2 protein expression in hPDLSCs. n = 3. Significant differences are presented relative to normal controls (* P < 0.05, ** P < 0.01, *** P < 0.005)

3.3 GroEL promotes the adipogenic capacity of hPDLSCs

Next, we investigated the effects of GroEL on the adipogenic capacity of hPDLSCs. The adipogenic-induced cells were stimulated with GroEL (0-10 μg/mL). Oil Red O staining showed that GroEL treatment promoted the formation of lipid clusters in a dose-dependent manner (Figure 2A). Western blotting further verified the expression of proteins (C/EBPα and PPARγ) was increased in the GroEL treatment groups (Figure 2B, C). These results suggested that GroEL enhanced the directed differentiation of hPDLSCs to adipogenic lineages.

The impact of GroEL on the adipogenic capacity of hPDLSCs. (A) Adipogenic-induced hPDLSCs were stimulated with 0, 1, and 10 μg/mL GroEL. Oil Red O staining on day 30 showed more Oil Red O-positive lipid clusters in hPDLSCs treated with GroEL than in the control group. (B) Representative western blots showing the protein expression of C/EBPα and PPARγ in hPDLSCs stimulated with 0, 1, and 10 μg/mL GroEL detected on day 21 of adipogenic induction. (C) Histograms showing the quantitative analysis of C/EBPα and PPARγ protein expression in hPDLSCs. n = 3. Significant differences are presented relative to normal controls (* P < 0.05, ** P < 0.01)

3.4 GroEL activates JNK/MAPK and NF-κB signaling in hPDLSCs

To investigate the potential mechanisms of GroEL-mediated changes of differentiation in hPDLSCs, we implemented Western blotting to measure the changes in potential signaling pathway proteins. The Western blot results showed that NF-κB (acetyl p65) and phosphorylated JNK (p-JNK) were increased after GroEL incubation. However, GroEL stimulation had no significant influence on other signaling pathway components, including total NF-κB, JNK, p38, p-p38, ERK, and p-ERK (Figure 3A, B). IF staining showed that p-JNK accumulation in the nucleus was observed after GroEL (10 μg/mL) treatment (Figure 3C). Moreover, GroEL (10 μg/mL) accelerated nuclear accumulation of NF-κB in hPDLSCs (Figure 3D), and we further measured NF-κB (acetyl p65), which is acetylated at K310 in the nucleus and function directly to the genome, and found that the levels of NF-κB (acetyl p65) were increased in hPDLSCs with 10 μg/mL GroEL stimulation (Figure 3D).

GroEL activates JNK/MAPK and NF-κB signaling in hPDLSCs. (A) Representative western blots showing the activated NF-κB (acetyl P65) and p-JNK, and other unvaried signaling pathway factors, including ERK/p-ERK and p38/p-p38, in hPDLSCs treated with 0, 1, and 10 μg/mL GroEL for 10 hours. (B) The graph showing the quantification of NF-κB (acetyl p65) and p-JNK protein expression in hPDLSCs. (C) Representative IF staining by CLSM showing the nuclear accumulation of activated p-JNK in hPDLSCs treated with GroEL (10 μg/mL) for 10 hours. p-JNK, red; nucleus, blue; cytoskeleton (F-actin), green. (D) Representative IF staining by CLSM showing the nuclear accumulation of activated NF-κB (C-terminal, total protein) and increased expression of nuclear NF-κB (acetyl K310, nuclear p65) in hPDLSCs treated with GroEL (10 μg/mL) for 10 hours. NF-κB (C-terminal, total protein) and NF-κB (acetyl K310, nuclear p65), red; nucleus, blue; cytoskeleton (F-actin), green. n = 3. Significant differences are presented relative to normal controls (** P < 0.01)

3.5 Inhibition of MyD88 blocks GroEL-induced JNK/MAPK signaling and NF-κB signaling

It is widely recognized that the TLRs family exert an enormous function on initial mammalian recognition of infectious pathogens.25, 26 In addition, TLR1-2, and TLR4-6 are mainly present in the cell membrane, whereas TLR3, and TLR7-9 are mainly expressed in the cytoplasm.25, 26 We first performed a q-PCR assay to identify changes of TLRs in hPDLSCs after treatment with GroEL (Figure 4A and Figure S3). q-PCR analysis demonstrated that the expression of membrane-bound TLR2 and TLR4 significantly increased after stimulation with GroEL for 6 hours (Figure 4A). Meanwhile, the expression of TLR3 also increased in hPDLSCs treated GroEL (Figure S3). There was no significant change in the expression of TLR1, TLR5-7, and TLR9 (Figure S3). ST2825, a specific inhibitor of MyD88 (which is an adaptor protein and exerts an essential function on the intracellular signaling mediated by all TLRs except TLR3), can block the TLRs signaling pathway.27 To confirm whether GroEL-stimulated cytoplasmic signaling were mediated by TLRs, we then used ST2825 to examine the changes of GroEL-mediated JNK/MAPK and NF-κB signaling in hPDLSCs. Western blot analysis showed that ST2825 pretreatment blocked GroEL-upregulated expression of p-JNK and NF-κB (acetyl-p65) in hPDLSCs (Figure 4B, C). IF staining further revealed that, after pretreatment with ST2825, GroEL did not increase the nuclear accumulation of NF-κB (total protein), the levels of NF-κB (acetyl p65) in the nucleus (Figure 4D), or enhance the nuclear accumulation of p-JNK (Figure 4E).

