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Research - (2022) Volume 8, Issue 7

Effect of Transforming Growth Factor-β2 on Smad-Independent Signaling in MC3T3-E1 Cells
Hiroyuki Komamura1, Akira Nakajima1,2*, Remi Sano1 and Mitsuru Motoyoshi1,2
 
1Department of Orthodontics, Nihon University, Japan
2Dental Research Center, Nihon University, Japan
 
*Correspondence: Akira Nakajima, Department of Orthodontics, Nihon University, Japan, Email:

Received: 01-Jul-2022, Manuscript No. IPBMBJ-22-14021; Editor assigned: 03-Jul-2022, Pre QC No. IPBMBJ-22-14021(PQ); Reviewed: 17-Jul-2022, QC No. IPBMBJ-22-14021; Revised: 22-Jul-2022, Manuscript No. IPBMBJ-22-14021(R); Published: 29-Jul-2022, DOI: 10.36648/2471-8084-22.8.81

Abstract

The aim of this study was to investigate the effects of bone formation on the Smad-independent signaling pathways in MC3T3-E1 cells stimulated with recombinant human TGF-β2 (rhTGF-β2). The concentration of TGF-β2 in cells subjected to an optimal compressive force of 1.0 g/cm2 (simulate as orthodontic force) was determined using an ELISA. After cell stimulation with 2.5 ng/mL rhTGF-β2 for 3 h, the expression of Smad-independent signaling factors (ERK1/2, TAK1, p38, and JNK) and the expression of osteogenic transcriptional factors (Msx2 and Dlx5) were determined. The expression levels of phosphorylated Smad-independent signaling and transcription factors were also significantly increased by rhTGF-β2 stimulation. These increased expression levels were significantly decreased by LY2109761, an inhibitor of TGF-β receptors. Our new findings suggest that the Smad-independent pathway defines cellular-specific responses to TGF-β2. In addition, rhTGF-β2 stimulation induces the osteogenic transcription factors, Msx2 and Dlx5, via MAPK phosphorylation in the Smad-independent signaling pathway in osteoblasts.

Keywords

TGF-β2 signalling; Bone formation; Mechanical stress; Smad-independent signalling; MAPK signalling; MC3T3-E1 cells

Abbreviations

(TGF) Transforming Growth Factor; (RH) Recombinant Human; (ELISA) Enzyme-Linked Immunosorbent Assay; (MAPKs) Mitogen-Activated Protein Kinases; (ERK) Ras-Raf-MEK-ERK; (TAK1) TGF-β-Activated Kinase; (JNK) c-Jun N-TERMINAL KINASE; (p38) p38-MAPK; (Msx2) Msh Homeobox 2; (Dlx5) Distal-Less Homeobox 5; (PCR) Polymerase Chain Reaction

Introduction

Orthodontic tooth movement is caused by bone remodeling. Various molecules are present in the periodontal tissue, including alveolar bone osteoblast/osteoclast interactions, during tooth movement [1-3]. Transforming growth factor-β (TGF-β) plays a role in cell proliferation, migration, and apoptosis through processes partially controlled by complex adhesive interactions between cellular receptors [3-5]. TGF-β has three main isoforms: TGF-β1, TGF-β2, and TGF-β3 [4,6,7]. TGF-β2 is most commonly involved in developmental processes that occur in affected bone tissues, such as epithelial-mesenchymal interactions, cell growth, extracellular matrix production, and remodeling [4,7,8]. The TGF-β2 null mutant exhibits perinatal mortality and a wide range of developmental defects due to single gene disruption [9].

Ligands of the TGF-β superfamily bind to type I and II receptors (Tβr1/2) [4,6,7]. The type I receptors then phosphorylate receptor- regulated Smads (signal transducers known as “mothers against decapentaplegic homolog”) (R-Smads; e.g., Smad2 and Smad3), which can bind to common partner Smads (Co-Smads; e.g., Smad4) [10,11]. This Smad-complex (Smad-dependent) signaling from TGF-β is shared by the bone morphogenetic protein (BMP) pathway [4,6,7,10-12]. R-Smad/Co-Smad complexes accumulate in the nucleus, where they act as transcription factors and participate in the regulation of target gene expression via Smad-dependent signaling [4,6,7].

