GSE192933
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. Overall design: 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