Inhibition of MyD88 blocks GroEL-induced JNK/MAPK signaling and NF-κB signaling. (A) Representative q-PCR result showing increased mRNA expression of TLR2 and TLR4 in hPDLSCs stimulated with 0, 1, and 10 μg/mL GroEL. (B) Western blot showing the expression of NF-κB (total p65)/NF-κB (acetyl-P65), and JNK/p-JNK in hPDLSCs treated with GroEL (10 μg/mL) for 10 hours in the presence or absence of ST2825 (5 μM). (C) Quantification of NF-kB (acetyl-P65) and p-JNK protein expression in (A) was analyzed by ImageJ software. (D) Typical IF staining by CLSM showing reduced nuclear translocation of NF-κB (C-terminal, total protein) and expression of NF-κB (acetyl K310, nuclear p65) in hPDLSCs after pretreatment with 5 μM ST2825 for 1 hour and then stimulation with 10 μg/mL GroEL for 10 hours compared with the group that was only treated with GroEL. NF-κB (C-terminal, total protein) and NF-κB (acetyl K310, nuclear p65), red; nucleus, blue; cytoskeleton (F-actin), green. (E) Representative IF staining by CLSM showing reduced translocation of p-JNK in hPDLSCs after pretreatment with 5 μM ST2825 for 1 hour and stimulation with 10 μg/mL GroEL for 10 hours in comparison with the group treated only with GroEL. p-JNK, red; nucleus, blue; cytoskeleton (F-actin), green. n = 3. Significant differences are presented relative to normal controls (* P < 0.05, ** P < 0.01, *** P < 0.005)

3.6 The inhibitors of MyD88, JNK/MAPK and NF-κB signaling partly restored GroEL-impaired osteogenic differentiation and reverted GroEL-promoted adipogenic differentiation

To verify the important role of TLRs/MyD88 in GroEL-mediated osteogenic and adipogenic differentiation in hPDLSCs, we used ST2825 to examine the impact on the osteogenic and adipogenic capacity of GroEL-stimulated hPDLSCs. ALP staining and Alizarin Red staining showed that ST2825 pretreatment restored the GroEL-impaired ALP expression (Day 4) (Figure 5A) and the formation of calcified nodules (Day 14) (Figure 5B). Oil Red O staining showed that ST2825 treatment reverted GroEL-promoted the formation of lipid clusters (Day 30) (Figure 5C).

The inhibitors of MyD88, JNK/MAPK and NF-κB signaling restore GroEL-induced osteogenic and adipogenic differentiation of hPDLSCs. (A, B) hPDLSCs were treated with osteogenic medium and osteogenic medium supplemented with DMSO, GroEL (10 μg/mL), ST2825 (5 μM), and GroEL (10 μg/mL) + ST2825 (5 μM). Then, ALP staining on day 4 (A) and Alizarin Red staining on day 14 (B) were detected. (C) hPDLSCs were treated with adipogenic medium and adipogenic medium supplemented with GroEL (10 μg/mL), ST2825 (5 μM) and GroEL (10 μg/mL) + ST2825 (5 μM). Then, Oil Red O staining on day 30 were detected. (D) Western blot showing the expression of NF-κB (total p65)/NF-κB (acetyl-P65) and JNK/p-JNK in hPDLSCs treated with GroEL (10 μg/mL) for 10 hours in the presence or absence of BAY117085 (5 μM) and SP600125 (10 μM). (E) Quantification of NF-kB (acetyl-P65) and p-JNK protein expression in (D) was analyzed by ImageJ software. (F, G) hPDLSCs were treated with osteogenic medium and osteogenic medium supplemented with GroEL (10 μg/mL), GroEL (10 μg/mL) + BAY117085 (5 μM) and GroEL (10 μg/mL) + SP600125 (10 μM). Then, ALP staining on day 4 (F) and Alizarin Red staining on day 14 (G) were detected. (H) hPDLSCs were treated with adipogenic medium and adipogenic medium supplemented with GroEL (10 μg/mL), GroEL (10 μg/mL) + BAY117085 (5 μM) and GroEL (10 μg/mL) + SP600125 (10 μM). Then, Oil Red O staining on day 30 were detected

