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  • Several functions of FAT have been suggested


    Several functions of FAT10 have been suggested. It has been shown that FAT10 binds noncovalently to the mitotic spindle checkpoint protein MAD2 and that this binding might cause chromosome instability in the cancer CB-5083 overexpressing FAT10 (Liu et al., 1999, Ren et al., 2006). In other studies, overexpression of the wild-type FAT10, but not its diglycine mutant, was found to cause apoptosis, suggesting that FAT10 conjugation of cellular targets may play a role in apoptosis (Raasi et al., 2001). This is potentially important, as FAT10 is strongly induced by stimulation with TNFα and IFNγ, which synergistically promote apoptosis of many cell types including pancreatic beta cells and cancer cells (Lee, 2002). Fusion of FAT10 to long-lived proteins such as GFP greatly facilitates the degradation of the fusion proteins by the proteasome through a ubiquitin-independent mechanism (Hipp et al., 2005). Thus, FAT10 conjugation may be another ubiquitin-like modification that targets protein degradation by the proteasome. Despite these ex vivo studies, the physiological functions of FAT10 remain unclear, as the FAT10-deficient mice displayed no obvious abnormality, except that these mice were more sensitive to endotoxin challenge than the wild-type mice (Canaan et al., 2006). As FAT10 is strongly induced by cytokines, phenotypic examination of FAT10-deficient cells following cytokine stimulation may help uncover the physiological function of FAT10.
    Experimental Procedures
    Introduction Osteoporosis, a well-known bone-health problem for elderly, is caused by the reduction in bone density that poses a high risk to bone fractures. It is caused by an imbalance between bone resorption and bone formation, namely the activities of osteoclast exceeds osteoblast. Hence, inhibiting the osteoclast and inducing osteoblasts is a common treatment for osteoporosis [1]. However, the drugs commonly used to prevent osteoporosis have some side effects such as oesophageal irritation, acute-phase reaction [2]. Thus, the development of natural and nontoxic treatment strategies is important. Eggs have been recognized as an important contributor of functional ingredients for humans [3]. Egg yolk is composed of various biologically active proteins. The water-soluble proteins of egg yolk have been examined for their functional effects on longitudinal bone growth during the growth period in adolescent rats [4]. Scholars reported that yolk water-soluble protein (YSP) stimulated bone formation in osteoblastic MC3T3-E1 cell line and inhibited bone resorption in osteoclast precursor cells [5]. Their further studies with the peptides prepared with a water-soluble fraction of egg yolk indicated that the peptides also had significant effects on bone metabolism in vitro as well as in vivo [6, 7]. Phosvitin (PV), which has the highest known level of phosphorylation among egg yolk proteins [8, 9], has been reported to possess a strong Ca-binding capacity and regulate the properties of biological mineralization [[10], [11], [12], [13]] or increase calcium incorporation into bones [14, 15]. Liu et al. [16] reported that PV showed bioactivities in connective tissues and bone organogenesis. Although PV has been reported to be a bone growth factor both in vitro and in vivo, current evidences about the regulation mechanisms of PV on bone calcification are not sufficient. Our previous work indicated that PV was involved in the bone formation of chicken embryo through the dephosphorylation [17]. Thus, we are interested in exploring whether PV with different degree of phosphorylation has a direct effect to the osteoblasts. Bone-forming osteoblasts involve the proliferation and differentiation of osteoblastic cells in response to some factors in vivo. During differentiation, pre-osteoblastic cells express proteins of extra-cellular matrix such as collagen, alkaline phosphatase (ALP), osteocalcin and other bone matrix proteins [18, 19], and then osteoblastic cells form mineralized bone. MC3T3-E1 cell line, a kind of pre-osteoblastic cell, is widely used in bone remodeling studies because it has the capacity to differentiate into osteoblasts [20]. The process of osteogenesis can be modulated through several signaling pathways (i.e. RANK/RANKL/OPG, Smads and MAPK pathway). Bone remodeling is mediated by osteoblast/osteoclast coordination, which converges at the RANK/RANKL/OPG [20, 21]. Molecular factors involved in the RANK/RANKL/OPG include the receptor activator of nuclear fact r-kappa B (RANK), the RANK ligand (RANKL) and osteoprotegerin (OPG) - a natural inhibitor of RANKL [22]. OPG is an important mediator in RANKL and RANK association, which could regulate osteoclast activity. OPG inhibits osteoclastic bone resorption by blocking RANKL, which reduces RANKL-RANK binding [23]. Therefore, the RANKL/OPG ratio is a key factor in bone remodeling. So, the objective of this study was to investigate whether PV with different degree of phosphorylation had any impacts to the RANKL/OPG ratio.