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Thymosin beta 4 is associated with RUNX2 expression through the Smad and Akt signaling pathways in mouse dental epithelial cells.

Someya H, Fujiwara H, Nagata K, Wada H, Hasegawa K, Mikami Y, Jinno A, Sakai H, Koyano K, Kiyoshima T - Int. J. Mol. Med. (2015)

Bottom Line: In previous studies by our group, we reported that thymosin beta 4 (Tb4) is closely associated with the initiation and development of the tooth germ, and can induce the expression of runt-related transcription factor 2 (RUNX2) during the development of the tooth germ.The protein expression of p-Smad1/5 and p-Akt in the mDE6 cells was reduced by treatment with Tb4-siRNA.This study provides putative information concerning the signaling pathway through which Tb4 induces RUNX2 expression, which may help to understand the regulation of tooth development and tooth regeneration.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Oral Pathology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.

ABSTRACT
In previous studies by our group, we reported that thymosin beta 4 (Tb4) is closely associated with the initiation and development of the tooth germ, and can induce the expression of runt-related transcription factor 2 (RUNX2) during the development of the tooth germ. RUNX2 regulates the expression of odontogenesis-related genes, such as amelogenin, X-linked (Amelx), ameloblastin (Ambn) and enamelin (Enam), as well as the differentiation of osteoblasts during bone formation. However, the mechanisms through which Tb4 induces the expression of RUNX2 remain unknown. In the present study, we employed a mouse dental epithelial cell line, mDE6, with the aim to elucidate these mechanisms. The mDE6 cells expressed odontogenesis-related genes, such as Runx2, Amelx, Ambn and Enam, and formed calcified matrices upon the induction of calcification, thus showing characteristics of odontogenic epithelial cells. The expression of odontogenesis-related genes, and the calcification of the mDE6 cells were reduced by the inhibition of phosphorylated Smad1/5 (p-Smad1/5) and phosphorylated Akt (p-Akt) proteins. Furthermore, we used siRNA against Tb4 to determine whether RUNX2 expression and calcification are associated with Tb4 expression in the mDE6 cells. The protein expression of p-Smad1/5 and p-Akt in the mDE6 cells was reduced by treatment with Tb4-siRNA. These results suggest that Tb4 is associated with RUNX2 expression through the Smad and PI3K-Akt signaling pathways, and with calcification through RUNX2 expression in the mDE6 cells. This study provides putative information concerning the signaling pathway through which Tb4 induces RUNX2 expression, which may help to understand the regulation of tooth development and tooth regeneration.

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The results of the analysis of signaling pathways upstream of runt-related transcription factor 2 (Runx2) expression in the mDE6 cells. (A) The mRNA and (B) protein expression levels of Runx2 were increased in the mDE6 cells cultured in calcified induction medium (CIM) for 21 days. (C–F) The expression level of each phosphorylated/non-phosphorylated protein is shown. The expression levels of (C) p-Smad1/5 and (D) p-Akt were significantly increased in the mDE6 cells treated with CIM for 21 days compared with those in the control cells not treated with CIM. There were no significant differences observed in the (E) p-ERK1/2 and (F) β-catenin protein expression levels between the treated mDE6 cells and the controls. The data are the means ± SD from triplicate samples. *P<0.05 and **P<0.01 vs. day 0; and #P<0.05 and ##P<0.01 vs. CIM- by a one-way ANOVA with the Tukey-Kramer comparison test.
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f2-ijmm-35-05-1169: The results of the analysis of signaling pathways upstream of runt-related transcription factor 2 (Runx2) expression in the mDE6 cells. (A) The mRNA and (B) protein expression levels of Runx2 were increased in the mDE6 cells cultured in calcified induction medium (CIM) for 21 days. (C–F) The expression level of each phosphorylated/non-phosphorylated protein is shown. The expression levels of (C) p-Smad1/5 and (D) p-Akt were significantly increased in the mDE6 cells treated with CIM for 21 days compared with those in the control cells not treated with CIM. There were no significant differences observed in the (E) p-ERK1/2 and (F) β-catenin protein expression levels between the treated mDE6 cells and the controls. The data are the means ± SD from triplicate samples. *P<0.05 and **P<0.01 vs. day 0; and #P<0.05 and ##P<0.01 vs. CIM- by a one-way ANOVA with the Tukey-Kramer comparison test.

Mentions: In order to confirm which signaling pathway(s) is/are involved in calcification as the upstream mediator of Runx2 expression, we examined the mRNA and protein expression levels of Runx2 and the protein expression of p-Smad1/5, p-Akt, p-ERK1/2 and β-catenin in the CIM+ cells in comparison to that observed in the CIM-cells and the cells on day 0. The mRNA and protein expression levels of Runx2 were significantly increased in the CIM+ cells in comparison to those observed in the CIM- and the cells on day 0 (Fig. 2A and B). The p-Smad1/5 protein level was also increased in the CIM+ cells in comparison to that observed in the CIM- cells and the cells on day 0 (Fig. 2C). Although the p-Akt protein expression level was increased in the CIM+ cells compared to that observed in the cells on day 0 (Fig. 2D), there were no significant differences in p-Akt protein expression between the CIM+ cells and CIM- cells (Fig. 2D). Although there was a decrease in the p-ERK1/2 protein expression in the CIM+ cells and no marked changes were observed in β-catenin protein expression, there were no significant differences observed in these levels between the CIM+ cells and the controls (Fig. 2E and F). These findings suggest that the Smad signaling pathway is associated with RUNX2 expression in the mDE6 cells.


