Hydrostatic Pressure promotes odontoblast differentiation via PIEZO1-dependent activation of RUNX2 and WNT16 in SHED

Hydrostatic Pressure promotes odontoblast differentiation via PIEZO1-dependent activation of RUNX2 and WNT16 in SHED | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hydrostatic Pressure promotes odontoblast differentiation via PIEZO1-dependent activation of RUNX2 and WNT16 in SHED Aya Miyazaki, Anrizandy Narwidina, Asuna Sugimoto, Rika Kurogoushi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8381409/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Mechanical stimulation plays a crucial role in odontoblast differentiation. However, the underlying molecular mechanisms remain unclear. We have previously shown that hydrostatic pressure (HP) applied to stem cells from human exfoliated deciduous teeth (SHED) promotes odontoblast differentiation by translocating RUNX2 and increasing WNT16 expression through PIEZO1 signaling. In this study, we further explored the downstream signaling cascade linking PIEZO1 activation and odontoblast differentiation. HP stimulation increased the expression of odontoblast differentiation markers PANX3 and DSPP , as shown by qPCR, and enhanced Alizarin Red staining—results significantly suppressed by siRNA targeting either PIEZO1 or WNT16 . RT-PCR analysis revealed that, among the two known human WNT16 isoforms, only WNT16b was expressed in SHED. qPCR demonstrated that HP-induced WNT16 expression was reduced by si PIEZO1 and further decreased by si RUNX2 . Promoter analysis identified four RUNX2-binding sites within the upstream region of WNT16 . A luciferase reporter assay using plasmids containing the WNT16 promoter showed that RUNX2 overexpression in HEK293 cells significantly increased luciferase activity. Moreover, HaloChIP assays with a HaloTag-RUNX2 expression vector confirmed RUNX2's binding to the WNT16 promoter. These findings suggest that PIEZO1-mediated mechanical stress promotes odontoblast differentiation through the RUNX2-dependent transcriptional activation of WNT16. Biological sciences/Cell biology Biological sciences/Molecular biology Biological sciences/Stem cells PIEZO1 RUNX2 WNT16 SHED odontoblast differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Odontoblasts, mechanosensitive cells located along the periphery of the dental pulp, play an important role in primary dentin formation and contribute to the formation of secondary and tertiary dentin in response to external stimuli. The long-established hydrodynamic theory has proposed that mechanical stimuli, such as thermal, osmotic, or tactile force, induce the movement of dentinal tubular fluid, leading to the activation of sensory nerves and pain perception in the dentin-pulp complex 1 , 2 . Cold stimuli causing sharp pain induce outward flow toward the enamel surface, while heat stimuli causing dull pain induce inward flow 3 . Interestingly, these changes in flow are thought to trigger the movement of odontoblast processes 1 , 4 , suggesting that the movement of dentinal fluid also imposes mechanical stress on odontoblasts themselves and may be converted into cellular responses. Importantly, secondary dentin formation often occurs in response to chronic mechanical loads such as occlusal forces and acts as a compensatory reinforcement mechanism 5 , whereas reparative tertiary dentin is also stimulated by more abrupt mechanical disturbances involving pressure changes 6 , suggesting that positive pressure, representing compressive stress toward the odontoblast, may act as the primary inductive stimulus. However, the molecular mechanisms by which the mechanical stimuli derived from dentinal fluid flow regulate odontoblast differentiation and dentin formation remain poorly understood. In particular, it is unclear how mechanotransduction in odontoblasts translates into specific gene expression programs and functional differentiation under physical conditions. Mechanical signals, converted biochemical signals, are increasingly recognized as crucial regulators of tissue homeostasis and regeneration across various organ systems. Mechanosensitive ion channels are central role in this process by acting as molecular mediators that convert physical forces into intracellular signaling. Among these, piezo-type mechanosensitive ion channel component 1 (PIEZO1) has been identified as a key mechanosensor involved in diverse biological processes, including vascular development, bone remodeling, and stem cell differentiation 7 , 8 . PIEZO1 is a large, mechanically activated cation channel that responds to stimuli such as membrane tension or hydrostatic pressure (HP), triggering calcium influx and downstream transcriptional responses 9 , 10 . We have previously demonstrated that PIEZO1 is expressed in stem cells from human exfoliated deciduous teeth (SHED) and the process of odontoblasts within predentin and mediates odontoblast differentiation of SHED under HP, partly through the nuclear translocation of runt-related transcription factor 2 (RUNX2) and upregulation of WNT16 expression 11 . Therefore, we hypothesized that PIEZO1 regulates WNT16 transcription via RUNX2, thereby forming a mechanotransduction axis crucial for odontoblast differentiation. In the present study, we aimed to elucidate whether RUNX2 directly regulates WNT16 transcriptional activity and how the PIEZO1-Wnt16 axis contributes to odontoblast activity under mechanical stress. Material and Methods Cell isolation and culture Exfoliated deciduous teeth without carious lesions were obtained from 24 healthy children (13 males and 11 females; aged 6–13 years) treated at the Tokushima University Hospital (Tokushima, Japan). Written informed consent was obtained from the parents of all minor donors, and informed assent was also obtained from the children, when appropriate. The experimental protocol was approved by the Ethics Committee of Tokushima University Hospital (approval no. 1799). All experimental procedures were performed in accordance with the relevant guidelines and regulations of the Institutional Review Boards in compliance with the Declaration of Helsinki. Isolation of stem cells from human exfoliated deciduous teeth (SHED) followed the general procedure described by Miura et al (2003) 12 . Briefly extracted teeth were washed with phosphate-buffered saline (PBS) and immersed in PBS supplemented with 2x antibiotic–antimycotic solution (Nacalai Tesque, Japan). The dental pulp tissue was carefully dissected, minced into small fragments, and enzymatically digested for 1 hour at 37°C using 3 mg/mL collagenase (Wako, Japan) and 4 mg/mL dispase (Wako, Japan). The resulting cell suspension was diluted with α-MEM, filtered through a 40 µm cell strainer (Falcon, USA), and centrifuged at 1,000 rpm for 5 min. The cell pellet was resuspended in α-MEM containing 10% heat-inactivated fetal bovine serum (FBS) and 1% Antibiotic–Antimycotic. Cells were cultured under standard conditions and used for experiments at passages 2–3 when they reached approximately 50–60% confluence. Hydrostatic pressure loading SHED were maintained in growth medium consisting of α-MEM supplemented with 10% FBS and 1% antibiotic–antimycotic. The application of hydrostatic pressure (HP) was applied as previously described methods 11 . In brief, cells were seeded onto 35-mm glass-bottom dishes (Matsunami) 1 day before HP exposure. To apply HP, each dish was placed at the bottom of a beaker filled with culture medium to a height of 5 cm, generating an approximate pressure of 0.5 kPa (3.7 mmHg). The control group was cultured under atmospheric conditions