No abstract

Estrogen is a crucial hormone for osteoclast inhibition and for preventing osteoporosis. However, the hormone's role in osteoblast growth and differentiation remains unclear. The complexity of estrogen's role in guiding osteoblast behavior arises partly from crosstalk with other signaling pathways, including Wnt signaling. In this study, we show that the Wnt agonist, LiCl, induced Fhl1 gene expression, which substantially enhanced osteoblast differentiation. Staining with alizarin red revealed that MC3T3-E1 mineralization was enhanced by overexpression of Fhl1. In addition, Fhl1 promoted the expression of the osteogenic markers, Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN), whereas MC3T3-E1 cells with gene knockdown of Fhl1 exhibited limited mineralization and expression of Runx2, OCN, and OPN. We further demonstrate evidences from quantitative reverse transcription real-time polymerase chain reaction and reporter assay that Fhl1 expression was synergistically stimulated by estrogen (E2) and LiCl, but reduced by the estrogen-receptor inhibitor fulvestrant (ICI 182,780). However, estrogen could not enhance osteogenesis while Fhl1 expression was knocked down. Because estrogen and Wnt signaling frequently interact in developmental processes, we propose that Fhl1 can be an acting molecule mediating both signaling pathways during osteogenesis.

Wnt signaling is well known playing dual roles during chondrogenesis. The overexpression of Fhl1 (Four-and-a-half LIM domain 1) induces myotube formation as a downstream event of Wnt signaling. Because Fhl1 is widely expressed in other mesenchyme-derived tissues, including chondrocytes, we investigated the role of Fhl1 in chondrogenesis and its relationship with Wnt signaling. We found that the expression of Fhl1 can be enhanced by β-catenin and LiCl (Lithium chloride) in chondrogenic ATDC5 cells. Overexpression of Fhl1 as well as canonical Wnt signaling inhibits chondrogenesis of ATDC5 cells. Moreover, shRNA-mediated knockdown of Fhl1 expression also inhibited ATDC5 cell differentiation, and this result is resembled to the mutant phenotype caused by deletion of β-catenin as was described previously. Because the endogenous Fhl1 expression remains constant during the middle and late phases of chondrogenesis, we propose that proper Fhl1 expression is necessary for the chondrogenic differentiation of ATDC5 cells and altered Fhl1 expression serves as an aberrant Wnt signal that impedes chondrogenesis.