Mechanical loading activates β-catenin signaling in periodontal ligament cells
To determine whether β-catenin signaling is responsive to mechanical loading in periodontal ligament (PDL) cells. To determine whether Wnt/β-catenin signaling pathway components are present and functional, PDL cells were treated with lithium chloride or Wnt3a-conditioned media. To determine whether mechanical strain activates β-catenin signaling, PDL cells were subjected to compressive loading. Activation of the β-catenin signaling pathway was determined by immunofluorescence, Western immunoblotting, and TOPflash assay. Mimicking Wnt signaling stimulates β-catenin nuclear translocation and T-cell factor/lymphoid enhancer binding factor–dependent transcriptional activation in PDL cells. Mechanical loading stimulates a transient accumulation of dephosphorylated β-catenin in the cytoplasm and its translocation to the nucleus. This effect of strain acts through activation of protein kinase B and phosphorylation of glycogen synthase kinase-3 beta. These strain-related changes do not involve the low-density lipoprotein receptor-related protein 5/Wnt receptor. The Wnt/β-catenin signaling pathway components are functional and activated by mechanical loading in PDL cells. β-catenin serves as an effector of mechanical signals in PDL cells.Abstract
Objective:
Materials and Methods:
Results:
Conclusions:
INTRODUCTION
Modeling of alveolar bone around a tooth subjected to orthodontic force is of fundamental importance in orthodontics. Periodontal ligament (PDL) cells are thought to be the transducers of orthodontic loads. The PDL consists of a heterogeneous cell population that includes fibroblasts, osteoblasts, and cementoblasts and contains mesenchymal stem cells that are capable of differentiating into osteoblast-like cells.1 PDL cells perceive mechanical signals and respond to them by signaling to the progenitor cells in the periodontium, which differentiate into osteoblasts and osteoclasts. Although it is clear that PDL cells are responsive to mechanical stimulation,2–4 the molecular pathway(s) associated with mechanotransduction remains unknown.
Recent studies have demonstrated that Wnt/β-catenin signaling is required for mechanotransduction in bone.5 Both mechanical strain and fluid shear stress induce nuclear translocation of β-catenin in primary calvarial osteoblasts, osteocytes, and cell lines such as MC3T3-E1, CIMC-4, UMR-106, and ROS 17/2.8.5–10 The cytoplasmic pool of β-catenin upon appropriate stimulation translocates to the nucleus and increases gene transcription by interacting with T-cell factor/lymphoid enhancer binding factor (TCF/LEF). The aim of this study was to determine the effect of mechanical loading on β-catenin signaling in PDL cells.
MATERIALS AND METHODS
Cell Culture and Reagents
Primary human PDL cells were obtained from ScienCell (Carlsbad, Calif) and cultured in minimum essential medium (MEM)-alpha modification (CellGro, Manassas, Va) supplemented with antibiotics and 10% fetal bovine serum (Thermo Scientific Hyclone, Logan, Ut) at 37°C in a humidified 5% CO2 atmosphere. Cells between the third and eighth passages were used in experiments. Lithium chloride (LiCl) was obtained from Sigma Aldrich (St Louis, Mo). AKT/Protein Kinase signaling inhibitor-2 (API-2) was obtained from Cayman Chemical (Ann Arbor, Mich). Human recombinant Dickkopf-related protein 1 (DKK1) was obtained from R&D Systems (Minneapolis, Minn).
Mechanical Loading
PDL cells were plated in six-well plates at a density of 50,000 cells/cm2 on the day before the experiment, and mechanical loading was applied by placing 25-mm-diameter cover glasses and/or customized glass cylinders over the cells as described previously.2 Force magnitude was adjusted by adding or removing lead granules. PDL cells were subjected to compressive forces of 0.2 g/cm2, 2.2 g/cm2, and 5 g/cm2.
Generation of Wnt3a-Conditioned Media
Mouse Wnt3a-overexpressing cells (L-Wnt3a) and control nontransfected L cells were obtained from ATCC (Manassas, Va) and cultured in Dulbecco MEM with 10% fetal bovine serum, penicillin, streptomycin, and G418. Conditioned media from L-Wnt3a and control L cells were collected according to the manufacturers' instructions.
