Calcium influx is crucial for T cell activation and differentiation. The detailed regulation of this process remains unclear. We report here that golli protein, an alternatively spliced product of the myelin basic protein gene, plays a critical role in regulating calcium influx in T cells. Golli-deficient T cells were hyperproliferative and showed enhanced calcium entry upon T cell receptor stimulation. We further found that golli regulates calcium influx in T cells through the inhibition of the store depletion-induced calcium influx. Mutation of the myristoylation site on golli disrupted its association with the plasma membrane and reversed its inhibitory action on Ca2+ influx, indicating that membrane association of golli was essential for its inhibitory action. These results indicate that golli functions in a unique way to regulate T cell activation through a mechanism involving the modulation of the calcium homeostasis.
Systemic administration of a sclerostin neutralizing antibody (Scl-Ab) has been shown to enhance fracture callus density and strength in several animal models. In order to further evaluate the potential of Scl-Ab to improve healing in a bone defect model, we evaluated Scl-Ab in a 3 mm femoral defect in young male outbred rats. Scl-Ab was given either continuously for 6 or 12 weeks after surgery or with 2 weeks of delay for 10 weeks. Bone formation was assessed by radiographs, m-CT, and histology. Complete bony union was achieved in only a few defects after 12 weeks of healing (Scl-Ab treated 5/30, vehicle treated 1/15). m-CT evaluation demonstrated a significant increase in the BV/TV in the defect in the delayed treatment group (65%, p < 0.05), but a non-significant increase in the continuous group (35%, p ¼ 0.11) compared to control. However, both regimens induced an anabolic response in the bone proximal and distal to the defect and in the un-operated femurs. We demonstrate that treatment with Scl-Ab can enhance bone repair in a bone defect and in the surrounding host bone, but lacks the osteoinductive activity to heal it. This agent seems to be most effective in bone repair scenarios where there is cortical integrity. The Wnt/b-catenin signaling pathway is known to be a key regulator of bone formation and its role in fracture healing has been established in several studies. 1-3This pathway is a target for developing strategies to promote bone fracture healing. 4 In pre-clinical studies activation of the Wnt pathway either by inhibiting Wnt inhibitors such as sclerostin [5][6][7] or DKK1 8 or more recently by liposomal delivery of Wnt3a 9 has been shown to enhance bone repair. Fracture non-union may occur in as many as 10% of all fractures. 10Treatment of non-unions may require multiple surgeries and prolonged hospital stays which leads to increased cost of treatment.11 Autologous bone graft remains the gold standard for management of nonunions, but there are disadvantages including donor site morbidity and limited availability. 12,13 BMPs are osteoinductive proteins that are FDA approved to treat open tibial shaft fractures 14,15 and to facilitate lumbar spine fusions. 16 The success of BMPs in humans has been variable and it is an expensive treatment associated with side effects such as local soft tissue edema 17 and ectopic bone formation.18 Therefore there is interest in identifying systemic agents that can enhance the healing of fractures.Sclerostin, a product of SOST gene, is primarily secreted by osteocytes and inhibits the Wnt pathway by interacting with the LRP5/6. 19 Mutations in SOST gene causing sclerostin loss of function are associated with a clinical syndrome of progressive bone thickening known as sclerosteosis. 20,21 Efficacy of a monoclonal sclerostin neutralizing antibody (Scl-Ab) to increase bone mass and strength and in promoting fracture healing has been demonstrated in several animal models including female osteoporotic rats, aged male rats, and gonad-intact non-human primates....
‘Ex vivo’ gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, Δtk (LV-Δtk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90–95% cell death at 12 days with GCV at 1 µg ml−1 in MBMCs vs 90–95% cell death at 5 days by 0.1 µg ml−1 of GCV in W-20-17 cells). Implantation of LV-Δtk-T2A-BMP-2 transduced MBMCs healed a 2 mm femoral defect at 4 weeks. Early GCV treatment (days 0–14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-Δtk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.
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