Identification of transforming growth factor-β3 signaling by compressive force in MC3T3- E1 cells reactions

Identification of transforming growth factor-β3 signaling by compressive force in MC3T3- E1 cells reactions | 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 Research Article Identification of transforming growth factor-β3 signaling by compressive force in MC3T3- E1 cells reactions Moeko Togawa, Akira Nakajima, Nichika Fukumashi, Takayuki Kawato This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6242390/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jun, 2025 Read the published version in Cell Biochemistry and Biophysics → Version 1 posted 19 You are reading this latest preprint version Abstract The function of Transforming growth factor (TGF) -β is reported to be associated with bone formation. However, the functional role of TGF-β3 in mediating the effects of orthodontic force such as the compressive force as a mechanical stress on osteoblasts remains unclear. We investigated the expression of TGF-β3 in osteoblasts and the effect of compressive force on downstream signaling pathways consisting of inflammatory cytokines. Cultured MC3T3-E1 and ATDC5 cells were subjected to continuous compressive forces, which are 0.5, 1.0, 2.0 g/cm 2 , for 30-minutes, 1-hour, and 3-hours. Western blot analysis was determined phosphorylation of Smad-dependent and MAPKs. Measurement of TGF-3, Cox2, and IL-6 expression levels was done by Western blot analysis and real-time polymerase chain reaction. The expression of TGF-β3 in both cell lines was significantly increased upon application of 1.0 g/cm 2 , but not 0.5 and 2.0 g/cm 2 compressive force in 1-hour, relative to the respective levels in unloaded control cells. At 1.0 g/cm 2 compressive force increased the phosphorylation of Smad2, Smad3, ERK1/2, p-38, and the expressions of COX-2 and IL-6. The increased expression was attenuated by pretreatment with siRNA of TGF-β3. These results indicate that a compressive force of 1.0 g/cm 2 induces the expression of inflammatory cytokines and bone-specific transcription factors via TGF-β3 signaling in the osteogenesis. TGF-β3 signaling tooth movement Smad-independent signaling MAPK signaling compressing force MC3T3-E1 cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In the orthodontic treatment, the optimal orthodontic force during tooth movement is clinically very important, which is caused by the balance of alveolar bone remodeling on the compressive and traction sides[ 1 – 6 ]. The mechanical stresses such as compressive force that simulate orthodontic forces associated with tooth movement induce an inflammatory reaction in the variable cells that comprise periodontal tissue and alveolar bone osteoblasts [ 1 – 4 ]. The function transforming growth factor-β (TGF-β) family, including BMP are known as the osteogenesis primarily through ligand-receptor complex adhesion interactions in the bone formation, even though during orthodontic tooth movement [ 5 – 9 ]. TGF-β ligands are reported mainly three isoforms which are TGF-β1, TGF-β2, and TGF-β3 [ 7 , 10 , 11 ]. In craniofacial bone formation, only TGF-β2-deficient mice showed severe skeletal abnormalities in both endochondral and intramembranous bone, while TGF-β1- and TGF-β3-null mice showed nearly a normal skeleton [ 7 , 12 ]. In addition, activation of TGF-β1 leads to phosphorylation of Smad2, causing transcriptional activation of downstream mediators and self-induction of TGF-β1 as autocrine mechanism [ 8 ]. Other previous study found that a compressive force of 1.0 g/cm 2 for 3-hour and 6-hour induces bone-specific transcription factors in osteoblasts via autocrine action of TGF-β1 and TGF-β2 signaling, respectively [ 5 , 6 ]. TGF-β3 is most frequently associated with developmental stages that influence palatal and pulmonary development, such as epithelial-mesenchymal interactions, cell proliferation, and remodeling [ 7 , 11 , 13 ]. However, it is not clear whether the optimal mechanical stress provided by compressive forces stimulates a specific functional role for TGF-β3 during tooth migration. It is also unclear whether Smad-dependent and/or Smad-independent pathways are involved in this regulatory mechanism, including the relationship between TGF-β3 and inflammatory cytokines such as cyclooxygenase-2 (COX-2) [ 14 ] and interleukin-6 (IL-6) [ 15 ] and bone formation during osteoblast function. TGF -β3stimulation mechanisms have not been clearly identified. In this study, we investigated the relation between optimal compressive force and TGF-β3 expression using the MC3T3-E1 cell line to determine the functional role of TGF-β3 in osteoblast function via Smad-dependent and Smad-independent signaling pathways. The aim of this study was to determine whether compressive force affects osteogenesis by modulating TGF-β3 expression via Smad-dependent or/and Smad-independent signaling pathways using small interfering RNA (siRNA) methods. Materials and methods Cell culture and compressive force loading The MC3T3-E1 cells (RIKEN BioResource Center) were cultured in the medium (α-minimal essential medium: Gibco) with 10% (v/v) heat-inactivated fetal bovine serum (FBS: Gibco), and in a humidified atmosphere containing 95% air and 5% CO 2 [ 1 – 5 ]. The cultured cells were seeded in 100 mm cell culture dishes (83 mm inner diameter) at a density of 2.0 × 104 cells/cm 2 and cultured overnight until confluent. The cells were then continuously compressed by applying the uniform compression method described above. To obtain the treatment samples, the thin round glass plate (78 mm inner diameter; Fig. 1 A) was placed over a layer of confluent cells without FBS, and a lead weight (Fig. 1 B) was placed on top of the round glass plate to adjust the compression force (0.5, 1.0, and 2.0 g/cm 2 ) [ 1 – 5 ]. Control cells were covered with round cover glass (Matunami) of the same diameter without lead weights, generating a compressive force of approximately 0.035 g/cm 2 [ 3 – 5 ]. Because removing the glass plate would generate static electricity that could affect the cells, glass plates were used for both compression forces-treated and control cells, and samples were taken under the same conditions in the control and stimulation groups (Fig. 1 ) [ 3 – 5 ]. siTGF-β3 transfection To identify the effect of compressive force on TGF-β3 function, we seeded MC3T3-E1 cells in 100 mm cell culture dishes and cells were cultured to 80% confluency. Experimental groups were treated with siRNA specific for TGF-β3 (Thermo Fisher Scientific, Silencer Select siRNA; Assay ID 186708, 186709) 1 day before stimulation with compressive force. the sequence of the TGF-β3 siRNA pair was 5′-UUA CCA AUU UGG UCA CUG UCA UGGA-3′ and 5′-UCC AUG ACA GUG ACC AAA UUG GUAA-3′ (Invitrogen, Stealth). Stimulation was examined for up to 1-hour with or without compressive force. For the effect of siRNA assessment, the cell samples received either siTGF-β3 or nontargeting control siRNA (siControl; Stealth RNAi™ siRNA Negative Control Lo GC Duplexes, Invitrogen) at a final concentration of 25 pM. siRNA complexes were formed using RNAiMAX (Invitrogen) in Opti-MEM (Gibco). For all the conditions, antibiotics were omitted, as recommended by the manufacturer (Invitrogen), to preserve cell viability during the transaction process. Quantitative Real-time reverse transcription polymerase chain reaction (RT-PCR) Total mRNA was extracted from cultured MC3T3-E1 cells using an mRNA extraction kit (NucleoSpin RNA; TaKaRa). First-strand cDNA synthesis was carried out according to protocol using a reverse transcription kit (PrimeScriptTM RT reagent kit; TaKaRa); the procedure for real-time RT-PCR using SYBR Green I dye is described in a previous report [ 1 – 6 ]. The primer sequences of target genes and GAPDH are listed in Table 1. PCR was performed on a thermal cycler (Smart Cycler; TaKaRa) and data were analyzed using the Smart Cycler software (ver. 1.2d; TaKaRa). The cycling conditions were as follows previous study [ 1 – 6 ]. The averages of each target mRNA expression were calculated and normalized GAPDH expression levels. The western blot hybridization (SDS-PAGE) Cultured cells from the control and treatment groups were washed several times with phosphate-buffered saline and homogenized in loading buffer (50 mm Tris-HCl, 0.1% Triton X-100, 0.1 mm EDTA, 1 mm vinyl-methylsulfonyl fluoride) [ 3 – 6 ]. The cells were into the above sample buffer solution, and gel electrophoresis (SDS-PAGE) and transfer membrane procedures for are described in previous reports [ 3 – 6 ]. Using SNAP i.d. 2.0 (Millipore Sigma), the 1.0% blocking solution (Roche) in Tres-buffered saline solution (pH 7.6) was performed for blocking and the antibodies diluted by 0.5% blocking solution in Tris-buffered saline solution. Membranes were probed with antibodies (Table 2), and the immunoreactive expressions were indicated using the western blot chemiluminescence detecting kit (Bio-rad: Clartity™ Western blot ECL substrate). The band intensities of the expressions were scanned images using the western blot imaging systems (ImageQuant™800, Amersham) and digital image analysis software (ImageJ, version 1.54d). Statistical analysis All experiments were performed at least five times independently. To compare between each experimental group, including control, a nonparametric procedure of Wilcoxon's signed rank test was used for statistical analysis. Data are expressed as mean ± standard error of the mean, and n represents the number of experimental samples. Statistical analysis was performed using SPSS software (IBM Corporation). Statistical significance was defined as a P value less than 0.05 (P < 0.05). Results Effect of compressive force (CF) and siRNA on the expression of TGF-β3 Real-time RT-PCR of unstimulated MC3T3-E1 cells or cells stimulated with a continuous compressive force (0.5 g/cm 2 CF, 1.0 g/cm 2 CF, and 2.0 g/cm 2 CF) indicated that the TGF-β3 mRNA expression gradually increased, peaked at 1-hour, and then gradually decreased (Fig. 2 A). The expression of TGF-β3 mRNA was increased in the 1.0 g/cm 2 CF group compared with that in the control, and 0.5 and 2.0 g/cm 2 CF groups at 1-hour ( P < 0.05, n = 5; Fig. 2 A). The western blot analysis detected that among cells stimulated for 1-hour with 0.5 g/cm 2 CF, 1.0 g/cm 2 CF, and 2.0 g/cm 2 CF groups. The TGF-β3 protein