title: Ectodermal-Neural Cortex 1 Isoforms Have Contrasting Effects on MC3T3-E1 Osteoblast Mineralization and Gene Expression.
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. © 2016 Wiley Periodicals, Inc.
authors.affiliation: The University of Queensland, Brisbane, Queensland, Australia.|Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, Washington.
authors.forename: Leah E
authors.initials: LE
authors.lastname: Worton
authors.affiliation: Garvan Institute of Medical Research, Sydney, New South Wales, Australia.|Faculty of Medicine, University of New South Wales, New South Wales, Australia.
authors.forename: Yan-Chuan
authors.initials: YC
authors.lastname: Shi
authors.affiliation: Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
authors.forename: Elisabeth J
authors.initials: EJ
authors.lastname: Smith
authors.affiliation: The University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Simon C
authors.initials: SC
authors.lastname: Barry
authors.affiliation: The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gonda
authors.affiliation: School of Life Sciences, University of Lincoln, England, UK.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Whitehead
authors.affiliation: The University of Queensland, Brisbane, Queensland, Australia.|Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, Washington.
authors.forename: Edith M
authors.initials: EM
authors.lastname: Gardiner
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002113:Calcification, Physiologic / Q000235:genetics / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D015151:Immunoblotting; D017403:In Situ Hybridization; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D010011:Osteocytes / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D060449:Wnt Signaling Pathway / Q000235:genetics / Q000502:physiology; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: D008840:Microfilament Proteins; D009479:Neuropeptides; D009687:Nuclear Proteins; D020033:Protein Isoforms; D051176:beta Catenin; C112302:ectodermal-neural cortex 1 protein; C510878:ALPL protein, mouse; D000469:Alkaline Phosphatase
keywords: ALPL; DIFFERENTIATION; ENC1; MC3T3-E1; MINERALIZATION; NRP/B; OSTEOBLAST