Journal: Frontiers in Cell and Developmental Biology

Article Title: Adiponectin as Well as Compressive Forces Regulate in vitro β-Catenin Expression on Cementoblasts via Mitogen-Activated Protein Kinase Signaling Activation

doi: 10.3389/fcell.2021.645005

Figure Lengend Snippet: Adiponectin as well as compression regulates the expression of adiponectin receptors (AdipoRs), mitogen-activated protein kinase (MAPK), and β-catenin. (A) Representative western blots showing the expression changes of AdipoR1 and AdipoR2 in the presence of adiponectin (100 ng/ml) or compression (1.2, 2.4, and 3.6 gf/cm 2 ). Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) analysis shows that 2.4 or 3.6 gf/cm 2 compressive forces significantly increase mRNA expression of adiponectin receptors ( AdipoR1 and AdipoR2 ) in mouse OCCM-30 cells ( ∗∗ p < 0.01). (B,C) Western blots showing the expression of p-P38, p-ERK1/2, and p-JNK induced by adiponectin or compression. (D) The OCCM-30 cells show down-regulated mRNA expression of GSK-3 β when exposed to adiponectin (100 ng/ml) or compression (3.6 gf/cm 2 ), whereas up-regulated mRNA expression of β- catenin ( ∗∗ p < 0.01). β-Catenin protein expression was increased after compression stimulation ( ∗ p < 0.05). Graphics show mRNA expression levels of P38 α, JNK1 , ERK1, ERK2, GSK-3 β, and β- catenin after 60-min stimulation with adiponectin or compression. Data were derived from three independent experiments. Data is normalized to 1, and values are expressed as means ± SD. Asterisks indicate significant differences compared to control cells ( ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05; ns, not significant).

Article Snippet: Membranes were blocked with 5% non-fat milk (T145.1, ROTH) for 1 h at room temperature and further incubated with the primary antibodies for ERK1/2 (1:1,000, MBS8241746, BIOZOL); phospho-ERK1/2 (p44/42, Thr202/Tyr204) (1:1,000, #4370, Cell Signaling Technology); p54/p56 JNK (1:1,000, #9252, Cell Signaling Technology); phosphor-SAPK/JNK (Thr183/Tyr185) (1:1,000, #4668, Cell Signaling Technology); P38 MAPK (1:1,000, #9212, Cell Signaling Technology); phospho-P38 MAPK alpha (1:1,000, #4511, Cell Signaling Technology); GSK-3β (1:1,000, #12456, Cell Signaling Technology); phospho-GSK-3β (Ser9) (1:1,000, #9323, Cell Signaling Technology); β-catenin (1:1,000, #8480, Cell Signaling Technology); phospho-β-catenin (Ser33/37/Thr41) (1:1,000, #9561, Cell Signaling Technology); and β-actin (1:2,000, ab8227, Abcam) followed by peroxidase-conjugated secondary antibodies including polyclonal goat anti-rabbit (1:2,000, P0448, Dako); rabbit anti-goat (1:2,000, P0160, Dako), and polyclonal goat anti-mouse (1:2,000, P0447, Dako) in 2.5% non-fat milk (T145.1, ROTH) for 1 h at room temperature.

Techniques: Expressing, Western Blot, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Adiponectin as Well as Compressive Forces Regulate in vitro β-Catenin Expression on Cementoblasts via Mitogen-Activated Protein Kinase Signaling Activation

doi: 10.3389/fcell.2021.645005

Figure Lengend Snippet: Adiponectin in combination with compression enhances MAPK signaling activation. (A,B) Compression promotes P38, ERK1/2, and JNK phosphorylation on OCCM-30 cells. The kinetic protein expression of P38, ERK1/2, and JNK as well as their phosphorylated forms in response to compressive forces of 2.4 gf/cm 2 were analyzed by western blots. β-Actin served as a loading control. (C,D) After stimulation with 2.4 gf/cm 2 compression and adiponectin (100 ng/ml), phosphorylated forms of P38, ERK1/2, and JNK were up-regulated at different time points. Graphics represent the relative expression values of p-P38, p-ERK1/2, and p-JNK normalized to control cells at time point 0 as protein fold changes, respectively. (E) Western blot showing the expression changes of MAPK protein induced by adiponectin (100 ng/ml), compression (2.4 gf/cm 2 ), or adiponectin combined with compression (1.2, 2.4, and 3.6 gf/cm 2 ). The quantification ratio of p-P38, p-ERK1/2, and p-JNK is shown as phosphorylated state unit/total unphosphorylated protein (Phospho/Total). The statistical analysis was based on three independent experiments. Values are shown as the means ± SD. Asterisks indicate significant differences compared to control cells (*** p < 0.001, ** p < 0.01, and * p < 0.05; ns, not significant).

