Ca2+/calmodulin-dependent protein kinase II regulates the inflammatory hDPSCs dentino-differentiation via BDNF/TrkB receptor signaling

Abstract

CaMKII is a serine/threonine-specific protein kinase that plays a crucial role in normal and pathological conditions. However, limited information is available regarding the roles of CaMKII in dentinogenesis, particularly in an inflammatory context. Previously, we demonstrated the pivotal role of TrkB in inflammation-induced differentiation of hDPSCs into odontoblast-like cells. Here, we investigate the interaction between CaMKII and TrkB during hDPSCs odontogenic differentiation. hDPSCs were cultured and subjected to CaMKII knockdown using siRNA, followed by treatment with dentinogenic media. TNFα-stimulated cells were treated with CaMKII- inhibitor, -protein, or TrkB antagonist, CTX-B. Immunocytochemistry and ARS were used to visualize targeted proteins and calcium deposits. Real-time PCR detected expression levels of odontogenic and mineralization markers such as DSPP and DMP-1. Our data indicate that CaMKII inhibition enhances TrkB protein levels and promotes TNFα-induced transcriptional activation of genes associated with odontogenic differentiation. CaMKII knockdown via siRNA and pharmacological inhibition elevated DSPP and DMP-1 protein levels, whereas CaMKII overexpression suppressed their expression. Notably, treatment with TNF-α and a CaMKII inhibitor upregulated DSPP and DMP-1 expression, while co-treatment with CTX-B abolished this effect. Similarly, mRNA expression of DSPP and DMP-1 was reduced at day 10. Mineralization activity exhibited a similar pattern to the expression of these markers. Our findings unveil a novel mechanism underlying the role of CaMKII via TrkB in dentinogenesis, which is vital for the success of hDPSCs engineering strategies.

Keywords: CaMKII; TNFα; TrkB; hDPSCs; inflammation; odontoblastic differentiation.

FIGURE 1

Molecular expression of CaMKII and p-CaMKII in hDPSCs. (A) Schematic timeline representation of odontogenic DPSCs differentiation until 14 days of odontoblastic hDPSCs differentiation with various treatments (TNF-α, CAMKII INH, CaMKII P, and CTX-B). (B) Expression of mesenchymal-stem-cell marker (STRO-1) and DAPI (A, B) with the merged image (C) in hDPSCs at day 4. (C) Images of early differentiated hDPSCs at day 4 (D4) and after 10 days of differentiation (D14) under the light microscope. Scale bars: (B) 50 μm; and (C) 100 μm. (D) The expression of odontoblastic markers ALP, BMP-2, COL1A1, and DMP-1 mRNA during the odontogenic differentiation was quantified by real-time PCR. The elevated level of these markers represents the odontoblast-like differentiation of DPSCs. The bar graph shows the mean ± SD of at least three independent experiments (n = 3) in duplicates. *p < 0.05 and **p < 0.01 vs fold change at day 0.

FIGURE 2

Expression of CaMKII-mediated DSPP and DMP-1 after hDSPCs odontoblastic differentiation. (A) Immunofluorescence double staining of DSPP and DMP-1 expression in CaMKII-mediated DPSC differentiation after 14 days. Anti-DSPP (green) expression of control (a), CaMKII inhibitor (f), and CaMKII protein (k). Anti-DMP-1 (red) expression of control (b), CaMKII inhibitor (g), and CaMKII protein (l). Staining nuclei with DAPI (blue; c, h, m). Co-localization of DSPP, DMP-1, and DAPI (d, i, n). Scale bar: 50 μm. Higher magnification of merged images (e, j, o). Scale bar: 50 μm. (B) Analyzed anti-DSPP (green) fluorescence intensity from different treatment groups: Control, CaMKII INH, and CaMKII protein. (C) Analyzed fluorescence intensity of anti-DMP-1 (red) from the different treatment groups. The bar graph shows the mean ± SD of at least three independent experiments performed in duplicate (n = 3). *p < 0.05, **p < 0.01 vs. control.

