ECTS Abstracts (2015) 1 OC3.1

Identification of chloride intracellular channel protein 3 as a novel gene affecting bone formation

Andrea Brum1, Cindy van der Leije1, Jeroen van de Peppel1, Mark Janssen2, Marco Eijken2, Johannes van Leeuwen1 & Bram van der Eerden1

1Erasmus MC, Internal Medicine, Rotterdam, The Netherlands; 2Arcarios BV, Rotterdam, The Netherlands.

Osteoporosis is a common skeletal disorder characterised by low bone mass leading to increased bone fragility and fracture susceptibility. Identification of specific factors that stimulate osteoblast differentiation from human mesenchymal stromal cells (hMSCs) may deliver therapeutic targets to treat osteoporosis. The aim is to determine novel factors and mechanisms involved in human osteoblast differentiation. Gene expression profiling was performed on hMSCs differentiated towards osteoblasts or adipocytes using Illumina microarrays. We selected genes that were differentially (twofold) regulated in the osteogenic vs the adipogenic condition, as well as up- or down-regulated (1.5-fold) vs time point zero. Based on bioinformatic analyses we identified the gene CLIC3 (Chloride Intracellular Channel Protein 3). Lentiviral overexpression of CLIC3 in hMSCs was used to assess the effect osteogenic differentiation. CLIC3 overexpression caused a 34% increase in both alkaline phosphatase activity (P=0.047) and mineralization (P=0.04). Next, we used an in vivo human bone formation model where hMSCs lentivirally transduced with the CLIC3 overexpression construct were loaded onto a scaffold (hydroxyapatite-tricalcium-phosphate) and implanted under the skin of NOD-SCID mice and analysed for bone formation after 8 weeks. CLIC3 overexpression led to a 15-fold increase in bone formation (0.33% vs 5.05% bone area relative to scaffold, P=0.0007). Knockdown of CLIC3 in hMSCs using two short hairpin RNAs against CLIC3 resulted in 89–96% reduction in CLIC3 mRNA expression (P=0.0037 and 0.0026, respectively) and 70–90% less mineralisation (P<0.0001 for both) compared with scrambled control. In conclusion, we successfully identified CLIC3 to be a lineage-specific gene regulating osteoblast differentiation and bone formation. CLIC3 encodes a membrane transport protein that may function in cell growth, vesicle transport, and integrin trafficking. We are currently using pull down and proteomic analysis to investigate the molecular mechanism underlying the CLIC3 control of osteoblast differentiation.

Disclosure: The authors declared no competing interests.

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