expression was significantly higher in cells treated with 1.0 g/cm 2 than in the other conditions (P < 0.05, n = 5; Fig. 2 B), which expressions were detected similar as mRNA expressions. To identify siRNA transfection, we examined the RT-PCR for TGF-β3 (Fig. 2 C). The mRNA expression in cells subjected to 1.0 g/cm 2 compressive force (1.0 g/cm 2 CF group) and in siControl-treated cells subjected to 1.0 g/cm 2 compressive force (siControl + 1.0 g/cm 2 CF group) was significantly higher than in control cells without compressive force ( P < 0.05, n = 5; Fig. 2 C). The TGF-β3 mRNA level in siTGF-β3-treated cells subjected to 1.0 g/cm 2 compressive force cell (siTGF-β3 + 1.0 g/cm 2 CF group) was significantly lower than that in 1.0 g/cm 2 CF and siControl + 1.0 g/cm 2 CF groups, which siTGF-β3-treated cells was no significant difference compared with control group ( P < 0.05, n = 5; Fig. 2 C). In the western blot results, the expression of TGF-β3 in control + 1.0 g/cm 2 CF and siControl + 1.0 g/cm 2 CF groups were higher than those in the control group, which was without CF. The increase in TGF-β3 expressed in siTGF-β3 + 1.0 g/cm 2 CF was significantly reduced by siRNA of TGF-β3 treatment ( P < 0.05, n = 5; Fig. 2 D). Influence of the downstream signaling by siTGF-β3 transfection on compressive force stimulation (CF) With regard to the effect of siTGF-β3 transfection on the CF-induced phosphorylation of proteins in Smad-dependent and Smad-independent signaling pathways, the relative levels of phosphorylated Smad2 and phosphorylated Smad3 in the 1.0 g/cm 2 CF and siControl + 1.0 g/cm 2 CF groups were significantly higher than in the control group (P < 0.05, n = 5; Fig. 3 A and B). The relative levels of phosphorylated Smad2 and Smad3 in the siTGF-β3 + 1.0 g/cm 2 CF group were significantly higher than in the control group (P < 0.05, n = 5; Fig. 3 A and B). There was no difference of the influence by CF stimulation and also reduce by siTGF-β3 transaction between Smad2 and Smad3 (Fig. 3 A and B). The relative levels of phosphorylated ERK-1/2 and phosphorylated p38 in 1.0 g/cm 2 CF and siControl + 1.0 g/cm 2 CF groups were significantly higher than in the control group, and there was no difference between the 1.0 g/cm 2 CF and siControl + 1.0 g/cm 2 CF groups. The increase in phosphorylation of ERK1/2 and p38 in the siTGF-β3 + 1.0 g/cm 2 CF group was significantly decreased compared with that in the 1.0 g/cm 2 CF and siControl + 1.0 g/cm 2 CF groups, and was similar level to that in these CF groups ( P < 0.05, n = 5; Fig. 3 C and 3 D). In contrast, the increase in phosphorylation of ERK1/2 and p38 by compressive force was significantly decreased via siTGF-β3 treatment to levels similar value in the control group (P < 0.05, n = 5; Fig. 3 C and 3 D). Influence COX-2 expression of stimulation on compressive force (CF) and siTGF-β3 transfection When the COX-2 expression as the inflammatory cytokines in osteoblasts was assessed using real-time RT-PCR and western blot analysis, both mRNA and protein levels of inflammatory cytokines COX-2 in the 1.0 g/cm 2 CF group after 1-hour, significantly higher than those in the control, 0.5 g/cm 2 CF and 2.0 g/cm 2 CF groups ( P < 0.05, n = 5; Fig. 4 A and B) and could be correlated with TGF-β3 expression. Regarding the effect of siTGF-β3 transfection in the real-time RT-PCR and the western blot analysis, the expression of COX-2 in the 1.0 g/cm 2 CF and siControl + 1.0 g/cm 2 CF groups was significantly increased compared to the control group, which was almost same level ( P < 0.05, n = 5; Fig. 4 C and D). These increased COX-2 expressions by 1.0 g/cm 2 CF stimulation were significantly reduced by siTGF-β3 treatment ( P < 0.05, n = 5; Fig. 4 A and B). These influences of COX-2 expression could be correlated with TGF-β3 expression (Fig. 4 A-D). Influence IL-6 expression of stimulation on compressive force (CF) and siTGF-β3 transfection The IL-6 is one of the inflammatory cytokines, which associated with CF 15 . The expression of stimulation on CF was examined using real-time RT-PCR and western blot analysis, both mRNA and protein levels of inflammatory cytokines IL-6 in the 1.0 g/cm 2 CF group after 1-hour, significantly higher than those in the control, 0.5 g/cm 2 CF and 2.0 g/cm 2 CF groups ( P < 0.05, n = 5; Fig. 4 A and B). These effects of IL-6 expressions could be correlated with TGF-β3 expression. Regarding the effect of siTGF-β3 transfection, the expressions of IL-6 in the 1.0 g/cm 2 CF and siControl + 1.0 g/cm 2 CF groups were significantly increased compared to the control group in the real-time RT-PCR and the western blot analysis, which was no significantly difference between these CF stimulation groups ( P < 0.05, n = 5; Fig. 5 C and D). These increased IL-6 expressions by 1.0 g/cm 2 CF were significantly reduced by siTGF-β3 treatment ( P < 0.05, n = 5; Fig. 5 A and B). The expression of IL-6 of the 1.0 g/cm 2 and siControl + 1.0 g/cm 2 CF groups was significantly higher than in the control group in both expression levels of the mRNA and the protein reaction ( P < 0.05, n = 5; Fig. 5 C and D). The increased expression by these 1.0 g/cm 2 CF stimulations groups were significantly decreased by siTGF-β3 transfection ( P < 0.05, n = 5; Fig. 5 C and D). Discussion The main novelty of this study is that a compressive force of 1.0 g/cm 2 of the optimal force stimulated the osteoblastic cells via TGF-β3 signaling pathway, which signaling were both Smad-dependent/-independent signaling pathway. The expression levels of COX-2 and IL-6 as the inflammatory cytokines were also increased upon application of 1.0 g/cm 2 compressive force (Fig. 8). These stimulatory effects were not observed in cells subjected to 0.5 or 2.0 g/cm 2 compressive force. The stimulatory effects of 1.0 g/cm 2 compressive force were significantly reduced by siTGF-β3 treatment of cells, and the expression levels were similar to those in control cells that were not subjected to compressive forces. These new findings on the function of TGF-β3 are possibility suggested that that a compressive force of 1.0 g/cm 2 , as an optimal mechanical stress, promotes bone formation osteoblasts by increasing the levels of type I collagen, bone sialoprotein, and bone morphogenetic protein (BMP). The sequence of TGF-β3 is 86% similar to the sequence of TGF-β1 and 91% similar to the sequence of TGF-β2.14 However, even though TGF-β2 and -β3 share the highest level of sequence similarity of the three isoforms, TGF-β2 binds to TGF-β1 and -β3 by binds to TGF-β receptor II (TβRII) by a different mechanism15. 15 Furthermore, while TGF-β1 and -β3 can bind directly to TβRII, presentation of TGF-β2 to the receptor requires the presence of a coreceptor (β-glycan or endoglin), which may explain the role of TGF-β2 and -β3, which may explain the different roles of TGF-β2 and -β3 in activity [ 16 ]. Previous studies have shown that TGF-β3 plays an important role in normal craniofacial developmental biology, and exhibits isoform-specific biology at both in vivo and in vitro levels [ 18 – 20 ]. To understand the role of TGF-β3 in osteoblastogenic mechanisms, it is important to understand the unique biology of TGF-β3. The different functional roles of TGF-β have been studied in some depth with respect to palatal development. During palatal development, TGF-β3 is strongly expressed in midline epithelial cells prior to contact and fusion of opposing palatal shelves [ 18 ]. During palatal fusion, it is also strongly expressed at the midline seam palatal epithelial cells, including mesenchymal cells undergoing epithelial-to-mesenchymal transition [ 21 ]. Notably, a complete cleft palate is observed in TGF-β3 null mice, even though the palatal mesenchymal shelves in this model have sufficient length and orientation to allow fusion [ 18 , 21 ]. Furthermore, unlike other null mutants such exhibit cleft palate, TGF-β3 null mice lack other craniofacial abnormalities [ 18 , 19 ]. In previous studies on osteoblast responses at an optimal compressive force, the expression of TGF-β1 at the 3-hour and TGF-β2 at the 6-hour time point gradually increased and then gradually decreased [ 22 ]. Therefore, in this result of new findings, the functional role of TGF-β3 when MC3T3-E1 cells are stimulated with the current 1.0 g/cm 2 compressive force shows a relatively early pattern of bone remodeling expression compared to other TGF-βs. Also, it could be specific to osteoblast localization in bone formation during tooth migration, and fine-tunes signaling inflammatory cytokines and may fine-tune signaling inflammatory cytokines. Given the positive modulatory effect of TGF-β3 on osteoblast differentiation [ 23 ], it is presumed that applying a compressive force of 1.0 g/cm 2 , but not 0.5 g/cm 2 and 2.0 g/cm 2 , promotes osteoblast differentiation. Osteoblast differentiation, including the bone formation was occurred to be affected not only by the Smad-dependent TGF-β3 signaling pathway, but also by the Smad-independent TGF-β3 signaling pathway [ 7 , 24 – 29 ]. For example, the association of TAK1 and TAK1-binding protein 1 induced by TGF-β leads to activation of the mitogen-activated protein kinase (MAPK) kinase 3-p38 MAPK signaling cascade and induction of type I collagen expression [ 30 – 33 ]. Notably, a recent study demonstrated that following TGF-β3 induction, the Smad and ERK/p38 MAPK pathways converged at Runt-related transcription factor (Runx2), which associated with osteoblast differentiation, in the control of mesenchymal precursor cell differentiation [ 34 ]. However, the issue of Smad-independent signaling induced by TGF-β, i.e., autocrine/paracrine inflammatory cytokines (IL-6 or COX-2 e.g.) activated by mitogen-activated protein kinase (MAPK) signaling cascade, remains to be addressed (Fig. 8). The such autocrine/paracrine signaling of TGF-β3 could be investigated as future planned. BMPs are members of the TGF-β superfamily, have 32–37% sequence homology with TGF-βs, and significantly affect osteoblast activity [ 1 , 2 ]. After BMP induction, Smad1/5 and MAPK cascades (such as ERK1/2 and p38) converge on Runx2 to regulate mesenchymal progenitor cell differentiation [ 38 ]. The optimal force has been reported to increase expression of BMPs and their receptors, phosphorylated Smad1, Runx2, and the homeobox protein DLX-5, which promotes calcification in vitro [ 1 , 2 ]. Thus, the results of the present study suggest that both BMP and TGF-β signaling pathways could be stimulated by optimal compressive forces to induce osteoblast differentiation through Smad-dependent and Smad-independent cascades, and that crosstalk between BMP and TGF-β signaling pathways may be responsible for these may be related to these phenomena. In this study, the results were the compressive force of 1.0 g/cm 2 of the optimal force stimulated the osteoblastic cells via TGF-β3 signaling pathway. And our results found that transfection with siTGF-β3 suppressed Smad2/3, ERK, and p38 phosphorylation and inflammatory cytokine expression of the initial compressive force. This supports the idea that Smad-dependent/-independent signaling also promotes osteoblast differentiation via expression of osteogenesis-related transcription factors (Fig. 8). Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Sources of funding This work was supported by JSPS KAKENHI (Grant Number: JP22K10280) and the Dental Research Center in Nihon University School of Dentistry, Graduate School of Dentistry (B). Statement form The authors declare that this research used experimental cell line subjects, so this study did not applicable the patient’s or guardian’s consent. Acknowledgements The authors thank Professor Yoshio Nakano for the suggestions of statistical analysis, and our colleagues at the Department of Orthodontics and the Department of Oral Health Sciences, University School of Dentistry, Japan for their continuous support. References Mitsui, N., Suzuki, N., Maeno, M., Mayahara, K., Yanagisawa, M., Otsuka, K., Shimizu, N. (2005). Optimal compressive force induces bone formation via increasing bone sialoprotein and prostaglandin E(2) production appropriately. Life Sci, 77(25), 3168–3182. Mitsui, N., Suzuki, N., Maeno M., Yanagisawa, M., Koyama, Y., Otsuka, K., Shimizu, N. (2006). Optimal compressive force induces bone formation via increasing bone morphogenetic proteins production and decreasing their antagonists production by Saos-2 cells. Life Sci, 78(23), 2697–2706. Sanuki R., Shionome C., Kuwabara A., Mitsui N., Koyama Y., Suzuki N., Zhang, F., Shimizu, N., Maeno, M. (2010). Compressive force induces osteoclast differentiation via prostaglandin E2 production in MC3T3-E1 cells. Connect Tissue Res, 51, 150–8. Zhang, F., Koyama, Y., Sanuki, R., Mitsui, N., Suzuki, N., Kimura, A., Nakajima, A., Shimizu, N., Maeno, M. (2010). IL-17A stimulates the expression of inflammatory cytokines via celecoxib-blocked prostaglandin in MC3T3-E1 cells. Arch Oral Biol, 55, 679–688. Sano, R., Nakajima, A., Kawato, T., Maeno, M., Shimizu, N. (2017). Effect of compressive force on TGF-β1/2 signaling pathway in MC3T3-E1 cells. J Hard Tissue Biol, 26, 177–186. Komamura, H., Nakajima, A., Sano, R., Motoyoshi, M. (2022). Effect of Transforming Growth Factor-β2 on Smad-Independent Signaling in MC3T3-E1 Cells. Biochem Mol Biol, 8, 2471–8084. Nakajima, A., Shuler, CF., Gulka, AOD., Hanai, JI. (2018). TGF-beta Signaling and the Epithelial-Mesenchymal Transition during Palatal Fusion. Int J Mol Sci, 19(11). Peters, AS., Brunner, G., Krieg, T., Eckes, B. (2015). Cyclic mechanical strain induces TGFbeta1-signalling in dermal fibroblasts embedded in a 3D collagen lattice. Arch Dermatol Res, 307(2), 191–197. Manokawinchoke, J., Limjeerajarus, N., Limjeerajarus, C., Sastravaha, P., Everts, V., Pavasant, P. (2015). Mechanical Force-induced TGFB1 Increases Expression of SOST/POSTN by hPDL Cells. J Dent Res, 94(7), 983–989. Grafe, I., Alexander, S., Peterson, JR., Snider, TN., Levi, B., Lee, B., Mishina, Y. (2018). TGF-beta Family Signaling in Mesenchymal Differentiation. Cold Spring Harb Perspect Biol, 10(5). Wu, M., Chen, G., Li, YP. (2016). TGF-beta and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res, 4, 16009. Mengrui, Wu., Guiqian, Chen., Yi-Ping, Li. (2016). TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res, 4, 16009. https://doi.org/10.1038/boneres.2016.9 Takeyama, K., Chatani, M., Inohaya, K., Kudo, A. (2016). TGFbeta-2 signaling is essential for osteoblast migration and differentiation during fracture healing in medaka fish. Bone, 86, 68–78. Hossain, MG., Iwata, T., Mizusawa, N., Shima, SWN., Okutsu, T., Ishimoto, K., Yoshimoto, K. (2010). Compressive force inhibits adipogenesis through COX-2-mediated down-regulation of PPARgamma2 and C/EBPalpha, J Biosci Bioeng, 109(3), 297–303. https://doi.org/10.1016/j.jbiosc.2009.09.003 Phusuntornsakul, P., Jitpukdeebodintra, S., Pavasant, P. (2108). Vibration enhances PGE2, IL-6, and IL-8 expression in compressed hPDL cells via cyclooxygenase pathway. J Periodontol, 89(9), 1131–1141. Laverty, HG., Wakefield, LM., Occleston, NL., O'Kane, S., Ferguson, MW. (2009). TGF-beta3 and cancer: a review. Cytokine Growth Factor Rev, 20(4), 305–317. Crescenzo, GD., Hinck, CS., Shu, Z., Zúñiga, J., Yang, J., Tang, Y., Baardsnes, J., Mendoza, V., Sun, L., López-Casillas, F., O'Connor-McCourt, M., Hinck. AP. (2006). Three key residues underlie the differential affinity of the TGFbeta isoforms for the TGFbeta type II receptor. J Mol Biol, 355, 47–62. Kaartinen, V., Cui, XM., Heisterkamp, N., Groffen, J., Shuler, CF. (1997). Transforming growth factor-beta3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane. Dev Dyn. 209, 255–260. Kaartinen, V., Voncken, JW., Shuler, C., Warburton, D., Bu, D., Heisterkamp, N., Groffen, J. (1995). Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction. Nat Genet. 11(4), 415–421. Taya, Y., O'Kane, S., Ferguson, MW. (1999). Pathogenesis of cleft palate in TGF-beta3 knockout mice. Development. 126(17), 3869–3879. Proetzel, G., Pawlowski, SA., Wiles, MV., Yin, M., Boivin, GP., Howles, PN., Ding, J., Ferguson, MW., Doetschman, T. (1995). Transforming growth factor-beta 3 is required for secondary palate fusion. Nat Genet. 11(4), 409–414. Nakajima, A., Ito, Y., Tanaka, E., Sano, R., Karasawa, Y., Maeno, M., Iwata, K., Shimizu, N., Shuler, CF. (2014). Functional role of TGF-beta receptors during palatal fusion in vitro. Arch Oral Biol. 59(11), 1192–1204. Kim, SI., Kwak, JH., Zachariah, M., He, Y., Wang, L., Choi, ME. (2007). TGF-beta-activated kinase 1 and TAK1-binding protein 1 cooperate to mediate TGF-beta1-induced MKK3-p38 MAPK activation and stimulation of type I collagen. Am J Physiol Renal Physiol. 292(5), F1471-1478. Derynck, R., Zhang, Y., Feng, XH. (1998). Smads: transcriptional activators of TGF-beta responses. Cell, 95(6), 737–740. Kretzschmar, M., Massague, J. (1998). SMADs: mediators and regulators of TGF-beta signaling. Curr Opin Genet Dev, 8, 103–111. Janssens, K., ten Dijke, P., Janssens, S., Van, Hul W. (2005). Transforming growth factor-beta1 to the bone. Endocr Rev, 26(6), 743–774. Shi, Y., Massague, J. (2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 113(6), 685–700. Massague, J., Seoane, J., Wotton, D. (2005). Smad transcription factors. Genes Dev, 19(23), 2783–2810. Kang, JS., Liu, C., Derynck, R. (2009). New regulatory mechanisms of TGF-beta receptor function. Trends Cell Biol, 19(8), 385–394. Kimura, N., Matsuo, R., Shibuya, H., Nakashima, K., Taga, T. (2000). BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6. J Biol Chem, 275, 17647–17652. Sorrentino, A., Thakur, N., Grimsby, S., Marcusson, A., Bulow, V., Schuster, N., Zhang, S., Heldin, CH., Landström, M. (2008). The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner. Nat Cell Biol, 10(10), 1199–1207. Yamashita, M., Fatyol, K., Jin, C., Wang, X., Liu, Z, Zhang, YE. (2008). TRAF6 mediates Smad-independent activation of JNK and p38 by TGF-beta. Mol Cell, 31(6), 918–924. Jung, SM., Lee, JH., Park, J., Oh, YS., Lee, SK., Park, JS., Lee, YS., Kim, JH., Lee, JY., Bae, YS., Koo, SH., Kim, SJ., Park, SH. (2013). Smad6 inhibits non-canonical TGF-beta1 signalling by recruiting the deubiquitinase A20 to TRAF6. Nat Commun, 4, 2562. Lee, KS., Hong, SH., Bae, SC. (2002). Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene, 21(47), 7156–7163. Lai, CF., Cheng, SL. (2002). Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells. J Biol Chem, 277(18), 15514–15522. Miyazono, K., Maeda, S., Imamura, T. (2005). BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine Growth Factor Rev. 16(3), 251–263. Chen G., Deng C., Li YP. 2012. TGF-beta and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci, 8(2), 272–288. Rahman, MS., Akhtar, N., Jamil, HM, Banik, RS., Asaduzzaman, SM. (2015). TGF-beta/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation. Bone Res, 3, 15005. Tables Table 1 to 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1PCRprimer.xlsx Table2Antibody.xlsx Cite Share Download PDF Status: Published Journal Publication published 24 Jun, 2025 Read the published version in Cell Biochemistry and Biophysics → Version 1 posted Editorial decision: Revision requested 17 Apr, 2025 Reviews received at journal 17 Apr, 2025 Reviews received at journal 17 Apr, 2025 Reviews received at journal 16 Apr, 2025 Reviews received at journal 12 Apr, 2025 Reviews received at journal 01 Apr, 2025 Reviewers agreed at journal 24 Mar, 2025 Reviewers agreed at journal 21 Mar, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers agreed at journal 19 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers invited by journal 18 Mar, 2025 Editor assigned by journal 18 Mar, 2025 Submission checks completed at journal 18 Mar, 2025 First submitted to journal 17 Mar, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6242390","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433031339,"identity":"25dd2236-7c33-4515-ad5f-684efe63492f","order_by":0,"name":"Moeko Togawa","email":"","orcid":"","institution":"Nihon University School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Moeko","middleName":"","lastName":"Togawa","suffix":""},{"id":433031347,"identity":"ef91cb83-dc10-4056-8c77-cb4da7c44bea","order_by":1,"name":"Akira Nakajima","email":"data:image/png;base64,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","orcid":"","institution":"Nihon University School of Dentistry","correspondingAuthor":true,"prefix":"","firstName":"Akira","middleName":"","lastName":"Nakajima","suffix":""},{"id":433031348,"identity":"4277e307-e3c4-4389-a556-73c428d3e714","order_by":2,"name":"Nichika Fukumashi","email":"","orcid":"","institution":"Nihon University School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Nichika","middleName":"","lastName":"Fukumashi","suffix":""},{"id":433031350,"identity":"1876e4d5-485a-46c4-be38-0c79fb6b2882","order_by":3,"name":"Takayuki Kawato","email":"","orcid":"","institution":"Nihon University School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Kawato","suffix":""}],"badges":[],"createdAt":"2025-03-17 08:08:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6242390/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6242390/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12013-025-01783-6","type":"published","date":"2025-06-24T16:05:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79389131,"identity":"624f9ef1-5be6-4ac5-9a33-23c0736c7f15","added_by":"auto","created_at":"2025-03-27 19:22:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagrammatic representation of the application of compressive force.