Article Snippet: Membranes were blocked with 5% non-fat milk (T145.1, ROTH) for 1 h at room temperature and further incubated with the primary antibodies for ERK1/2 (1:1,000, MBS8241746, BIOZOL); phospho-ERK1/2 (p44/42, Thr202/Tyr204) (1:1,000, #4370, Cell Signaling Technology); p54/p56 JNK (1:1,000, #9252, Cell Signaling Technology); phosphor-SAPK/JNK (Thr183/Tyr185) (1:1,000, #4668, Cell Signaling Technology); P38 MAPK (1:1,000, #9212, Cell Signaling Technology); phospho-P38 MAPK alpha (1:1,000, #4511, Cell Signaling Technology); GSK-3β (1:1,000, #12456, Cell Signaling Technology); phospho-GSK-3β (Ser9) (1:1,000, #9323, Cell Signaling Technology); β-catenin (1:1,000, #8480, Cell Signaling Technology); phospho-β-catenin (Ser33/37/Thr41) (1:1,000, #9561, Cell Signaling Technology); and β-actin (1:2,000, ab8227, Abcam) followed by peroxidase-conjugated secondary antibodies including polyclonal goat anti-rabbit (1:2,000, P0448, Dako); rabbit anti-goat (1:2,000, P0160, Dako), and polyclonal goat anti-mouse (1:2,000, P0447, Dako) in 2.5% non-fat milk (T145.1, ROTH) for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Adiponectin as Well as Compressive Forces Regulate in vitro β-Catenin Expression on Cementoblasts via Mitogen-Activated Protein Kinase Signaling Activation

doi: 10.3389/fcell.2021.645005

Figure Lengend Snippet: MAPK inhibition blocks β-catenin, whereas adiponectin addition effectively rescues its expression. (A,B) Western blots indicate that GSK-3β expression was influenced by MAPK inhibitors 1 h after adiponectin addition: pictures show that addition of SB203580 (P38) and SP600125 (JNK) inhibitors to OCCM-30 cells up-regulate GSK-3β protein expression. The expression of β-catenin was reduced after P38 and ERK1/2 inhibition. The suppressed β-catenin signaling could be rescued by adiponectin (100 ng/ml) in different degrees. (C,D) Compressive forces of 2.4 gf/cm 2 decrease the expression of total GSK-3β protein in cells pretreated with SB203580 (P38), SP600125 (JNK), and FR180204 (ERK1/2) inhibitors and increase the expression of cellular β-catenin. Single suppression of P38, ERK, and JNK blocks β-catenin, but its expression was rescued by compression stimulation. (E,F) The negative effect that ERK1/2 and JNK inhibition exerts on the expression of total GSK-3β protein on OCCM-30 cells exposed to compression is not altered by adiponectin addition for 1 h of co-stimulation. P38 inhibitor combined with compression decreases the expression of GSK-3β. This effect was enhanced by adiponectin addition. The expression of β-catenin was not significantly altered in the presence of MAPK inhibitors and compression despite adiponectin. Graphics show the variations of GSK-3β and β-catenin protein expression as fold change when cells were exposed to MAPK inhibitors in cells cultivated under compressive forces (2.4 gf/cm 2 ) and/or adiponectin (100 ng/ml) compared to controls. (G) OCCM-30 transfected with siRNA ( AdipoR1 , AdipoR2 , P38 α, JNK1 , ERK1 , and ERK2 ) as well as TCF/LEF luciferase reporter vector were subsequently treated with adiponectin for 1 h. Asterisks indicate statistical significance ( ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05; ns, not significant).

