Limits...
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.

Show MeSH

Related in: MedlinePlus

The calcification and expression of odontogenesis-related genes in the mDE6 cells upon the induction of calcification. (A) The mDE6 cells incubated with calcified induction medium (CIM) for 21 days showed the formation and calcification of matrix. The calcified matrices in the mDE6 cells were positive for Alizarin red S (ALZ; upper panel) and von Kossa (Kossa; lower panel) staining. The control without CIM treatment (CIM-) was negative for ALZ and Kossa staining, as were the cells on day 0. (B) The enzymatic activity of alkaline phosphatase (ALP) was significantly increased in the mDE6 cells treated with CIM for 21 days. (C–E) The expression levels of (C) amelogenin, X-linked (Amelx), (D) ameloblastin (Ambn) and (E) enamelin (Enam) were increased in the mDE6 cells treated with CIM compared with those in CIM- cells and the cells on day 0. The data are the means ± SD from triplicate samples. *P<0.05 and **P<0.01 vs. day 0; and ##P<0.01 vs. CIM- by a one-way ANOVA with the Tukey-Kramer comparison test. ND, not detectable.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4380193&req=5

f1-ijmm-35-05-1169: The calcification and expression of odontogenesis-related genes in the mDE6 cells upon the induction of calcification. (A) The mDE6 cells incubated with calcified induction medium (CIM) for 21 days showed the formation and calcification of matrix. The calcified matrices in the mDE6 cells were positive for Alizarin red S (ALZ; upper panel) and von Kossa (Kossa; lower panel) staining. The control without CIM treatment (CIM-) was negative for ALZ and Kossa staining, as were the cells on day 0. (B) The enzymatic activity of alkaline phosphatase (ALP) was significantly increased in the mDE6 cells treated with CIM for 21 days. (C–E) The expression levels of (C) amelogenin, X-linked (Amelx), (D) ameloblastin (Ambn) and (E) enamelin (Enam) were increased in the mDE6 cells treated with CIM compared with those in CIM- cells and the cells on day 0. The data are the means ± SD from triplicate samples. *P<0.05 and **P<0.01 vs. day 0; and ##P<0.01 vs. CIM- by a one-way ANOVA with the Tukey-Kramer comparison test. ND, not detectable.

Mentions: We wished to determine whether the formation of calcified material was induced in mDE6 cells by the use of CIM, as well as whether the activity of alkaline phosphatase (ALP) is altered during calcification and whether the cells express odontogenesis-related genes, such as Amelx, Ambn and Enam. Calcification, as indicated by positive ALZ and Kossa staining, was observed in the mDE6 cells cultured in CIM for 21 days (CIM+ cells) (Fig. 1A), while no calcification was noted in the cells cultured without CIM (CIM-cells). ALP activity was significantly increased in the CIM+ cells (Fig. 1B). The mRNA expression levels of Amelx and Ambn were significantly increased in the CIM+ cells (Fig. 1C and D). Although there were no significant differences observed in Enam mRNA expression between the CIM+ cells and the controls (CIM-cells) and the cells just before the induction of calcification (cells on day 0), the Enam mRNA expression appeared to be increased in the CIM+ cells (Fig. 1E). There were no marked differences observed in the expression levels of these genes in the CIM- cells compared with those observed on day 0 (Fig. 1C–E). These results indicate that the mDE6 cells have the ability to express odontogenesis-related genes and form calcified material, depending on the culture conditions, and partially show the characteristics of odontogenic epithelial cells in vivo.


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 calcification and expression of odontogenesis-related genes in the mDE6 cells upon the induction of calcification. (A) The mDE6 cells incubated with calcified induction medium (CIM) for 21 days showed the formation and calcification of matrix. The calcified matrices in the mDE6 cells were positive for Alizarin red S (ALZ; upper panel) and von Kossa (Kossa; lower panel) staining. The control without CIM treatment (CIM-) was negative for ALZ and Kossa staining, as were the cells on day 0. (B) The enzymatic activity of alkaline phosphatase (ALP) was significantly increased in the mDE6 cells treated with CIM for 21 days. (C–E) The expression levels of (C) amelogenin, X-linked (Amelx), (D) ameloblastin (Ambn) and (E) enamelin (Enam) were increased in the mDE6 cells treated with CIM compared with those in CIM- cells and the cells on day 0. The data are the means ± SD from triplicate samples. *P<0.05 and **P<0.01 vs. day 0; and ##P<0.01 vs. CIM- by a one-way ANOVA with the Tukey-Kramer comparison test. ND, not detectable.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-ijmm-35-05-1169: The calcification and expression of odontogenesis-related genes in the mDE6 cells upon the induction of calcification. (A) The mDE6 cells incubated with calcified induction medium (CIM) for 21 days showed the formation and calcification of matrix. The calcified matrices in the mDE6 cells were positive for Alizarin red S (ALZ; upper panel) and von Kossa (Kossa; lower panel) staining. The control without CIM treatment (CIM-) was negative for ALZ and Kossa staining, as were the cells on day 0. (B) The enzymatic activity of alkaline phosphatase (ALP) was significantly increased in the mDE6 cells treated with CIM for 21 days. (C–E) The expression levels of (C) amelogenin, X-linked (Amelx), (D) ameloblastin (Ambn) and (E) enamelin (Enam) were increased in the mDE6 cells treated with CIM compared with those in CIM- cells and the cells on day 0. The data are the means ± SD from triplicate samples. *P<0.05 and **P<0.01 vs. day 0; and ##P<0.01 vs. CIM- by a one-way ANOVA with the Tukey-Kramer comparison test. ND, not detectable.
Mentions: We wished to determine whether the formation of calcified material was induced in mDE6 cells by the use of CIM, as well as whether the activity of alkaline phosphatase (ALP) is altered during calcification and whether the cells express odontogenesis-related genes, such as Amelx, Ambn and Enam. Calcification, as indicated by positive ALZ and Kossa staining, was observed in the mDE6 cells cultured in CIM for 21 days (CIM+ cells) (Fig. 1A), while no calcification was noted in the cells cultured without CIM (CIM-cells). ALP activity was significantly increased in the CIM+ cells (Fig. 1B). The mRNA expression levels of Amelx and Ambn were significantly increased in the CIM+ cells (Fig. 1C and D). Although there were no significant differences observed in Enam mRNA expression between the CIM+ cells and the controls (CIM-cells) and the cells just before the induction of calcification (cells on day 0), the Enam mRNA expression appeared to be increased in the CIM+ cells (Fig. 1E). There were no marked differences observed in the expression levels of these genes in the CIM- cells compared with those observed on day 0 (Fig. 1C–E). These results indicate that the mDE6 cells have the ability to express odontogenesis-related genes and form calcified material, depending on the culture conditions, and partially show the characteristics of odontogenic epithelial cells in vivo.

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