Endothelin Signaling Promotes Osteogenesis via IGF1 Induction

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 199-233-Bone Biology
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-207
Michael G. Johnson*1, Jasmin Kristianto2, Anne Gustavson3, Kathryn Koenicke1, Robert Daniel Blank4 and Baozhi Yuan1
1University of Wisconsin, Madison, WI, 2University of wisconsin- Madison, Madison, WI, 3University of Wisconsin, Madison, 4Univ of Wisconsin Sch of Medicin, Madison, WI
Endothelin (ET1) promotes the growth of osteoblastic breast and prostate cancer metastases, an effect previously shown to be due in part to derepression of canonical WNT signaling. Conversion of big ET1 to mature ET1, catalyzed primarily by endothelin converting enzyme 1 (ECE1), is necessary for ET1’s biological activity. We previously identified Ece1, encoding ECE1, as a positional candidate gene for a pleiotropic quantitative trait locus affecting femoral size, shape, mineralization, and biomechanical performance. To test the hypothesis that ET1 signaling regulates osteogenesis in the normal state as well as in cancer, we exposed TMOb osteoblasts to 25 ng/ml big ET1 prior to and over the course of in vitro differentiation. Cells were grown for 6 days in growth medium and then switched to mineralization medium for an additional 15 days, by which time they form mineralized nodules. Cells were harvested every three days following the switch to mineralization medium. We measured mRNA levels of genes involved in the ET1 signaling axis, production of paracrine factors involved in osteogenesis, and miRNA expression. Data were analyzed by the rank sum test or nonparametric ANOVA. TMOb cells exposed to big ET showed greater mineralization than control cells (N = 6, p = 0.008). The mineralization difference was specific to ET1 signaling, as it was blocked by pharmacological inhibition of ECE1 or endothelin receptor A. Under normal mineralization conditions, Ece1 mRNA expression showed no change over the course of mineralization, ET1 was repressed and endothelin receptor A was induced. Addition of big ET1 repressed expression of all three genes. IGF-1 production was analyzed by 2 way repeated measures ANOVA and IGF-1 levels were significantly (1.3-1.8X) higher over time in the presence of big ET (p<0.001). Big ET1 repressed anti-osteogenic miRNAs including miRNA 335-5p, which targets Igf1 and Runx2 transcripts, while miRNAs that target proteins involved in bone catabolism were induced by big ET1 exposure. Modulation of canonical WNT signaling could not fully account for ET1’s osteogenic effects, as big ET1 produced a greater mineralization than treatment with LiCl. Our data show that osteoblasts express all the elements needed for ET1 signaling over the course of differentiation and that ET1 signaling promotes mineralization. Moreover, they suggest that ET1’s osteogenic effects are mediated in part via IGF-1 induction, a previously unrecognized ET1 osteogenic mechanism.

Nothing to Disclose: MGJ, JK, AG, KK, RDB, BY

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm