The G-protein-coupled receptor GPR55 has been implicated in multiple biological activities, which has fuelled interest in its functional targeting. Its controversial pharmacology and often species-dependent regulation have impacted upon the potential translation of preclinical data involving GPR55. With the aim to identify novel GPR55 regulators, we have investigated lysophosphatidylinositol (LPI)-induced GPR55-mediated signal transduction. The expression system for wild-type and mutated GPR55 was HeLa cells silenced for their endogenous receptor by stable expression of a short-hairpin RNA specific for GPR55 5′-UTR, which allowed definition of the requirement of GPR55 Lys80 for LPI-induced MAPK activation and receptor internalisation. In RAW264.7 macrophages, GPR55 pathways were investigated by Gpr55 silencing using small-interfering RNAs, which demonstrated that LPI increased intracellular Ca2+ levels and induced actin filopodium formation through GPR55 activation. Furthermore, the LPI/GPR55 axis was shown to have an active role in osteoclastogenesis of precursor RAW264.7 cells induced by ‘receptor-activator of nuclear factor kappa-β ligand’ (RANKL). Indeed, this differentiation into mature osteoclasts was associated with a 14-fold increase in Gpr55 mRNA levels. Moreover, GPR55 silencing and antagonism impaired RANKL-induced transcription of the osteoclastogenesis markers: ‘nuclear factor of activated T-cells, cytoplasmic 1′, matrix metalloproteinase-9, cathepsin-K, tartrate-resistant acid phosphatase, and the calcitonin receptor, as evaluated by real-time PCR. Phage display was previously used to identify peptides that bind to GPR55. Here, the GPR55-specific peptide-P1 strongly inhibited osteoclast maturation of RAW264.7 macrophages, confirming its activity as a blocker of GPR55-mediated functions. Although osteoclast syncytium formation was not affected by pharmacological regulation of GPR55, osteoclast activity was dependent on GPR55 signalling, as shown with resorption assays on bone slices, where LPI stimulated and GPR55 antagonists inhibited bone erosion. Our data indicate that GPR55 represents a target for development of novel therapeutic approaches for treatment of pathological conditions caused by osteoclast-exacerbated bone degradation, such as in osteoporosis or during establishment of bone metastases.

中文翻译：

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溶血磷脂酰肌醇感应 GPR55 的肽靶向用于破骨细胞生成调节

G 蛋白偶联受体 GPR55 与多种生物活性有关，这激发了人们对其功能靶向的兴趣。其有争议的药理学和通常依赖于物种的调节影响了涉及 GPR55 的临床前数据的潜在转化。为了识别新的 GPR55 调节剂，我们研究了溶血磷脂酰肌醇 (LPI) 诱导的 GPR55 介导的信号转导。野生型和突变型 GPR55 的表达系统是 HeLa 细胞，通过稳定表达对 GPR55 5'-UTR 特异的短发夹 RNA 使其内源性受体沉默，这允许定义 GPR55 Lys80 对 LPI 诱导的 MAPK 激活的要求和受体内化。在 RAW264.7 巨噬细胞中，通过使用小干扰 RNA 进行 Gpr55 沉默来研究 GPR55 通路，这表明 LPI 通过 GPR55 激活增加细胞内 Ca2+ 水平并诱导肌动蛋白丝状体形成。此外，LPI/GPR55 轴显示在由“核因子 kappa-β 配体的受体激活剂”(RANKL) 诱导的前体 RAW264.7 细胞的破骨细胞生成中具有积极作用。事实上，这种向成熟破骨细胞的分化与 Gpr55 mRNA 水平增加 14 倍有关。此外，GPR55 的沉默和拮抗作用会损害 RANKL 诱导的破骨细胞生成标志物的转录：“活化 T 细胞的核因子、细胞质 1”、基质金属蛋白酶 9、组织蛋白酶 K、抗酒石酸酸性磷酸酶和降钙素受体，如通过实时 PCR 评估。噬菌体展示以前用于鉴定与 GPR55 结合的肽。这里，GPR55 特异性肽-P1 强烈抑制 RAW264.7 巨噬细胞的破骨细胞成熟，证实其作为 GPR55 介导功能的阻断剂的活性。尽管破骨细胞合胞体的形成不受 GPR55 药理调节的影响，但破骨细胞活性依赖于 GPR55 信号传导，如骨切片上的再吸收测定所示，其中 LPI 刺激而 GPR55 拮抗剂抑制骨侵蚀。我们的数据表明，GPR55 代表了开发新的治疗方法的目标，用于治疗由破骨细胞加剧的骨降解引起的病理状况，例如在骨质疏松症或骨转移建立期间。尽管破骨细胞合胞体的形成不受 GPR55 药理调节的影响，但破骨细胞活性依赖于 GPR55 信号传导，如骨切片上的再吸收测定所示，其中 LPI 刺激而 GPR55 拮抗剂抑制骨侵蚀。我们的数据表明，GPR55 代表了开发新的治疗方法的目标，用于治疗由破骨细胞加剧的骨降解引起的病理状况，例如在骨质疏松症或骨转移建立期间。尽管破骨细胞合胞体的形成不受 GPR55 药理学调节的影响，但破骨细胞活性依赖于 GPR55 信号传导，如骨切片上的再吸收测定所示，其中 LPI 刺激和 GPR55 拮抗剂抑制骨侵蚀。我们的数据表明，GPR55 代表了开发用于治疗由破骨细胞加剧的骨降解引起的病理状况的新治疗方法的目标，例如在骨质疏松症或骨转移建立期间。