with a medium height of 0.3 cm. The HP value was calculated using the formula P = pgh , where p is the medium density, g is the gravitational acceleration, and h is the liquid height. Atmospheric pressure served as the reference (zero), and all HP values indicated gauge pressure. For odontogenic differentiation, the induction medium was composed of growth medium supplemented with 10 mM β-glycerophosphate, 150 µg/mL ascorbic acid, and 10 − 8 M dexamethasone 12 , 13 . The medium was refreshed every 2 days. Mineral deposition was visualized using Alizarin Red S staining, and the stained mineralized areas were quantified using ImageJ software (NIH, USA). Alizarin Red S staining An Alizarin Red S staining kit (PG Research, Tokyo, Japan) was used to evaluate mineralized matrix formation. SHED were cultured in odontogenic induction medium for 7 days with or without HP. After differentiation, cells were fixed with 4% paraformaldehyde and rinsed with PBS. Mineral deposition was then visualized using an Alizarin Red S staining. The stained samples were observed under a phase-contrast microscope, and the areas positive for Alizarin Red S staining were quantified using ImageJ software (NIH, USA). RT-PCR and quantitative PCR Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 2 µg of total RNA using PrimeScript RT Master Mix (Takara, Japan). Conventional RT-PCR was performed using the KOD-Plus Ver. 2 polymerase (TOYOBO, Japan) under the following thermal cycling conditions: 94°C for 3 min; 33 cycles of 94°C for 40 s, 62°C for 30 s, and 72°C for 60 s; and a final extension step was at 72°C for 5 min. The resulting amplicons were separated on 2% agarose gels. Quantitative PCR (qPCR) was performed using TB Green Premix Ex Taq II (Takara, Japan) on a CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The reaction program consisted of 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, with a final step of 95°C for 5 s and 60°C for 30 s. Each reaction was performed in triplicate and repeated independently at least three times. The primer sequences are listed in Supplementary Table S1. siRNA transfection When the cells reached approximately 60–80% confluence, siRNA transfection was performed using Lipofectamine RNAiMAX (Invitrogen, USA) following the manufacturer’s instructions. The siRNA oligonucleotides used in this study were purchased from Dharmacon (USA) and included ON-TARGETplus Human PIEZO1 siRNA (J-020870-11, -12, -21), ON-TARGETplus Human WNT16 siRNA (J-010821-06, -08, -09), ON-TARGETplus Human RUNX2 siRNA (J-012665-06, -07, -08), and the ON-TARGETplus Non-targeting siRNA pool (D-001810-1005) as a negative control. Luciferase reporter assay Human embryonic kidney 293 (HEK293) cells, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were seeded into 96-well plates at a density of 1 × 10 4 cells per well and cultured overnight. The following day, the cells were transiently transfected with a luciferase reporter plasmid (pNL2.1_pWnt16B_NLuc Vector) together with either a HaloTag-RUNX2 expression vector or a HaloTag control vector using Lipofectamine 3000 (Thermo Fisher Scientific, USA). After 24 h of transfection, cell lysates were collected, and luciferase activity was quantified using a GloMax Discover luminometer (Promega, USA). Chromatin immunoprecipitation (ChIP) assay HEK293 cells were seeded in 6-well plates at a density of 4 × 10 5 cells/well and cultured overnight. Cells were transfected with either the HaloTag control vector or HaloTag-RUNX2 expression plasmid (pFN21AB9739) using a transfection reagent. After incubation, the cells were lysed, and chromatin was sheared by sonication for 48 cycles of 30 s on and 30 s off. The lysates were centrifuged, and the supernatant containing soluble chromatin was collected. HaloTag fusion proteins and their associated DNA fragments were captured using the HaloLink Resin (Promega, USA). Following immunoprecipitation, the cross-links were reversed, and the DNA was purified. Enrichment of the RUNX2-binding motif in the WNT16 promoter region was quantified using qPCR. HaloLink Resin was used to recover the HaloTag fusion protein and its associated DNA fragments. The collected samples were de-fixed, the DNA was purified, and the recovery of the RUNX2-binding motif present in the WNT16 promoter was determined by quantitative PCR. The primers for amplification are listed in Supplementary Table S2. Statistical analysis The data presented in Figs. 1 A, 1 B, 1 E, 2 E, 3 , 4 a, and 4 c are from the three independent experiments that yielded similar results. Error bars indicate the standard deviation. Statistical significance was evaluated using the Student’s t-test, with *p < 0.05 and **p < 0.01 considered statistically significant. Results PIEZO1 is essential for hydrostatic pressure-induced cell differentiation of SHED To investigate the role of endogenous PIEZO1 function in the cell differentiation of stem cells from human exfoliated deciduous teeth (SHED) in response to hydrostatic pressure (HP), we transfected PIEZO1 siRNA into SHED and induced cell differentiation under differentiation conditions. Three days after culturing, we examined the pre-odontoblast marker Pannexin 3 ( PANX3 ) 14 , 15 and the odontoblast marker dentin sialophosphoprotein ( DSPP ) 16 by qPCR. The expression of both PANX3 (Fig. 1 A) and DSPP (Fig. 1 B) was substantially reduced by PIEZO1 siRNA transfection compared to that in non-targeting control siRNA (si Scramble ) transfected cells. Furthermore, Alizarin Red S staining was performed to evaluate mineralization levels after 7 days under the differentiation conditions. We found that HP significantly promoted mineralized nodule formation in the control group (siScramble). In contrast, HP-induced mineralization was markedly suppressed in cells transfected with si PIEZO1 (Fig. 1 C–E). These results indicate that PIEZO1 is essential for SHED-mediated odontoblast differentiation, suggesting that PIEZO1 plays a crucial role as a mechanosensing receptor in odontoblasts. WNT16 is involved in hydrostatic pressure-induced mineralization of SHED Among the WNT family members, WNT16 is the most strongly induced by HP in SHED, and its expression is also predominantly upregulated by the PIEZO1 agonist Yoda1, suggesting that WNT16 is the key factor induced by mechanical stimulation in SHED 11 . However, the role of endogenous WNT16 in HP-induced mineralization remains unclear. Since WNT16 has two variants that differ in their 5’ structure, we examined which variant is expressed in SHED using RT-PCR. The result revealed that SHED did not express WNT16a but predominantly expressed WNT16b (Fig. 2 A). Next, to investigate the functional role of endogenous WNT16, we performed siRNA-mediated knockdown of its expression (Fig. 2 B) and subsequently induced mineralization under differentiation conditions for 7 days. Alizarin Red S staining showed that the HP-induced formation of mineralized nodules was significantly suppressed by si WNT16 (Fig. 2 C, D, and E). These findings suggest that WNT16 is involved in the HP-induced mineralization of SHED. PIEZO1-RUNX2 axis mediates WNT16 expression in SHED under hydrostatic pressure In SHED, the expression of WNT16 is induced by HP loading as well as by stimulation with Yoda1, a PIEZO1 activator 11 . However, the mechanism through which PIEZO1 regulates WNT16 expression remains unclear. To clarify this role, SHED were transfected with PIEZO1 siRNA and subjected to HP loading for 3 days. Analysis of quantitative gene expression showed that the HP-induced upregulation of WNT16 expression was significantly