TGF-βs activate Smad-independent signaling cascades that induce Smad signaling and Smad-independent TGF-β responses. The latter activates the mitogen-activated protein kinases (MAPKs) pathway, including the Ras-Raf-MEK-ERK (ERK) pathway, the TGF-β-activated kinase (TAK) 1/LK3/MEKK1 pathway and the c-Jun N-terminal Kinase (JNK) and p38-MAPK (p38) pathways [4,13,14]. JNK and p38-MAPK activation by TGF-βs is accompanied by Tβr-kinase activity- independent TRAF6-TAK1 phosphorylation [4]. However, the precise Smad-independent signaling pathways involved in bone formation are unclear.

A previous study suggested that cyclic mechanical strain results in a significant increase in active TGF-β1 levels, with no effects on the amount of total TGF-β1. TGF-β1 activation also leads to the phosphorylation of Smad2-mediated transcriptional elevation of downstream mediators and auto-induction of TGF-β1 [5]. As a result, TGF-β2 is considered to promote the differentiation of cells into osteoblasts to promote bone formation. Previously, we determined the effects of compressive force on the TGF-β1 and TGF-β2 signaling pathways [15]. Using MC3T3-E1 osteoblast-like cells, we observed increased TGF-β1 and TGF-β2 expression levels in cells subjected to 1.0 g/cm2 of compressive force for 3–6 h. The 1.0 g/cm2 compressive force-induced phosphorylation of Smad2/3 (Smad-dependent signaling) and upregulation of osteogenic transcription factors, such as Runt-related transcription factor 2 (Runx2) and Osterix (known as Sp7 transcription factor), were attenuated by pretreatment with a Tβr inhibitor [15]. This finding indicated that 1.0 g/cm2 compressive force could induce bone-specific transcription factors via the autocrine action of TGF-β1/2 signaling in osteoblasts [15].

To date, whether the specific functional role of TGF-β2 including the Smad-independence signaling during tooth movement is stimulated by the optimal mechanical stress of compressive force is unclear. Further, whether the two main Smad-independent pathways are specified by the exact regulatory mechanism and functions of TGF-β2 stimulation for bone formation during osteoblastic cell function, including the expression of osteogenetic transcription factors, such as Msh homeobox 2 (Msx2) and distal-less homeobox 5 gene (Dlx5), is unknown. To identify the functional role of TGF-β2 in osteoblastic function via the Smad-independent signaling pathway (e.g., ERK1/2 and TAK1), we opted to conduct experimental stimulation of TGF-β2 using an optimum compressive force in the MC3T3-E1 cell line. The objective of the present study was to determine whether osteoplasty is performed via the Smad-independent signaling pathway with TGF-β2 under an optimal compressive force.

Materials and Methods

Cell Culture and Enzyme-Linked Immunosorbent Assay (ELISA)

MC3T3-E1 cells from a mouse calvarial cell line were obtained from the RIKEN Bio Resource Center (Tsukuba, Japan) and used as osteoblast-like cells. The cells were maintained in α-minimal essential medium (α-MEM; Gibco BRL, Rockville, MD, USA) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Cells were subjected to 1.0 g/ cm2 optimal compressive force for 3 h, based on a previous report that described elevated TGF-β2 expression under these conditions [15]. Thereafter, the amounts of TGF-β2 in the culture medium were determined using an ELISA kit (R and vD Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The assays were performed in triplicate for each specimen, and the data were converted to ng/mL [2]. To evaluate the TGF-β2 downstream signaling pathway, cells were seeded in a 100 mm2 dish at a density of 1.0 × 104 cells/cm2 and treated for up to 3 h with 2.5 ng/ mL recombinant human (rh) TGF-β2 (R and D Systems, Inc. Minneapolis, MN, USA), based on the ELISA quantitation. Cells treated without rhTGF-β2 were used as a control.

Cell Culture with Exogenous Tβr1/2 Inhibitor (LY2109761)

LY2109761, an orally active Tβr1/2 kinase dual inhibitor (I, Ki=38 nmol/L; II, Ki=300 nmol/L [15]) was used to indicate Smad-independent signaling of TGF-β2. MC3T3-E1 cells were seeded in 6-well cell culture dishes at a density of 1.0 × 104 cells/cm2. After overnight incubation, the cells were treated for up to 3 h with 10 nM and 25 nM exogenous LY2109761 (G-T, Minneapolis, MN) 30 minutes before rhTGF-β2 stimulation.

Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Total mRNA was isolated from cultured MC3T3-E1 cells using a commercially available kit (RNeasy Mini Kit; Qiagen, Valencia, CA). Aliquots containing equal amounts of mRNA were subjected to real- time RT-PCR. First-strand cDNA synthesis was carried out using 1 μg of DNase-treated total mRNA in 20 μL of a solution containing first-strand buffer, 50 ng random primers, 10 mM dNTP mixture, 1 mM DTT, and 0.5 U reverse transcriptase at 42°C for 60 min. The cDNA mixtures were diluted five-fold using sterile distilled water, and 2-μL aliquots were subjected to real-time RT-PCR using SYBR Green I dye. Real-time RT-PCR was performed using a 25 μL final reaction volume containing 1X R-PR buffer, 1.5 mM dNTP mixture, 1X SYBR Green I, 15 mM MgCl2, 0.25 U ExTaq polymerase real-time RT-PCR version (TaKaRa, Tokyo Japan), and 20 mM specific primers (TaKaRa, Tokyo Japan); the primer sequences are listed in (Table 1). PCR was performed using a thermal cycler (Smart Cycler, Cepheid, and Sunnyvale, CA) and the data were analyzed using Smart Cycler software (ver. 1.2d). The cycling conditions were 95°C for 3 s and 68°C for 20 s for 35 cycles. Measurements were performed at the end of the annealing step at 68°C in each cycle. The specificity of the RT-PCR products was verified by adding melting curve analysis between 68 and 94°C. All real-time RT-PCR runs were performed in triplicate, and the mRNA expression levels were calculated and normalized to the level of GAPDH mRNA [1,2].

Table 1: Primer sequences used for PCR.

Primer Forward Reverse GenBank A/c No.
Msx2 5’-TGCAAGCGGCATCCATATACA-3’ 5’-GCGTGGCATAGAGTCCCACA-3’ NM_013601
Dlx5 5'-CCGCTTTACAGAGAAGGTTTCA-3' 5-TCTTCTTGATCTTGGATCTTTTGTT-3' NM_010056
GAPDH 5’-CAATGACCCCTTCATTGACC-3’ 5’-GACAAGCTTCCCGTTCTCAG-3’ XM_001473623

Western Blot Analysis

To obtain whole-cell extracts, MC3T3-E1 cells were cultured with or without rhTGF-β2 stimulation, rinsed with phosphate buffered saline, and then exposed to a lysis buffer comprising 50 mM Tris– HCl, 0.1% Triton X-100, 0.1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Cells in lysis buffer were sonicated three times (10 s each time). Aliquots containing equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Conditioned medium from western blots was collected, and aliquots containing equal amounts of protein were analyzed by SDSPAGE. Samples were loaded on 10–12% polyacrylamide gels and transferred to PVDF membranes using a semidry transfer unit. The membranes were probed with anti-p-TAK1, anti-p-ERK1/2, p-p38, and p-JNK (Cell Signaling Technology Japan, Tokyo, Japan; dilution 1:1000, polyclonal rabbit antibodies), anti-Msx2 and anti-Dlx5 (Santa Cruz Biotechnology, CA, USA; dilution 1:500), or anti-GAPDH antibodies as internal standards (Millipore, MA, USA; monoclonal mouse antibody, dilution 1:500) followed by a biotin-conjugated secondary antibody (Invitrogen, CA, USA; dilution 1:10,000). The membranes were then treated with horseradish peroxidase-conjugated streptavidin. Immunoreactive proteins were visualized using a chemiluminescence kit (Amersham Life Science, Buckinghamshire, UK), according to the manufacturer’s instructions. The intensities of the blots were quantified using a computer scanner (Epson PX-603F, Seiko Epson, Tokyo, Japan) and digital image analysis software (Scion Image, version Beta 4.0.3, Scion Corporation, NIH, USA) [16].

Statistical Analysis

Statistical analysis was conducted using the statistical Package for Social Sciences (version 8.0 for Windows, SPSS Japan Inc, Tokyo, Japan). Student’s t-test was used to compare differences between the control and 1.0 g/cm2 compressive force groups for the enzyme- linked immunosorbent assay results. The other data of Western blot and real-time revers transcription polymerase chain reaction for the statistical analysis were performed one-way analysis of variance (ANOVA) followed by of Tukey’s honestly significant difference tests using for multiple comparison between each data. Statistical significance was defined as a P-value of less than 0.05.

