Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the preosteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E 2 (PGE 2 ) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-b1 treatment of preosteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE 2 increased apoptosis threefold, while treatment of pre-osteocytes with TGF-b1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival.
Secreted frizzled-related proteins (sFRPs) are glycoproteins that are recognized as Wnt antagonists. To identify the functional domains that are involved in Wnt antagonist function, several sFRP-1 mutants and sFRP-1/sFRP-2 chimeras were generated. These mutants were characterized in an optimized T-cell factor (TCF)-luciferase based assay in U2OS human osteosarcoma cells. Deletions of the sFRP-1 cysteine rich domain (CRD) lead to the complete loss of Wnt antagonist function. A region between amino acids 73-86 within the second loop of the CRD of sFRP-1 was necessary for the optimal Wnt inhibitory function. Within this region, a conserved tyrosine residue played a critical role, and its change to neutral or polar amino acids lead to decreased Wnt inhibitory activity. The sFRP-1/sFRP-2 chimeras with the netrin domain of sFRP-1 replaced by corresponding sFRP-2 sequences showed 40-70% loss of Wnt antagonist function. The sFRP-1/sFRP-2 chimera with the replacement of C-terminal 19 amino acids of sFRP-1 with 11 amino acids of sFRP-2 resulted in 70% loss of activity indicating that carboxyl-terminal region of sFRP-1 is important for its Wnt inhibitory activity. The structure-function analysis studies of sFRP-1 clearly demonstrate the interaction of several functional domains for its optimal Wnt antagonist function.
Inhibitor of secreted frizzled related protein-1 (sFRP-1) would be a novel potential osteogenic agent, since loss of sFRP-1 affects osteoblast proliferation, differentiation, and activity, resulting in improved bone mineral density, quality, and strength. We have identified small molecule diarylsulfone sulfonamide derivatives as sFRP-1 inhibitors. Structure-activity relationship generated for various regions of the scaffold was utilized to improve the biochemical profile, resulting in the identification of potent selective analogues, such as 16 with desirable pharmaceutical profile.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.