Analysis on Wnt signaling phospho antibody array in the process of rat bone marrow mesenchymal stem cells differentiated into odontoblast-like cells

doi:10.3877/cma.j.issn.1674-1366.2017.02.001

Abstract

Abstract:

Objective

To investigate the changes of protein phosphorylation level of Wnt signaling in the process of rat bone marrow mesenchymal stem cells (BMSCs) differentiated into odontoblast-like cell and preliminarily discuss the mechanism.

Methods

We seperated, cultured and purified BMSCs by the method of whole bone marrow adherence. Tooth germ cell-conditioned medium was used to induce the differentiation of rats bone marrow mesenchymal stem cells into odontoblast-like cells. A phospho-specific antibody microarray (PNT227) targeting the Wnt Phospho signaling pathway was used to find the alterations of the downstream signaling events. Western blot was carried out to validate the antibody array results. The SPSS 20.0 software was used as the data processing tool. T-test and t'-test were used to analyze the difference between the groups.

Results

BMSCs obtained by whole bone marrow adherence method had the potential of self-renew and multilineage differentiation. After co-culture with TGC-CM for 2 days, BMSCs began to exhibit long spindle process. After 7 days, the cells had a long spindle shape and a tightly packed arrangement. The expression of DSPP (t = 3.700, P= 0.021) , DMP1 (t = 3.237, P= 0.032) , RUNX2 (t = 12.09, P<0.001) , ALP (t = 62.83, P<0.001) were enhanced after induction for 7 days. Results of Wnt signaling phospho antibody array showed 27 phosphorylation sites were up-regulated or down-regulated. Western blot analysis showed that nuclear expression of β-catenin (t= 4.588, P= 0.044) and the phosphorylation level of PKCδ (t= 5.438, P= 0.032) were enhanced, while the phosphorylation level of AKT (t= 30.85, P= 0.001) was reduced, which was consistent with the array.

Conclusion

Changes of phosphorylation site of Wnt signaling in the process of BMSCs differentiated into odontoblast-like cells were found.

Key words: Bone marrow, Mesenchymal stem cells, Odontoblast, Cell differentiation, Wnt signaling phospho antibody array

Yanling Huang, Jianmao Zheng, Junqi Ling. Analysis on Wnt signaling phospho antibody array in the process of rat bone marrow mesenchymal stem cells differentiated into odontoblast-like cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(02): 65-72.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhkqyxyjzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-1366.2017.02.001

https://zhkqyxyjzz.cma-cmc.com.cn/EN/Y2017/V11/I02/65

Figures/Tables 8

References 23

[1]	Shahdadfar A, Fronsdal K, Haug T，et al. In vitro expansion of human mesenchymal stem cells：choice of serum is a determinant of cell proliferation，differentiation，gene expression，and transcriptome stability[J]. Stem Cells，2005，23(9):1357-1366.
[2]	Ye L, Chen L, Feng F，et al. Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells[J]. Cell Biol Int，2015，39(10):1151-1161.
[3]	Liu F, Chu EY, Watt B，et al. Wnt/beta-catenin signaling directs multiple stages of tooth morphogenesis[J]. Dev Biol，2008，313(1):210-224.
[4]	Zhang YM, Dai BL, Zheng L，et al. A novel angiogenesis inhibitor impairs lovo cell survival via targeting against human VEGFR and its signaling pathway of phosphorylation[J]. Cell Death Dis，2012(3):e406.
[5]	Pittenger MF, Mackay AM, Beck SC，et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science，1999，284(5411):143-147.
[6]	Krause DS, Theise ND, Collector MI，et al. Multi-organ，multi-lineage engraftment by a single bone marrow-derived stem cell [J]. Cell，2001，105(3):369-377.
[7]	Long X, Olszewski M, Huang W，et al. Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells[J]. Stem Cells Dev，2005，14(1):65-69.
[8]	Makino S, Fukuda K, Miyoshi S，et al. Cardiomyocytes can be generated from marrow stromal cells in vitro[J]. J Clin Invest，1999，103(5):697-705.
[9]	Smith AJ, Lesot H. Induction and regulation of crown dentinogenesis：embryonic events as a template for dental tissue repair？[J]. Crit Rev Oral Biol Med，2001，12(5):425-437.
[10]	Wang J, Liu B, Gu S，et al. Effects of Wnt/β-catenin signalling on proliferation and differentiation of apical papilla stem cells[J]. Cell Prolif，2012，45(2):121-131.
[11]	Scheller EL, Chang J, Wang CY. Wnt/beta-catenin inhibits dental pulp stem cell differentiation[J]. J Dent Res，2008，87(2):126-130.
[12]	Shan T, Zhou C, Yang R，et al. Lithium chloride promotes the odontoblast differentiation of hair follicle neural crest cells by activating Wnt/β-catenin signaling[J]. Cell Biol Int，2015，39(1):35-43.
[13]	Etheridge SL, Spencer GJ, Heath DJ，et al. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells[J]. Stem Cells，2004，22(5):849-860.
[14]	Gao Q, Xia Y, Liu L，et al. Galectin-3 Enhances Migration of Minature Pig Bone Marrow Mesenchymal Stem Cells Through Inhibition of RhoA-GTP Activity[J]. Sci Rep，2016(6):26577.
[15]	Sheng L, Mao X, Yu Q，et al. Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells[J]. Exp Ther Med，2017，13(1):55-62.
[16]	He H, Zhao ZH, Han FS，et al. Activation of protein kinase C ε enhanced movement ability and paracrine function of rat bone marrow mesenchymal stem cells partly at least independent of SDF-1/CXCR4 axis and PI3K/AKT pathway[J]. Int J Clin Exp Med，2015，8(1):188-202.
[17]	Lee S, Cho HY, Bui HT，et al. The osteogenic or adipogenic lineage commitment of human mesenchymal stem cells is determined by protein kinase C delta[J]. BMC Cell Biol，2014(15):42.
[18]	Wang Y, Yi XD, Li CD. Suppression of mTOR signaling pathway promotes bone marrow mesenchymal stem cells differentiation into osteoblast in degenerative scoliosis：in vivo and in vitro[J]. Mol Biol Rep，2017，44(1):129-137.
[19]	Zhang LL, Liu JJ, Liu F，et al. MiR-499 induces cardiac differentiation of rat mesenchymal stem cells through wnt/β-catenin signaling pathway[J]. Biochem Biophys Res Commun，2012，420(4):875-881.
[20]	董金山，段晴月，文军，等.骨髓间充质干细胞向成牙本质样细胞分化的体外实验研究[J].牙体牙髓牙周病学杂志，2010，20(4):192-195.
[21]	Yu Y, Wang L, Yu J，et al. Dentin matrix proteins（DMPs）enhance differentiation of BMMSCs via ERK and P38 MAPK pathways[J]. Cell Tissue Res，2014，356(1):171-182.
[22]	Thornton TM, Pedraza-Alva G, Deng B，et al. Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation[J]. Science，2008，320(5876):667-670.
[23]	Chang J, Sonoyama W, Wang Z，et al. Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK[J]. J Biol Chem，2007，282(42):30938-30948.

