Autophagy mediates cementoblast mineralization through periostin/β-catenin signaling axis under compression

doi:10.21203/rs.3.rs-2098760/v1

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Autophagy mediates cementoblast mineralization through periostin/β-catenin signaling axis under compression

https://doi.org/10.21203/rs.3.rs-2098760/v1

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Repair of orthodontic external root resorption and periodontal tissue dysfunction induced by mechanical force remains a clinical challenge. Cementoblasts are vital in cementum mineralization, a process important for restoring damaged cementum and regaining healthy periodontal function. Autophagy is a vital self-renewal process for cellular homeostasis under various environmental stimuli. However, how autophagy mediated cementoblast mineralization remains unclear. Here we verified that murine cementoblasts exhibit compromised mineralization under compressive force. Autophagy was indispensable for cementoblast mineralization, and autophagic activation markedly reversed the capacity for cementoblast mineralization and cementum damage in mice. Subsequently, mRNA sequencing analyses identified periostin (Postn) as a regulator of autophagy and cementoblast mineralization. Cementoblast mineralization was significantly inhibited following knockdown of Postn. Furthermore, Postn silencing downregulated Wnt transcriptional activity by promoting ubiquitination of β-catenin. Together our results highlight autophagy as a mediator of cementoblast mineralization via Postn/β-catenin signaling under compressive force and may provide a new strategy for the remineralization of cementum and regeneration of periodontal tissue.

autophagy

mineralization

cementum

periostin

β-catenin

ubiquitination

Cementum, a thin layer of mineralized tissue covering the root surface, connects the periodontal ligament (PDL) to the tooth. The integrity of cementum is important to dental stability and periodontal function¹. However, mechanical force–induced cementum damage often occurs during orthodontic tooth movement, resulting in external root resorption and periodontal tissue dysfunction^{2, 3}. Cementoblasts secrete mineralized materials to form cementum tissue and thus are responsible for repairing cementum and restoring periodontal function^{2, 4}. Cementoblast functions are modulated by compressive force, but the effects of compressive force on cementoblast mineralization remain controversial^{5, 6}. Investigations into how compressive force impacts cementoblast mineralization are important for the development of therapeutics to repair external root resorption and achieve integrated periodontal function following orthodontic treatment.

Autophagy is a process of self-degradation in which dysfunctional macromolecules and organelles are degraded and recycled⁷. Although autophagy is required to maintain cellular homeostasis, insufficient or excessive autophagy induced by environmental stimuli can influence cell survival and differentiation^{8, 9}. Accumulating data have confirmed that autophagic vesicles are responsible for transporting mineralized materials to the extracellular matrix (ECM), highlighting the role of autophagy in the remolding of mineralized tissue^{7, 10}. However, the effects of autophagy on cementoblast mineralization subjected to compression remain unclear, and the downstream mechanism that regulate autophagy-mediated changes in cementoblast require further exploration.

Mineralization-related signaling pathways, including the TGF-β, Wnt, and Hippo pathways, have attracted interest as therapeutic targets for root resorption^11–13. Increasing evidence has shown that autophagy regulates many signaling pathways, including TGF-β, Wnt, and PI3K signaling^14–16. The cooperation between autophagy and Wnt/β-catenin signaling remains poorly understood. Autophagosome maturation in autophagy flux occurs via Wnt transcriptional complex recruitment¹⁷. Components of the Wnt signaling can be degraded via the formation of an autophagic complex with β-catenin¹⁸. Thus, it is important to identify the molecules or signaling pathways responsible for cementoblast mineralization.

Here we elucidate the effects of compressive force on cementoblast mineralization and autophagy of cementoblasts. The role of autophagy in cementoblast mineralization under compression was determined in vitro and in vivo. Then we investigated the downstream mechanism of autophagic effects on cementoblast mineralization. We performed gene expression profiling of autophagy inhibitor–treated cementoblasts to identify molecules involved in cementoblast mineralization and further verified the related signaling pathways. These findings broaden understanding of the cellular and molecular events underlying cementoblast mineralization subjected to compressive force and will aid in the development of new therapeutic targets for the repair of root resorption in orthodontic treatment.