To prove the important role of JNK/MAPK and NF-κB signaling in GroEL-induced osteogenic and adipogenic differentiation in hPDLSCs, we used BAY-117085 and SP600125 to blocking NF-κB and JNK/MAPK signaling to measure the changes of GroEL-induced osteogenic and adipogenic differentiation. Western blot analysis showed that after pretreatment with BAY-117085 and SP600125, GroEL no longer upregulated the expression of NF-κB (acetyl-p65) and p-JNK in hPDLSCs (Figure 5D, E). ALP staining and Alizarin Red staining showed that BAY-117085 and SP600125 partly restored GroEL-impaired ALP expression on days 4 (Figure 5F) and the formation of calcified nodules on days 14 (Figure 5G). Oil Red O staining showed that BAY-117085 and SP600125 partly reverted GroEL-promoted the formation of lipid clusters (Day 30) (Figure 5H).

4 DISCUSSION

In the current study, we harvested hPDLSCs from the extracted young human premolars, and used recombinant GroEL to stimulate hPDLSCs to investigate the impact of bacterial GroEL on the differentiation potential of hPDLSCs for the first time. In this study, we found that GroEL impaired the osteogenic capacity and promoted the adipogenic capacity of hPDLSCs through the involvement of JNK/MAPK and NF-κB signaling (Figure 6).

Schematic of GroEL-mediated osteogenic and adipogenic differentiation of hPDLSCs by regulating JNK/MAPK and NF-κB signaling. GroEL activates cytoplasmic JNK/MAPK and NF-κB signaling and promotes the nuclear accumulation of functionally acetylated NF-κB (p65) and phosphorylated JNK, which ultimately initiate changes in the osteogenic and adipogenic differentiation of hPDLSCs

The dynamic imbalance between plaque biofilms and the host leads to periodontal tissue destruction and more absorption of alveolar bone than formation.1-3 The reconstruction of periodontal tissue damaged by periodontal disease is a main goal of periodontal therapy.13 PDLSCs maintain PDL and alveolar bone homeostasis and have been indicated to be excellent target cells for periodontal tissue regeneration.12, 28 However, the inflammatory microenvironment hampers the osteogenic capacity of PDLSCs,12, 29, 30 although there are some controversies.19, 21 Many previous studies have demonstrated that LPS inhibits the osteogenic capacity of PDLSCs.16, 17, 23 GroEL, as one of the major stimulators of inflammation, contributes to the destruction of periodontal tissue.6-9, 31 It is crucial to investigate the effect of GroEL on the differentiation potential of hPDLSCs and its underlying mechanism for periodontal tissue regeneration. In this study, we showed that GroEL treatment significantly suppressed ALP expression and the formation of calcified nodules in a dose-dependent manner. This finding is consistent with the q-PCR and Western blot results, which showed that GroEL reduced the mRNA and protein expression of osteogenic transcription factors. These results indicated that GroEL could restrain directed differentiation of hPDLSCs to osteogenic lineages. Moreover, the impact of GroEL on adipogenic induced hPDLSCs was also examined. The results showed that GroEL treatment promoted the formation of lipid clusters and the protein expression of adipogenic transcription factors. Thus, we conclude that GroEL may promote directed adipogenic differentiation and competitively suppress directed osteogenic differentiation of hPDLSCs.

TLRs are the key proteins to detect microbes. Some papers have reported that TLRs identify endogenous molecules such as heat shock proteins and other peptides.25 Argueta JG et al. reported that P. gingivalis GroEL activates NF-κB signaling pathway through TLR2 and TLR4 and that anti-hTLR2 and anti-hTLR4 antibodies obviously reduced NF-κB transcriptional activity in THP-1 cells, which indicated the downstream signaling of GroEL was activated through TLR2 and TLR4.32 Ohashi K et al. showed that macrophages from C3H/HeJ mice (carrying a mutant TLR4) showed no response to hsp60 (GroEL), whereas hsp60 induced secretion of TNF-α and NO formation in macrophages from C57BL/6 and C3H/HeN mice, suggesting that TLR4 mediated hsp60 signaling.33 Ueki K et al. reported that anti-CD14 antibody effectively inhibited hsp60-induced TNF-α production and anti-TLR4 antibodies clearly down-regulated the stimulatory effect of hsp60 by 75% in THP-1 cells, which indicated CD14 and TLR4 mediated hsp60 signaling.34 Thus, it can be inferred that GroEL activates intracellular signaling via TLR2 and/or TLR4 in hPDLSCs. In this study, we measured the expression of TLRs in hPDLSCs treated with GroEL and showed that TLR2 and TLR4 expression significantly increased after GroEL treatment. Meanwhile, the expression of TLR3, which are expressed in the cytoplasm, also increased after GroEL treatment. We speculate that the downstream cascade signal transduction of activated TLR2 and TLR4 activated TLR3, but the specific activation of TLR3 has not been further explored in this experiment. There was no significant change in the expression of TLR1, TLR5-7, and TLR9. Besides, the expression of TLR8 and TLR10 was very low, so it was not detected by q-PCR assay. Then we measured the NF-κB and MAPK signaling pathways, which are associated with TLRs.25, 32 The results revealed that GroEL induced NF-κB and JNK/MAPK signaling in hPDLSCs. Previous studies have demonstrated that after stimulation, the intracellular signaling cascade causes the release of NF-κB dimer, and NF-κB dimer is further activated by various post-translational modifications and transferred to the nucleus, where it binds to the target gene and become acetylated to initiate the transcription of the target gene.35, 36 It is known that JNK/SAPKs are associated with metabolism, motility, apoptosis and proliferation; when stimulated, some activated JNK transfers into the nucleus to regulate a variety of transcription factors, such as c-Jun, activator protein 1 (AP-1) and p53.37 In this study, the IF staining results showed that GroEL treatment accelerated the nuclear accumulation of p-JNK and total NF-κB in hPDLSCs, and the level of acetylated NF-κB in the nucleus increased.