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfluent cells in a culture dish (inner diameter: 83 mm) were covered with a glass plate (inner diameter: 78 mm) \u003cstrong\u003e(A)\u003c/strong\u003e [20].\u003csup\u003e \u003c/sup\u003eThe weight \u003cstrong\u003e(B)\u003c/strong\u003e was positioned such that the force was evenly distributed across the cell monolayer.\u003c/p\u003e","description":"","filename":"Figure18.png","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/954ec91482c727fdaaa380c9.png"},{"id":79389140,"identity":"6466eae3-5f1a-4b70-8a59-08c6b9845a2f","added_by":"auto","created_at":"2025-03-27 19:22:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of compressive force (CF) and siTGF-β3 transfection at 1-hour on the expression of TGF-β3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eThe expression of TGF-β3 mRNA increased gradually, peaked at 1-hour, and decreased gradually, thereafter. The expression of TGF-β3 mRNA was higher in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force group than in the control, and 0.5 and 2.0 g/cm\u003csup\u003e2 \u003c/sup\u003ecompressive force groups at 1-hour (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5). \u0026nbsp;\u003cstrong\u003eB:\u003c/strong\u003e Western blot analysis confirmed that in cells stimulated with 0.5, 1.0, and 2.0 g/cm\u003csup\u003e2 \u003c/sup\u003efor 1-hour, the expression levels of TGF-β3 were significantly higher in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force group than under any other condition in the western blot analysis (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). \u003cstrong\u003eC:\u003c/strong\u003e In the effect of siTGF-β3 transfection, the expression of TGF-β3 mRNA in cells subjected to 1.0 g/cm\u003csup\u003e2 \u003c/sup\u003eCF (1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) and in siControl-treated cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force (siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) was increased significantly compared with that in cells not subjected to CF compressive force (control group) (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). The expression of TGF-β3 mRNA in siTGF-β3-treated cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF (siTGF-β3 + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) was significantly decreased compared with that both in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups, and was similar to that in the control group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). \u003cstrong\u003eD:\u003c/strong\u003e The expression levels of TGF-β3 in the control + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups was increased compared with that in the control group. The increase in expression caused by compressive force was significantly reduced by siTGF-β3 treatment in the western blot analysis (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure27.png","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/8b4e1da5708aa5280081d597.png"},{"id":79389452,"identity":"c9825af0-647c-4d19-af9d-4a90ad70aafc","added_by":"auto","created_at":"2025-03-27 19:30:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of siTGF-β3 transfection on compressive force (CF)-induced phosphorylation of proteins in Smad-dependent and Smad-independent signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e The levels of phosphorylated Smad2 subjected to 1.0 g/cm\u003csup\u003e2 \u003c/sup\u003ecompressive force (control + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) and in siControl-treated cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force (siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) were increased significantly compared with that in cells not subjected to compressive forces (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). The phosphorylation of Smad2 in siTGF-β3-treated cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force (siTGF-β3 + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) was significantly decreased compared with that in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups, and was similar value to that in the control group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). \u003cstrong\u003eB:\u003c/strong\u003e The phosphorylated Smad3 subjected to 1.0 g/cm\u003csup\u003e2 \u003c/sup\u003eCF and in siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF was increased significantly compared with that in cells not subjected to the compressive force (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). The phosphorylation of Smad3 in siTGF-β3 + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group was significantly decreased compared with that in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups, and was similar value to that in the control group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). These phosphorylation influences of Smad3 in the siTGF-β3 transaction could be similar as the Smad2 expressions (A).\u0026nbsp; \u003cstrong\u003eC and D:\u003c/strong\u003e The phosphorylation of ERK-1/2 and p38 as Smad-independent pathway, in the control + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e groups was as significantly increased compared with that in the control group, and there was no difference between the control + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e groups (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). \u003cstrong\u003e(C, D)\u003c/strong\u003e The increase in phosphorylation of ERK1/2 and p38 in the siTGF-β3 + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e group was significantly decreased compared with that in the control + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e groups, and was similar value to that in the control group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). In contrast, the increase in phosphorylation of ERK1/2 and p38 by compressive force was significantly decreased by siTGF-β3 treatment to levels similar expression to those in the control group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test).\u003c/p\u003e","description":"","filename":"Figure37.png","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/c20b95f6ec6fb771a44e032e.png"},{"id":79389451,"identity":"4e74943a-69e1-4bcb-bbb1-c7e3ef843193","added_by":"auto","created_at":"2025-03-27 19:30:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":85088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of compressive force and siTGF-β3 transfection on the mRNA and protein expression of COX-2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA and B:\u003c/strong\u003e The 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force group exhibited significantly higher mRNA levels and protein level of the inflammatory cytokines, COX-2 (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5) than did the control, and 0.5 and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force groups at 1-hour, the time point at which the stimulation of TGF-β3 expression by compressive force was maximum (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003en\u003c/em\u003e = 5; Wilcoxon signed rank test).\u0026nbsp; \u003cstrong\u003eC and D:\u003c/strong\u003e The expression of COX-2 in cells subjected to 1.0 g/cm\u003csup\u003e2 \u003c/sup\u003ecompressive force (control + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) and in siControl-treated cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force (siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) was increased significantly compared with control group in the results of real-time RT-PCR and the western blot (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). The increase in the expression of COX-2 by 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF was significantly reduced by siTGF-β3 treatment to a level similar level to that in the control group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test).\u003c/p\u003e","description":"","filename":"Figure46.png","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/373cfef2e986b1cf959a45b5.png"},{"id":79389134,"identity":"93efab34-143f-4d94-bc08-df7ff8dc367d","added_by":"auto","created_at":"2025-03-27 19:22:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of compressive force and siTGF-β3 transfection on the mRNA and protein expression of IL-6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA and B\u003c/strong\u003e The IL-6 expressions in the results of the real-time RT-PCR and the western blot cells subjected to 1.0 g/cm\u003csup\u003e2 \u003c/sup\u003ecompressive force (control + 1.0 g/cm\u003csup\u003e2 \u003c/sup\u003eCF group) and in siControl-treated cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force (siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) was increased significantly compared with that in cells not subjected to control group\u003cstrong\u003e \u003c/strong\u003e(*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). The increase in the expression of IL-6 by 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF was significantly reduced by siTGF-β3 treatment to a level similar level to that in the without CF group (control group)\u003cstrong\u003e \u003c/strong\u003e(*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed rank test). \u003cstrong\u003eC and D:\u003c/strong\u003e The both mRNA and protein expressions of IL-6 in the control + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e groups was significantly higher than that in the control (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed-rank test). The increase in expression caused by 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force in the control + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl + 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups was significantly reduced by siTGF-β3 transfection (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, n = 5; Wilcoxon signed-rank test). These influences of IL-6 expression change could be coincident with the tendency of TGF-β3 expressions.