Protein Extraction
Whole cell lysates were prepared with cell lysis buffer (Cell Signaling, Beverly, Mass). For isolation of cytoplasmic and nuclear fractionates, cells were trypsinized, washed with phosphate-buffered saline, and centrifuged to obtain the pellet. The cell pellet was lysed in cytoplasmic lysis buffer for 15 minutes. The cytoplasmic fractionate was collected following centrifugation, and the residual pellet was lysed in nuclear lysis buffer for 30 minutes. The nuclear fractionate was collected after centrifugation. Protein concentration was estimated by BCA Protein Assay (Thermo Fisher, Rockford, Ill).
Western Blot Analysis
Equal amounts of protein were run on polyacrylamide gel and transferred onto nitrocellulose membrane. Membranes were incubated with primary antibodies overnight. On the following day, the membranes were incubated with Alexa Fluor 680 conjugated secondary antibody (Invitrogen, Carlsbad, Calif). Proteins were detected with LiCor Odyssey System (LiCor, Lincoln, Neb). The primary antibodies used were against: Active β-catenin (Clone 8E7; Millipore, Billerica, Mass); total β-catenin (gift from Dr James Wahl, University of Nebraska Medical Center, Omaha, Neb); phosphorylated glycogen synthase kinase-3-beta (pGSK-3β) (serine9; Cell Signaling); total GSK-3β (Cell Signaling); and phosphorylated protein kinase B (pAkt) (serine473; Cell Signaling).
Immunofluorescence Microscopy
PDL cells were grown overnight on cover glasses. Following application of mechanical loading or LiCl treatment, strained, LiCl-treated, and control PDL cells were fixed in Histochoice (Electron Microscopy Sciences, Hatfield, Penn). Cells were incubated with primary antibodies (mouse anti-active β-catenin) for 1 hour. The secondary antibody was fluorescein isothiocyanate goat anti-mouse immunoglobulin G. Cover glasses were mounted in Vectashield media containing DAPI (Vector Laboratories, Burlingame, Calif) and viewed using a Zeiss Axiovert microscope (Jena, Germany).
Luciferase Assay
TOPflash (Millipore) luciferase reporter containing the TCF/LEF consensus sequence was used to assess the functional response to nuclear translocation of active β-catenin. PDL cells were transiently transfected with TOPflash or FOPflash with mutated TCF binding sites (Millipore) together with Renilla luciferase plasmid (Promega, Madison, Wisc) using Fugene transfection reagent (Roche, Indianapolis, Ind). Twenty-four hours after transfection, cells were treated with LiCl. Cell lysates were measured for reporter activity (Dual luciferase reporter assay, Promega). Firefly luciferase activity derived from TOPflash or FOPflash was standardized based on Renilla luciferase activity.
Statistical Analyses
Differences between the two groups were determined by Student's t-test. The level of significance was set at P < .05.
RESULTS
Wnt/β-Catenin Signaling Is Functional in PDL Cells
To determine whether Wnt/β-catenin signaling is functional in PDL cells, cell cultures were exposed to LiCl or Wnt3A-conditioned medium. LiCl is an inhibitor of GSK-3β kinase activity and activates β-catenin by phosphorylating GSK-3β at Ser9.11 Changes in cellular distribution and levels of dephosphorylated β-catenin were determined by immunohistochemistry and Western immunoblotting, respectively. The level of dephosphorylated β-catenin was determined using an antibody to detect β-catenin that is dephosphorylated at Ser37 and threonine.41 There was an increase in the amount of staining for dephosphorylated β-catenin in the nucleus of cells treated with LiCl (Figure 1B) compared to that of NaCl treatment (Figure 1A). This active form of β-catenin was increased in response to Wnt stimulation (Figure 2A). Mimicking Wnt signaling in PDL cells increased the amount of phosphorylated GSK-3β (Figure 2A). GSK-3β phosphorylation is associated with the translocation of active β-catenin to the nucleus and an increase in TCF/LEF-dependent transcriptional activation in PDL cells (Figure 2B).
