Rad and other Ras superfamily members have been shown to be activated by diverse extracellular stimulating factors and have been reported to be involved in a wide spectrum of cellular functions, including cell proliferation, differentiation, morphology, and apoptosis [19]. Rad was initially identified via subtractive cloning as an mRNA that is overexpressed in the skeletal muscle of a subset of patients with type II diabetes; however, it is normally highly expressed in the heart and lungs [10]. Previous studies have shown that Rad is regulated by TNF-α and PDGF [11] and that it interacts with skeletal muscle β-tropomyosin and the cytoskeleton of muscle cells [5] to inhibit insulin-stimulated glucose uptake in a variety of cultured cell lines [20]. Another study reported that Rad is temporarily regulated within myogenic progenitor cells during skeletal muscle regeneration [21]. Together, these reports suggest that Rad may mediate skeletal muscle function and regeneration, and/or cytoskeletal organization. Till date, however, its expression and role(s) in fracture healing and nonunion development have not been elucidated.
The RGK subfamily of small GTP-binding proteins comprises of four members, namely, Rad, Rem1, Rem2, and Gem (mouse homolog, also referred to as Kir) [22]. Of these, the present study selected Rem1 as a comparative protein for reference while investigating the relevance of Rad in nonunion development. Real-time PCR showed that, at the earliest analyzed time point (i.e., post-fracture day 7), there was no significant difference in Rad mRNA expression between the standard healing fractures and nonunions; however, Rad expression was significantly higher in the nonunions than in the standard healing fractures at later time points (i.e., post-fracture days 14, 21, and 28). In contrast, there was no significant difference in Rem1 gene expression between the two treatment groups at any of the analyzed time points. This suggests that Rad, but not Rem1, may be important in nonunion development. Notably, the time points at which Rad expression was increased in the nonunions coincided with angiogenesis progression and endochondral ossification initiation in the current rat fracture model, thereby suggesting that Rad may impact or modulate these processes to mediate nonunion development. Further research is needed to examine the underlying mechanism(s) and significance of the observed increase in Rad expression in nonunions (compared to standard healing fractures) at the later time points.
Till date, Rad has been the focus of significant research in a range of contexts. For example, Fu et al. previously published a cardiology study showing that Rad is a critical inhibitor of vascular lesion formation, which acts by suppressing vascular smooth muscle cell migration [11]. Conversely, previous oncology studies have suggested that Rad inactivation may promote both hepatocellular carcinoma metastasis and nasopharyngeal carcinoma development [23, 24]. Another recent report suggested that Rad may play an important role in the regulation of bone homeostasis [25]; however, to our knowledge, there is no report describing the role of Rad in bone fracture healing and nonunion development.
As discussed here, Rad is highly expressed in the skeletal muscle of patients with type II diabetes [10]. Interestingly, clinical studies have suggested that patients with diabetes mellitus are more likely to experience complications during fracture healing, including delayed union and nonunion [26], to the extent that fracture healing is estimated to take approximately twice as long in patients with diabetes than in those without [27]. Tyndall et al. previously suggested that fracture healing may be impeded in these patients, since they exhibit decreased PDGF expression, which in turn, inhibits cell proliferation [28]. Consistent with this hypothesis, high TNF-α production has been reported in fracture calluses in animals with diabetes, and as shown by Kayal et al., high TNF-α levels both promote chondrocyte apoptosis and inhibit fracture healing [29]. As mentioned previously herein, TNF-α and PDGF have been reported to be involved in the regulation of Rad gene expression [11]. In the present study, TNF-α gene expression was significantly higher in the nonunions than in the standard healing fractures and the pattern of gene expression closely resembled that of Rad. In contrast, PDGF gene expression was significantly lower in the nonunions as overserved in the diabetic condition. These results suggested that the regulation of Rad gene expression levels in nonunions is associated with TNF-α but not with PDGF. Chronic inflammation, which involves potent pro-inflammatory cytokines such as TNF-α, is well known as a factor that can lead to the development of nonunion [30]. High concentrations of TNF-α have been reported to inhibit osteoblast differentiation [31], and one of the mechanisms is thought to be the downregulation of preosteoblast epidermal growth factor-like repeat protein by meprin, A5 protein, and receptor protein-tyrosine phosphatase l domain (POEM) [32]. We speculate that there is another unknown pathway by which TNF-α inhibits fracture healing via Rad upregulation. High levels of Rad are often observed in diabetic skeletal muscle; thus, elucidating this detailed mechanism may explain reasons for delayed fracture healing in patients with diabetes; however, more research is required to confirm the presence of this relationship.
The present study also investigated Rad and Rem1 protein expression (immunolocalization) in standard healing fractures and nonunions. Interestingly, while Rad and Rem1 gene expression differed significantly in the standard healing fractures and nonunions, their protein expression patterns were very similar. For example, on post-fracture days 14 and 21, Rad and Rem1 in the standard healing fractures were detected at robust levels in fibroblast-like spindle cells, proliferating chondrocytes, and osteoblasts lining the trabecular bone, but only at very low levels in hypertrophic chondrocytes. Given that the differentiation of proliferating cells to hypertrophic chondrocytes is a vitally important process for endochondral ossification, this result suggests that the observed decrease in Rad expression may be associated with the progression of normal fracture healing process. This hypothesis was supported both by the real-time PCR results. As another possible explanation, because less hypertrophic chondrocytes, which seem to lack the expression of Rad, are present in the standard fracture than in the nonunion, the gene expression of Rad in the standard fracture might be lower than that in the nonunion.
Thus, results of the present study reveal that Rad expression patterns in standard healing fractures and nonunions are different from those of Rem1, despite their similar patterns of protein localization. This suggests that although both Rad and Rem1 are expressed in the fracture callus and fibrous tissues of standard healing fractures and nonunions respectively, their differential gene expression patterns may be reflective of their divergent roles in, and/or impacts on, nonunion development.
We have still some issues to be addressed. As one of the future directions, experiments investigating the effect of inhibiting PDGF and TNF-α using a Rad-overexpressing model should be considered. This might help to figure out how Rad affects nonunion development. Further, it is necessary to carry out investigations using a Rad-knock out model to determine how suppression of Rad influences the fracture healing process and nonunion development. As further in vitro experiment, studying the influences of Rad on MSC, osteoblasts, and chondrocytes would be helpful to understand the role of Rad in nonunion development. Furthermore, it would be necessary to conduct similar studies using female rats to determine whether Rad overexpression in nonunions occurs in only male rats. All of these are subjects of our future study. Rad is one of the genes or proteins that have not been fully explored and thus continuous research is necessary.