D us to assess the relative expression of GPCRs in mature

D us to assess the relative expression of GPCRs in mature OBs. We identified 27 Gs-and/or Gi-coupled GPCRs whose expressions on OBs were validated by Taqman real-time GPCR Array at Ct number below 30. Many of these receptors have known or suspected roles in OB and osteocyte differentiation and function. Not surprisingly, parathyroid Scutellarein biological activity hormone 1 receptor was highly expressed in this OB population. For many of the identified OB GPCRs, their role in regulating normal bone development and skeletal function is not known. Definitive assessment of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850275 role of specific GPCRs in mature OBs will require evaluation of the effects of targeted deletion. Several groups investigated gene expression profiles by microarray analysis in OB cell lines and in whole-bone samples in response to exogenous parathyroid hormone. This is the first study to date examining alterations in gene expression in vivo in enhanced Author Manuscript Author Manuscript Author Manuscript Author Manuscript Exp Cell Res. Author manuscript; available in PMC 2016 May 01. Wattanachanya et al. Page 12 OB Gs signaling. We found only a small number of differentially expressed genes that overlap with their results, consistent with the notion that there are different OB transcriptome effects, depending on the timing and source of Gs activation. In conclusion, our study provides a useful tool to evaluate the in vivo gene expression specifically occurring in OBs with activated Gs-GPCR signaling, at the cellular level rather than in a whole bone. The study revealed that cell cycle and transcriptional regulation were the most highly enriched pathways. Complete set of GPCRs expressed in OBs and new candidate paracrine mediators in response to chronic Gs activation in OBs were also indentified. All these regulated genes could be used for further functional studies. Biological validation of the role of these mediators may lead to new therapeutic targets for enhancing bone formation in osteoporosis and for reversing bone disease associated with excess Gs signaling. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Ct Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments Funding support: This work was supported by grants to RAN from NIH and the Department of Veterans Affairs, to ECH from the NIH and the National Osteoporosis Foundation. We thank Richard Kao, Zhiqiang cheng and Yongmei Wang for valuable technical assistance and discussion. The prevailing model that explains the role of Aurora B in regulating the SAC posits that under low tension, Aurora B phosphorylates microtubule-binding kinetochore proteins resulting in reduced affinity for microtubules leading to dissociation of kinetochore-microtubule interactions 1014. As chromosomes become bi-oriented and BQ123 sister kinetochores are under high tension, Aurora B is spatially separated from the kinetochore and can no longer destabilize kinetochoremicrotubule interactions. According to this spatial gradient model, inner centromere localization of the CPC is paramount for error correction 15. In yeast, however, INCENP can regulate chromosome segregation independent of centromere localization 16. CPC localization during metaphase is thought to rely on the intersection of two histone modifications at the centromere. Haspin kinase phosphorylates histone H3 on threonine 3 along chromosome arms and at centromeres, which is bound by Survivin 17,18. The kinetochore-localized kinase B.D us to assess the relative expression of GPCRs in mature OBs. We identified 27 Gs-and/or Gi-coupled GPCRs whose expressions on OBs were validated by Taqman real-time GPCR Array at Ct number below 30. Many of these receptors have known or suspected roles in OB and osteocyte differentiation and function. Not surprisingly, parathyroid hormone 1 receptor was highly expressed in this OB population. For many of the identified OB GPCRs, their role in regulating normal bone development and skeletal function is not known. Definitive assessment of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850275 role of specific GPCRs in mature OBs will require evaluation of the effects of targeted deletion. Several groups investigated gene expression profiles by microarray analysis in OB cell lines and in whole-bone samples in response to exogenous parathyroid hormone. This is the first study to date examining alterations in gene expression in vivo in enhanced Author Manuscript Author Manuscript Author Manuscript Author Manuscript Exp Cell Res. Author manuscript; available in PMC 2016 May 01. Wattanachanya et al. Page 12 OB Gs signaling. We found only a small number of differentially expressed genes that overlap with their results, consistent with the notion that there are different OB transcriptome effects, depending on the timing and source of Gs activation. In conclusion, our study provides a useful tool to evaluate the in vivo gene expression specifically occurring in OBs with activated Gs-GPCR signaling, at the cellular level rather than in a whole bone. The study revealed that cell cycle and transcriptional regulation were the most highly enriched pathways. Complete set of GPCRs expressed in OBs and new candidate paracrine mediators in response to chronic Gs activation in OBs were also indentified. All these regulated genes could be used for further functional studies. Biological validation of the role of these mediators may lead to new therapeutic targets for enhancing bone formation in osteoporosis and for reversing bone disease associated with excess Gs signaling. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Ct Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments Funding support: This work was supported by grants to RAN from NIH and the Department of Veterans Affairs, to ECH from the NIH and the National Osteoporosis Foundation. We thank Richard Kao, Zhiqiang cheng and Yongmei Wang for valuable technical assistance and discussion. The prevailing model that explains the role of Aurora B in regulating the SAC posits that under low tension, Aurora B phosphorylates microtubule-binding kinetochore proteins resulting in reduced affinity for microtubules leading to dissociation of kinetochore-microtubule interactions 1014. As chromosomes become bi-oriented and sister kinetochores are under high tension, Aurora B is spatially separated from the kinetochore and can no longer destabilize kinetochoremicrotubule interactions. According to this spatial gradient model, inner centromere localization of the CPC is paramount for error correction 15. In yeast, however, INCENP can regulate chromosome segregation independent of centromere localization 16. CPC localization during metaphase is thought to rely on the intersection of two histone modifications at the centromere. Haspin kinase phosphorylates histone H3 on threonine 3 along chromosome arms and at centromeres, which is bound by Survivin 17,18. The kinetochore-localized kinase B.