attenuated in PIEZO1 knockdown cells (Fig. 3 A). Furthermore, both HP loading and Yoda1 stimulation promote the nuclear translocation of RUNX2, a transcription factor critical for odontoblast differentiation 11 . To investigate the relationship between WNT16 expression and RUNX2, SHED were transfected with RUNX2 siRNA and exposed to HP. Although HP increased WNT16 expression in si Scramble siRNA-transfected cells, this upregulation was suppressed in RUNX2 knockdown cells (Fig. 3 B). In contrast, the knockdown of endogenous WNT16 did not affect the expression of RUNX2 (data not shown). These results suggest that the PIEZO1–RUNX2 axis as the principal signaling pathway for WNT16 expression in SHED. RUNX2 directly regulates WNT16 transcription To elucidate the regulatory relationship between RUNX2 and WNT16, we performed luciferase reporter assays and chromatin immunoprecipitation (ChIP). HEK293 cells were transfected with a WNT16 promoter-driven luciferase reporter plasmid together with a RUNX2 expression vector at two different concentrations (10 ng and 40 ng per well). The introduction of 10 ng of the HaloTag- RUNX2 resulted in a 2.4-fold increase in luciferase activity, whereas 40 ng induced a 5.6-fold increase compared with the HaloTag control (Fig. 4 A). These results indicate that RUNX2 significantly enhanced the transcriptional activity of the WNT16 promoter in a concentration-dependent manner. To further confirm the direct RUNX2 binding to the WNT16 promoter, ChIP assays were conducted on HEK293 cells transfected with either a HaloTag control vector or a HaloTag-RUNX2 expression vector. As a positive control, the enrichment of a RUNX2 binding motif within the BRD2 promoter 17 was assessed. The recovered DNA was subjected to qPCR to evaluate the enrichment at the WNT16 promoter and the BRD2 promoters. HaloTag-RUNX2-transfected cells showed approximately a 7-fold enrichment of multiple RUNX2 binding motifs within the WNT16 promoter compared with the HaloTag control vector-transfected cells (Fig. 4 B and C). Collectively, these findings demonstrated that RUNX2 directly binds to the WNT16 promoter and markedly enhances its transcriptional activity, establishing RUNX2 as an upstream regulator of WNT16 expression. Discussion In the present study, we demonstrated that PIEZO1 and WNT16 are essential for hydrostatic pressure (HP)-induced odontoblast differentiation and mineralization in stem cells from human exfoliated deciduous teeth (SHED). In particular, WNT16 was directly regulated by RUNX2, a master transcription factor involved in odontoblast differentiation. Luciferase reporter assays and ChIP experiments consistently showed that RUNX2 acts as a positive regulator of WNT16 expression. These findings suggest that the PIEZO1–RUNX2–WNT16 axis plays a pivotal role in HP-induced dentin formation. Previous studies have reported that WNT16 plays a critical role in bone metabolism, although its function appears to differ across developmental stages. For example, WNT16 knockout mice do not exhibit impaired bone formation during embryonic or early postnatal development; however, WNT16 is essential for maintaining cortical bone homeostasis during aging 18 . Moreover, osteocyte-specific WNT16 transgenic mice display markedly increased bone mass and strength during adulthood 19 . In line with these findings, mechanical loading induces WNT16 expression in osteoblasts and bone tissue 20 , 21 . Since WNT16 is strongly associated with bone mineral density, cortical thickness, and bone strength, it is considered to be a positive regulator of bone mass 22 . These findings suggest that WNT16 is dispensable for initial bone formation but crucial for maintaining bone homeostasis and facilitating mechanical adaptation. Dentin, like bone, undergoes secondary and tertiary matrix formation in response to external mechanical and pathological stimuli; therefore, WNT16 may also contribute to dentinogenesis under mechanical stress conditions in teeth. RUNX2 is a well-known master transcription factor essential for odontoblast differentiation and dentinogenesis 23 , 24 . Previously, we demonstrated that HP and the PIEZO1 activator Yoda1 promoted the nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased the HP-induced nuclear translocation of RUNX2 in SHED 11 . Interestingly, two independent studies using human third molar-derived dental pulp cells have reported contradictory results on the effects of PIEZO1 activation on odontoblast differentiation: one demonstrated inhibition 25 , while the other showed promotion 26 . Our earlier findings 11 were consistent with this promoting effect, and the differences in mechanical stimulation protocols or culture conditions may explain these discrepancies 27 . Considering these inconsistent results regarding PIEZO1, it is important to identify the downstream pathways that mediate its effects. The present study revealed that the siRNA-mediated knockdown of RUNX2 significantly suppressed HP-induced WNT16 expression, whereas silencing of WNT16 did not affect RUNX2 expression. These results indicate that RUNX2 functions upstream of WNT16, suggesting that WNT16-dependent odontoblast differentiation is a central driver mediated by the PIEZO1–RUNX2 pathway. Collectively, our findings identify the PIEZO1–RUNX2–WNT16 signaling as a previously unrecognized pathway that links mechanical stimulation to odontoblast differentiation in SHED. Promoter and chromatin immunoprecipitation assays confirmed that RUNX2 directly binds to and activates the WNT16 promoter. Furthermore, although two transcript variants of WNT16 have been reported 28 , differing at the 5’ end, only WNT16b was detected in SHED, suggesting that this isoform represents the functionally relevant target of the PIEZO1–RUNX2 pathway in odontoblast differentiation. Although WNT16 has been extensively studied in the context of bone homeostasis and mechanical adaptation 18 , 19 , the present study is the first to demonstrate its direct regulation by RUNX2 in SHED. This establishes the molecular mechanisms of mechanically responsive cells and tissues in bone and dentin biology, suggesting that WNT16 may serve as a critical mediator of secondary and tertiary dentin formation under mechanical stress. In conclusion, this study identified a novel HP-mediated signaling pathway involving PIEZO1, RUNX2, and WNT16 in odontoblast differentiation. Our findings highlight the critical role of mechanical stimuli in odontoblast regulation, provide mechanistic insights into secondary and tertiary dentin formation, and offer a foundation for future research aimed at developing innovative therapies for dental tissue repair and regeneration. Declarations Conflict of Interest The authors declare no conflicts of interest associated with the contents of this article. Funding This research was supported by the JSPS KAKENHI (grant numbers 20H03898, 23K27802, 23H03112, and 24K22179 to T.I. and 23K16207 to A.M.). Author Contribution **A.M.:** Data curation, Formal analysis, Investigation, Writing – original draft, **A.N.:** Data curation, Formal analysis, Investigation, **A.S.:** Investigation, **R.K.** : Investigation, **Y.N.:** Investigation, **N.H.:** Investigation, **A.Y.:** Data curation, validation, **T.I.:** Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, review and editing Acknowledgement We would like to express our appreciation to Dr. Ryo Miyazaki for his support with the promoter experiments. Data Availability The datasets are available from the corresponding author upon reasonable request. References Brannstrom, M. The elicitation of pain in human dentine and pulp by chemical stimuli. Arch. Oral Biol. 7 , 59–62. 