Compressive force suppresses mineralization and autophagy of cementoblasts

Cementoblasts incubated in proliferative and mineralized environments were designated the PM and MM groups, respectively. Cementoblast mineralization at different time points was confirmed by qRT-PCR (Fig. S1A). Compressive force inhibited mRNA expression of OCN and OSX, which are related to cementoblast mineralization, in both the PM and MM groups (Fig. 1A). Consistent with these results, Western blotting and ALP staining showed a decreased mineralization capacity in cementoblasts under compressive force (Fig. 1B, C).

To evaluate the effects of compressive force on autophagy of cementoblasts, two reliable markers of autophagy, expression of the autophagic receptor P62 and the conversion of LC3-I to LC3-II, were examined¹⁹. Western blotting showed that expression of P62 increased but conversion of LC3-I to LC3-II decreased following the exposure of cementoblasts to compressive force (Fig. 1D). Furthermore, immunofluorescence staining showed that the formation of autophagosome-positive LC3 was significantly decreased in cells stimulated with compressive force (Fig. 1E), which indicates that compressive force inhibits autophagic activity in cementoblasts. To assess changes in autophagy under compressive force in vivo, we established a mouse root resorption model (Fig. S1B). With increasing mechanical force-loading time, obvious resorption lacunae were observed on the compressed side of the distal buccal root after the application of force for 3 weeks (Fig. 1F). IHC showed that mechanical compressive force led to significant downregulation of LC3 on the compressed side of the root (Fig. 1G).

Autophagy is required for cementoblast mineralization

To determine the role of autophagy in cementoblast mineralization, we first detected changes in autophagy in cementoblasts under a mineralized environment and found that expression of P62 decreased but conversion of LC3-I to LC3-II increased with induction time (Fig. S2A). Then we treated cementoblasts with the autophagy activator Rapa to enhance autophagy or the autophagy inhibitor CQ to suppress autophagy. The effects of Rapa (1 µg/mL) and CQ (5 µM) on autophagy were confirmed (Fig. S2B). qRT-PCR analyses indicated that treatment with Rapa significantly increased expression of genes OSX and OCN related to cementoblast mineralization, whereas treatment with CQ decreased expression of mineralization-related genes in both the PM and MM groups (Fig. 2A). Western blotting and immunofluorescence analyses also showed enhanced capacity for cementoblast mineralization when cells were treated with Rapa and decreased mineralization ability when cells were treated with CQ (Fig. 2B, C). ALP staining and activity confirmed these results (Fig. 2D). To verify these findings in vivo, we injected mice intraperitoneally for 3 weeks with Rapa to activate, or CQ to block, autophagy. IHC showed that OCN expression on the surface of the root was significantly increased in the Rapa treatment group and decreased in the CQ treatment group (Fig. 2E).

Compression-suppressed cementoblast mineralization is partially dependent on autophagy

Next we determined whether compressive force regulates cementoblast mineralization via autophagy. Compression-inhibited expression of the mineralization-related genes OSX and OCN was rescued by Rapa in the PM and MM groups (Fig. 3A). Western blotting also revealed that the decrease in OSX and OCN protein levels under compressive force was reversed following treatment with Rapa (Fig. 3B). To further confirm the effects of autophagy on cementoblast mineralization in vivo, we systemically injected mice with Rapa and applied force for 3 weeks. Root resorption was observed by micro-CT; force-induced root resorption was partially rescued in Rapa-injected mice (Fig. 3C). The compressed side of the root exhibited reduced cementoblast mineralization, as reflected by expression of OCN; this effect was reversed following treatment with Rapa (Fig. 3D). Because the quality of PDL attachment is vital for restoring periodontal function after the application of force, PSR staining was used to evaluate fibers attached to the cementum. PDL attachment to the compressed side of root surfaces was significantly decreased; this was partially reversed by Rapa injection (Fig. 3E).

