GRK2 desensitizes flow-induced responses in osteoblasts

Abstract

ABSTRACT. 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 Ca 2+ and phosphorylated ERK1/2 activities, detected using a fluorescent Ca 2+ -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 flowinduced 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.

Purinergic Signaling Mediates PTH and Fluid Flow-Induced Osteoblast Proliferation

“…After subjected to oscillatory fluid flow for 5 minutes, MC3T3-E1 osteoblast may take up to 120 minutes to completely recover its sensitivity to respond to another oscillatory fluid flow according to our previous experiments [ 28 ]. In this study, MC3T3-E1 cells were treated with or without PTH for 30 minutes after first fluid flow stimulation, then exposed to second fluid flow stimulation.…”

“…Since both PTH receptor and P2Y2 receptor are GPCRs, they can be desensitized by GPCR kinases (GRKs). We have previous demonstrated that GRKs may be involved in fluid flow-induced osteoblast desensitization [ 28 ]. If PTH receptors are desensitized by the same GRK as P2Y2 receptors, then the administration of PTH may lead to recruitment of GRK to PTH receptors.…”