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Transcription Factor FOXC1 Deregulates BMP-SMAD Signalling During Osteoblast Differentiation Open Access


Other title
Type of item
Degree grantor
University of Alberta
Author or creator
Caddy, Jordan F
Supervisor and department
Berry, Fred (Medical Genetics/Surgery)
Examining committee member and department
Walter, Michael (Medical Genetics)
Graf, Daniel (Medical Genetics/Dentistry)
Lehmann, Ordan (Ophthalmology and Visual Sciences)
Medical Sciences-Medical Genetics

Date accepted
Graduation date
2016-06:Fall 2016
Master of Science
Degree level
Skeletal development is a tightly regulated process that continues through adulthood in the form of bone remodeling. Many bones of the appendicular and axial skeleton develop using a cartilage intermediate in a process called endochondral ossification. The cranial and flat bones of the skeleton develop directly from osteoblasts through intramembranous ossification. Among the important signalling pathways regulating bone formation is BMP-SMAD signalling. BMP ligands bind transmembrane receptors that activate receptor SMAD proteins. These activated proteins enter the nucleus and bind various cofactors to increase target specificity and regulate gene expression during bone development. Forkhead box (FOX) transcription factors all contain a forkhead domain capable of DNA binding in the major and minor groove of the DNA double helix. FOX proteins are involved in a diverse array of biological processes from homeostasis to organ and tissue development and cell proliferation. TGF-B and BMP signalling directly interact with FOX proteins for target specificity and regulation. FOXC proteins are a subfamily of FOX proteins with overlapping functions throughout development. Vascular, renal, and eye development proceed optimally through tightly regulated FOXC activity. FOXC proteins also contribute to proper bone development and patterning by interaction with TGF-B-SMAD and BMP-SMAD signalling. FOXC1 and FOXC2 are both capable of directly interacting with common SMAD4. When FOXC1 is ablated in mice, errors in endochondral and intramembranous ossification develop leading to small, misshapen endochondral bone while many intramembranous bones are completely missing. Despite understanding that effective BMP-induced bone development depends on expression of FOXC1, the nature and mechanism of the relationship is not known. The present research aims to discover the mechanism through which FOXC1 impacts bone formation. This work explores FOXC1's impact on a SMAD binding site isolated from the SMAD target gene Id1 called the BMP Responsive Element (BRE) with and without BMP-4 induction. FOXC1 constructs missing functional regions are tested against wildtype FOXC1 function on BRE activation to determine regions crucial to BRE regulation. Finally, mouse myoblast cells stably expressing FOXC1 are used to evaluate what effect FOXC1 has on BMP-induced osteoblast transdifferentiation of myoblast cells. This work reports FOXC1 inhibits BMP-induced BRE activation through interaction with the BMP-SMAD pathway. The N-terminal activation, inhibitory, and DNA binding domains are all important for this inhibitory activity. Ectopic FOXC1 production in myoblasts likewise inhibits endogenous Id1 expression, though not other BMP-SMAD target genes. Osteoblast markers Alpl and Col1a1 are upregulated in cells stably expressing FOXC1 independent of BMP-4 treatment. FOXC1 appears to inhibit basal and BMP-4 induced Id1 expression. FOXC1 otherwise may not directly impact BMP-SMAD target expression, suggesting FOXC1 specifically targets Id1 expression and not BMP-SMAD signalling globally. Transdifferentiation of myoblast cells ectopically expressing FOXC1 can proceed without BMP-4 induction, indicated by Col1a1 and Alpl upregulation. These findings suggest FOXC1 may play an important role in the early stages of osteoblastogenesis.
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