Identification and validation of periostin as a key molecule associated with autophagy and mineralization of cementoblasts

To identify the molecular mechanism by which the inhibition of autophagy downregulates cementoblast mineralization, we performed high-throughput RNA-seq to identify differentially expressed mRNAs of cementoblasts treated with or without CQ. The results were visualized in volcano plots (Fig. 4A). A total of 73 mRNAs were significantly upregulated and 88 mRNAs were significantly downregulated in cementoblasts treated with CQ compared to the control group (P < 0.05 and fold change > 1.5). To further identify the mRNAs correlated with cementoblast mineralization, we subjected all differentially expressed mRNAs to Gene Ontology (GO) enrichment analyses. The GO results indicated that bone mineralization (GO:0030500), and ECM organization (GO:0030198) were enriched among biological process (BP) and cellular component (CC) terms (Fig. 4B). Among these GO terms, four mRNAs (periostin [Postn], tenascin-C [Tnc], osteomodulin [Omd], and matrix Gla protein [Mgp]) were significantly downregulated and thus may be involved in cementoblast mineralization. These mRNAs were selected for qRT-PCR verification. We treated cells with the autophagy inhibitor CQ or 3-MA (2.5 mM) and found that two mRNAs (Postn and Tnc) were significantly decreased (Fig. 4C). Postn binds Tnc and fibronectin (FN) and transports them to the ECM, which is fundamental to the construction of the Tnc and FN meshwork structure for mineralized ECM organization²⁰. In light of this, we selected Postn for further validation and mechanistic investigation. Protein levels of Postn were significantly inhibited in CQ- and 3-MA-treated cementoblasts, as determined by Western blotting and immunofluorescence staining (Fig. 4D, E). Furthermore, IHC analyses of CQ-injected mice showed that protein expression of Postn was significantly decreased on the root surface compared to the control group (Fig. 4F).

Knockdown of Postn inhibits cementoblast mineralization

To determine the effects of Postn on cementoblast mineralization, we infected cells with siRNAs against Postn (si Postn-1/2) or a scrambled control (si NC). Expression of Postn was potently reduced following transfection with si Postn-1 and si Postn-2, as determined by qRT-PCR and Western blotting (Fig. S5A, B). Note that after 4 days of induction, knockdown of Postn significantly decreased gene expression of BSP, OSX, OCN, and COL1 compared to si NC (Fig. 5A). Western blotting of BSP, OSX, and OCN and immunofluorescence staining of BSP and OSX showed that cementoblast mineralization was inhibited following knockdown of Postn (Fig. 5B, C). ALP staining and activity showed similar inhibition of cementoblast mineralization following si Postn-1/2 treatment (Fig. 5D). Collectively our data indicate that knockdown of Postn inhibits cementoblast mineralization in vitro. Growing evidence suggests that Postn has a regulatory effect on autophagy^{21, 22}; thus, we examined the effects of Postn on autophagy in cementoblasts. Autophagy was upregulated with knockdown of Postn, as reflected by decreased levels of P62 and increased conversion of LC3-I to LC3-II (Fig. S5C).

Knockdown of Postn regulates Wnt signaling by promoting ubiquitination of β-catenin

Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the Wnt signaling pathway was influenced in cementoblasts when autophagic activity was suppressed (Fig. S6A). mRNA expression of Wnt target genes (CCND1, LEF1, and c-MYC) decreased when cementoblasts were treated with CQ or 3-MA (Fig. S6B). Postn regulates Wnt ligands and the major signal transducer, β-catenin^{23, 24}. Therefore, we examined whether Postn is involved in Wnt/β-catenin signaling and found that Postn knockdown decreased mRNA levels of Wnt target genes, whereas β-catenin transcript levels were only slightly inhibited (Fig. 6A). At the protein level, Postn knockdown reduced total β-catenin accumulation (Fig. 6B). Immunofluorescence analyses further confirmed the inhibition of nuclear accumulation of β-catenin following knockdown of Postn (Fig. 6C).

