Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Multi-Targeted Kinase Inhibitor PKC412 Diminishes Osteoclastogenesis and Counteracts Osteoclast Mediated Bone Resorption In Vitro.

Sykoutri1,  Despoina, Hayer3,  Silvia, Smolen4,  Josef S., Redlich2,  Kurt

Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna
Krankenhaus Lainz, Vienna, Austria

Purpose:

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by osteoclast mediated bone erosions. Small molecule multi-kinase inhibitors are being explored as potential therapeutic targets in RA. PKC412 is a small molecule multi-kinase inhibitor targeting class III tyrosine-protein-kinases such as FMS-like tyrosine kinase 3 (FLT-3) and multiple isoforms of serine/threonine protein kinase C. PKC412 has been shown to inhibit macrophage function in vitro. However, the role of PKC412 in modulating the commitment of the monocyte/macrophage lineage to osteoclast precursors, their differentiation into pre-osteoclasts and their differentiation into mature osteoclasts has not been fully elucidated. We aimed to investigate the effect of PKC412 on osteoclast differentiation and function.

Methods:

We differentiated mouse bone marrow derived cells in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL) into tartrate-resistant acid phosphatase positive (TRAP+) mononuclear osteoclasts (pre-osteoclasts) and TRAP+ multinucleated mature osteoclasts, and added PKC412 in increasing concentrations (10nM to 10mM) to the culture. We also assessed the role of PKC412 in the bone resorbing capacity of osteoclasts by culturing osteoclasts on dentine slices. To further characterize the effect of PKC412 on cell proliferation we performed MTT assays. We used quantitative PCR to evaluate expression levels of mRNA encoding for osteoclast specific markers such as nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), matrix metalloproteinase 9 (MMP-9), Cathepsin K and TRAP. Flow cytometry analysis for Annexin V and 7-AAD was performed to determine potential apoptotic effects of PKC412 on pre-osteoclasts and osteoclasts.

Results:

We found that increasing concentrations of PKC412 (IC50: 250nM) dose-dependently reduced osteoclast numbers. Furthermore, numbers of pre-osteoclasts were also significantly decreased after addition of PKC412, indicating an effect of PKC412 on early stages of osteoclastogenesis. In line with this finding, we could show a dose-dependent reduction of pre-osteoclast proliferation using MTT proliferation assays. Moreover, we detected a significant time- and dose-dependent increase in the ratio of apoptotic cells in the PKC412-treated cells by Annexin V and 7-AAD staining. Additionally, in the presence of PKC412 we obtained a significant reduction in osteoclast size and nuclei number, as well as in the size of resorption pits on dentin slices. This indicates that the bone resorbing capacity of osteoclasts is altered by PKC412. Consistently, we were able to demonstrate a dose dependent downregulation of mRNA coding for osteoclast markers, such as NFATc1, MMP-9, Cathepsin K and TRAP in the presence of PKC412.

Conclusion:

These results suggest a regulatory role of PKC412 in pre-osteoclast differentiation und osteoclastogenesis through apoptosis induction.

To cite this abstract, please use the following information:
Sykoutri, Despoina, Hayer, Silvia, Smolen, Josef S., Redlich, Kurt; Multi-Targeted Kinase Inhibitor PKC412 Diminishes Osteoclastogenesis and Counteracts Osteoclast Mediated Bone Resorption In Vitro. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1493
DOI: 10.1002/art.29259

Abstract Supplement

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