Results

Effect of Compressive Force on TGF-β2 Expression

In a previous study, TGF-β2 expression in MC3T3-E1 cells occurred at an optimal compressive force of 1.0 g/cm2 for 3 h [15]. In order to identify Smad-independent pathway via a specific TGF-β2 signaling, the amount of TGF-β2 in MC3T3-E1 cells subjected to the optimum force was determined using ELISA. Compared with the control group (not subjected to compressive force), the concentration of TGF-β2 was detected 2.54 ng/mL (Figure 1).

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Figure 1: Expression of TGF-β2 in cells subjected to 1.0 g/cm2 of compressive force

MC3T3-E1 cells were cultured with or without a continuous compressive force of 1.0 g/cm2 for up to 3 h. After 24 h, TGF-β2 protein levels in cells was measured from the culture medium using an enzyme-linked immunosorbent assay (n=5, *p<.05).

To confirm the stimulation induced by rhTGF-β2 and the effect of the Tβr1/2 inhibitor (LY2157299), western blot analysis was performed. After stimulation with rhTGF-β2, the expression of TGF-β2 was significantly increased compared to that in the control. This increased TGF-β2 expression was significantly decreased by LY2157299 in a dose-dependent manner (Figure 2).

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Figure 2: Effect of rhTGF-β2 stimulation and Tβr inhibition on TGF-β2 expression

To confirm the stimulation of recombinant human (rh) TGF-β2 (2.5 ng/mL) and the effect of the TGF-β receptor inhibitor (LY2157299; 10 nM or 25 nM), western blot analysis was performed. The expression of TGF-β2 was significantly increased compared to that in the control after stimulation with rhTGF-β2. This increased TGF-β2 expression was significantly decreased by LY2157299 in a dose-dependent manner (n=5, *p<.05).

Effect of ERK1/2 Phosphorylation by TGF-β2 Stimulation

Smad-independent signaling mainly involves two MAPKs pathways, one of which is the ERK1/2 cascade [4,17]. The phosphorylation of ERK1/2 was found to be significantly increased by rhTGF- β2 stimulation (p<0.05). In fact, this increase in expression was approximately 1.5-fold higher than that found in the control.

Phosphorylation of ERK1/2 was also decreased by LY2109761 in a dose-dependent manner (p<0.05) (Figure 3).

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Figure 3: Effect of rhTGF-β2 stimulation and Tβr inhibition on ERK1/2 and TAK1 phosphorylation

The expression of phosphorylated ERK1/2 (p-ERK1/2; A) and TAK1 (p-TAK1; B) was significantly increased by recombinant human TGF-β2 stimulation. p-ERK1/2 and p-TAK1 expression levels were approximately 1.5 and 1.8-fold higher, respectively, than that in the control (n=5, *p<0.05). This upregulation was significantly reduced by the Tβr inhibitor LY2157299 in a dose-dependent manner.

Thus, the relative density of p-ERK1/2 expression in MC3T3-E1 cells was significantly increased by stimulation with rhTGF-β2 compared to that in control cells.

Effects of TAK1, p38, and JNK Phosphorylation by TGF-β2 Stimulation

Another Smad-independent signaling pathway is the TAK1 cascade [4,18]. The phosphorylation of TAK1 was significantly increased by rhTGF-β2 stimulation (approximately 1.8-fold higher than that in the control; p<0.05). The phosphorylation of TAK1 was decreased by LY2109761 in a dose-dependent manner (p<0.05) (Figure 3B).

The downstream molecules of TAK1 signaling include p38 and JNK [4,18]. The expression of p-p38 was significantly increased by rhTGF-β2 stimulation, with an approximate 1.75-fold increase compared to that in the control (p<0.05). This upregulated p-p38 expression was significantly reduced by LY2157299 in a dose-dependent manner (Figure 4A).

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Figure 4: Effect of rhTGF-β2 stimulation and Tβr inhibition on p38 and JNK phosphorylation

The expression of p-JNK was also significantly increased by TGF-β2 stimulation. In fact, p-JNK expression was approximately 1.5-fold higher than that in the control (p<0.05), but was decreased by the inhibitor (LY2109761) in a dose dependent manner (p<0.05) (Figure 4B).