We further determined whether Postn regulates protein stabilization of β-catenin. We treated cells with CHX (10 µM), a protein synthesis inhibitor, to determine the half-life of β-catenin. Knockdown of Postn significantly prolonged the half-life of β-catenin compared to si NC (Fig. 6D). Next we treated cells with the Wnt signaling agonist SKL2001 (30 µM), which disrupts formation of the β-catenin destruction complex. SKL2001 significantly increased expression of β-catenin in control cells, whereas only a slight upregulation of β-catenin was observed in cells in which Postn was knocked down (Fig. 6E), which indicates that Postn may play a role in the degradation of β-catenin. Ubiquitination is required for the degradation of β-catenin. We treated cementoblasts with the proteasome inhibitor MG132 (5 µM) and found that β-catenin levels decreased after Postn knockdown; this decrease was reversed following treatment with MG132, which suggests that Postn regulates the degradation of β-catenin in a proteasome-dependent manner (Fig. 6F). IP analyses confirmed that Postn knockdown in cementoblasts significantly increased the ubiquitination of β-catenin compared to si NC (Fig. 6G). Taken together, as shown in the schematic diagram (Fig. 6H), autophagy restores cementoblast mineralization through Postn/β-catenin signaling axis under compressive force.

The orthodontic mechanical force modulates the function of cementoblasts, which is closely correlated with cementum remineralization and periodontal regeneration. In this study, we determined the role of autophagy in the modulation of cementoblast mineralization under compressive force and explored how autophagy-regulated Postn is involved in cementoblast mineralization. Several studies have indicated that mechanical force regulates the osteogenic differentiation of PDL cells^{25, 26}, but the effects remain controversial. In particular, the effects of compressive force on cementoblast mineralization are ambiguous. Our previous study found that orthodontic force inhibits cementoblast mineralization in vivo²⁷. Consistent with previous findings, the present study also showed an adverse effect of compressive stimulation on cementoblast mineralization.

Autophagy is a crucial survival mechanism of cells under stress²⁸. Our data demonstrated that autophagic activity was inhibited when cementoblasts were subjected to compressive force in vitro and in vivo. Recently, evidence has emerged in support of a vital role of autophagy in promoting osteoblast differentiation and bone mineralization^{7, 14, 29}. Based on insights from these studies, we investigated whether autophagy is involved in cementoblast mineralization. We found that activation of autophagy enhanced cementoblast mineralization, whereas inhibition of autophagy inhibited cementoblast mineralization in normal or mineralized environments. Furthermore, inhibition of cementoblast mineralization induced by compressive force was markedly reversed by autophagic activation with Rapa in vitro and in vivo. These data suggest that the decreased autophagy in cementoblasts under compressive force is linked to their compromised mineralization ability. It is worth noting that reversing the inhibition of autophagy under compressive force with Rapa injection rescued not only cementoblast mineralization but also PDL attachment to the compressed root surface. Together these data indicate that increased autophagy helps to restrain force-induced external root resorption and restore cementum mineralization.

To identify molecules involved in autophagy and cementoblast mineralization, we used transcriptional profiling analyses to compare gene expression in cementoblasts treated with or without the autophagy inhibitor CQ. After screening and validation, Postn was selected for further study. As a secreted matricellular protein, Postn is preferentially expressed at sites of the hard tissue–soft tissue interface of teeth, where it mediates cell–matrix interactions³⁰. The biological functions of Postn in collagen fibrillogenesis are of great importance in ECM mineralization and maintenance of the periodontal functional unit^{31, 32}. However, the effects of autophagy on Postn remain largely unknown. We found that the expression of Postn in cementoblasts was downregulated following inhibition of autophagy in vitro and in vivo.

Postn promotes cell adhesion, migration, and differentiation^{24, 30, 32}. Postn is also responsible for the integrity of the PDL and promotes osteogenesis of PDL cells during periodontal tissue healing^{33, 34}. Based on this, we knocked down Postn to confirm its role in cementoblast mineralization. Levels of mineralization-related genes and proteins decreased after Postn knockdown. Immunofluorescence staining and ALP activity were consistent with these results, which suggests that knockdown of Postn leads to impaired mineralization capacity of cementoblasts. In addition, decreased levels of LC3 have been observed following exposure to Postn²¹. The autophagic function of osteoblasts is enhanced when Postn is knocked down²². Consistent with previous studies, we demonstrated that Postn knockdown enhanced autophagic activity in cementoblasts. Our results indicate that autophagy regulates Postn to participate in cementoblast mineralization and that negative feedback of Postn on autophagy is beneficial for maintaining autophagic balance in stressful conditions.