10.1016/0003-9969(62)90048-1 (1962). Liu, X. X. et al. Pathogenesis, diagnosis and management of dentin hypersensitivity: an evidence-based overview for dental practitioners. 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Supplementary Files Appendix.docx Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 23 Jan, 2026 Reviews received at journal 19 Jan, 2026 Reviews received at journal 07 Jan, 2026 Reviews received at journal 07 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers agreed at journal 06 Jan, 2026 Reviewers agreed at journal 06 Jan, 2026 Reviewers invited by journal 23 Dec, 2025 Editor invited by journal 23 Dec, 2025 Editor assigned by journal 23 Dec, 2025 Submission checks completed at journal 22 Dec, 2025 First submitted to journal 22 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Iwamoto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIie3QPQuCQBjA8UeCWg5d75DqKwiCFNh3uQiuxbn5piaj1Y/RJI2Cg4s0Ky5F1NRgBOGYEvaynLVF3B8O7oYfz90ByGQ/WPuxCxBADqDw6oAbCb0TxaMfEHglLVQTUSqeHFbnwgY1WoSXUeF3eSfcwnAtuBhmVupRBiTeMN2hmckRM8qTgOjUyhANwUgcq1WSMQcHgMxFZHqtiXkZVEQ7NRHnMcXQoSK4aUr/NEs9xhCJY4u4LDPn+GgEorf03chPctvuqZFr5oWddZfaZLcngh+rQ8+55QoJbybvKeeviUwmk/1xNy6JUXHk+nrbAAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Tsutomu","middleName":"","lastName":"Iwamoto","suffix":""}],"badges":[],"createdAt":"2025-12-17 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16:19:08","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87065,"visible":true,"origin":"","legend":"","description":"","filename":"0857c8edfeea4c3aa3426fedaac1d8c11structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/b21ea69f71dea4605e768ac0.xml"},{"id":98994611,"identity":"98023f9a-6028-4bf6-9adf-2f2599171669","added_by":"auto","created_at":"2025-12-25 11:51:58","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98230,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/cc8fb50a6c27faf2757de3c2.html"},{"id":99312246,"identity":"d28363ec-5472-47a5-8b19-030a17633912","added_by":"auto","created_at":"2025-12-31 16:18:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2315831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePIEZO1 is essential for hydrostatic pressure-induced calcification in SHED.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression levels of odontogenic marker genes such as \u003cem\u003ePANX3 \u003c/em\u003e(A) and \u003cem\u003eDSPP \u003c/em\u003e(B) in SHED. Total RNA was extracted after 72 h of odontogenic induction with or without the hydrostatic pressure (HP). Gene expression was analyzed by qPCR. Alizarin Red S staining for odontoblast differentiation in SHED cultured in induction medium with or without\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003ePIEZO1\u003c/em\u003e-specific siRNA for 7 days. The cells were subjected to hydrostatic pressure (HP). Microscope View (C) and Plate view (D). Alizarin Red-positive areas were quantified using ImageJ (E). Scale bar indicates 150 μm. The error bars indicate the standard deviations. Statistical analysis was performed using the Student’s t-test. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/0bf13d0e5ae721994790fa37.png"},{"id":98994595,"identity":"be2f8f1c-3a67-46ac-8d26-e91e0c67950c","added_by":"auto","created_at":"2025-12-25 11:51:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1994108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eWNT16b\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression is a key for HP-induced calcification in SHED.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cem\u003eWNT16a\u003c/em\u003e and \u003cem\u003eWNT16b\u003c/em\u003eare categorized in \u003cem\u003eWNT16\u003c/em\u003e, and only the expression of \u003cem\u003eWNT16b\u003c/em\u003e was demonstrated in total RNA extracted from SHED by RT-PCR. (B) Expression levels of \u003cem\u003eWNT16b\u003c/em\u003e in SHED with\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eWNT16\u003c/em\u003e-specific siRNA were transfected, and total RNA was extracted from the cells. Gene expression was analyzed by RT-PCR. The gel images shown in A and B represent full-length, uncropped gels and were not digitally manipulated. (C) Alizarin Red S staining for odontoblast differentiation in SHED cultured in induction media with or without\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eWnt16\u003c/em\u003e-specific siRNA for 7 days. Cells were also subjected with or without HP. The scale bar indicates 150 μm. (D) Plate view of C. (E) The Alizarin Red-positive areas were quantified using ImageJ. Cells were also subjected with or without hydrostatic pressure (HP). The Alizarin Red-positive areas were quantified using ImageJ. Scale bar indicates 150 μm. The error bars indicate standard deviations. Statistical analysis was performed using Student’s t-test. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/a6e62b7dc0f2c4d07511c614.png"},{"id":98994594,"identity":"139c83e6-39ff-4d57-a943-66d7006fa8e5","added_by":"auto","created_at":"2025-12-25 11:51:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":326255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of HP-induced \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eWNT16\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene expression is augmented by PIEZO1 and RUNX2 inhibitors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression levels of \u003cem\u003eWNT16\u003c/em\u003e in SHED transfected with\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003ePIEZO1\u003c/em\u003e-specific (A) and \u003cem\u003eRUNX2-\u003c/em\u003especific (B) siRNAs. Total RNA was extracted 72 h after odontogenic induction, with or without the HP. Gene expression was analyzed using qPCR. The error bars indicate the standard deviations. Statistical analysis was performed using the Student’s t-test. *p \u0026lt; 0.05; n.s., not significant.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/1933598e05d1238bc180b6e1.png"},{"id":99312013,"identity":"331f6221-1b85-4303-80e4-900ca4762cda","added_by":"auto","created_at":"2025-12-31 16:17:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":698538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular mechanism underlying RUNX2-mediated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eWNT16\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HEK293 cells were transfected with \u003cem\u003eWNT16\u003c/em\u003epromoter-driven luciferase reporter plasmid together with RUNX2 expression vector (10 ng or 40 ng/well). After 24 h, cell lysates were prepared and assayed for luciferase activity. (B) Schematic representation depicting the positions of RUNX2 binding motifs in the WNT16 promoter and primers used for ChIP assay. (C) HEK293 cells were transfected with each plasmid (HaloTag control vector, pFN21AB9739 (HaloTag-RUNX2)), and the cells were cultured for 24 h. The treated cells were subjected to reversal of cross-linking, sonication, immunoprecipitated, and PCR using the primers listed in Supplementary Table S2. Statistical analysis was performed using the Student’s t-test. **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/b9e061971e0208e18fd13c99.png"},{"id":99313296,"identity":"fc45c479-457d-45e4-8d9c-556c909814cb","added_by":"auto","created_at":"2025-12-31 16:19:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":236908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram illustrating the effects of mechanotransduction on odontoblast differentiation via PIEZO1 and WNT16 in SHED.