磷酸化位点	磷酸化比值（TGC-CM组/对照组）
磷酸化位点	样本1	样本2
AKT(Phospho-Ser473)	0.90	0.81
APC1(Phospho-Ser355)	1.12	1.21
CaMK2 alpha(Phospho-Thr286)	0.84	0.78
Catenin beta(Phospho-Tyr654)	0.83	0.71
GSK3 beta(Phospho-Ser9)	1.12	1.25
MAP3K1/MEKK1(Phospho-Thr1402)	1.18	1.18
MAP3K7/TAK1(Phospho-Thr187)	1.19	1.19
MAP3K8/COT(Phospho-Ser400)	1.19	1.12
MEK1(Phospho-Ser298)	1.14	1.71
mTOR(Phospho-Ser2448)	0.88	0.77
mTOR(Phospho-Thr2446)	0.80	0.71
NFAT1(Phospho-Ser326)	0.84	0.74
NFAT3(Phospho-Ser168/Ser170)	0.76	0.76
P38 MAPK(Phospho-Thr179/Tyr181)	0.88	0.87
P38 MAPK(Phospho-Tyr182)	0.82	0.69
PI3-kinase p85-subunit alpha/gamma (Phospho-Tyr467/Tyr199)	1.24	1.38
PKC delta(Phospho-Tyr52)	1.34	1.59
PKC pan(Phospho-Thr497)	1.22	1.24
PKC theta(Phospho-Thr538)	1.24	1.68
PLCG1(Phospho-Tyr1253)	0.72	0.67
PLCG1(Phospho-Tyr783)	1.12	1.10
PLCG2(Phospho-Tyr1217)	0.87	0.86
PLCG2(Phospho-Tyr759)	0.71	0.87
Rac1/cdc42(Phospho-Ser71)	0.72	0.58
RhoA(Phospho-Ser188)	0.89	0.63
Src(Phospho-Tyr418)	1.34	1.15

磷酸化位点	磷酸化比值（TGC-CM组/对照组）
磷酸化位点	样本1	样本2
AKT(Phospho-Ser473)	0.90	0.81
APC1(Phospho-Ser355)	1.12	1.21
CaMK2 alpha(Phospho-Thr286)	0.84	0.78
Catenin beta(Phospho-Tyr654)	0.83	0.71
GSK3 beta(Phospho-Ser9)	1.12	1.25
MAP3K1/MEKK1(Phospho-Thr1402)	1.18	1.18
MAP3K7/TAK1(Phospho-Thr187)	1.19	1.19
MAP3K8/COT(Phospho-Ser400)	1.19	1.12
MEK1(Phospho-Ser298)	1.14	1.71
mTOR(Phospho-Ser2448)	0.88	0.77
mTOR(Phospho-Thr2446)	0.80	0.71
NFAT1(Phospho-Ser326)	0.84	0.74
NFAT3(Phospho-Ser168/Ser170)	0.76	0.76
P38 MAPK(Phospho-Thr179/Tyr181)	0.88	0.87
P38 MAPK(Phospho-Tyr182)	0.82	0.69
PI3-kinase p85-subunit alpha/gamma (Phospho-Tyr467/Tyr199)	1.24	1.38
PKC delta(Phospho-Tyr52)	1.34	1.59
PKC pan(Phospho-Thr497)	1.22	1.24
PKC theta(Phospho-Thr538)	1.24	1.68
PLCG1(Phospho-Tyr1253)	0.72	0.67
PLCG1(Phospho-Tyr783)	1.12	1.10
PLCG2(Phospho-Tyr1217)	0.87	0.86
PLCG2(Phospho-Tyr759)	0.71	0.87
Rac1/cdc42(Phospho-Ser71)	0.72	0.58
RhoA(Phospho-Ser188)	0.89	0.63
Src(Phospho-Tyr418)	1.34	1.15

Please choose a citation manager

Content to export