\u003c/p\u003e","description":"","filename":"Figure56.png","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/5b49d94a68d41d7d27f068a9.png"},{"id":79389138,"identity":"a603cc7f-2347-48f4-bd18-cbe7609802e2","added_by":"auto","created_at":"2025-03-27 19:22:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":147262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagrammatically show TGF-β3 signaling cascade events related with Smad-dependent and -independent with their interactions including inflammatory cytokines and osteogenic transcription factors by mechanical stress stimulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene abbreviations are listed below. Transforming growth factor: TGF, TGF receptor: TβR, Cyclooxygenase-2: COX-2, Interleukin 6: IL-6, Extracellular signal-regulated kinases: ERK, p38 MAP Kinase: p38.\u003c/p\u003e","description":"","filename":"Figure63.png","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/1328065270a96be7a7ab643a.png"},{"id":85686732,"identity":"c578ef00-c18f-4d13-abd4-290d9ef8f936","added_by":"auto","created_at":"2025-06-30 16:08:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1554328,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/af76f8f0-ac21-4d7f-8e38-635d08584eea.pdf"},{"id":79389146,"identity":"c464de3c-38ff-4780-854d-50399bc58ac9","added_by":"auto","created_at":"2025-03-27 19:22:41","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":13155,"visible":true,"origin":"","legend":"","description":"","filename":"Table1PCRprimer.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/7f3bf27432809c3bc9125777.xlsx"},{"id":79389453,"identity":"d9bda20a-657a-4643-8285-28324ffe6e7e","added_by":"auto","created_at":"2025-03-27 19:30:41","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":11682,"visible":true,"origin":"","legend":"","description":"","filename":"Table2Antibody.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6242390/v1/02cb40034a67709e20a092c1.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of transforming growth factor-β3 signaling by compressive force in MC3T3- E1 cells reactions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the orthodontic treatment, the optimal orthodontic force during tooth movement is clinically very important, which is caused by the balance of alveolar bone remodeling on the compressive and traction sides[\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The mechanical stresses such as compressive force that simulate orthodontic forces associated with tooth movement induce an inflammatory reaction in the variable cells that comprise periodontal tissue and alveolar bone osteoblasts [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe function transforming growth factor-β (TGF-β) family, including BMP are known as the osteogenesis primarily through ligand-receptor complex adhesion interactions in the bone formation, even though during orthodontic tooth movement [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. TGF-β ligands are reported mainly three isoforms which are TGF-β1, TGF-β2, and TGF-β3 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In craniofacial bone formation, only TGF-β2-deficient mice showed severe skeletal abnormalities in both endochondral and intramembranous bone, while TGF-β1- and TGF-β3-null mice showed nearly a normal skeleton [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, activation of TGF-β1 leads to phosphorylation of Smad2, causing transcriptional activation of downstream mediators and self-induction of TGF-β1 as autocrine mechanism [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Other previous study found that a compressive force of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e for 3-hour and 6-hour induces bone-specific transcription factors in osteoblasts via autocrine action of TGF-β1 and TGF-β2 signaling, respectively [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTGF-β3 is most frequently associated with developmental stages that influence palatal and pulmonary development, such as epithelial-mesenchymal interactions, cell proliferation, and remodeling [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, it is not clear whether the optimal mechanical stress provided by compressive forces stimulates a specific functional role for TGF-β3 during tooth migration. It is also unclear whether Smad-dependent and/or Smad-independent pathways are involved in this regulatory mechanism, including the relationship between TGF-β3 and inflammatory cytokines such as cyclooxygenase-2 (COX-2) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and interleukin-6 (IL-6) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and bone formation during osteoblast function. TGF -β3stimulation mechanisms have not been clearly identified.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the relation between optimal compressive force and TGF-β3 expression using the MC3T3-E1 cell line to determine the functional role of TGF-β3 in osteoblast function via Smad-dependent and Smad-independent signaling pathways. The aim of this study was to determine whether compressive force affects osteogenesis by modulating TGF-β3 expression via Smad-dependent or/and Smad-independent signaling pathways using small interfering RNA (siRNA) methods.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and compressive force loading\u003c/h2\u003e \u003cp\u003eThe MC3T3-E1 cells (RIKEN BioResource Center) were cultured in the medium (α-minimal essential medium: Gibco) with 10% (v/v) heat-inactivated fetal bovine serum (FBS: Gibco), and in a humidified atmosphere containing 95% air and 5% CO\u003csup\u003e2\u003c/sup\u003e [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The cultured cells were seeded in 100 mm cell culture dishes (83 mm inner diameter) at a density of 2.0 \u0026times; 104 cells/cm\u003csup\u003e2\u003c/sup\u003e and cultured overnight until confluent. The cells were then continuously compressed by applying the uniform compression method described above. To obtain the treatment samples, the thin round glass plate (78 mm inner diameter; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) was placed over a layer of confluent cells without FBS, and a lead weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) was placed on top of the round glass plate to adjust the compression force (0.5, 1.0, and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e) [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Control cells were covered with round cover glass (Matunami) of the same diameter without lead weights, generating a compressive force of approximately 0.035 g/cm\u003csup\u003e2\u003c/sup\u003e [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Because removing the glass plate would generate static electricity that could affect the cells, glass plates were used for both compression forces-treated and control cells, and samples were taken under the same conditions in the control and stimulation groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003esiTGF-β3 transfection\u003c/h3\u003e\n\u003cp\u003eTo identify the effect of compressive force on TGF-β3 function, we seeded MC3T3-E1 cells in 100 mm cell culture dishes and cells were cultured to 80% confluency. Experimental groups were treated with siRNA specific for TGF-β3 (Thermo Fisher Scientific, Silencer Select siRNA; Assay ID 186708, 186709) 1 day before stimulation with compressive force. the sequence of the TGF-β3 siRNA pair was 5\u0026prime;-UUA CCA AUU UGG UCA CUG UCA UGGA-3\u0026prime; and 5\u0026prime;-UCC AUG ACA GUG ACC AAA UUG GUAA-3\u0026prime; (Invitrogen, Stealth). Stimulation was examined for up to 1-hour with or without compressive force.\u003c/p\u003e \u003cp\u003eFor the effect of siRNA assessment, the cell samples received either siTGF-β3 or nontargeting control siRNA (siControl; Stealth RNAi\u0026trade; siRNA Negative Control Lo GC Duplexes, Invitrogen) at a final concentration of 25 pM. siRNA complexes were formed using RNAiMAX (Invitrogen) in Opti-MEM (Gibco). For all the conditions, antibiotics were omitted, as recommended by the manufacturer (Invitrogen), to preserve cell viability during the transaction process.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-time reverse transcription polymerase chain reaction (RT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal mRNA was extracted from cultured MC3T3-E1 cells using an mRNA extraction kit (NucleoSpin RNA; TaKaRa). First-strand cDNA synthesis was carried out according to protocol using a reverse transcription kit (PrimeScriptTM RT reagent kit; TaKaRa); the procedure for real-time RT-PCR using SYBR Green I dye is described in a previous report [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The primer sequences of target genes and GAPDH are listed in Table\u0026nbsp;1. PCR was performed on a thermal cycler (Smart Cycler; TaKaRa) and data were analyzed using the Smart Cycler software (ver. 1.2d; TaKaRa). The cycling conditions were as follows previous study [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The averages of each target mRNA expression were calculated and normalized GAPDH expression levels.\u003c/p\u003e\n\u003ch3\u003eThe western blot hybridization (SDS-PAGE)\u003c/h3\u003e\n\u003cp\u003eCultured cells from the control and treatment groups were washed several times with phosphate-buffered saline and homogenized in loading buffer (50 mm Tris-HCl, 0.1% Triton X-100, 0.1 mm EDTA, 1 mm vinyl-methylsulfonyl fluoride) [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The cells were into the above sample buffer solution, and gel electrophoresis (SDS-PAGE) and transfer membrane procedures for are described in previous reports [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Using SNAP i.d. 2.0 (Millipore Sigma), the 1.0% blocking solution (Roche) in Tres-buffered saline solution (pH 7.6) was performed for blocking and the antibodies diluted by 0.5% blocking solution in Tris-buffered saline solution. Membranes were probed with antibodies (Table\u0026nbsp;2), and the immunoreactive expressions were indicated using the western blot chemiluminescence detecting kit (Bio-rad: Clartity\u0026trade; Western blot ECL substrate). The band intensities of the expressions were scanned images using the western blot imaging systems (ImageQuant\u0026trade;800, Amersham) and digital image analysis software (ImageJ, version 1.54d).