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon application of HP, the mechanosensitive ion channel PIEZO1 is activated, leading to an influx of mechanical stimuli. This process involves the primary cilia, which act as antennas for these mechanical signals and play a crucial role in modulating cellular responses. The activation of PIEZO1 subsequently leads to the translocation of the transcription factor RUNX2 to the nucleus. Within the nucleus, RUNX2 activates WNT16 expression, which further enhances cellular differentiation through both autocrine and paracrine signaling pathways. The interaction between WNT16 and the Frizzled receptors on the cell surface initiates a signaling cascade that promotes differentiation. This mechanistic pathway underscores the intricate balance between mechanical forces and biochemical signals in promoting odontoblast differentiation and emphasizes the pivotal roles of PIEZO1, RUNX2, and WNT16 in this complex biological process.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/c11525432fa9d1e4ada67306.png"},{"id":106343423,"identity":"c919296a-2067-43a2-9724-c85acdd85f12","added_by":"auto","created_at":"2026-04-07 16:05:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7746398,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/b05bfdf8-61fe-4bdd-90ec-79f5fc0c8e82.pdf"},{"id":98994592,"identity":"88972da1-b733-486c-ad77-5079e168a83d","added_by":"auto","created_at":"2025-12-25 11:51:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19309,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-8381409/v1/43d446f44f8ca53c4cfefa01.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hydrostatic Pressure promotes odontoblast differentiation via PIEZO1-dependent activation of RUNX2 and WNT16 in SHED","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOdontoblasts, mechanosensitive cells located along the periphery of the dental pulp, play an important role in primary dentin formation and contribute to the formation of secondary and tertiary dentin in response to external stimuli. The long-established hydrodynamic theory has proposed that mechanical stimuli, such as thermal, osmotic, or tactile force, induce the movement of dentinal tubular fluid, leading to the activation of sensory nerves and pain perception in the dentin-pulp complex\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Cold stimuli causing sharp pain induce outward flow toward the enamel surface, while heat stimuli causing dull pain induce inward flow\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Interestingly, these changes in flow are thought to trigger the movement of odontoblast processes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, suggesting that the movement of dentinal fluid also imposes mechanical stress on odontoblasts themselves and may be converted into cellular responses. Importantly, secondary dentin formation often occurs in response to chronic mechanical loads such as occlusal forces and acts as a compensatory reinforcement mechanism\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, whereas reparative tertiary dentin is also stimulated by more abrupt mechanical disturbances involving pressure changes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, suggesting that positive pressure, representing compressive stress toward the odontoblast, may act as the primary inductive stimulus. However, the molecular mechanisms by which the mechanical stimuli derived from dentinal fluid flow regulate odontoblast differentiation and dentin formation remain poorly understood. In particular, it is unclear how mechanotransduction in odontoblasts translates into specific gene expression programs and functional differentiation under physical conditions.\u003c/p\u003e \u003cp\u003eMechanical signals, converted biochemical signals, are increasingly recognized as crucial regulators of tissue homeostasis and regeneration across various organ systems. Mechanosensitive ion channels are central role in this process by acting as molecular mediators that convert physical forces into intracellular signaling. Among these, piezo-type mechanosensitive ion channel component 1 (PIEZO1) has been identified as a key mechanosensor involved in diverse biological processes, including vascular development, bone remodeling, and stem cell differentiation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. PIEZO1 is a large, mechanically activated cation channel that responds to stimuli such as membrane tension or hydrostatic pressure (HP), triggering calcium influx and downstream transcriptional responses\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We have previously demonstrated that PIEZO1 is expressed in stem cells from human exfoliated deciduous teeth (SHED) and the process of odontoblasts within predentin and mediates odontoblast differentiation of SHED under HP, partly through the nuclear translocation of runt-related transcription factor 2 (RUNX2) and upregulation of WNT16 expression\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesized that PIEZO1 regulates WNT16 transcription via RUNX2, thereby forming a mechanotransduction axis crucial for odontoblast differentiation.\u003c/p\u003e \u003cp\u003eIn the present study, we aimed to elucidate whether RUNX2 directly regulates WNT16 transcriptional activity and how the PIEZO1-Wnt16 axis contributes to odontoblast activity under mechanical stress.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell isolation and culture\u003c/h2\u003e \u003cp\u003eExfoliated deciduous teeth without carious lesions were obtained from 24 healthy children (13 males and 11 females; aged 6\u0026ndash;13 years) treated at the Tokushima University Hospital (Tokushima, Japan). Written informed consent was obtained from the parents of all minor donors, and informed assent was also obtained from the children, when appropriate. The experimental protocol was approved by the Ethics Committee of Tokushima University Hospital (approval no. 1799). All experimental procedures were performed in accordance with the relevant guidelines and regulations of the Institutional Review Boards in compliance with the Declaration of Helsinki.\u003c/p\u003e \u003cp\u003eIsolation of stem cells from human exfoliated deciduous teeth (SHED) followed the general procedure described by Miura et al (2003)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Briefly extracted teeth were washed with phosphate-buffered saline (PBS) and immersed in PBS supplemented with 2x antibiotic\u0026ndash;antimycotic solution (Nacalai Tesque, Japan). The dental pulp tissue was carefully dissected, minced into small fragments, and enzymatically digested for 1 hour at 37\u0026deg;C using 3 mg/mL collagenase (Wako, Japan) and 4 mg/mL dispase (Wako, Japan). The resulting cell suspension was diluted with α-MEM, filtered through a 40 \u0026micro;m cell strainer (Falcon, USA), and centrifuged at 1,000 rpm for 5 min. The cell pellet was resuspended in α-MEM containing 10% heat-inactivated fetal bovine serum (FBS) and 1% Antibiotic\u0026ndash;Antimycotic. Cells were cultured under standard conditions and used for experiments at passages 2\u0026ndash;3 when they reached approximately 50\u0026ndash;60% confluence.