title: Endothelin signaling regulates mineralization and posttranscriptionally regulates SOST in TMOb cells via miR 126-3p.
abstract: Previously, our laboratory identifiedECE-1, encoding endothelin-converting enzyme-1 (ECE-1), as a positional candidate for a pleiotropic quantitative trait locus affecting femoral size, shape, and biomechanical performance. We hypothesized that endothelin-1 (ET-1) signaling promotes osteogenesis. Exposure of immortalized mouse osteoblast (TMOb) cells to big ET-1 increased mineralization. Following big ET-1 treatment, we measured the secretion of insulin-like-growth factor-1 (IGF1), dickkopf-homolog-1 protein 1 (DKK1), and sclerostin (SOST). In each case, big ET-1 signaling changed secretion in a manner that favored increased osteogenic activity. Treatment with ECE-1, endothelin receptor A (EDNRA), or WNT receptor antagonists inhibited the big ET-1-mediated increase in mineralization. In the presence of big ET-1, message levels ofRunx2,Igf1,Dkk1,andSostare uncoupled from protein production, suggesting posttranscriptional regulation. To evaluate the role of big ET-1 in normal bone physiology, we inhibited EDNRA signaling during mineralization in the absence of exogenous ET-1. EDNRA blockade reduced mineralization, decreased IGF1, and increased DKK1 and SOST secretion, responses opposite to those induced by exogenous big ET-1. Pharmacological and siRNA knockdown to inhibit ECE-1 reduced mineralization and IGF1 secretion with decreasing DKK1 and decreasing or stable SOST secretion, suggesting a further, unknown role of ECE-1 in osteoblast maturation. Previously we identified miR 126-3p as a potential ET-1-responsive regulator of SOST in murine cells. Overexpression of miR126-3p increased mineralization in TMOb cells and decreased SOST secretion. Osteoblasts express the ET-1 signaling pathway and ET-1 signaling is necessary for normal osteoblast differentiation and mineralization, acting through regulation of miRs that target osteogenic molecules.
authors.affiliation: Geriatrics Research, Education and Clinical Center, William S. Middleton Veterans Affairs Hospital, Madison, Wisconsin mgjohns2@medicine.wisc.edu.|Division of Endocrinology, Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Johnson
authors.affiliation: Medical Service, Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin.|Division of Endocrinology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Konicke
authors.affiliation: Geriatrics Research, Education and Clinical Center, William S. Middleton Veterans Affairs Hospital, Madison, Wisconsin.|Division of Endocrinology, Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Kristianto
authors.affiliation: Geriatrics Research, Education and Clinical Center, William S. Middleton Veterans Affairs Hospital, Madison, Wisconsin.|Division of Endocrinology, Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: Anne
authors.initials: A
authors.lastname: Gustavson
authors.affiliation: Geriatrics Research, Education and Clinical Center, William S. Middleton Veterans Affairs Hospital, Madison, Wisconsin.|Division of Endocrinology, Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: Rachel
authors.initials: R
authors.lastname: Garbo
authors.affiliation: Medical Service, Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin.|Division of Endocrinology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin.
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Geriatrics Research, Education and Clinical Center, William S. Middleton Veterans Affairs Hospital, Madison, Wisconsin.|Division of Endocrinology, Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: Baozhi
authors.initials: B
authors.lastname: Yuan
authors.affiliation: Geriatrics Research, Education and Clinical Center, William S. Middleton Veterans Affairs Hospital, Madison, Wisconsin.|Medical Service, Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin.|Division of Endocrinology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Blank
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019332:Endothelin-1 / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C501127:Dkk1 protein, mouse; D019332:Endothelin-1; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; C545201:MIRN126 microRNA, mouse; D035683:MicroRNAs; C486030:Sost protein, mouse; D007334:Insulin-Like Growth Factor I
keywords: Endothelin‐1; endothelin‐converting enzyme‐1; miR 126‐3p; mineralization; osteoblast