Abstract
BackgroundRad is the prototypic member of a subfamily of Ras-related small G-proteins and is highly expressed in the skeletal muscle of patients with type II diabetes. Our previous microarray analysis suggested that Rad may mediate fracture nonunion development. Thus, the present study used rat experimental models to investigate and compare the gene and protein expression patterns of both Rad and Rem1, another RGK subfamily member, in nonunions and standard healing fractures.MethodsStandard healing fractures and nonunions (produced via periosteal cauterization at the fracture site) were created in the femurs of 3-month-old male Sprague-Dawley rats. At post-fracture days 7, 14, 21, and 28, the fracture callus and fibrous tissue from the standard healing fractures and nonunions, respectively, were harvested and screened (via real-time PCR) for Rad and Rem1 expression. The immunolocalization of both encoded proteins was analyzed at post-fracture days 14 and 21. At the same time points, hematoxylin and eosin staining was performed to identify the detailed tissue structures.ResultsResults of real-time PCR analysis showed that Rad expression increased significantly in the nonunions, compared to that in the standard healing fractures, at post-fracture days 14, 21, and 28. Conversely, immunohistochemical analysis revealed the immunolocalization of Rad to be similar to that of Rem1 in both fracture types at post-fracture days 14 and 21.ConclusionsRad may mediate nonunion development, and thus, may be a promising therapeutic target to treat these injuries.
Highlights
Ras associated with diabetes (Rad) is the prototypic member of a subfamily of Ras-related small G-proteins and is highly expressed in the skeletal muscle of patients with type II diabetes
We investigated the gene expression patterns of tumor necrosis factor-α (TNF-α) and platelet-derived growth factor (PDGF), which are considered involved in the regulation of Rad gene expression [11]
Rad and Rem1 gene expression in nonunions and standard healing fractures No significant difference in Rad gene expression was observed between the standard healing fractures and nonunions at post-fracture day 7; Rad expression was significantly (6.2, 2.2, and 5.6-fold) higher in the nonunions than in the standard healing fractures at the later time points, i.e., post-fracture days 14, 21, and 28, respectively (Fig. 1a)
Summary
Rad is the prototypic member of a subfamily of Ras-related small G-proteins and is highly expressed in the skeletal muscle of patients with type II diabetes. The present study used rat experimental models to investigate and compare the gene and protein expression patterns of both Rad and Rem, another RGK subfamily member, in nonunions and standard healing fractures. We have previously investigated and compared gene expression patterns in nonunions and standard healing fractures, using rat experimental models. The fracture callus and fibrous tissue of the standard healing fractures and nonunions, respectively, were harvested and subjected to a microarray analysis. Results of this analysis indicated that the expression of Ras associated with diabetes (Rad, RRAD) was higher in nonunion tissues than in fracture calluses in a standard fracturehealing model [3]. The specific function of Rad in fracture healing and/or nonunion development is still unknown
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