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHydrostatic pressure loading\u003c/h3\u003e\n\u003cp\u003eSHED were maintained in growth medium consisting of α-MEM supplemented with 10% FBS and 1% antibiotic\u0026ndash;antimycotic. The application of hydrostatic pressure (HP) was applied as previously described methods \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In brief, cells were seeded onto 35-mm glass-bottom dishes (Matsunami) 1 day before HP exposure. To apply HP, each dish was placed at the bottom of a beaker filled with culture medium to a height of 5 cm, generating an approximate pressure of 0.5 kPa (3.7 mmHg). The control group was cultured under atmospheric conditions with a medium height of 0.3 cm. The HP value was calculated using the formula \u003cem\u003eP\u0026thinsp;=\u0026thinsp;pgh\u003c/em\u003e, where \u003cem\u003ep\u003c/em\u003e is the medium density, \u003cem\u003eg\u003c/em\u003e is the gravitational acceleration, and \u003cem\u003eh\u003c/em\u003e is the liquid height. Atmospheric pressure served as the reference (zero), and all HP values indicated gauge pressure. For odontogenic differentiation, the induction medium was composed of growth medium supplemented with 10 mM β-glycerophosphate, 150 \u0026micro;g/mL ascorbic acid, and 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M dexamethasone\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The medium was refreshed every 2 days. Mineral deposition was visualized using Alizarin Red S staining, and the stained mineralized areas were quantified using ImageJ software (NIH, USA).\u003c/p\u003e\n\u003ch3\u003eAlizarin Red S staining\u003c/h3\u003e\n\u003cp\u003eAn Alizarin Red S staining kit (PG Research, Tokyo, Japan) was used to evaluate mineralized matrix formation. SHED were cultured in odontogenic induction medium for 7 days with or without HP. After differentiation, cells were fixed with 4% paraformaldehyde and rinsed with PBS. Mineral deposition was then visualized using an Alizarin Red S staining. The stained samples were observed under a phase-contrast microscope, and the areas positive for Alizarin Red S staining were quantified using ImageJ software (NIH, USA).\u003c/p\u003e\n\u003ch3\u003eRT-PCR and quantitative PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer\u0026rsquo;s instructions. Complementary DNA (cDNA) was synthesized from 2 \u0026micro;g of total RNA using PrimeScript RT Master Mix (Takara, Japan). Conventional RT-PCR was performed using the KOD-Plus Ver. 2 polymerase (TOYOBO, Japan) under the following thermal cycling conditions: 94\u0026deg;C for 3 min; 33 cycles of 94\u0026deg;C for 40 s, 62\u0026deg;C for 30 s, and 72\u0026deg;C for 60 s; and a final extension step was at 72\u0026deg;C for 5 min. The resulting amplicons were separated on 2% agarose gels. Quantitative PCR (qPCR) was performed using TB Green Premix Ex Taq II (Takara, Japan) on a CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The reaction program consisted of 95\u0026deg;C for 10 s, followed by 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s, with a final step of 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s. Each reaction was performed in triplicate and repeated independently at least three times. The primer sequences are listed in Supplementary Table S1.\u003c/p\u003e\n\u003ch3\u003esiRNA transfection\u003c/h3\u003e\n\u003cp\u003eWhen the cells reached approximately 60\u0026ndash;80% confluence, siRNA transfection was performed using Lipofectamine RNAiMAX (Invitrogen, USA) following the manufacturer\u0026rsquo;s instructions. The siRNA oligonucleotides used in this study were purchased from Dharmacon (USA) and included ON-TARGETplus Human PIEZO1 siRNA (J-020870-11, -12, -21), ON-TARGETplus Human WNT16 siRNA (J-010821-06, -08, -09), ON-TARGETplus Human RUNX2 siRNA (J-012665-06, -07, -08), and the ON-TARGETplus Non-targeting siRNA pool (D-001810-1005) as a negative control.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eHuman embryonic kidney 293 (HEK293) cells, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were seeded into 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and cultured overnight. The following day, the cells were transiently transfected with a luciferase reporter plasmid (pNL2.1_pWnt16B_NLuc Vector) together with either a HaloTag-RUNX2 expression vector or a HaloTag control vector using Lipofectamine 3000 (Thermo Fisher Scientific, USA). After 24 h of transfection, cell lysates were collected, and luciferase activity was quantified using a GloMax Discover luminometer (Promega, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChromatin immunoprecipitation (ChIP) assay\u003c/h3\u003e\n\u003cp\u003eHEK293 cells were seeded in 6-well plates at a density of 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and cultured overnight. Cells were transfected with either the HaloTag control vector or HaloTag-RUNX2 expression plasmid (pFN21AB9739) using a transfection reagent. After incubation, the cells were lysed, and chromatin was sheared by sonication for 48 cycles of 30 s on and 30 s off. The lysates were centrifuged, and the supernatant containing soluble chromatin was collected. HaloTag fusion proteins and their associated DNA fragments were captured using the HaloLink Resin (Promega, USA). Following immunoprecipitation, the cross-links were reversed, and the DNA was purified. Enrichment of the RUNX2-binding motif in the WNT16 promoter region was quantified using qPCR. HaloLink Resin was used to recover the HaloTag fusion protein and its associated DNA fragments. The collected samples were de-fixed, the DNA was purified, and the recovery of the RUNX2-binding motif present in the WNT16 promoter was determined by quantitative PCR. The primers for amplification are listed in Supplementary Table S2.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec are from the three independent experiments that yielded similar results. Error bars indicate the standard deviation. Statistical significance was evaluated using the Student\u0026rsquo;s t-test, with *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePIEZO1 is essential for hydrostatic pressure-induced cell differentiation of SHED\u003c/h2\u003e \u003cp\u003eTo investigate the role of endogenous \u003cem\u003ePIEZO1\u003c/em\u003e function in the cell differentiation of stem cells from human exfoliated deciduous teeth (SHED) in response to hydrostatic pressure (HP), we transfected \u003cem\u003ePIEZO1\u003c/em\u003e siRNA into SHED and induced cell differentiation under differentiation conditions. Three days after culturing, we examined the pre-odontoblast marker Pannexin 3 (\u003cem\u003ePANX3\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and the odontoblast marker dentin sialophosphoprotein (\u003cem\u003eDSPP\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e by qPCR. The expression of both \u003cem\u003ePANX3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and \u003cem\u003eDSPP\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) was substantially reduced by \u003cem\u003ePIEZO1\u003c/em\u003e siRNA transfection compared to that in non-targeting control siRNA (si\u003cem\u003eScramble\u003c/em\u003e) transfected cells. Furthermore, Alizarin Red S staining was performed to evaluate mineralization levels after 7 days under the differentiation conditions. We found that HP significantly promoted mineralized nodule formation in the control group (siScramble). In contrast, HP-induced mineralization was markedly suppressed in cells transfected with si\u003cem\u003ePIEZO1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;E). These results indicate that \u003cem\u003ePIEZO1\u003c/em\u003e is essential for SHED-mediated odontoblast differentiation, suggesting that PIEZO1 plays a crucial role as a mechanosensing receptor in odontoblasts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWNT16 is involved in hydrostatic pressure-induced mineralization of SHED\u003c/h2\u003e \u003cp\u003eAmong the WNT