2011), and fractured bones (Abdelmagid et al. 2010). In vitro studies have shown that Gpnmb induces osteoblast and osteoclast differentiation (Selim et al. 2003; Selim et al. 2007; Abdelmagid et al. 2008; Sheng et al. 2008). In Selleckchem SB715992 denervated mouse skeletal muscles, Gpnmb upregulates MMP-3 and MMP-9 in infiltrating fibroblasts (Ogawa et al. 2005). Gpnmb also functions as an inhibitor of T lymphocytes (Chung et al. 2007) and macrophages (Ripoll et al. 2007). These findings demonstrated the multiple roles of Gpnmb in normal tissues. However, with regard to
the nervous system, Gpnmb expression has been exclusively investigated in glioblastomas. Its expression in the normal brain is still unclear. Some studies steadily detected Gpnmb mRNA (Safadi et al. Inhibitors,research,lifescience,medical 2002; Onaga et al. 2003; Owen et al. 2003), but others not (Loging, et al. 2000; Shikano et al. 2001; Kuan et al. 2006). Moreover, little is known about the regional distribution and cellular localization of Gpnmb in the normal central nervous system (CNS). Therefore, we examined Gpnmb expression in Inhibitors,research,lifescience,medical CNS regions of normal adult rats by reverse transcription-polymerase chain reaction (RT-PCR)
and immunohistochemical analyses. Furthermore, to gain insight into the role of Gpnmb in the non-tumorous CNS, we studied changes in Gpnmb expression in inflamed brains. Materials and Methods Experimental animals Adult Wister rats (200–300 g) were purchased from Charles River Japan (Yokohama, Inhibitors,research,lifescience,medical Japan) and New Zealand white rabbits (approximately 4 kg) from CLEA Japan, Inc. (Tokyo, Japan). The experimental procedures approved by the Guideline for the Care and Use of Laboratory Animals in Kanazawa University. These animals
were maintained in the Institute for Experimental Animals of Kanazawa University Advanced Science Research Center. Inhibitors,research,lifescience,medical Injection of lipopolysaccharide (LPS) LPS from Escherichia coli serotype O127:B8 (Sigma, St. Louis, MO) was dissolved in sterile phosphate-buffered saline (PBS; pH Inhibitors,research,lifescience,medical 7.4) and intraperitoneally injected at a dose of 0.1 mg/kg of body weight. RT-PCR cDNA encoding the entire protein-coding sequence of rat Gpnmb was obtained by RT-PCR using the following set of primers: 5′-AGAGTCAAGCCCTGACTGGC-3′ (forward 1) and 5′-GAAGAGTGGGTTCCCAGTCA-3′ (reverse 1). PCR was performed using a 50-μl reaction mixture containing cDNA prepared from injured sciatic nerve (Osamura et al. 2005; corresponding to 50 ng of total RNA), 1 × KOD FX buffer second (Toyobo, Osaka, Japan), 200 μM dNTPs, 200 nM of each primer, and 1 unit of KOD FX DNA polymerase (Toyobo). The amplification consisted of 35 cycles of 10-sec denaturation at 98°C, 30-sec annealing at 60°C, and 2-min extension at 68°C. For TA cloning, 3′-A overhangs were added to the amplified product by treating it for 10 min at 72°C in a reaction mixture containing 1 × ExTaq buffer (Takara Shuzo, Otsu, Japan), 75 μM dNTPs, 2.5 mM MgCl2, and 2.5 units of ExTaq DNA polymerase (Takara Shuzo). The resulting fragment was cloned into a pCR2.