Next we evaluated the therapeutic potential of compound

Next, we evaluated the therapeutic potential of compound () using the mouse model of collagen-induced arthritis (CIA model), which is the pathological model for RA. Consequently, oral once-daily dosing of 3 mg/kg reduced the overall progression of the clinical score, including inflammation and bone erosion. Therefore, we expected that (), which is , would be the most promising clinical candidate from these results. We are submitting reports that describe the therapeutic potential of in detail.
Introduction Proto-oncogene c-fms (encoding a receptor tyrosine kinase, CSF-1R) is expressed in breast cancer [1], and serves as an important mediator of breast cancer development [2], invasion [3], [4], metastasis [5], [6], recurrence [7] and survival [8]. The binding of colony stimulating factor-1 (CSF-1) to CSF-1R activates downstream signal transduction pathways involved in these breast cancer related biological phenotypes. We previously reported that vigilin binds a 69 nt pyrimidine-rich element in the CSF-1R mRNA 3′UTR (3499–3567) and suppresses CSF-1R mRNA expression in breast cancer Fludara [9]. The binding of mRNA with RNA-binding proteins can form an mRNP spatial structure whose formation is crucial in determining mRNA stability and translation efficiency [10].Vigilin suppresses the expression of c-fms mRNA by down-regulating mRNA stability as well as inhibiting translation [9]. While vigilin, a large nucleo-cytoplasmic RNA binding protein, was first described decades ago, renewed interest has focused on its role in cardiovascular disease [11]. Data on its role in cancer and the underlying mechanism is still limited. Further, it is recognized that while many of vigilin\'s known actions occur in the cytoplasm, vigilin\'s nuclear functions are less well understood [12]. Next, we explored the in vitro phenotypes of vigilin expressing breast cancer cells by competition of protein binding to CSF-1R mRNA by excess 3′UTR CSF-1R sequences containing the vigilin binding site, as well as by use of a deletional mutant of the 69 nt element. Lastly, we are the first to show vigilin expression in human breast tissues, and study in particular the expression and cellular localization in its transition to ductal carcinoma-in-situ.
Materials and Methods
Results
Discussion We first confirmed that translational repression of target mRNAs by vigilin (Figure 1). It is known that vigilin binds a large number of mRNAs (estimated 700 mRNAs; [12]). We demonstrate that vigilin directly interacts with the pyrimidine-rich element in the CSF-1R mRNA 3′UTR, and requires the full-length 69 nt for stable binding (Figure 2). We show that mutation of the 69 nt CSF-1R element dramatically alters CSF-1R translational efficiency, and obviates vigilin effect on translational repression of CSF-1R (Figure 3). Our data does not rule out a contribution of vigilin on mRNA decay to these experiments on translation, as the mechanisms of mRNA stability and translation are closely coupled. In fact, we have previously shown that vigilin OE can enhance CSF-1R mRNA decay by approximately 1.8-fold, while vigilin silencing had less of an effect on mRNA stability [9]. What is clear collectively is that the primary effect of vigilin is on translation, with a more minor component on mRNA decay. This pyrimidine-rich 69 nt CSF-1R element is a target for binding by proteins other than vigilin. Translational repression and mRNA decay may be reversible by competing CSF-1R mRNA with other RNA binding proteins. We have previously characterized one of them, HuR, with a competition between vigilin and HuR demonstrated for binding this element, with opposing effects on CSF-1R expression [9], [13], [19]. The binding of vigilin to the pyrimidine-rich sequence significantly decreases CSF-1R expression (Figures 2 and 4). In contrast, the binding of HuR to this pyrimidine rich region increases the CSF-1R expression [13]. The present data indicates that the 69 nt CSF-1R binding element confers net translational repression, and that the repression effect is very significant in the presence of cellular RNA binding proteins, including vigilin and HuR (Figure 3). We have shown that the affinity of vigilin to this pyrimidine-rich region in the CSF-1R mRNA 3′UTR is at least 3-fold stronger than that of HuR in BT20 breast cancer cells by in vitro competition assay [19]. Thus, this specificity of binding of the 69 nt pyrimidine-rich region to vigilin, may explain why disruption of the pyrimidine-rich sequence appears to more significantly affect vigilin-specific actions, rather than those of HuR.