The phosphorylation of p38 (A) and JNK (B) was determined using western blot analysis to identify the balance in Smad-independent signaling. The phosphorylation of p38 (p-38) and JNK (p-JNK) was significantly increased by rhTGF-β2 stimulation. p-p38 and p-JNK protein levels were approximately 1.75 and 1.5-fold higher, respectively, than those of the control (n=5, *p<0.05). Phosphorylation was significantly decreased by the Tβr inhibitor LY2109761 in a dose-dependent manner (n=5, *p<0.05; A and B).

Therefore, TAK1, p38, and JNK phosphorylation were increased by TGF-β2 stimulation, and decreased by 25 nM LY2157299, leading to the same level found for the control.

Changes in the Expression of Msx2 and Dlx5

To identify the expression of osteogenic transcription factors following rhTGF-β2 stimulation and/or LY2157299 inhibition, the mRNA expression of Msx2 and Dlx5 was determined using real- time PCR and the protein levels were observed using western blot. Msx2 gene (Figure 5A) and protein (Figure 5B) expression levels were significantly increased compared with those of the control (p<0.05). Further, this increased expression was significantly decreased by LY2157299.

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Figure 5: Effect of rhTGF-β2 stimulation and Tβr inhibition on Msx2 gene and protein expression

The gene (A) and protein (B) expression levels of Msx2 were significantly increased by rhTGF-β2 stimulation, but significantly reduced by the Tβr inhibitor LY2157299 (n=5, *p<0.05).

Dlx5 expression was significantly increased in cells stimulated with rhTGF-β2 compared to control cells (p<0.05) (Figures 6A and 6B). The Dlx5 gene and protein expressions were also significantly reduced by LY2157299. These increased expression levels may not be influenced by the Smad-dependent signaling pathway, but may be mediated via the Smad-independent pathway. Therefore, these osteogenic transcription factors could be associated with the TGF-β2 signaling pathway, which could be mediated via the Smad-independent pathway.

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Figure 6: Effect of rhTGF-β2 stimulation and Tβr inhibition on Dlx5 gene and protein expression

The gene (A) and protein (B) expression levels of Dlx5 were significantly increased by rhTGF-β2 stimulation and decreased by the Tβr inhibitor LY2157299 in a dose-dependent manner (n=5, *p<0.05).

Discussion

The Smad-independent effects of rhTGF-β2 stimulation upon compressive force were evaluated in MC3T3-E1 cells and the expression level of osteogenic transcription factors owing to TGF-β2 signaling was determined. To accurately demonstrate the relationship between TGF-β2 and the Smad-independent signaling pathways using compressive force, the amount of TGF-β2 was first determined. Thereafter, the cells were stimulated using a specific amount of rhTGF-β2. Consequently, TGF-β2, which is expressed due to optimum compressive force, and TGF-β2 stimulation in osteoblasts could clarify the influence of Smad-independent signaling and the expression of osteogenic transcription factors.

In terms of MAPK signaling, osteoblast differentiation and bone formation may be influenced by the Smad-independent TGF-β signaling pathway [11,18,19]. The association of TAK1 (known as MAP3K7) is induced by TGF-β2, resulting in the activation of the JNK or the MAPK-p38 signaling cascades. TAK1 regulates the steady-state protein levels of these three kinases [11,18]. According to a previous study, exposure to either dynamic or static pressure induces the initial osteogenic differentiation of mesenchymal stem cells [14]. Particularly, both types of pressure strongly stimulated the expression of osteogenesis-related factors in undifferentiated mesenchymal stem cells. Extracellular signal-regulated kinases (ERKs) signaling participates in early osteo-differentiation and plays a positive but non-critical role in mechano-transduction [14]. Indeed, the present results indicate that TGF-β inhibition prevents rhTGF-β2-induced phosphorylation of ERK1/2 and TAK1 expression. Further, the current data support the notion that Smad-independent signaling can promote osteoblast differentiation via the expression of osteogenesis-related transcription factors.

Following Smad-independent phosphorylation, the expression of Msx2 and Dlx5 as osteogenetic transcription factors was induced by an effect exhibited by the TGF-β2 specific signaling pathways. As a result, their expression levels were significantly increased by rhTGF-β2 stimulation. Msx2 and Dlx5 are homeobox genes that encode homeodomain protein products that share a characteristic protein fold structure that binds DNA to regulate the expression of target genes [20-23]. Homeodomain proteins regulate gene expression and cell differentiation during early embryonic development; therefore, mutations in homeobox genes can cause developmental disorders [21,23,24].