Citation: The Angle Orthodontist 81, 4; 10.2319/090310-519.1
Citation: The Angle Orthodontist 81, 4; 10.2319/090310-519.1
Mechanical Loading Induces Nuclear Translocation of β-Catenin in PDL Cells
To determine whether mechanical loading induces nuclear translocation of dephosphorylated β-catenin in PDL cells, we exposed PDL cell cultures to mechanical loading and determined the cellular distribution of dephosphorylated β-catenin by immunohistochemistry. There was an increase in the amount of staining for dephosphorylated β-catenin in the nucleus of the cells that had been subjected to mechanical loading (Figure 3B) compared to unloaded controls (Figure 3A). This correlated with our Western blot analysis (Figure 4A,B). Mechanical loading increased the levels of dephosphorylated β-catenin in the nucleus, but it had no effect on the levels in the cytoplasm (Figure 4C). The absence of any increase in dephosphorylated β-catenin in the cytoplasm suggests its rapid translocation to the nucleus.
Citation: The Angle Orthodontist 81, 4; 10.2319/090310-519.1
Citation: The Angle Orthodontist 81, 4; 10.2319/090310-519.1
Mechanical Loading Causes Phosphorylation of GSK-3β
Phosphorylation of GSK-3β at Ser9 inhibits its ability to phosphorylate β-catenin, which marks β-catenin for degradation.6 The possibility that this pathway was involved in strain-mediated activation of β-catenin in PDL cells was considered. The levels of Ser9 pGSK-3β were increased 6 hours after application of loading (Figure 5). These changes in pGSK-3β levels correlated with increased levels of dephosphorylated β-catenin in the PDL cells subjected to mechanical loading (Figure 5).
Citation: The Angle Orthodontist 81, 4; 10.2319/090310-519.1
Mechanical Loading–Induced Dephosphorylation of β-Catenin Does Not Involve LRP5/Frizzled/Wnt Receptor
Studies in osteoblasts have stated that phosphorylation of GSK-3β and subsequent activation of β-catenin could possibly work through Wnt-dependent and/or Wnt-independent pathways.6,9 DKK1 was used to evaluate whether the Wnt/low-density lipoprotein receptor-related protein 5 (LRP5) pathway was required for strain-mediated activation of β-catenin signaling. DKK1 is an inhibitor of Wnt signaling. It competes with Wnt ligands for LRP5/frizzled receptors. PDL cells either were strained or exposed to Wnt3a-conditioned media in the presence of DKK1, and expression of dephosphorylated β-catenin and p-GSK-3β was determined by Western blotting. The data shown in the Western blot (Figure 6A) indicate that DKK1 treatment did inhibit Wnt/LRP5–mediated activation of β-catenin in PDL cells. This demonstrated that DKK1 could be effectively used to block LRP5/frizzled receptors in PDL cells. On the other hand, DKK1 did not disrupt strain-mediated phosphorylation of GSK-3β and activation of β-catenin (Figure 6B).
Citation: The Angle Orthodontist 81, 4; 10.2319/090310-519.1
Mechanical Loading of PDL Cells Increases Levels of Active Akt, Which Correlates with Phosphorylation of GSK-3β
Since Akt is a serine/threonine kinase capable of phosphorylating GSK-3β at Ser9, we wanted to determine whether mechanical loading–induced phosphorylation of GSK-3β was mediated by activation of Akt. PDL cells were subjected to mechanical loading, and levels of Ser phosphorylat Akt, inactive GSK-3β (phosphorylated at Ser9), and active β-catenin were measured by Western blotting. The Western blot indicated that Akt became phosphorylated and the levels of total Akt remained constant after application of mechanical strain (Figure 7A). Pretreatment of the PDL cells with API-2 (which is an inhibitor of Akt) prevented the strain-mediated activation of Akt, phosphorylation of GSK-3β, and activation of β-catenin (Figure 7B).
