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Modeling Paracrine Noncanonical Wnt Signaling In Vitro
Published on: December 10, 2021
The orphan receptor tyrosine kinase Ror2 modulates canonical Wnt signaling in osteoblastic cells
Julia Billiard1, Deana S Way, Laura M Seestaller-Wehr
1Women's Health Research Institute, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426, USA. billiaj@wyeth.com
Abstract:
Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic beta-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.
Insights
Ror2 regulates bone formation by modulating Wnt signaling in osteoblasts. This receptor tyrosine kinase enhances Wnt1 but antagonizes Wnt3, impacting beta-catenin stabilization and reporter gene activity during bone development.
Area of Science:
- Cell Biology
- Developmental Biology
- Biochemistry
Background:
- Ror2 (receptor tyrosine kinase) is vital for skeletal development.
- Canonical Wnt signaling is crucial for osteoblast function and bone formation.
- The precise role of Ror2 in Wnt signaling within bone cells remains unclear.
Purpose of the Study:
- To investigate the role of human Ror2 in regulating canonical Wnt signaling in osteoblastic cells.
- To determine the functional interactions between Ror2 and Wnt ligands (Wnt1, Wnt3).
- To examine the expression pattern of Ror2 during osteoblast differentiation.
Main Methods:
- Immunoprecipitation to assess physical interactions between Ror2, Wnt1, and Wnt3.
- Western blotting to analyze beta-catenin stabilization.
- Reporter gene assays to measure Wnt signaling activity.
- Quantitative RT-PCR to determine Ror2 mRNA expression levels.
Main Results:
- Ror2 physically interacts with both Wnt1 and Wnt3 in osteoblastic cells.
- Ror2 differentially regulates Wnt signaling: it enhances Wnt1 but antagonizes Wnt3.
- Ror2 expression is tightly regulated during osteoblast differentiation, peaking in preosteoblasts.
- Ror2 expression is suppressed by secreted frizzled-related protein 1.
Conclusions:
- Ror2 acts as a novel regulator of canonical Wnt signaling in osteoblasts.
- Ror2's distinct modulation of Wnt1 and Wnt3 pathways suggests a complex role in bone formation.
- The regulated expression of Ror2 during osteoblast differentiation implies its physiological importance in bone development and homeostasis.
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