family members, WNT16 is the most strongly induced by HP in SHED, and its expression is also predominantly upregulated by the PIEZO1 agonist Yoda1, suggesting that WNT16 is the key factor induced by mechanical stimulation in SHED\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the role of endogenous WNT16 in HP-induced mineralization remains unclear. Since WNT16 has two variants that differ in their 5\u0026rsquo; structure, we examined which variant is expressed in SHED using RT-PCR. The result revealed that SHED did not express WNT16a but predominantly expressed WNT16b (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eNext, to investigate the functional role of endogenous WNT16, we performed siRNA-mediated knockdown of its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and subsequently induced mineralization under differentiation conditions for 7 days. Alizarin Red S staining showed that the HP-induced formation of mineralized nodules was significantly suppressed by si\u003cem\u003eWNT16\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D, and E). These findings suggest that WNT16 is involved in the HP-induced mineralization of SHED.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePIEZO1-RUNX2 axis mediates WNT16 expression in SHED under hydrostatic pressure\u003c/h2\u003e \u003cp\u003eIn SHED, the expression of \u003cem\u003eWNT16\u003c/em\u003e is induced by HP loading as well as by stimulation with Yoda1, a PIEZO1 activator\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the mechanism through which PIEZO1 regulates WNT16 expression remains unclear. To clarify this role, SHED were transfected with \u003cem\u003ePIEZO1\u003c/em\u003e siRNA and subjected to HP loading for 3 days. Analysis of quantitative gene expression showed that the HP-induced upregulation of \u003cem\u003eWNT16\u003c/em\u003e expression was significantly attenuated in \u003cem\u003ePIEZO1\u003c/em\u003e knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, both HP loading and Yoda1 stimulation promote the nuclear translocation of RUNX2, a transcription factor critical for odontoblast differentiation \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To investigate the relationship between WNT16 expression and RUNX2, SHED were transfected with \u003cem\u003eRUNX2\u003c/em\u003e siRNA and exposed to HP. Although HP increased \u003cem\u003eWNT16\u003c/em\u003e expression in si\u003cem\u003eScramble\u003c/em\u003e siRNA-transfected cells, this upregulation was suppressed in \u003cem\u003eRUNX2\u003c/em\u003e knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, the knockdown of endogenous \u003cem\u003eWNT16\u003c/em\u003e did not affect the expression of \u003cem\u003eRUNX2\u003c/em\u003e (data not shown). These results suggest that the PIEZO1\u0026ndash;RUNX2 axis as the principal signaling pathway for WNT16 expression in SHED.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRUNX2 directly regulates\u003c/b\u003e \u003cb\u003eWNT16\u003c/b\u003e \u003cb\u003etranscription\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the regulatory relationship between RUNX2 and WNT16, we performed luciferase reporter assays and chromatin immunoprecipitation (ChIP). HEK293 cells were transfected with a \u003cem\u003eWNT16\u003c/em\u003e promoter-driven luciferase reporter plasmid together with a RUNX2 expression vector at two different concentrations (10 ng and 40 ng per well). The introduction of 10 ng of the HaloTag-\u003cem\u003eRUNX2\u003c/em\u003e resulted in a 2.4-fold increase in luciferase activity, whereas 40 ng induced a 5.6-fold increase compared with the HaloTag control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These results indicate that \u003cem\u003eRUNX2\u003c/em\u003e significantly enhanced the transcriptional activity of the \u003cem\u003eWNT16\u003c/em\u003e promoter in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003eTo further confirm the direct RUNX2 binding to the \u003cem\u003eWNT16\u003c/em\u003e promoter, ChIP assays were conducted on HEK293 cells transfected with either a HaloTag control vector or a HaloTag-RUNX2 expression vector. As a positive control, the enrichment of a RUNX2 binding motif within the \u003cem\u003eBRD2\u003c/em\u003e promoter\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e was assessed. The recovered DNA was subjected to qPCR to evaluate the enrichment at the \u003cem\u003eWNT16\u003c/em\u003e promoter and the \u003cem\u003eBRD2\u003c/em\u003e promoters. HaloTag-RUNX2-transfected cells showed approximately a 7-fold enrichment of multiple RUNX2 binding motifs within the \u003cem\u003eWNT16\u003c/em\u003e promoter compared with the HaloTag control vector-transfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrated that RUNX2 directly binds to the \u003cem\u003eWNT16\u003c/em\u003e promoter and markedly enhances its transcriptional activity, establishing RUNX2 as an upstream regulator of WNT16 expression.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we demonstrated that PIEZO1 and WNT16 are essential for hydrostatic pressure (HP)-induced odontoblast differentiation and mineralization in stem cells from human exfoliated deciduous teeth (SHED). In particular, \u003cem\u003eWNT16\u003c/em\u003e was directly regulated by RUNX2, a master transcription factor involved in odontoblast differentiation. Luciferase reporter assays and ChIP experiments consistently showed that RUNX2 acts as a positive regulator of \u003cem\u003eWNT16\u003c/em\u003e expression. These findings suggest that the PIEZO1\u0026ndash;RUNX2\u0026ndash;WNT16 axis plays a pivotal role in HP-induced dentin formation.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that WNT16 plays a critical role in bone metabolism, although its function appears to differ across developmental stages. For example, \u003cem\u003eWNT16\u003c/em\u003e knockout mice do not exhibit impaired bone formation during embryonic or early postnatal development; however, WNT16 is essential for maintaining cortical bone homeostasis during aging\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Moreover, osteocyte-specific \u003cem\u003eWNT16\u003c/em\u003e transgenic mice display markedly increased bone mass and strength during adulthood\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In line with these findings, mechanical loading induces WNT16 expression in osteoblasts and bone tissue\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Since WNT16 is strongly associated with bone mineral density, cortical thickness, and bone strength, it is considered to be a positive regulator of bone mass\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These findings suggest that WNT16 is dispensable for initial bone formation but crucial for maintaining bone homeostasis and facilitating mechanical adaptation. Dentin, like bone, undergoes secondary and tertiary matrix formation in response to external mechanical and pathological stimuli; therefore, WNT16 may also contribute to dentinogenesis under mechanical stress conditions in teeth.