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed at least five times independently. To compare between each experimental group, including control, a nonparametric procedure of Wilcoxon's signed rank test was used for statistical analysis. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean, and n represents the number of experimental samples. Statistical analysis was performed using SPSS software (IBM Corporation). Statistical significance was defined as a P value less than 0.05 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffect of compressive force (CF) and siRNA on the expression of TGF-β3\u003c/h2\u003e \u003cp\u003eReal-time RT-PCR of unstimulated MC3T3-E1 cells or cells stimulated with a continuous compressive force (0.5 g/cm\u003csup\u003e2\u003c/sup\u003e CF, 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF, and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF) indicated that the TGF-β3 mRNA expression gradually increased, peaked at 1-hour, and then gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The expression of TGF-β3 mRNA was increased in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group compared with that in the control, and 0.5 and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups at 1-hour (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe western blot analysis detected that among cells stimulated for 1-hour with 0.5 g/cm\u003csup\u003e2\u003c/sup\u003e CF, 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF, and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups. The TGF-β3 protein expression was significantly higher in cells treated with 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e than in the other conditions (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), which expressions were detected similar as mRNA expressions.\u003c/p\u003e \u003cp\u003eTo identify siRNA transfection, we examined the RT-PCR for TGF-β3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The mRNA expression in cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force (1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) and in siControl-treated cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force (siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) was significantly higher than in control cells without compressive force (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The TGF-β3 mRNA level in siTGF-β3-treated cells subjected to 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force cell (siTGF-β3\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group) was significantly lower than that in 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups, which siTGF-β3-treated cells was no significant difference compared with control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn the western blot results, the expression of TGF-β3 in control\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups were higher than those in the control group, which was without CF. The increase in TGF-β3 expressed in siTGF-β3\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF was significantly reduced by siRNA of TGF-β3 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInfluence of the downstream signaling by siTGF-β3 transfection on compressive force stimulation (CF)\u003c/h3\u003e\n\u003cp\u003eWith regard to the effect of siTGF-β3 transfection on the CF-induced phosphorylation of proteins in Smad-dependent and Smad-independent signaling pathways, the relative levels of phosphorylated Smad2 and phosphorylated Smad3 in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups were significantly higher than in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). The relative levels of phosphorylated Smad2 and Smad3 in the siTGF-β3\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group were significantly higher than in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). There was no difference of the influence by CF stimulation and also reduce by siTGF-β3 transaction between Smad2 and Smad3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relative levels of phosphorylated ERK-1/2 and phosphorylated p38 in 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups were significantly higher than in the control group, and there was no difference between the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups. The increase in phosphorylation of ERK1/2 and p38 in the siTGF-β3\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group was significantly decreased compared with that in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups, and was similar level to that in these CF groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In contrast, the increase in phosphorylation of ERK1/2 and p38 by compressive force was significantly decreased via siTGF-β3 treatment to levels similar value in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInfluence COX-2 expression of stimulation on compressive force (CF) and siTGF-β3 transfection\u003c/h2\u003e \u003cp\u003eWhen the COX-2 expression as the inflammatory cytokines in osteoblasts was assessed using real-time RT-PCR and western blot analysis, both mRNA and protein levels of inflammatory cytokines COX-2 in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group after 1-hour, significantly higher than those in the control, 0.5 g/cm\u003csup\u003e2\u003c/sup\u003e CF and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B) and could be correlated with TGF-β3 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the effect of siTGF-β3 transfection in the real-time RT-PCR and the western blot analysis, the expression of COX-2 in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups was significantly increased compared to the control group, which was almost same level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). These increased COX-2 expressions by 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF stimulation were significantly reduced by siTGF-β3 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). These influences of COX-2 expression could be correlated with TGF-β3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInfluence IL-6 expression of stimulation on compressive force (CF) and siTGF-β3 transfection\u003c/h2\u003e \u003cp\u003eThe IL-6 is one of the inflammatory cytokines, which associated with CF\u003csup\u003e15\u003c/sup\u003e. The expression of stimulation on CF was examined using real-time RT-PCR and western blot analysis, both mRNA and protein levels of inflammatory cytokines IL-6 in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF group after 1-hour, significantly higher than those in the control, 0.5 g/cm\u003csup\u003e2\u003c/sup\u003e CF and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). These effects of IL-6 expressions could be correlated with TGF-β3 expression.\u003c/p\u003e \u003cp\u003eRegarding the effect of siTGF-β3 transfection, the expressions of IL-6 in the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups were significantly increased compared to the control group in the real-time RT-PCR and the western blot analysis, which was no significantly difference between these CF stimulation groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). These increased IL-6 expressions by 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF were significantly reduced by siTGF-β3 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). The expression of IL-6 of the 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e and siControl\u0026thinsp;+\u0026thinsp;1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF groups was significantly higher than in the control group in both expression levels of the mRNA and the protein reaction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). The increased expression by these 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e CF stimulations groups were significantly decreased by siTGF-β3 transfection (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main novelty of this study is that a compressive force of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e of the optimal force stimulated the osteoblastic cells via TGF-β3 signaling pathway, which signaling were both Smad-dependent/-independent signaling pathway. The expression levels of COX-2 and IL-6 as the inflammatory cytokines were also increased upon application of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force (Fig.\u0026nbsp;8). These stimulatory effects were not observed in cells subjected to 0.5 or 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force. The stimulatory effects of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force were significantly reduced by siTGF-β3 treatment of cells, and the expression levels were similar to those in control cells that were not subjected to compressive forces. These new findings on the function of TGF-β3 are possibility suggested that that a compressive force of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e, as an optimal mechanical stress, promotes bone formation osteoblasts by increasing the levels of type I collagen, bone sialoprotein, and bone morphogenetic protein (BMP).\u003c/p\u003e \u003cp\u003eThe sequence of TGF-β3 is 86% similar to the sequence of TGF-β1 and 91% similar to the sequence of TGF-β2.14 However, even though TGF-β2 and -β3 share the highest level of sequence similarity of the three isoforms, TGF-β2 binds to TGF-β1 and -β3 by binds to TGF-β receptor II (TβRII) by a different mechanism15. 