Citation: The Angle Orthodontist 81, 4; 10.2319/090310-519.1
Mechanical Loading–Induced Nuclear Translocation of β-Catenin Requires Akt Activation
To determine whether mechanical loading–induced nuclear translocation of β-catenin in PDL cells is dependent upon Akt activity, PDL cells pretreated with API-2 or dimethyl sulfoxide were subjected to mechanical loading, and cellular distribution of dephosphorylated β-catenin was determined by immunohistochemistry. Pretreatment of the cells with API-2 before mechanical loading resulted in a decrease in the amount of dephosphorylated β-catenin in the nucleus (Figure 8).
Citation: The Angle Orthodontist 81, 4; 10.2319/090310-519.1
DISCUSSION
Orthodontic tooth movement occurs when orthodontic forces are converted into biological events. According to the pressure-tension model of tooth movement, the orthodontic force is transduced by PDL cells. Progenitor cells in the periodontium then differentiate into osteoclasts and osteoblasts, thereby causing bone resorption and apposition, respectively. Osteoclast formation and differentiation are regulated by a balance between levels of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG).12,13 Mechanical strain up-regulates RANKL expression in PDL cells.2,14,15 In this respect, significant changes in RANKL and OPG levels have been demonstrated in the periodontal tissues during orthodontic tooth movement.3,16 However, the molecular mechanism by which PDL cells transduce mechanical force into biological mediators such as OPG and RANKL has remained unclear.
OPG is a direct target gene of Wnt/β-catenin signaling, and β-catenin signaling controls osteoclastogenesis by directly regulating RANKL/OPG levels.17,18 Mechanical strain has been shown to activate Wnt/β-catenin target genes5 in osteosarcoma and osteoblastic cells. β-catenin nuclear translocation has been shown to up-regulate cyclooxygenase-2 expression in multiple cell types.6,7,19,20 The aim of the present study was to explore whether similar pathways are involved in the PDL cell response to mechanical loading.
Using Western immunoblotting and TOP flash reporter assay, we demonstrated that Wnt/β-catenin signaling is functional in PDL cells. We chose to use LiCl and Wnt3a-conditioned media to mimic Wnt signaling in PDL cells. LiCl is a widely used mimetic of canonical Wnt signaling and induces dephosphorylation of β-catenin.21 In PDL cells, LiCl-induced dephosphorylation of β-catenin at levels comparable to Wnt3a-conditioned media and TCF/LEF mediated transcription. This indicates that PDL cells have the ability to send and receive Wnt signals. The results of our study are consistent with the findings of recent studies in PDL cells.22–24
In this study, we report a novel finding that mechanical loading activates β-catenin signaling in PDL cells. The data presented here clearly showed changes in the phosphorylation status and intracellular location of β-catenin in PDL cells subjected to 2.2 g/cm2 of compressive loading for 6 hours. The value 2.2 g/cm2 was chosen because it is within the range of estimated strain in the PDL tissues of a tooth that is subjected to orthodontic loading in clinical situations.25 The system used here to apply compressive loads to PDL cells has been shown to be effective in many studies.2,25 GSK-3β constitutively phosphorylates the N-terminus of β-catenin cyclin-dependent kinase at Ser45, which marks β-catenin for degradation. When GSK-3β is phosphorylated at Ser9, its ability to phosphorylate β-catenin is blocked. We have shown that mechanical loading induced an increase in Ser9 phosphorylation of GSK-3β that correlated with the changes in β-catenin levels, demonstrating inactivation of GSK-3β by mechanical loading.
Our studies have shown that mechanical loading–induced activation of β-catenin in PDL cells is not mediated by Wnt/LRP5/frizzled receptors. Pretreatment with DKK1 failed to inhibit mechanical loading–induced dephosphorylation of β-catenin in PDL cells, thus agreeing with the findings of recent studies in osteoblasts.6,9 Akt is known to phosphorylate and inactivate GSK-3β.26 Mechanical strain can activate Akt in multiple cell types.6,9,27 The data presented here demonstrate that Akt phosphorylation was induced by mechanical loading. This strain-mediated activation of Akt occurred in parallel with phosphorylation of GSK-3β. Mechanical loading–induced dephosphorylation of β-catenin and its nuclear translocation was dependent on Akt activation, as evidenced by the blocked response following pretreatment with API-2.