KEGG analyses revealed that the inhibition of autophagy influences Wnt signaling. The Wnt/β-catenin pathway contributes to osteogenic differentiation and bone formation^{35, 36}. We thus investigated whether Postn regulates cementoblast mineralization via Wnt/β-catenin signaling. We discovered that Wnt transcriptional activity was suppressed with Postn knockdown. Decreased protein levels and nuclear accumulation of β-catenin following knockdown of Postn were observed. Decreased nuclear translocation of β-catenin inhibits T-cell factor/lymphoid enhancing factor (TCF/LEF) family–mediated Wnt target gene transcription³⁷. As a core signal transducer of the Wnt pathway, regulation of β-catenin stabilization is complex³⁸. Proteasome-dependent ubiquitination is the principal mechanism behind the degradation of β-catenin³⁹. Here our data revealed that treatment with an activator of the Wnt pathway targeted to the destructive complex of β-catenin (consisting of Axin, Gsk3β, and APC), or proteasomal inhibitor reversed Postn knockdown–mediated β-catenin downregulation. More important, Postn knockdown promoted the ubiquitination of β-catenin in cementoblasts. These findings highlight the role of Postn in aberrant inactivation of Wnt signaling in cementoblast mineralization.

Overall, our data provide strong evidence that autophagy is indispensable to the restoration of compression-suppressed cementoblast mineralization in vitro and in vivo. We further demonstrate that autophagy regulates Postn expression and that Postn modulates cementoblast mineralization partially through the ubiquitination of β-catenin. This newly discovered function of the Postn/β-catenin axis could explain how autophagy mediates cementoblast mineralization under compressive force. Our findings also identify a new therapeutic target for the restoration of cementum mineralization and periodontal tissue regeneration.

Reagents

Chloroquine (CQ) and rapamycin (Rapa) were purchased from ApexBio (Shanghai, China). 3-methyladenine (3-MA), SKL2001 and MG132 were purchased from MedChem Express (Monmouth Junction, NJ). Cycloheximide (CHX) was purchased from AZKKA (Beijing, China).

Cell culture

OCCM-30, an immortalized murine cementoblast cell line, was provided by Dr. Martha J. Somerman (National Institutes of Health, Bethesda, MD, USA). The cell proliferation medium (PM) was prepared with Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA), 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere at 37°C with 5% CO₂ as described previously⁴⁰. To induce cementoblast mineralization, we prepared mineralized medium (MM) with DMEM, 10% FBS, 1% penicillin-streptomycin, 10 mM sodium β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), and 50 µg/mL ascorbic acid (Sigma-Aldrich).

Application of compressive force

To apply compressive force in vitro, we used a mechanical force-loading device. Briefly, a cover glass was placed over a confluent cell layer in the well, and the compressive force was adjusted by changing the number of steel balls in the plastic bottle. Based on our previously established protocols, OCCM-30 cells were subjected to compressive force at a magnitude of 1.5 g/cm² for 12 h to mimic in vivo conditions⁴¹. For experiments assessing the effects of compressive force on cementoblast mineralization, we removed the force-loading device after the application of compression and incubated cells in PM or MM for 4 or 7 days.

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

Total RNA was isolated from primary cells with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. mRNA was reverse-transcribed to complementary DNA with a cDNA transcription kit (Takara, Tokyo, Japan). qRT-PCR was performed with gene-specific primers and SYBR Green (Roche) and an ABI Prism 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences are listed in Appendix Table 1 in the Supplementary Information.

