GRK2 desensitizes flow-induced responses in osteoblasts.

Bone desensitization after mechanical loading is essential for bone to adapt to its mechanical environment. However, the desensitization mechanism is unknown. Previous studies suggest that G protein-coupled receptors (GPCRs), including P2Y and parathyroid hormone receptors, play important roles in osteoblast mechanobiology. Thus, for the present research, we examined the role of G protein-coupled receptor kinase 2 (GRK2) in osteoblast desensitization after exposure to mechanical stimulation. We first showed the existence of osteoblast desensitization after mechanical stimulation based on cytosol Ca2+ and phosphorylated ERK1/2 activities, detected using a fluorescent Ca2+-sensitive dye and western blotting, respectively. We then demonstrated that GRK2 overexpression in MC3T3-E1 cells inhibits flow-induced ERK1/2 phosphorylation, while siRNA knockdown of GRK2 enhances ERK1/2 phosphorylation. Additionally, we found that GRK2 overexpression in MC3T3-E1 cells inhibits cyclooxygenase-2 mRNA expression in the short term and alkaline phosphatase activity in the long term. More importantly, we discovered that GRK2 translocated to the cell membrane shortly after flow stimulation - a step necessary for GPCR desensitization. Previously, we have demonstrated that P2Y2 purinergic receptors, one type of GPCRs, are involved in various flow-induced osteoblastic responses. In this research, we also showed that GRK2 overexpression does not affect ATP release. Accordingly, GRK2 is able to inhibit flow-induced osteoblast responses possibly through desensitizing P2Y2 receptors.