Manipulating WNT Receptors to Affect Osteogenesis
Location
Hager-Lubbers Exhibition Hall
Description
BACKGROUND AND PURPOSE: WNT signaling is one of the most important pathways necessary for development; from tissue regeneration, to stem cell differentiation. However, dysregulation of the pathway is associated with numerous human cancers and human diseases such as osteoporosis, cardiovascular disease and diabetes. The WNT/ß-catenin signaling plays a crucial role in osteoblast differentiation and bone homeostasis. WNT receptors, known as Frizzled (Fzd), are engaged by WNT ligands to activate ß-catenin and TCF-dependent gene transcription. METHODS: Relative expression of 10 Fzds in different tissues and cells was surveyed within publically available RNA-sequencing datasets. The dominant Fzd (Fzd 2) in a mesenchymal cell line, C3H10T1/2, was knocked out by CRISPR-Cas9 technology. Znrf3/Rnf43 (RZ) knockout was achieved by infecting Rnf43-flox/flox; Znrf3-flox/flox calvarial cells with Cre-expressing adenovirus. Osteoblast differentiation and mineralization were studied with 21-day in vitro osteogenic differentiation. RESULTS: Fzd2-deficient cell clones revealed significantly suppressed responses to WNT treatment, including decreased WNT target gene (Axin2) transcription and decreased osteogenic marker alkaline phosphatase (ALP) activity. In contrast, RZ knockout calvarial cells showed accelerated osteoblast maturation and mineralization in vitro. CONCLUSIONS: Fzd2 is critical for early osteoblast commitment in mesenchymal cells. Targeting RZ in bone to increase Fzd receptors available on cell membranes may be a therapeutic strategy to increase bone mass.
Manipulating WNT Receptors to Affect Osteogenesis
Hager-Lubbers Exhibition Hall
BACKGROUND AND PURPOSE: WNT signaling is one of the most important pathways necessary for development; from tissue regeneration, to stem cell differentiation. However, dysregulation of the pathway is associated with numerous human cancers and human diseases such as osteoporosis, cardiovascular disease and diabetes. The WNT/ß-catenin signaling plays a crucial role in osteoblast differentiation and bone homeostasis. WNT receptors, known as Frizzled (Fzd), are engaged by WNT ligands to activate ß-catenin and TCF-dependent gene transcription. METHODS: Relative expression of 10 Fzds in different tissues and cells was surveyed within publically available RNA-sequencing datasets. The dominant Fzd (Fzd 2) in a mesenchymal cell line, C3H10T1/2, was knocked out by CRISPR-Cas9 technology. Znrf3/Rnf43 (RZ) knockout was achieved by infecting Rnf43-flox/flox; Znrf3-flox/flox calvarial cells with Cre-expressing adenovirus. Osteoblast differentiation and mineralization were studied with 21-day in vitro osteogenic differentiation. RESULTS: Fzd2-deficient cell clones revealed significantly suppressed responses to WNT treatment, including decreased WNT target gene (Axin2) transcription and decreased osteogenic marker alkaline phosphatase (ALP) activity. In contrast, RZ knockout calvarial cells showed accelerated osteoblast maturation and mineralization in vitro. CONCLUSIONS: Fzd2 is critical for early osteoblast commitment in mesenchymal cells. Targeting RZ in bone to increase Fzd receptors available on cell membranes may be a therapeutic strategy to increase bone mass.