Msx2 is localized on human chromosome 5, which encodes a transcriptional repressor and activator responsible for craniofacial and limb-bud development [23]. Cells express Msx2 when exposed to signaling molecules, such as BMP-2 and BMP-4, which are members of the TGF-β family [24,25]. The expression of Msx2 leads to the proliferation, migration, and osteogenic differentiation of neural crest cells during embryogenesis and bone fracture. Germline knockout mice have been created for this gene (Msx2 ±) to enable evaluations of functional loss [26]. Msx2 null mice exhibit intramembranous and endochondral ossification defects [27] and a decreased number of chondrocytes in their resting, proliferating and hypertrophic zones. In our study, the cell numbers in these three zones decreased, suggesting that specific TGF-β2 signaling is involved in chondrogenesis by controlling Msx2 expression in undifferentiated cells that induce endochondral bone.

As previously mentioned, Dlx5 is a member of the homeobox transcription factor gene family [20-22]. Previous studies have shown that the homeobox gene family is important for appendage development [21]. DLX5 is necessary for proper craniofacial, axial, and appendicular skeleton development [21,22]. A previous study found that the expression of Dlx5 is enhanced in Tgfbr2fl/fl; Wnt1- Cre mice and that its deletion resulted in a partial rescue of the cartilage phenotypes [21]. Therefore, we propose that TGF-β2 and Dlx5 induce osteoblastic functions via Smad-independent signaling, which determines the optimal compressive force stimulation.

Based on the relation between mechanical stress and the expression of Msx2 and Dlx5, which are osteogenic transcription factors, a previous study reported that a compressive force of 1.0 g/cm2 significantly increased the mRNA and protein expression of Msx2 and Dlx5, which are critical for osteoblast differentiation in the osteoblastic cell line (ROS 17/2.8 cells) [28]. In the present study, the expression of these osteogenesis-related transcription factors could also be strongly associated with the TGF-β2 signaling pathway via a Smad-independent signaling cascade. Interestingly, a recent study demonstrated that, following TGF-β induction, both TAK1 and ERK MAPK pathways converge at Msx2 and Dlx5 in their control of mesenchymal precursor cell differentiation [29]. Future studies should investigate the expression of osteogenic transcription factors via the TGF-β2 signaling pathway during the inhibition of either ERK1/2 or TAK1 signaling. As TGF-β can have positive or negative regulatory effects on osteoblast differentiation [3,30,31], our findings suggest that rhTGF-β2 is appropriate for promoting osteogenesis.

Conclusion

rhTGF-β2 stimulated the expression of components of the Smad-independent TGF-β signaling pathway, including osteogenic transcription factors, such as Msx2 and Dlx5, in MC3T3-E1 cells. These osteoblastic differentiation phenomena were reflected by specific Smad-independent signaling pathway molecules, suggesting that TGF-β2 stimulation induces osteogenic bone formation, at least under the present experimental conditions. Overall, the results of this study could be used to specify the TGF-β2 signaling pathway under optimum compressive force, and the Smad-independent pathways may define cellular-specific responses to TGF-β2.

Acknowledgements

The authors are thankful to their colleagues at the Department of Orthodontics and the Department of Oral Health Sciences, Nihon University School of Dentistry, for their continuous support.

Author Contributions

The contributions of all authors are described in the following statement:

1. Study conception and design of study: Remi Sano, Akira Nakajima, and Mitsuru Motoyoshi.

2. Analysis and interpretation of results: Hiroyuki Komamura, Remi Sano, Akira Nakajima and Mitsuru Motoyoshi.

3. Draft manuscript preparation: Hiroyuki Komamura, Remi Sano, Akira Nakajima and Mitsuru Motoyoshi.

4. The results and approved the final version of the manuscript: Hiroyuki Komamura, Remi Sano, Akira Nakajima and Mitsuru Motoyoshi.

Declaration of Competing Interests

Declarations of interest: none

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 (1 and 2).

REFERENCES

Citation: Komamura H, Nakajima A, Sano R, Motoyoshi M (2022) Effect of Transforming Growth Factor-?2 on Smad-Independent Signaling in MC3T3-E1 Cells. Biochem Mol Biol J. 8:81.

Copyright: © 2022 Komamura H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.