Western blotting analysis

Cells were lysed with radioimmunoprecipation buffer (Solarbio, Beijing, China) for 30 min on ice. Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Solarbio). Equal amounts of protein were electrophoresed on 10% or 15% sodium dodecyl sulfate polyacrylamide gels, and separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich) and incubated overnight at 4°C with the indicated primary antibodies. After washing, the membrane was incubated with the appropriate secondary antibody at room temperature for 1 h. Protein bands were visualized with a chemiluminescence kit (Applygen, Beijing, China), and band intensities were quantified with ImageJ. Antibodies used for Western blotting are listed in Appendix Table 2.

Alkaline phosphatase (ALP) staining and activity

ALP staining was performed with a NBT/BCIP staining kit (Beyotime, Beijing, China) according to the manufacturer’s instructions. After the induction of mineralization for 7 days, cells were fixed in 4% paraformaldehyde and incubated in alkaline solution for 60 min. ALP activity was analyzed with a colorimetric assay kit (Beyotime). Briefly, cells were washed with phosphate-buffered saline (PBS), lysed with 1% Triton X-100 (Sigma-Aldrich), and scraped into distilled water. We detected ALP activity at 405 nm using p-nitro-phenyl phosphate as a substrate. ALP activity relative to the control group was calculated following normalization to protein content.

Immunofluorescence staining

Cell samples were rinsed, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% goat serum (Zhongshan Golden Bridge, Beijing, China). Proteins were visualized with the primary antibodies indicated in Appendix Table 2. Secondary antibodies conjugated with fluorescent dyes were used to visualize target proteins. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Zhongshan Golden Bridge). Images were captured with a confocal imaging system (Carl Zeiss, Jena, Germany).

Mouse root resorption model

Adult male C57BL/6 mice (22–25 g, 6–7 weeks old) were purchased from Vital River Laboratory Animal Technology for use in this study. The experimental protocols were approved by the Animal Use and Care Committee of Peking University (LA2021076).

Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate. The root resorption model was established in the right maxilla of each mouse, and the left side was designated the negative control site. Application of orthodontic mechanical force was performed as described previously²⁷. A nickel–titanium coil spring (wire size, 0.2 mm; Smart Technology, Beijing, China) was used to connect the right maxillary first molar and maxillary incisors and provided a nearly constant force of approximately 20 g. Mice were randomly divided into different groups according to the duration of force applied (0, 1, 2, or 3 weeks), with five mice in each group.

Injection of the autophagy inhibitor CQ or activator Rapa

Mice were subjected to intraperitoneal injection of Rapa (3 mg/kg; ApexBio, Shanghai, China) to activate autophagy, CQ (25 mg/kg; ApexBio) to block autophagy, or PBS as a negative control. Mice were injected every other day for 3 weeks, with five mice in each group.

Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)

Maxillae were collected, fixed in 4% paraformaldehyde for 24 h, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 3 weeks, and embedded with paraffin. Consecutive sections (4 µm) were cut horizontally from the middle to apical third of the maxillary first molar. Sections from a similar position in the roots were used for histological study. Sections were stained with H&E for descriptive histology. After antigen repair, we stained sections for IHC by incubating them with the serial reagents of the UltraSensitive SP IHC Kit (Zhongshan Golden Bridge) according to the manufacturer’s instructions and with primary antibody (anti-LC3, OCN, Postn; 1:200) overnight at 4°C. Root sections were observed with a microscope.

Micro-computed tomography (micro-CT) scanning

Maxillae of mice were scanned with a micro-CT system (skyscan1272; Bruker, Belgium). The following parameters were used for micro-CT: voltage, 50 kV; current, 200 µA; pixel size, 3.5 µm. The average scan time per mouse was 90 min. Micro-CT scanning data were transferred to Mimics 21.0 (Materialise, Leuven, Belgium) for three-dimensional image processing and reconstruction.

Picrosirius red (PSR) staining

For PDL analyses, sections were deparaffinized and stained with PSR dye according to the manufacturer’s instructions. Collagen fibers were analyzed via polarizing microscopy. In this technique, Col-I fibers appear yellow.