Thymosin beta 4 is associated with RUNX2 expression through the Smad and Akt signaling pathways in mouse dental epithelial cells.

Someya H, Fujiwara H, Nagata K, Wada H, Hasegawa K, Mikami Y, Jinno A, Sakai H, Koyano K, Kiyoshima T - Int. J. Mol. Med. (2015)

The results of the analysis of signaling pathways upstream of runt-related transcription factor 2 (Runx2) expression in the mDE6 cells. (A) The mRNA and (B) protein expression levels of Runx2 were increased in the mDE6 cells cultured in calcified induction medium (CIM) for 21 days. (C–F) The expression level of each phosphorylated/non-phosphorylated protein is shown. The expression levels of (C) p-Smad1/5 and (D) p-Akt were significantly increased in the mDE6 cells treated with CIM for 21 days compared with those in the control cells not treated with CIM. There were no significant differences observed in the (E) p-ERK1/2 and (F) β-catenin protein expression levels between the treated mDE6 cells and the controls. The data are the means ± SD from triplicate samples. *P<0.05 and **P<0.01 vs. day 0; and #P<0.05 and ##P<0.01 vs. CIM- by a one-way ANOVA with the Tukey-Kramer comparison test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380193&req=5

f2-ijmm-35-05-1169: The results of the analysis of signaling pathways upstream of runt-related transcription factor 2 (Runx2) expression in the mDE6 cells. (A) The mRNA and (B) protein expression levels of Runx2 were increased in the mDE6 cells cultured in calcified induction medium (CIM) for 21 days. (C–F) The expression level of each phosphorylated/non-phosphorylated protein is shown. The expression levels of (C) p-Smad1/5 and (D) p-Akt were significantly increased in the mDE6 cells treated with CIM for 21 days compared with those in the control cells not treated with CIM. There were no significant differences observed in the (E) p-ERK1/2 and (F) β-catenin protein expression levels between the treated mDE6 cells and the controls. The data are the means ± SD from triplicate samples. *P<0.05 and **P<0.01 vs. day 0; and #P<0.05 and ##P<0.01 vs. CIM- by a one-way ANOVA with the Tukey-Kramer comparison test.
Mentions: In order to confirm which signaling pathway(s) is/are involved in calcification as the upstream mediator of Runx2 expression, we examined the mRNA and protein expression levels of Runx2 and the protein expression of p-Smad1/5, p-Akt, p-ERK1/2 and β-catenin in the CIM+ cells in comparison to that observed in the CIM-cells and the cells on day 0. The mRNA and protein expression levels of Runx2 were significantly increased in the CIM+ cells in comparison to those observed in the CIM- and the cells on day 0 (Fig. 2A and B). The p-Smad1/5 protein level was also increased in the CIM+ cells in comparison to that observed in the CIM- cells and the cells on day 0 (Fig. 2C). Although the p-Akt protein expression level was increased in the CIM+ cells compared to that observed in the cells on day 0 (Fig. 2D), there were no significant differences in p-Akt protein expression between the CIM+ cells and CIM- cells (Fig. 2D). Although there was a decrease in the p-ERK1/2 protein expression in the CIM+ cells and no marked changes were observed in β-catenin protein expression, there were no significant differences observed in these levels between the CIM+ cells and the controls (Fig. 2E and F). These findings suggest that the Smad signaling pathway is associated with RUNX2 expression in the mDE6 cells.

Bottom Line: In previous studies by our group, we reported that thymosin beta 4 (Tb4) is closely associated with the initiation and development of the tooth germ, and can induce the expression of runt-related transcription factor 2 (RUNX2) during the development of the tooth germ.The protein expression of p-Smad1/5 and p-Akt in the mDE6 cells was reduced by treatment with Tb4-siRNA.This study provides putative information concerning the signaling pathway through which Tb4 induces RUNX2 expression, which may help to understand the regulation of tooth development and tooth regeneration.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Oral Pathology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.

ABSTRACT
In previous studies by our group, we reported that thymosin beta 4 (Tb4) is closely associated with the initiation and development of the tooth germ, and can induce the expression of runt-related transcription factor 2 (RUNX2) during the development of the tooth germ. RUNX2 regulates the expression of odontogenesis-related genes, such as amelogenin, X-linked (Amelx), ameloblastin (Ambn) and enamelin (Enam), as well as the differentiation of osteoblasts during bone formation. However, the mechanisms through which Tb4 induces the expression of RUNX2 remain unknown. In the present study, we employed a mouse dental epithelial cell line, mDE6, with the aim to elucidate these mechanisms. The mDE6 cells expressed odontogenesis-related genes, such as Runx2, Amelx, Ambn and Enam, and formed calcified matrices upon the induction of calcification, thus showing characteristics of odontogenic epithelial cells. The expression of odontogenesis-related genes, and the calcification of the mDE6 cells were reduced by the inhibition of phosphorylated Smad1/5 (p-Smad1/5) and phosphorylated Akt (p-Akt) proteins. Furthermore, we used siRNA against Tb4 to determine whether RUNX2 expression and calcification are associated with Tb4 expression in the mDE6 cells. The protein expression of p-Smad1/5 and p-Akt in the mDE6 cells was reduced by treatment with Tb4-siRNA. These results suggest that Tb4 is associated with RUNX2 expression through the Smad and PI3K-Akt signaling pathways, and with calcification through RUNX2 expression in the mDE6 cells. This study provides putative information concerning the signaling pathway through which Tb4 induces RUNX2 expression, which may help to understand the regulation of tooth development and tooth regeneration.

Show MeSH
Related in: MedlinePlus