\u003c/p\u003e \u003cp\u003eRUNX2 is a well-known master transcription factor essential for odontoblast differentiation and dentinogenesis\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Previously, we demonstrated that HP and the PIEZO1 activator Yoda1 promoted the nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased the HP-induced nuclear translocation of RUNX2 in SHED \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Interestingly, two independent studies using human third molar-derived dental pulp cells have reported contradictory results on the effects of PIEZO1 activation on odontoblast differentiation: one demonstrated inhibition\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, while the other showed promotion\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Our earlier findings\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e were consistent with this promoting effect, and the differences in mechanical stimulation protocols or culture conditions may explain these discrepancies\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Considering these inconsistent results regarding PIEZO1, it is important to identify the downstream pathways that mediate its effects. The present study revealed that the siRNA-mediated knockdown of RUNX2 significantly suppressed HP-induced WNT16 expression, whereas silencing of WNT16 did not affect RUNX2 expression. These results indicate that RUNX2 functions upstream of WNT16, suggesting that WNT16-dependent odontoblast differentiation is a central driver mediated by the PIEZO1\u0026ndash;RUNX2 pathway.\u003c/p\u003e \u003cp\u003eCollectively, our findings identify the PIEZO1\u0026ndash;RUNX2\u0026ndash;WNT16 signaling as a previously unrecognized pathway that links mechanical stimulation to odontoblast differentiation in SHED. Promoter and chromatin immunoprecipitation assays confirmed that RUNX2 directly binds to and activates the \u003cem\u003eWNT16\u003c/em\u003e promoter. Furthermore, although two transcript variants of \u003cem\u003eWNT16\u003c/em\u003e have been reported\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, differing at the 5\u0026rsquo; end, only \u003cem\u003eWNT16b\u003c/em\u003e was detected in SHED, suggesting that this isoform represents the functionally relevant target of the PIEZO1\u0026ndash;RUNX2 pathway in odontoblast differentiation. Although WNT16 has been extensively studied in the context of bone homeostasis and mechanical adaptation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, the present study is the first to demonstrate its direct regulation by RUNX2 in SHED. This establishes the molecular mechanisms of mechanically responsive cells and tissues in bone and dentin biology, suggesting that WNT16 may serve as a critical mediator of secondary and tertiary dentin formation under mechanical stress.\u003c/p\u003e \u003cp\u003eIn conclusion, this study identified a novel HP-mediated signaling pathway involving PIEZO1, RUNX2, and WNT16 in odontoblast differentiation. Our findings highlight the critical role of mechanical stimuli in odontoblast regulation, provide mechanistic insights into secondary and tertiary dentin formation, and offer a foundation for future research aimed at developing innovative therapies for dental tissue repair and regeneration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest associated with the contents of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the JSPS KAKENHI (grant numbers 20H03898, 23K27802, 23H03112, and 24K22179 to T.I. and 23K16207 to A.M.).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**A.M.:** Data curation, Formal analysis, Investigation, Writing \u0026ndash; original draft, **A.N.:** Data curation, Formal analysis, Investigation, **A.S.:** Investigation, **R.K.** : Investigation, **Y.N.:** Investigation, **N.H.:** Investigation, **A.Y.:** Data curation, validation, **T.I.:** Conceptualization, Funding acquisition, Project administration, Supervision, Writing \u0026ndash; original draft, review and editing\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to express our appreciation to Dr. Ryo Miyazaki for his support with the promoter experiments.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrannstrom, M. 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However, the underlying molecular mechanisms remain unclear. We have previously shown that hydrostatic pressure (HP) applied to stem cells from human exfoliated deciduous teeth (SHED) promotes odontoblast differentiation by translocating RUNX2 and increasing WNT16 expression through PIEZO1 signaling. In this study, we further explored the downstream signaling cascade linking PIEZO1 activation and odontoblast differentiation. HP stimulation increased the expression of odontoblast differentiation markers \u003cem\u003ePANX3\u003c/em\u003e and \u003cem\u003eDSPP\u003c/em\u003e, as shown by qPCR, and enhanced Alizarin Red staining\u0026mdash;results significantly suppressed by siRNA targeting either \u003cem\u003ePIEZO1\u003c/em\u003e or \u003cem\u003eWNT16\u003c/em\u003e. RT-PCR analysis revealed that, among the two known human \u003cem\u003eWNT16\u003c/em\u003e isoforms, only \u003cem\u003eWNT16b\u003c/em\u003e was expressed in SHED. qPCR demonstrated that HP-induced \u003cem\u003eWNT16\u003c/em\u003e expression was reduced by si\u003cem\u003ePIEZO1\u003c/em\u003e and further decreased by si\u003cem\u003eRUNX2\u003c/em\u003e. Promoter analysis identified four RUNX2-binding sites within the upstream region of \u003cem\u003eWNT16\u003c/em\u003e. A luciferase reporter assay using plasmids containing the \u003cem\u003eWNT16\u003c/em\u003e promoter showed that RUNX2 overexpression in HEK293 cells significantly increased luciferase activity. Moreover, HaloChIP assays with a HaloTag-RUNX2 expression vector confirmed RUNX2's binding to the \u003cem\u003eWNT16\u003c/em\u003e promoter. These findings suggest that PIEZO1-mediated mechanical stress promotes odontoblast differentiation through the RUNX2-dependent transcriptional activation of WNT16.\u003c/p\u003e","manuscriptTitle":"Hydrostatic Pressure promotes odontoblast differentiation via PIEZO1-dependent activation of RUNX2 and WNT16 in SHED","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-25 11:51:53","doi":"10.21203/rs.3.rs-8381409/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-23T06:10:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T12:09:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T02:11:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T02:02:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167949158676132440747881248432660954325","date":"2026-01-07T10:44:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23024415658576779091623682462007871210","date":"2026-01-07T01:22:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190277651067485234847382884354464594476","date":"2026-01-06T12:56:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-24T02:30:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-23T16:16:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T06:17:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-22T05:13:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-22T05:05:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"67fef0da-1f1a-449a-8a0d-e239271fda74","owner":[],"postedDate":"December 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":60192759,"name":"Biological sciences/Cell biology"},{"id":60192760,"name":"Biological sciences/Molecular biology"},{"id":60192761,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-04-07T16:03:01+00:00","versionOfRecord":{"articleIdentity":"rs-8381409","link":"https://doi.org/10.1038/s41598-026-46415-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-01 15:59:39","publishedOnDateReadable":"April 1st, 2026"},"versionCreatedAt":"2025-12-25 11:51:53","video":"","vorDoi":"10.1038/s41598-026-46415-y","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46415-y","workflowStages":[]},"version":"v1","identity":"rs-8381409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8381409","identity":"rs-8381409","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}