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We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr-EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with β-arrestins (Arrbs), the small GTPase Rab5, importin-β (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors. T he oxytocin (Oxt) receptor (Oxtr), a member of the rhodopsin-type (class I) family of G protein-coupled receptors (GPCRs), responds to the neurohypophyseal hormone Oxt to stimulate lactation and social bonding (1, 2). More recently, we and others have shown that Oxt also stimulates bone formation directly by interacting with an osteoblastic Oxtr (3, 4). The genetic deletion of Oxt or Oxtr in mice thus decreases osteoblast differentiation and bone formation, causing osteopenia (5, 6). We believe that this action supports maternal skeletal remineralization after the intergenerational transfer of calcium during pregnancy and lactation (5). However, the precise mechanism through which Oxtr activation translates into increased bone formation remains unclear.Oxt binding to Oxtrs is known to activate both G protein G αi -and G αq/11 -mediated phospholipase C pathways (7). However, in common with other GPCRs, prolonged or repetitive agonist stimulation by Oxt results in Oxtr internalization through β-arrestin (Arrb). GPCR internalization can activate signaling pathways quite distinct from those activated by the same receptors resident at the cell surface. Hence, desensitization of primary G protein-dependent signaling can be followed by a second wave of Arrb-mediated signaling (8). β-Arrestins can also recruit signaling proteins that connect GPCRs to various cytoplasmic effector pathways, such as mitogen-activated protein kinase (MAPK) and protein kinase B/phosphatidylinositol-4,5-bisphosphate 3-kinase (Akt/PI3K) (9, 10). Such mechanisms can elicit delayed genomic responses.After being internalized, GPCRs are either recycled back to the plasma m...

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Background: Effective screening for colorectal cancer can reduce mortality by early detection of tumours and colonic polyps. An altered pattern of volatile organic compounds (VOCs) in exhaled breath has been proposed as a potential non-invasive diagnostic tool for detection of cancer. The aim of this study was to evaluate the reliability of breath-testing for colorectal cancer screening and early diagnosis using an advanced breath sampler. Methods: The exhaled breath of patients with colorectal cancer and non-cancer controls with negative findings on colonoscopy was collected using the ReCIVA ® Breath Sampler. This portable device is able to capture the alveolar breath fraction without environmental contamination. VOCs were desorbed thermally and analysed by gas chromatography-mass spectrometry. The discriminatory ability of VOCs in detecting colorectal cancer was evaluated by receiver operating characteristic (ROC) curve analysis for each VOC, followed by cross-validation by the leave-one-out method, and by applying stepwise logistic regression analysis. Results: The study included 83 patients with colorectal cancer and 90 non-cancer controls. Fourteen VOCs were found to have significant discriminatory ability in detecting patients with colorectal cancer. The model with the diagnosis of cancer versus no cancer resulted in a statistically significant likelihood of discrimination of 173⋅45 (P < 0⋅001), with an area under the ROC curve of 0⋅979. Cross-validation of the model resulted in a true predictive value for colorectal cancer of 93 per cent overall. Reliability of the breath analysis was maintained irrespective of cancer stage. Conclusion: This study demonstrated that analysis of exhaled VOCs can discriminate patients with colorectal cancer from those without. This finding may eventually lead to the creation of a smart online sensory device, capable of providing a binary answer (cancer/no cancer) and directing to further screening.