Previous studies have shown that Wnt signaling is involved in postnatal mammalian myogenesis; however, the downstream mechanism of Wnt signaling is not fully understood. This study reports that the murine four-and-a-half LIM domain 1 (Fhl1) could be stimulated by β-catenin or LiCl treatment to induce myogenesis. In contrast, knockdown of the Fhl1 gene expression in C2C12 cells led to reduced myotube formation. We also adopted reporter assays to demonstrate that either β-catenin or LiCl significantly activated the Fhl1 promoter, which contains four putative consensus TCF/LEF binding sites. Mutations of two of these sites caused a significant decrease in promoter activity by luciferase reporter assay. Thus, we suggest that Wnt signaling induces muscle cell differentiation, at least partly, through Fhl1 activation.

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.

In this study, we developed a new purification method using chondroitin sulfate C (CSC) and protamine sulfate (PS) to concentrate lentivirus. To evaluate the efficiency of this new method, we compared it with several previously described purification protocols, including virus concentrated by ultracentrifugation (Ultra), precipitated by polyethylene glycol (PEG), and sedimented by CSC combined with polybrene (PB). After using the different methods to purify and concentrate equivalent amounts of lentivirus supernatant, the virus pellets precipitated by the different methods were resuspended using the equivalent volumes of DMEM. Subsequently, 10 μl of each lentivirus stock carrying EGFP gene was used to transduce two types of cells, human embryonic kidney 293T (HEK293T) cells and mouse mesenchymal stem cells (mMSC). It was obvious that HEK293T and mMSC appeared much intensiver green fluorescence through virus transduction from PS method than from other methods. To quantitate the transduction efficiency of the viruses, we examined virus titer in the cells after transduction using a real-time PCR-based analysis. Accordingly, we verified that PS precipitation could generate virus with a higher titer (4.39 × 10 IU/ml) than PB (2.43 × 10 IU/ml), Ultra (1.16 × 10 IU/ml), and PEG (0.56 × 10 IU/ml) in HEK293T cells. As for HEK293T cells in mMSC, the PS method also generated virus with a higher titer (4.66 × 10 IU/ml) than the Ultra method (2.36 × 10 IU/ml), and a much higher titer than those of the other chemical-based precipitation methods using PB (4.82 × 10 IU/ml) and PEG (8.98 × 10 IU/ml). Furthermore, the HEK293T cells and mMSC transduced by PS(1X)-virus appeared to have higher cell growth ratios, respectively, than the HEK293T cells and mMSC transduced by lentivirus using the other methods. We conclude that our new method for purifying lentivirus is cost-effective, time-saving, and highly efficient, and that lentivirus purification by this means could possibly be used to transduce a variety of cells, including stem cells.

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.