WT versus Girk3-/- Chondrocytes

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein coupled receptor (GPCR) signaling regulates skeletal development and long bone growth. G protein-coupled Inward-Rectifying potassium (K+) channel (GIRK) genes are key functional components of the GPCR in excitable tissues (e.g., neurons and cardiomyocytes) but their roles in the non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study we analyzed skeletal phenotypes of Girk2-/-, Girk3-/- and Girk2/3-/- mice. Bones from 12-week-old Girk2-/- mice were normal in length, but femurs and tibiae from Girk3-/- and Girk2/3-/- mice were longer. Epiphyseal chondrocytes from five-day-old Girk3-/- mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly and lower levels of VEGF receptors and produced larger micromasses than wildtype chondrocytes in vitro. Girk3-/- chondrocytes were also more responsive to the GPCR ligand, dynorphin, as evidenced by greater cAMP and pCREB expression, greater GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3-/- mice, supporting a role for delayed vasculogenesis during postnatal growth. Together these data identify Girk3 as a GPCR effector that controls bone lengthening. Six samples of total RNA, n=3 biological replicates of wildtype (WT) mice and n=3 biological replicates of Girk3-null (Girk3-/-) five-day-old mouse chondrocytes长骨通过软骨内成骨（endochondral ossification）过程形成并修复。G蛋白偶联受体（G protein-coupled receptor, GPCR）信号通路的激活可调控骨骼发育与长骨生长。G蛋白偶联内向整流钾（K+）通道（GIRK）基因是可兴奋组织（如神经元与心肌细胞）中GPCR的关键功能组分，但它们在直接参与软骨内成骨的非兴奋细胞中的作用尚未得到研究。本研究对Girk2基因敲除（Girk2-/-）、Girk3基因敲除（Girk3-/-）以及Girk2/3双基因敲除（Girk2/3-/-）小鼠的骨骼表型进行了分析。12周龄Girk2-/-小鼠的骨骼长度正常，但Girk3-/-与Girk2/3-/-小鼠的股骨与胫骨长度更长。与野生型软骨细胞相比，5日龄Girk3-/-小鼠的骨骺软骨细胞在体外培养时，胶原链三聚化与胶原纤维组装相关基因的表达水平更高，血管内皮生长因子（vascular endothelial growth factor, VEGF）受体的表达水平更低，且形成的细胞微团更大。Girk3-/-软骨细胞对GPCR配体强啡肽（dynorphin）的应答也更强，具体表现为环磷酸腺苷（cyclic adenosine monophosphate, cAMP）与磷酸化环腺苷酸应答元件结合蛋白（phosphorylated cAMP response element-binding protein, pCREB）的表达水平更高、糖胺聚糖（glycosaminoglycan, GAG）合成量更多，以及Col2a1与Sox9转录本的表达上调。成像研究显示，幼年Girk3-/-小鼠骨骼中的Kdr（Vegfr2）与内皮黏蛋白（endomucin）表达水平显著降低，这支持其在出生后生长过程中存在血管生成延迟的表型。综上，本研究数据确定Girk3作为GPCR效应分子调控骨延长。本研究共收集6份总RNA样本：包含3份野生型（wildtype, WT）5日龄小鼠软骨细胞生物学重复样本，以及3份Girk3基因敲除（Girk3-null, Girk3-/-）5日龄小鼠软骨细胞生物学重复样本。