High-throughput RNA sequencing (RNA-seq)

OCCM-30 cells were cultured and treated without or with CQ for 24 h and subjected to high-throughput RNA-seq. RNA was extracted from each sample, and ribosomal RNA was depleted with a TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). An RNA library was prepared according to the manufacturer’s instructions, and paired-end sequencing was performed on a HiSeq X system (Illumina). After sequencing, transcriptome sequencing data were filtered by domestic Java code to remove adapter sequences and low-quality reads and mapped to the mouse genome with HISAT2. The raw RNA-seq data were deposited in the Gene Expression Omnibus database.

Small interfering RNA (siRNA) knockdown

All siRNA products were purchased from GenePharma (Shanghai, China). The following RNAi oligonucleotide sequences were used in this study: Postn-1: 5ʹ-GGAGAACAAUGUCAAUGUUTT-3ʹ (sense) and 5ʹ-AACAUUGACAUUGUUCUCCTT-3ʹ (antisense); Postn-2: 5ʹ-GCAGAAGACGACCUUUCAUTT-3ʹ (sense) and 5ʹ-AUGAAAGGUCGUCUUCUGCTT-3ʹ (antisense). When cells reached 60–70% confluence, transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.

Immunoprecipitation (IP)

β-catenin was immunoprecipitated to detect ubiquitination using IP kits (Proteintech, Wuhan, Hubei, China) according to the manufacturer’s instructions. Briefly, we harvested cells by adding lysis buffer, collected the supernatant, and used it to determine the total protein concentration. Protein samples (1–2 mg) were incubated overnight with β-catenin antibody (Proteintech) or control rabbit IgG (Proteintech) at 4°C, followed by the addition of a protein A sepharose bead slurry and incubation for 4 h at 4°C. After bead binding, lysates bound to antibodies were centrifuged, and the pellets were resuspended and prepared for Western blotting. Horseradish peroxidase-conjugated anti-rabbit/mouse IgG secondary antibody was used to detect ubiquitination of β-catenin.

Statistical analysis

Statistical analyses were performed with SPSS 13.0 and GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). All data are presented as means ± standard deviations (SDs) and were evaluated by independent two-tailed Student’s t tests or one-way analysis of variance (ANOVA). Significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001. P > 0.05 was considered not significant (ns).

Data Availability

The data that supported the findings of this study are this study are available within the published article and its Supplementary Information files. Additional data are available from the corresponding author on reasonable request.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81801010) and State Key Laboratory of Oral Diseases Open Fund (SKLOD2022OF04).

Author Contributions

Yuhui Yang: conception, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript. Hao Liu: data acquisition, analysis, and interpretation, and critically revised the manuscript. Ruoxi Wang and Yi Zhao: data acquisition. Yunfei Zheng: data analysis. Yiping Huang and Weiran Li: design, funding acquisition, supervision, project administration, and critically revised the manuscript.

Conflict of Interest

The authors declare no conflicts of interest related to this study.

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Autophagy mediates cementoblast mineralization through periostin/β-catenin signaling axis under compression

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Abstract

Figures

Introduction

Results

Compressive force suppresses mineralization and autophagy of cementoblasts

Autophagy is required for cementoblast mineralization

Compression-suppressed cementoblast mineralization is partially dependent on autophagy

Identification and validation of periostin as a key molecule associated with autophagy and mineralization of cementoblasts

Knockdown of Postn inhibits cementoblast mineralization

Knockdown of Postn regulates Wnt signaling by promoting ubiquitination of β-catenin

Discussion

Materials And Methods

Reagents

Cell culture

Application of compressive force

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

Western blotting analysis

Alkaline phosphatase (ALP) staining and activity

Immunofluorescence staining

Mouse root resorption model

Injection of the autophagy inhibitor CQ or activator Rapa

Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)

Micro-computed tomography (micro-CT) scanning

Picrosirius red (PSR) staining

High-throughput RNA sequencing (RNA-seq)

Small interfering RNA (siRNA) knockdown

Immunoprecipitation (IP)

Statistical analysis

Data Availability

Declarations

References

Additional Declarations

Supplementary Files

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