K. Igari1, M. J. Kelly1, B. Darwich1, D. Yamanouchi1 1University Of Wisconsin,Division Of Vascular Surgery, Department Of Surgery,Madison, WI, USA
Introduction:
We have previously reported the role of osteoclastogenic macrophage activation in abdominal aortic aneurysms (AAAs). Previous reports indicated Wnt signaling has the dual effect of proliferation and differentiation during osteoclatogenesis. Wnt/β-Catenin pathway is a critical regulator of cell pluripotency, cell survival, and cell fate decision in both embryos and adults. The inhibition of β-catenin suppressed proliferation but induced differentiation of osteoclast precursor cells. The aim of this study is to examine the effect of the Wnt signaling inhibitor, ICG-001, under the hypothesis that ICG-001 inhibits osteoclastogenesis through the inhibition of proliferation without induction of differentiation.
Methods:
RAW 264.7 macrophages were stimulated with soluble receptor activator of NF-kB ligand (RANKL) (30ng/ml) to induce the osteoclastogenesis. To examine the effect of the inhibition of the CBP co-activator in Wnt signaling, macrophages were treated with or without ICG-001 (10mM) during RANKL stimulation. The activation and differentiation of macrophages were examined by western blotting, quantitative PCR, and tartrate-resistant acid phosphate (TRAP) staining in vitro. Data are reported as the means ± standard deviation. P values less than 0.05 were accepted as statistically significant.
Results:
The relative expression level of protein of nuclear factor of activated T-cells cytoplasmic 1, a key transcription factor during osteoclastogeneis, was significantly suppressed by ICG-001 treatment compared to the non-treated group (4.46±0.74 versus 7.13±0.50, P < 0.05). Even though there was not a statistically significant difference in TRAP expression between the ICG-001 treated group and non-treated group (1.78±0.55 versus 2.35±0.38, P = 0.24), we showed the trend that ICG-001 decreased TRAP protein expression. Furthermore, the expression of cathepsin K was significantly suppressed in the ICG-001 treated group compared to the non-treated group (4.62±1.60 versus 12.2±1.73, P < 0.05). The relative expression levels of mRNA of TRAP, cathepsin K, and matrix metalloproteinase-9, were significantly lower in the ICG-001 treated group compared to the non-treated group (72.4±5.3 versus 167.1±5.6, P < 0.05, 38.6±7.2 versus 149.3±14.3, P < 0.05, and 175.5±10.5 versus 323.6±70.0, P < 0.05, respectively). Furthermore, TRAP-staining demonstrated the suppressive effect of ICG-001 on osteoclastogenesis. The number of TRAP-positive decreased in the ICG-001 treated group relative to the non-treated group (24.4±7.0 versus 131.2±19.4, P < 0.05).
Conclusion:
The inhibition of Wnt signaling pathway via ICG-001 suppressed osteoclastogenic macrophage activation. Our previous studies showed the importance of osteoclastogenic macrophage activation in AAA, therefore, further studies to examine the therapeutic potential of ICG-001 on AAA are warranted.