gms | German Medical Science

Objectives: Osteoporosis continues to be a significant cause of bone fractures in elderly patients and an overactivity of the bone resorbing osteoclasts plays a relevant role in the pathophysiology of the disease. Fusion of monocytic precursor cells is typically observed during osteoclastogenesis [1]. The transmembrane protein CD9 is in general essential for fusion of plasma membranes [2] and therefore also in the development of osteoclasts. It is known that high-dose RANKL, a cytokine necessary for osteoclastogenesis, leads to increased CD9-expression [3]. Here we tested, if a stable, initial CD9-overexpression itself can lead to enlarged and/or overactive osteoclasts.

Methods: The stable human cell line THP-1 was transduced with a lentiviral vector containing CD9 and GFP. The overexpression of CD9 was shown in gene expression and western blot analysis. Using RANKL and M-CSF the cells were differentiated to mature osteoclasts on plastic and on bovine bone slices. Morphology and activity were analyzed with TRAP+DAPI-staining after 7 days and toluidine blue staining of bone slices after 28 days.

hPBCMs were isolated from human buffy coats and magnetically sorted for CD14 to increase the number of monocytes. The same lentiviral vector was used to transduce CD9-GFP into the monocytes for 72 h. Analysis of morphology and activity followed an analogue procedure to the THP-1-cells after 28 days. For the determination of cell size and the area of resorption per bone slice an interactive machine learning tool was trained (ilastik: interactive machine learning for (bio)image analysis).

Results and conclusion: Transducted THP-1-cells showed a significant CD9-overexpression in fluorescence microscopy, on RNA level (mean effect: 8.47; p<0.0001) and protein level (mean effect: 13.8; p<0.0001). They differentiatied to TRAP+, multinuclear cells. Cells with CD9 overexpression displayed a higher number of nuclei than the control cells (p=0.01) while the cell surface area remained unchanged. Neither the THP-1-wildtype nor the transduced cells showed any signs of resorption after cultivation on bone slices.

After CD9-GFP lentiviral transduction of CD14+ hPBMCs a strong GFP signal was observed in fluorescence microscopy. After 28 days of stimulation with RANKL and M-CSF multinucleated and TRAP+ cells as well as bone resorption were visible in both cell types. The quantitative analysis showed no significant difference in total resorbed area, the average resorption area size or the average diameter of the resorption pits, which supports the result of the unchanged cell size of the THP-1-cells.

CD9 seems to promote fusion and increase the number of nuclei per osteoclast but has no influence on the activity of the cells. This suggests that the number of nuclei is not directly connected to the activity of osteoclasts. Further research will be focused on the influence of CD9 on other osteoclast properties like cathepsin K or carboanhydrase II expression and the mechanism behind the increased multinuclearity.