CONCLUSIONS
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The data presented here demonstrate for the first time that exposure of PDL cells to mechanical loading is associated with translocation of active β-catenin to the nucleus. This effect of strain operates through activation of Akt and phosphorylation of GSK-3β and does not involve the LRP5/Wnt receptor.
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Our results indicate that β-catenin signaling pathway components are functional and activated by mechanical strain in PDL cells. β-catenin plays a role in the general response to mechanical stimulation of PDL cells.
Mimicking Wnt signaling in PDL cells induced nuclear translocation of β-catenin. LiCl stimulated nuclear translocation of dephosphorylated β-catenin in PDL cells (B) compared with NaCl controls (A). DAPI was used to stain the nuclei (blue) (C and D).
Mimicking Wnt signaling in PDL cells induced dephosphorylation of β-catenin and TCF/LEF–dependent transcriptional activation. Cells were treated with LiCl or Wnt3a-conditioned media. Western blots showed an increase in the levels of dephosphorylated β-catenin and phosphorylated GSK-3β in cells treated with LiCl or Wnt3a-CM (A) compared with cells incubated with NaCl or L cell–conditioned media. Glyceraldehyde phosphate dehydrogenase (GAPDH) is shown as a control for equal protein loading. Mimicking Wnt signaling with LiCl stimulated TCF/LEF–mediated gene transcription. Mean values are shown ± SEMs (n = 4). *P < .05 (B).
Mechanical loading induces nuclear translocation of β-catenin. PDL cells were subjected to compressive loading and immunohistochemistry was performed to detect dephosphorylated β-catenin. Unloaded controls (A); nuclear translocation of dephosphorylated β-catenin (arrows) stimulated by mechanical loading (B). DAPI was used to stain the nuclei (blue) (C and D).
Mechanical loading induces dephosphorylation and nuclear translocation of β-catenin. PDL cells were subjected to mechanical loading. Western blots showed an increase in the levels of dephosphorylated β-catenin in whole cell lysates and nuclear fractionate from cells subjected to mechanical loading (A,B). Cytoplasmic extracts showed no expression of dephosphorylated β-catenin (C). Expression of lamin B and GAPDH are shown as controls for equal protein loading.
Mechanical loading induces phosphorylation of GSK-3β. PDL cells were subjected to mechanical loading for 6 hours. Western immunoblotting showed an increase in the levels of phosphorylated GSK-3β and dephosphorylated β-catenin. GAPDH is shown as a control for equal protein loading.
Mechanical loading–induced dephosphorylation of β-catenin does not involve LRP5/Frizzled/Wnt receptors. PDL cells were treated with Wnt3a-conditioned media (CM) or L cell–CM in the presence of DKK1. Western blots showed increased levels of dephosphorylated β-catenin and phosphorylated GSK-3β in the cells treated with Wnt3a CM (A). The presence of DKK1 inhibited the Wnt3a-induced phosphorylation of GSK-3β and dephosphorylation of β-catenin (A). Cells were pretreated with DKK1 and subjected to mechanical loading. DKK1 did not block the mechanical loading–induced phosphorylation of GSK-3β or dephosphorylation of β-catenin (B). GAPDH is shown as a control for equal protein loading.
Mechanical loading–induced phosphorylation of GSK-3β requires Akt phosphorylation. PDL cells were pretreated with API-2 and subjected to mechanical loading. Western blots of whole cell lysates showed that API-2 inhibited the mechanical loading–induced phosphorylation of Akt and GSK-3β. Levels of total Akt and GSK-3β remained constant (A). Western blot of nuclear fractionate showed that API-2 prevented mechanical loading–induced dephosphorylation of β-catenin (B).
Mechanical loading–induced nuclear translocation of β-catenin requires Akt activation. PDL cells were pretreated with API-2 and subjected to compressive loading. Immunohistochemistry was performed to detect dephosphorylated β-catenin. Pretreatment of cells with API-2 inhibited the mechanical loading–induced nuclear translocation of dephosphorylated β-catenin. DAPI was used to stain the nuclei (blue).
Contributor Notes