Schnurri-3 regulates ERK downstream of WNT signaling in osteoblasts
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
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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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 3

SHN3 inhibits ERK MAPK activity in osteoblasts via the D-domain 3 lysine motif.

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SHN3 inhibits ERK MAPK activity in osteoblasts via the D-domain 3 lysine...
(A) Immortalized Shn3+/+ and Shn3–/– COBs were lysed, immunoprecipitation was performed with anti-ERK1/2 antibody and protein A–agarose, and the resulting complex was immunoblotted with an anti-SHN3 antibody. (B and C) HA-ERK2 (WT or DN [D319N]) was incubated with recombinant GST or with GST-tagged BAS, KA-BAS, or KR-BAS. GST-containing proteins were immunoprecipitated with glutathione-agarose, and the resulting complex was immunoblotted with anti-HA antibody. Input indicates loading controls of HA-ERK2, GST, or GST-BAS protein. (D) Primary COBs were isolated from Shn3+/+ and Shn3–/– mice, lysed, and immunoblotted with anti–phospho-ERK1/2 antibody. GAPDH was used as a cytosolic protein control. (E) Immortalized Shn3+/+ and Shn3–/– COBs were stimulated with PMA for 30 minutes, and then ERK1/2 was immunoprecipitated. The immunoprecipitates were mixed with recombinant ELK1, and ERK kinase activity was analyzed by in vitro kinase assay. (F) Recombinant ERK2 (rERK2) was incubated with or without the indicated recombinant His-SHN3 (rSHN3) along with GST-ELK1, and ERK kinase activity was analyzed by in vitro kinase assay. Coomassie blue staining indicates loading control of recombinant His-SHN3. (G) μCT analysis of 5-week-old WT (Shn3fl/+), Shn3-Het, Mek1/2-Het, and Shn3/Mek1/2-DHet mouse femurs. Bone volume fraction and cortical thickness are shown. Results are presented as mean + SD. *P < 0.05, **P < 0.005, Student’s t test.