Article Snippet: Membranes were blocked with 5% non-fat milk (T145.1, ROTH) for 1 h at room temperature and further incubated with the primary antibodies for ERK1/2 (1:1,000, MBS8241746, BIOZOL); phospho-ERK1/2 (p44/42, Thr202/Tyr204) (1:1,000, #4370, Cell Signaling Technology); p54/p56 JNK (1:1,000, #9252, Cell Signaling Technology); phosphor-SAPK/JNK (Thr183/Tyr185) (1:1,000, #4668, Cell Signaling Technology); P38 MAPK (1:1,000, #9212, Cell Signaling Technology); phospho-P38 MAPK alpha (1:1,000, #4511, Cell Signaling Technology); GSK-3β (1:1,000, #12456, Cell Signaling Technology); phospho-GSK-3β (Ser9) (1:1,000, #9323, Cell Signaling Technology); β-catenin (1:1,000, #8480, Cell Signaling Technology); phospho-β-catenin (Ser33/37/Thr41) (1:1,000, #9561, Cell Signaling Technology); and β-actin (1:2,000, ab8227, Abcam) followed by peroxidase-conjugated secondary antibodies including polyclonal goat anti-rabbit (1:2,000, P0448, Dako); rabbit anti-goat (1:2,000, P0160, Dako), and polyclonal goat anti-mouse (1:2,000, P0447, Dako) in 2.5% non-fat milk (T145.1, ROTH) for 1 h at room temperature.

Techniques: Inhibition, Expressing, Western Blot, Transfection, Luciferase, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Adiponectin as Well as Compressive Forces Regulate in vitro β-Catenin Expression on Cementoblasts via Mitogen-Activated Protein Kinase Signaling Activation

doi: 10.3389/fcell.2021.645005

Figure Lengend Snippet: Adiponectin/AdipoR1/P38α cascade is particularly involved in adiponectin-induced cementogenesis. (A) The efficacy of siRNA transfections was analyzed by RT-PCR analysis. (B) Single knocking down of AdipoR2 in the presence of adiponectin has a positive effect on GSK-3 β gene expression and significantly increases β- catenin expression ( ∗ p < 0.05). (C) The single silencing of ERK2 causes increased gene expression of GSK-3 β after adiponectin addition ( ∗∗ p < 0.01). Silencing of P38 α and JNK1 slightly activates GSK-3 β gene expression. After adiponectin addition, its expression was significantly decreased ( ∗ p < 0.05). Single suppression of P38 α or JNK1 significantly decreased β- catenin expression ( ∗∗ p < 0.01 and ∗ p < 0.05, respectively), an effect that was restored after adiponectin addition in both groups. (D) Single knocking down of AdipoR1 or AdipoR2 has a down-regulating effect on OCN ( ∗ p < 0.05) and OPG ( ∗∗ p < 0.01) mRNA expression. The single silencing of P38α causes a significant down-regulation of OCN ( ∗ p < 0.05) and OPG gene expression in the present of adiponectin (100 ng/ml) ( ∗∗ p < 0.01). These effects were not observed by ERK1 , ERK2 , and JNK1 single suppression. Asterisks indicate statistical significance ( ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05; ns, not significant).

Article Snippet: Membranes were blocked with 5% non-fat milk (T145.1, ROTH) for 1 h at room temperature and further incubated with the primary antibodies for ERK1/2 (1:1,000, MBS8241746, BIOZOL); phospho-ERK1/2 (p44/42, Thr202/Tyr204) (1:1,000, #4370, Cell Signaling Technology); p54/p56 JNK (1:1,000, #9252, Cell Signaling Technology); phosphor-SAPK/JNK (Thr183/Tyr185) (1:1,000, #4668, Cell Signaling Technology); P38 MAPK (1:1,000, #9212, Cell Signaling Technology); phospho-P38 MAPK alpha (1:1,000, #4511, Cell Signaling Technology); GSK-3β (1:1,000, #12456, Cell Signaling Technology); phospho-GSK-3β (Ser9) (1:1,000, #9323, Cell Signaling Technology); β-catenin (1:1,000, #8480, Cell Signaling Technology); phospho-β-catenin (Ser33/37/Thr41) (1:1,000, #9561, Cell Signaling Technology); and β-actin (1:2,000, ab8227, Abcam) followed by peroxidase-conjugated secondary antibodies including polyclonal goat anti-rabbit (1:2,000, P0448, Dako); rabbit anti-goat (1:2,000, P0160, Dako), and polyclonal goat anti-mouse (1:2,000, P0447, Dako) in 2.5% non-fat milk (T145.1, ROTH) for 1 h at room temperature.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing