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Bone Abstracts (2014) 3 PP150 | DOI: 10.1530/boneabs.3.PP150

ECTS2014 Poster Presentations Cell biology: osteoblasts and bone formation (48 abstracts)

PRKG1: A novel regulator of human skeletal (mesenchymal) stem cell differentiation

Abbas Jafari 1, , Majken Siersbaek 1, , Matthias Dobbelstein 3 & Moustapha Kassen 1,

1Danish Stem Cell Center (DanStem), Institute of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 2Department of Endocrinology and Metabolism, Endocrine Research Laboratory (KMEB), Odense University Hospital, Odense, Denmark; 3Department of Molecular Oncology, Göttingen Center of Molecular Biosciences, GZMB, University of Göttingen, Göttingen, Germany.

Protein kinases are an important class of regulatory elements and pharmacological targets for treatment of a number of diseases including cancer, heart and lung diseases. However, their role in regulation of the skeletal (mesenchymal) stem cell (MSC) functions and bone formation is not fully understood. Thus, we performed functional screening of human kinome, using three siRNAs for each of the ~700 known kinases represented in the human genome. Activity of alkaline phosphatase (ALP) was quantified to assess the effect of each siRNA on OB differentiation of human MSC (hMSC). We identified PRKG1 as a negative regulator of OB differentiation of hMSC. Follow up studies showed that siRNA-mediated loss of PRKG1 function (siPRKG1) enhanced OB differentiation of hMSC as shown by increased ALP activity, gene expression of the osteogenic markers ALP and Collagen type1 and ex vivo matrix mineralization and suppressed adipocyte differentiation of hMSC as shown by Oil Red O staining of mature adipocytes. Furthermore, activation of PRKG1 function using 8-pCPT-cGMP suppressed OB differentiation as shown by reduced ALP activity. We also identified the molecular mechanism of PRKG1 action mediated by phosphorylation and inactivation of the small GTPase RhoA. Thus, loss of PRKG1 function increased RhoA activity and Akt signaling, as shown by G-LISA RhoA activity assay and western blot analysis. To investigate the therapeutic benefits of targeting PRKG1 in vivo, we employed a small molecule kinase inhibitor of PRKG1 (H-8). Enhancement of heterotopic bone formation (96% as compared to vehicle) was observed when mice containing hMSC-loaded subcutaneous implants, received H-8 intraperitoneal injections (90 mg/kg per day for 4 weeks). Necropsy of the test animals, did not show any toxic effects of H-8 treatment. PRKG1 is a novel negative regulator of OB differentiation of hMSC and small molecule inhibition of PRKG1 enhances bone formation and has the potential to be developed as a novel anabolic therapy for treatment of bone-loss diseases.

Volume 3

European Calcified Tissue Society Congress 2014

Prague, Czech Republic
17 May 2014 - 20 May 2014

European Calcified Tissue Society 

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