Probes for INS

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K+ (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in 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 than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3-/- mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3-/- chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and 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 and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features _in vivo_, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and _in situ_ mRNA hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form _bona fide_ bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like formation with chondrocyte-to-osteoblast trans differentiation. The two non-mineralizing cell-ECM structures may mimic periosteal cambium and pathological, non-mineralizing osteoid. Importantly, the most commonly used culture conditions (10 mM β-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of _bona fide_ bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research.

Bone growth requires a specialised, highly angiogenic blood vessel subtype, so-called type H vessels, which pave the way for osteoblasts surrounding these vessels. At the end of adolescence, type H vessels differentiate into quiescent type L endothelium lacking the capacity to promote bone growth. Until now, the signals that switch off type H vessel identity and thus limit adolescent bone growth have remained ill defined. Here we show that mechanical forces, associated with increased body weight at the end of adolescence, trigger the mechanoreceptor PIEZO1 and thereby mediate enhanced production of the kinase FAM20C in osteoblasts. FAM20C, the major kinase of the secreted phosphoproteome, phosphorylates dentin matrix protein 1, previously identified as a key factor in bone mineralization. Thereupon, dentin matrix protein 1 is secreted from osteoblasts in a burst-like manner. Extracellular dentin matrix protein 1 inhibits vascular endothelial growth factor signalling by preventing phosphorylation of vascular endothelial growth factor receptor 2. Hence, secreted dentin matrix protein 1 transforms type H vessels into type L to limit bone growth activity and enhance bone mineralization. The discovered mechanism may suggest new options for the treatment of diseases characterised by aberrant activity of bone and vessels such as osteoarthritis, osteoporosis and osteosarcoma.