Ectodermal-neural cortex 1 isoforms have contrasting effects on MC3T3-E1 osteoblast mineralization and gene expression

Worton, Leah E., Shi, Yan-Chuan, Smith, Elisabeth J., Barry, Simon C., Gonda, Thomas J., Whitehead, Jonathan P. and Gardiner, Edith M. (2017) Ectodermal-neural cortex 1 isoforms have contrasting effects on MC3T3-E1 osteoblast mineralization and gene expression. Journal of Cellular Biochemistry, 118 8: 2141-2150. doi:10.1002/jcb.25851


Author Worton, Leah E.
Shi, Yan-Chuan
Smith, Elisabeth J.
Barry, Simon C.
Gonda, Thomas J.
Whitehead, Jonathan P.
Gardiner, Edith M.
Title Ectodermal-neural cortex 1 isoforms have contrasting effects on MC3T3-E1 osteoblast mineralization and gene expression
Journal name Journal of Cellular Biochemistry   Check publisher's open access policy
ISSN 1097-4644
0730-2312
Publication date 2017-04-18
Sub-type Article (original research)
DOI 10.1002/jcb.25851
Open Access Status Not yet assessed
Volume 118
Issue 8
Start page 2141
End page 2150
Total pages 10
Place of publication Hoboken, NJ, United States
Publisher John Wiley & Sons
Language eng
Subject 1303 Biochemistry
1312 Molecular Biology
1307 Cell Biology
Abstract The importance of Wnt pathway signaling in development of bone has been well established. Here we investigated the role of a known Wnt target, ENC1 (ectodermal-neural cortex 1; NRP/B), in osteoblast differentiation. Enc1 expression was detected in mouse osteoblasts, chondrocytes, and osteocytes by in situ hybridization, and osteoblastic expression was verified in differentiating primary cultures and MC3T3-E1 pre-osteoblast cells, with 57 kDa and 67 kDa ENC1 protein isoforms detected throughout differentiation. Induced knockdown of both ENC1 isoforms reduced alkaline phosphatase staining and virtually abolished MC3T3-E1 mineralization. At culture confluence, Alpl (alkaline phosphatase liver/bone/kidney) expression was markedly reduced compared with control cells, and there was significant and coordinated alteration of other genes involved in cellular phosphate biochemistry. In contrast, with 67 kDa-selective knockdown mineralized nodule formation was enhanced and there was a two-fold increase in Alpl expression at confluence. There was enhanced expression of Wnt/β-catenin target genes with knockdown of both isoforms at this time-point and a five-fold increase in Frzb (Frizzled related protein) with 67 kDa-selective knockdown at mineralization, indicating possible ENC1 interactions with Wnt signaling in osteoblasts. These results are the first to demonstrate a role for ENC1 in the control of osteoblast differentiation. Additionally, the contrasting mineralization phenotypes and transcriptional patterns seen with coordinate knockdown of both ENC1 isoforms vs selective knockdown of 67 kDa ENC1 suggest opposing roles for the isoforms in regulation of osteoblastic differentiation, through effects on Alpl expression and phosphate cellular biochemistry. This study is the first to report differential roles for the ENC1 isoforms in any cell lineage. J. Cell. Biochem. 118: 2141–2150, 2017.
Formatted abstract
The importance of Wnt pathway signaling in development of bone has been well established. Here we investigated the role of a known Wnt target, ENC1 (ectodermal-neural cortex 1; NRP/B), in osteoblast differentiation. Enc1 expression was detected in mouse osteoblasts, chondrocytes, and osteocytes by in situ hybridization, and osteoblastic expression was verified in differentiating primary cultures and MC3T3-E1 pre-osteoblast cells, with 57 kDa and 67 kDa ENC1 protein isoforms detected throughout differentiation. Induced knockdown of both ENC1 isoforms reduced alkaline phosphatase staining and virtually abolished MC3T3-E1 mineralization. At culture confluence, Alpl (alkaline phosphatase liver/bone/kidney) expression was markedly reduced compared with control cells, and there was significant and coordinated alteration of other genes involved in cellular phosphate biochemistry. In contrast, with 67 kDa-selective knockdown mineralized nodule formation was enhanced and there was a two-fold increase in Alpl expression at confluence. There was enhanced expression of Wnt/β-catenin target genes with knockdown of both isoforms at this time-point and a five-fold increase in Frzb (Frizzled related protein) with 67 kDa-selective knockdown at mineralization, indicating possible ENC1 interactions with Wnt signaling in osteoblasts. These results are the first to demonstrate a role for ENC1 in the control of osteoblast differentiation. Additionally, the contrasting mineralization phenotypes and transcriptional patterns seen with coordinate knockdown of both ENC1 isoforms vs selective knockdown of 67 kDa ENC1 suggest opposing roles for the isoforms in regulation of osteoblastic differentiation, through effects on Alpl expression and phosphate cellular biochemistry. This study is the first to report differential roles for the ENC1 isoforms in any cell lineage.
Keyword ALPL
Differentiation
ENC1
MC3T3-E1
Mineralization
NRP/B
Osteoblast
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID 455907
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
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