15 Furthermore, while TGF-β1 and -β3 can bind directly to TβRII, presentation of TGF-β2 to the receptor requires the presence of a coreceptor (β-glycan or endoglin), which may explain the role of TGF-β2 and -β3, which may explain the different roles of TGF-β2 and -β3 in activity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have shown that TGF-β3 plays an important role in normal craniofacial developmental biology, and exhibits isoform-specific biology at both in vivo and in vitro levels [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To understand the role of TGF-β3 in osteoblastogenic mechanisms, it is important to understand the unique biology of TGF-β3. The different functional roles of TGF-β have been studied in some depth with respect to palatal development. During palatal development, TGF-β3 is strongly expressed in midline epithelial cells prior to contact and fusion of opposing palatal shelves [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. During palatal fusion, it is also strongly expressed at the midline seam palatal epithelial cells, including mesenchymal cells undergoing epithelial-to-mesenchymal transition [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Notably, a complete cleft palate is observed in TGF-β3 null mice, even though the palatal mesenchymal shelves in this model have sufficient length and orientation to allow fusion [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, unlike other null mutants such exhibit cleft palate, TGF-β3 null mice lack other craniofacial abnormalities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn previous studies on osteoblast responses at an optimal compressive force, the expression of TGF-β1 at the 3-hour and TGF-β2 at the 6-hour time point gradually increased and then gradually decreased [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, in this result of new findings, the functional role of TGF-β3 when MC3T3-E1 cells are stimulated with the current 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force shows a relatively early pattern of bone remodeling expression compared to other TGF-βs. Also, it could be specific to osteoblast localization in bone formation during tooth migration, and fine-tunes signaling inflammatory cytokines and may fine-tune signaling inflammatory cytokines.\u003c/p\u003e \u003cp\u003eGiven the positive modulatory effect of TGF-β3 on osteoblast differentiation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], it is presumed that applying a compressive force of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e, but not 0.5 g/cm\u003csup\u003e2\u003c/sup\u003e and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e, promotes osteoblast differentiation. Osteoblast differentiation, including the bone formation was occurred to be affected not only by the Smad-dependent TGF-β3 signaling pathway, but also by the Smad-independent TGF-β3 signaling pathway [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For example, the association of TAK1 and TAK1-binding protein 1 induced by TGF-β leads to activation of the mitogen-activated protein kinase (MAPK) kinase 3-p38 MAPK signaling cascade and induction of type I collagen expression [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Notably, a recent study demonstrated that following TGF-β3 induction, the Smad and ERK/p38 MAPK pathways converged at Runt-related transcription factor (Runx2), which associated with osteoblast differentiation, in the control of mesenchymal precursor cell differentiation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, the issue of Smad-independent signaling induced by TGF-β, i.e., autocrine/paracrine inflammatory cytokines (IL-6 or COX-2 e.g.) activated by mitogen-activated protein kinase (MAPK) signaling cascade, remains to be addressed (Fig.\u0026nbsp;8). The such autocrine/paracrine signaling of TGF-β3 could be investigated as future planned.\u003c/p\u003e \u003cp\u003eBMPs are members of the TGF-β superfamily, have 32\u0026ndash;37% sequence homology with TGF-βs, and significantly affect osteoblast activity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. After BMP induction, Smad1/5 and MAPK cascades (such as ERK1/2 and p38) converge on Runx2 to regulate mesenchymal progenitor cell differentiation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The optimal force has been reported to increase expression of BMPs and their receptors, phosphorylated Smad1, Runx2, and the homeobox protein DLX-5, which promotes calcification in vitro [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Thus, the results of the present study suggest that both BMP and TGF-β signaling pathways could be stimulated by optimal compressive forces to induce osteoblast differentiation through Smad-dependent and Smad-independent cascades, and that crosstalk between BMP and TGF-β signaling pathways may be responsible for these may be related to these phenomena.\u003c/p\u003e \u003cp\u003eIn this study, the results were the compressive force of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e of the optimal force stimulated the osteoblastic cells via TGF-β3 signaling pathway. And our results found that transfection with siTGF-β3 suppressed Smad2/3, ERK, and p38 phosphorylation and inflammatory cytokine expression of the initial compressive force. This supports the idea that Smad-dependent/-independent signaling also promotes osteoblast differentiation via expression of osteogenesis-related transcription factors (Fig.\u0026nbsp;8).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSources of funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI (Grant Number: JP22K10280) and the Dental Research Center in Nihon University School of Dentistry, Graduate School of Dentistry (B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement form\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this research used experimental cell line subjects, so this study did not applicable the patient\u0026rsquo;s or guardian\u0026rsquo;s consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Professor Yoshio Nakano for the suggestions of statistical analysis, and our colleagues at the Department of Orthodontics and the Department of Oral Health Sciences, University School of Dentistry, Japan for their continuous support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMitsui, N., Suzuki, N., Maeno, M., Mayahara, K., Yanagisawa, M., Otsuka, K., Shimizu, N. (2005). Optimal compressive force induces bone formation via increasing bone sialoprotein and prostaglandin E(2) production appropriately. Life Sci, 77(25), 3168\u0026ndash;3182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitsui, N., Suzuki, N., Maeno M., Yanagisawa, M., Koyama, Y., Otsuka, K., Shimizu, N. (2006). Optimal compressive force induces bone formation via increasing bone morphogenetic proteins production and decreasing their antagonists production by Saos-2 cells. Life Sci, 78(23), 2697\u0026ndash;2706.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanuki R., Shionome C., Kuwabara A., Mitsui N., Koyama Y., Suzuki N., Zhang, F., Shimizu, N., Maeno, M. (2010). Compressive force induces osteoclast differentiation via prostaglandin E2 production in MC3T3-E1 cells. Connect Tissue Res, 51, 150\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, F., Koyama, Y., Sanuki, R., Mitsui, N., Suzuki, N., Kimura, A., Nakajima, A., Shimizu, N., Maeno, M. (2010). IL-17A stimulates the expression of inflammatory cytokines via celecoxib-blocked prostaglandin in MC3T3-E1 cells. Arch Oral Biol, 55, 679\u0026ndash;688.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSano, R., Nakajima, A., Kawato, T., Maeno, M., Shimizu, N. (2017). Effect of compressive force on TGF-β1/2 signaling pathway in MC3T3-E1 cells. J Hard Tissue Biol, 26, 177\u0026ndash;186.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKomamura, H., Nakajima, A., Sano, R., Motoyoshi, M. (2022). Effect of Transforming Growth Factor-β2 on Smad-Independent Signaling in MC3T3-E1 Cells. Biochem Mol Biol, 8, 2471\u0026ndash;8084.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakajima, A., Shuler, CF., Gulka, AOD., Hanai, JI. (2018). TGF-beta Signaling and the Epithelial-Mesenchymal Transition during Palatal Fusion. Int J Mol Sci, 19(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeters, AS., Brunner, G., Krieg, T., Eckes, B. (2015). Cyclic mechanical strain induces TGFbeta1-signalling in dermal fibroblasts embedded in a 3D collagen lattice. Arch Dermatol Res, 307(2), 191\u0026ndash;197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManokawinchoke, J., Limjeerajarus, N., Limjeerajarus, C., Sastravaha, P., Everts, V., Pavasant, P. (2015). Mechanical Force-induced TGFB1 Increases Expression of SOST/POSTN by hPDL Cells. J Dent Res, 94(7), 983\u0026ndash;989.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrafe, I., Alexander, S., Peterson, JR., Snider, TN., Levi, B., Lee, B., Mishina, Y. (2018). TGF-beta Family Signaling in Mesenchymal Differentiation. Cold Spring Harb Perspect Biol, 10(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, M., Chen, G., Li, YP. (2016). TGF-beta and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res, 4, 16009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMengrui, Wu., Guiqian, Chen., Yi-Ping, Li. (2016). TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res, 4, 16009. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/boneres.2016.9\u003c/span\u003e\u003cspan address=\"10.1038/boneres.2016.9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeyama, K., Chatani, M., Inohaya, K., Kudo, A. (2016). TGFbeta-2 signaling is essential for osteoblast migration and differentiation during fracture healing in medaka fish. Bone, 86, 68\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHossain, MG., Iwata, T., Mizusawa, N., Shima, SWN., Okutsu, T., Ishimoto, K., Yoshimoto, K. (2010). Compressive force inhibits adipogenesis through COX-2-mediated down-regulation of PPARgamma2 and C/EBPalpha, J Biosci Bioeng, 109(3), 297\u0026ndash;303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jbiosc.2009.09.003\u003c/span\u003e\u003cspan address=\"10.1016/j.jbiosc.2009.09.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhusuntornsakul, P., Jitpukdeebodintra, S., Pavasant, P. (2108). Vibration enhances PGE2, IL-6, and IL-8 expression in compressed hPDL cells via cyclooxygenase pathway. J Periodontol, 89(9), 1131\u0026ndash;1141.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaverty, HG., Wakefield, LM., Occleston, NL., O'Kane, S., Ferguson, MW. (2009). TGF-beta3 and cancer: a review. Cytokine Growth Factor Rev, 20(4), 305\u0026ndash;317.