FIGURE 3

Expression of CaMKII-mediated DSPP and DMP-1 after hDSPCs odontoblastic differentiation. (A) Immunofluorescence double staining of DSPP and DMP-1 expression in CaMKII-mediated DPSC differentiation after 14 days. Anti-DSPP (green) expression of control (a), CaMKII inhibitor (f), and CaMKII protein (k). Anti-DMP-1 (red) expression of control (b), CaMKII inhibitor (g), and CaMKII protein (l). Staining nuclei with DAPI (blue; c, h, m). Co-localization of DSPP, DMP-1, and DAPI (d, i, n). Scale bar: 50 μm. Higher magnification of merged images (e, j, o). Scale bar: 50 μm. (B) Analyzed anti-DSPP (green) fluorescence intensity from different treatment groups: Control, CaMKII INH, and CaMKII protein. (C) Analyzed fluorescence intensity of anti-DMP-1 (red) from the different treatment groups. The bar graph shows the mean ± SD of at least three independent experiments performed in duplicate (n = 3). *p < 0.05, **p < 0.01 vs. control.

FIGURE 4

mRNA expression of CaMKII-mediated DSPP and DMP-1 after odontoblastic differentiation and siRNA of CaMKII at days 10 and 14. (A, B) Immunopositivity of anti-DSPP expression at day 10 in control and siCaMKII treated odontoblastic differentiated hDPSCs. Scale bar: 50 μm. (C–F) mRNA expression of DSPP and DMP-1 in odontoblastic differentiated hDPSCs at day 10 and day 14 from different treatment groups: control, CaMKII INH, CaMKII protein, and siRNA of CaMKII. The bar graph shows the mean ± SD of at least three independent experiments performed in duplicate (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 vs control.

FIGURE 5

CaMKII-mediated DSPP and DMP-1 expression in TNFα-induced odontogenic hDPSCs differentiation via TrkB receptor. (A) Detection of anti-DSPP (green) and anti-DMP-1 (red) in various treatment groups. Anti-DSPP showed in control, CaMKII INH, TNFα, TNFα + CaMKII INH, and TNFα + CaMKII INH + CTX-B (a, f, k, p, u). Anti-DMP-1 is shown in the different treatment groups (b, g, l, q, v). Immuno-positivity of nucleus staining of DAPI (c, h, m, r, w). Merge images of anti-DSPP and DMP-1 (d, i, n, s, x). Scale bars: 50 μm. Higher magnification from the white box of merged images (e, j, o, t, y). Scale bars: 25 μm. (B, C) Analyzed fluorescence intensity of DSPP and DMP-1. (D) The regulation of the CaMKII affects the odontogenic differentiation of DPSCs. ELISA has compared DMP-1 levels among several treatment groups at day 10 of hDPSCs odontoblastic differentiation. (E–H) mRNA expression of CaMKII-mediated DSPP and DMP-1 after TNFα-induced odontogenic hDPSCs differentiation via TrkB at day 10 and day 14. The bar graphs show the mean ± SD of at least three independent experiments performed in duplicate (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 vs control.

FIGURE 6

Phosphorylation of CaMKII in DPSC differentiated odontoblast-like cells. (A) in-cell western assay showing the expression of CaMKII and p-CaMKII in DPSC differentiated odontoblast-like cells after 10 days of differentiation with various treatments. The expression of CaMKII and p-CaMKII (a) and their corresponding fluorescence intensity distribution in a 3D surface plot (b). The β-actin signal, used as a loading control, showed consistent fluorescence intensity across all samples (Figure A-c), with its 3D surface plot (Figure A-d). (B) Bar graph showing the integrated fluorescence intensity. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control. #p < 0.05 vs CaMKII and p-CaMKII.

FIGURE 7

Mineralization activity of CaMKII-mediated odontogenic hDPSCs differentiation by TNFα via TrkB at day 14. (A) Representative images of ARS assay from different treatment groups; control, CaMKII INH, CaMKII P, CaMKII INH + CTX-B, TNFα, TNFα + CaMKII INH, and TNFα + CaMKII INH + CTX-B at 14 days of inflammation-induced odontogenic differentiation in hDPSCs. The white arrow indicates calcium crystallization. (B) Analysis of the percentages of the stained areas in the ARS assay. The bar graph shows the mean ± SD of at least three independent experiments performed in duplicate (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 vs control.

FIGURE 8

Summarized effects of TNFα-stimulation and BDNF/TrkB signaling in DPSCs odontoblast-like differentiation.