Schnurri-3 regulates ERK downstream of WNT signaling in osteoblasts
Jae-Hyuck Shim, … , Laurie H. Glimcher, Dallas C. Jones
Jae-Hyuck Shim, … , Laurie H. Glimcher, Dallas C. Jones
Published August 15, 2013
Citation Information: J Clin Invest. 2013;123(9):4010-4022. https://doi.org/10.1172/JCI69443.
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Research Article Bone biology

Schnurri-3 regulates ERK downstream of WNT signaling in osteoblasts

Abstract

Mice deficient in Schnurri-3 (SHN3; also known as HIVEP3) display increased bone formation, but harnessing this observation for therapeutic benefit requires an improved understanding of how SHN3 functions in osteoblasts. Here we identified SHN3 as a dampener of ERK activity that functions in part downstream of WNT signaling in osteoblasts. A D-domain motif within SHN3 mediated the interaction with and inhibition of ERK activity and osteoblast differentiation, and knockin of a mutation in Shn3 that abolishes this interaction resulted in aberrant ERK activation and consequent osteoblast hyperactivity in vivo. Additionally, in vivo genetic interaction studies demonstrated that crossing to Lrp5–/– mice partially rescued the osteosclerotic phenotype of Shn3–/– mice; mechanistically, this corresponded to the ability of SHN3 to inhibit ERK-mediated suppression of GSK3β. Inducible knockdown of Shn3 in adult mice resulted in a high–bone mass phenotype, providing evidence that transient blockade of these pathways in adults holds promise as a therapy for osteoporosis.

Authors

Jae-Hyuck Shim, Matthew B. Greenblatt, Weiguo Zou, Zhiwei Huang, Marc N. Wein, Nicholas Brady, Dorothy Hu, Jean Charron, Heather R. Brodkin, Gregory A. Petsko, Dennis Zaller, Bo Zhai, Steven Gygi, Laurie H. Glimcher, Dallas C. Jones

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Figure 4

SHN3 inhibits the WNT-mediated β-catenin pathway via ERK regulation.

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SHN3 inhibits the WNT-mediated β-catenin pathway via ERK regulation. (A ...
(A and B) C3H10T1/2 cells were infected by lentivirus expressing vector or SHN3-WT (A) or SHN3-KA (B) and transfected with top-flash luc and Renilla along with a xWNT8/Fz5 fusion protein, a constitutively active mutant of LRP5 (LRP5-CA), or xWNT8/Fz5 fusion protein plus LRP5. Relative luciferase activity normalized to Renilla is shown. (C) Shn3+/+ and Shn3–/– COBs were transfected with top-flash luc along with Renilla. Luciferase activity was analyzed 6 days after culture in differentiation medium. Values are normalized to Renilla. (D and E) μCT analysis of 8-week-old Shn3+/+, Lrp5–/–, Shn3–/–, and Lrp5–/–;Shn3–/– mouse femurs. (D) 3-dimensional reconstructions of proximal femur. (E) Bone volume fraction, trabecular number, trabecular thickness, cortical bone volume fraction (C.BV/TV), trabecular space, and cortical thickness. (F) Primary Shn3–/– COBs were infected by control or Lrp5 shRNA–expressing lentivirus and cultured in differentiation medium, and mineralization activity was analyzed by alizarin red staining. (G) Primary Mek1fl/fl;Mek2–/– COBs were infected by lentivirus expressing vector (Mek1/2+/+) or Cre recombinase (Mek1/2–/–), cultured in differentiation medium, lysed, and immunoblotted with the indicated antibodies. (H) Primary Mek1fl/fl;Mek2–/– COBs infected as in G were transfected with top-flash luc and Renilla. Luciferase activity was analyzed 6 days after culture in differentiation medium. Values are normalized to Renilla. Results are presented as mean + SD. **P < 0.005, ***P < 0.0005, Student’s t test.