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrescenzo, GD., Hinck, CS., Shu, Z., Z\u0026uacute;\u0026ntilde;iga, J., Yang, J., Tang, Y., Baardsnes, J., Mendoza, V., Sun, L., L\u0026oacute;pez-Casillas, F., O'Connor-McCourt, M., Hinck. AP. (2006). Three key residues underlie the differential affinity of the TGFbeta isoforms for the TGFbeta type II receptor. J Mol Biol, 355, 47\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaartinen, V., Cui, XM., Heisterkamp, N., Groffen, J., Shuler, CF. (1997). Transforming growth factor-beta3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane. Dev Dyn. 209, 255\u0026ndash;260.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaartinen, V., Voncken, JW., Shuler, C., Warburton, D., Bu, D., Heisterkamp, N., Groffen, J. (1995). Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction. Nat Genet. 11(4), 415\u0026ndash;421.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaya, Y., O'Kane, S., Ferguson, MW. (1999). Pathogenesis of cleft palate in TGF-beta3 knockout mice. Development. 126(17), 3869\u0026ndash;3879.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProetzel, G., Pawlowski, SA., Wiles, MV., Yin, M., Boivin, GP., Howles, PN., Ding, J., Ferguson, MW., Doetschman, T. (1995). Transforming growth factor-beta 3 is required for secondary palate fusion. Nat Genet. 11(4), 409\u0026ndash;414.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakajima, A., Ito, Y., Tanaka, E., Sano, R., Karasawa, Y., Maeno, M., Iwata, K., Shimizu, N., Shuler, CF. (2014). Functional role of TGF-beta receptors during palatal fusion in vitro. Arch Oral Biol. 59(11), 1192\u0026ndash;1204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, SI., Kwak, JH., Zachariah, M., He, Y., Wang, L., Choi, ME. (2007). TGF-beta-activated kinase 1 and TAK1-binding protein 1 cooperate to mediate TGF-beta1-induced MKK3-p38 MAPK activation and stimulation of type I collagen. Am J Physiol Renal Physiol. 292(5), F1471-1478.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDerynck, R., Zhang, Y., Feng, XH. (1998). Smads: transcriptional activators of TGF-beta responses. Cell, 95(6), 737\u0026ndash;740.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKretzschmar, M., Massague, J. (1998). SMADs: mediators and regulators of TGF-beta signaling. Curr Opin Genet Dev, 8, 103\u0026ndash;111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanssens, K., ten Dijke, P., Janssens, S., Van, Hul W. (2005). Transforming growth factor-beta1 to the bone. Endocr Rev, 26(6), 743\u0026ndash;774.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, Y., Massague, J. (2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 113(6), 685\u0026ndash;700.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMassague, J., Seoane, J., Wotton, D. (2005). Smad transcription factors. Genes Dev, 19(23), 2783\u0026ndash;2810.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang, JS., Liu, C., Derynck, R. (2009). New regulatory mechanisms of TGF-beta receptor function. Trends Cell Biol, 19(8), 385\u0026ndash;394.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKimura, N., Matsuo, R., Shibuya, H., Nakashima, K., Taga, T. (2000). BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6. J Biol Chem, 275, 17647\u0026ndash;17652.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSorrentino, A., Thakur, N., Grimsby, S., Marcusson, A., Bulow, V., Schuster, N., Zhang, S., Heldin, CH., Landstr\u0026ouml;m, M. (2008). The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner. Nat Cell Biol, 10(10), 1199\u0026ndash;1207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamashita, M., Fatyol, K., Jin, C., Wang, X., Liu, Z, Zhang, YE. (2008). TRAF6 mediates Smad-independent activation of JNK and p38 by TGF-beta. Mol Cell, 31(6), 918\u0026ndash;924.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung, SM., Lee, JH., Park, J., Oh, YS., Lee, SK., Park, JS., Lee, YS., Kim, JH., Lee, JY., Bae, YS., Koo, SH., Kim, SJ., Park, SH. (2013). Smad6 inhibits non-canonical TGF-beta1 signalling by recruiting the deubiquitinase A20 to TRAF6. Nat Commun, 4, 2562.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, KS., Hong, SH., Bae, SC. (2002). Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene, 21(47), 7156\u0026ndash;7163.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLai, CF., Cheng, SL. (2002). Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells. J Biol Chem, 277(18), 15514\u0026ndash;15522.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyazono, K., Maeda, S., Imamura, T. (2005). BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine Growth Factor Rev. 16(3), 251\u0026ndash;263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen G., Deng C., Li YP. 2012. TGF-beta and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci, 8(2), 272\u0026ndash;288.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman, MS., Akhtar, N., Jamil, HM, Banik, RS., Asaduzzaman, SM. (2015). TGF-beta/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation. Bone Res, 3, 15005.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-biochemistry-and-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbbi","sideBox":"Learn more about [Cell Biochemistry and Biophysics](http://link.springer.com/journal/12013)","snPcode":"12013","submissionUrl":"https://submission.nature.com/new-submission/12013/3","title":"Cell Biochemistry and Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TGF-β3 signaling, tooth movement, Smad-independent signaling, MAPK signaling, compressing force, MC3T3-E1 cells","lastPublishedDoi":"10.21203/rs.3.rs-6242390/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6242390/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe function of Transforming growth factor (TGF) -β is reported to be associated with bone formation. However, the functional role of TGF-β3 in mediating the effects of orthodontic force such as the compressive force as a mechanical stress on osteoblasts remains unclear. We investigated the expression of TGF-β3 in osteoblasts and the effect of compressive force on downstream signaling pathways consisting of inflammatory cytokines. Cultured MC3T3-E1 and ATDC5 cells were subjected to continuous compressive forces, which are 0.5, 1.0, 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e, for 30-minutes, 1-hour, and 3-hours. Western blot analysis was determined phosphorylation of Smad-dependent and MAPKs. Measurement of TGF-3, Cox2, and IL-6 expression levels was done by Western blot analysis and real-time polymerase chain reaction. The expression of TGF-β3 in both cell lines was significantly increased upon application of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e, but not 0.5 and 2.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force in 1-hour, relative to the respective levels in unloaded control cells. At 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e compressive force increased the phosphorylation of Smad2, Smad3, ERK1/2, p-38, and the expressions of COX-2 and IL-6. The increased expression was attenuated by pretreatment with siRNA of TGF-β3. These results indicate that a compressive force of 1.0 g/cm\u003csup\u003e2\u003c/sup\u003e induces the expression of inflammatory cytokines and bone-specific transcription factors via TGF-β3 signaling in the osteogenesis.\u003c/p\u003e","manuscriptTitle":"Identification of transforming growth factor-β3 signaling by compressive force in MC3T3- E1 cells reactions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 19:22:36","doi":"10.21203/rs.3.rs-6242390/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-17T18:53:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T14:00:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T07:16:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-16T14:41:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-12T12:09:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-01T06:38:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136506219712029340257847451367641639326","date":"2025-03-24T05:57:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316488107674269648545793825206901138318","date":"2025-03-21T13:20:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189564103017246100382710905813532307275","date":"2025-03-20T12:21:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148608235274839939270396297916542389417","date":"2025-03-20T05:50:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271696872635525392504419603952800632690","date":"2025-03-19T12:19:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30463666146891631011923393129199706464","date":"2025-03-19T01:02:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187384813139385209403101702767681945788","date":"2025-03-18T23:19:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98040288843549789696236421253671779350","date":"2025-03-18T21:47:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300380744092021255310295344842417763964","date":"2025-03-18T18:31:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-18T18:25:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-18T07:50:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-18T07:48:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Biochemistry and Biophysics","date":"2025-03-17T08:01:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-biochemistry-and-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbbi","sideBox":"Learn more about [Cell Biochemistry and Biophysics](http://link.springer.com/journal/12013)","snPcode":"12013","submissionUrl":"https://submission.nature.com/new-submission/12013/3","title":"Cell Biochemistry and Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"62332220-7054-4669-8335-1dbaf93fb267","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-30T16:08:13+00:00","versionOfRecord":{"articleIdentity":"rs-6242390","link":"https://doi.org/10.1007/s12013-025-01783-6","journal":{"identity":"cell-biochemistry-and-biophysics","isVorOnly":false,"title":"Cell Biochemistry and Biophysics"},"publishedOn":"2025-06-24 16:05:38","publishedOnDateReadable":"June 24th, 2025"},"versionCreatedAt":"2025-03-27 19:22:36","video":"","vorDoi":"10.1007/s12013-025-01783-6","vorDoiUrl":"https://doi.org/10.1007/s12013-025-01783-6","workflowStages":[]},"version":"v1","identity":"rs-6242390","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6242390","identity":"rs-6242390","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}