ST2825 can block TLRs’ downstream signaling by interfering with MyD88 homodimerization.27 Our results showed that GroEL-mediated NF-κB and p-JNK transcriptional activity was significantly inhibited by ST2825. Next, we examined the involvement of MyD88, NF-κB and JNK/MAPK signaling in the effect of GroEL on the osteogenic and adipogenic capacity of hPDLSCs. ST2825 treatment restored GroEL-impaired osteogenic differentiation and reverted GroEL-promoted adipogenic differentiation of hPDLSCs. Blocking JNK/MAPK or NF-κB signaling can partly restore GroEL-impaired osteogenic differentiation and revert GroEL-promoted adipogenic differentiation of hPDLSCs. The results confirmed that MyD88, NF-κB and JNK/MAPK signaling are involved in the impact of GroEL on the differentiation of hPDLSCs.

We admit some limitations in the present study. First, the GroEL used in this study was from E. coli, and GroEL from different organisms may be different in structure, which may lead to different cellular responses. The reason we used GroEL from E. coli is that bacterial GroEL is evolutionarily highly conserved in terms of sequence, function, and structure, and GroEL from E. coli is representative and easily accessible. It has been reported that bacterial GroEL has 51% sequence homology with human hsp60, and amino acid sequencing of E. coli GroEL indicates 85% homology with Actinobacillus actinomycetemcomitans GroEL.38 We cannot rule out the possibility that different results may be because of different sources of GroEL. Therefore, the impact of GroEL from different pathogens on host cells needs to be further studied. Second, we only measured the activation of TLR2 and TLR4 and ST2825 blocks the downstream signaling of TLRs but cannot specifically block TLR2 and TLR4. The role of TLR2 and TLR4 in GroEL-mediated signaling pathway needs additional experiments to confirm.

5 CONCLUSION

In the current study, we demonstrated a mutual effect between bacteria and host cells by exhibiting the effect of bacterial GroEL on the differentiation potential of hPDLSCs for the first time. We concluded that: 1) GroEL impaired the osteogenic capacity of hPDLSCs 2) GroEL enhanced the adipogenic capacity of hPDLSCs; 3) GroEL upregulated JNK/MAPK and NF-κB signaling, which can be blocked by inhibition of MyD88; 4) Inhibiting MyD88 restored GroEL-impaired osteogenic differentiation and reverted GroEL-promoted adipogenic differentiation of hPDLSCs 5) Blocking JNK/MAPK or NF-κB signaling partly restored GroEL effects. This study provides evidence that bacterial products might impact on hPDLSCs differentiation shedding light on a potential relevance of GroEL for tissue regeneration.

ACKNOWLEDGMENTS

We acknowledge the funding provided by the National Natural Science Foundation of China (81600840, 81771047 to Jing Xie, 81901040 to Chenchen Zhou), China Postdoctoral Science Foundation (2019M653440), and Postdoctoral Foundation of Sichuan University Grant (20826041C4102).

CONFLICTS OF INTEREST

The authors report no potential conflicts of interest.

AUTHOR CONTRIBUTIONS

Li Zhang, Chenchen Zhou and Jing Xie designed the studies. Li Zhang, Yujia Cui, Zuping Wu, Linyi Cai, Liu Yang, and Mengmeng Duan performed the experiments. Li Zhang, Lei Cheng and Jing Xie performed data collection and analysis and preparation of the manuscript. All authors contributed to the review of the manuscript, and Jing Xie gave a final approval of the version to be published.