Volume 14, Issue 7, Pages 860-869 (July 2007) A Triterpene Glycoside from Black Cohosh that Inhibits Osteoclastogenesis by Modulating RANKL and TNFα Signaling Pathways  Samuel X. Qiu, Chun Dan, Li-Sheng Ding, Shulin Peng, Shao-Nong Chen, Norman R. Farnsworth, Jan Nolta, Michael L. Gross, Ping Zhou  Chemistry & Biology  Volume 14, Issue 7, Pages 860-869 (July 2007) DOI: 10.1016/j.chembiol.2007.06.010 Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 1 ACCX Inhibits Osteoclastogenesis In Vitro (A) Structure of ACCX. (B and C) BMMs were grown in the presence of 10 ng/ml M-CSF and 100 ng/ml RANKL for 4 days to generate mature osteoclasts. ACCX was supplied to the medium at the indicated concentration for (B) 4 days (upper panels), for (B) 1 day at the beginning before being withdrawn for the remaining 3 days (lower panels), or (C) at 10 μM at the indicated time. Cells were stained for TRAP. Chemistry & Biology 2007 14, 860-869DOI: (10.1016/j.chembiol.2007.06.010) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 2 Effects of ACCX on Proliferation and Cell Death of BMMs (A) BMMs were grown in the presence of 10 ng/ml M-CSF with or without 100 ng/ml RANKL for 24 hr. Cells were simultaneously exposed to ACCX at the indicated concentrations. Cells were labeled with BrdU for 6 hr and were then assayed for BrdU incorporation (∗p < 0.01 versus nontreated). (B) BMMs were cultured with 10 ng/ml M-CSF and were exposed to ACCX at the indicated concentration for 24 hr. DNA fragmentation was assayed by cell death ELISA (∗p < 0.05, ∗∗p < 0.0005 versus nontreated; the error bars show standard deviations). Chemistry & Biology 2007 14, 860-869DOI: (10.1016/j.chembiol.2007.06.010) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 3 ACCX Alters RANKL Signaling Events and Suppresses Osteoclast Gene Markers (A) BMMs were grown in serum-free medium for 6 hr with or without exposure to 10 μM ACCX for 4 hr. Cells were subsequently stimulated with 100 ng/ml RANKL for the indicated time periods. Cell lysates were subjected to immunoblotting with antibodies as indicated. (B) BMMs were treated in a manner similar to that described in (A), but with indicated doses of ACCX, and were stimulated with RANKL for 15 min. (C) BMMs were treated with 20 μM ACCX for the indicated time periods and were stimulated with RANKL for 15 min. (D) Cells were treated with or without 20 μM ACCX for 4 hr, followed by RANKL (100 ng/ml) stimulation for 0 or 30 min. Nuclear extracts were allowed to bind biotin-labeled oligonucleotides bearing a κB site. Bound proteins were pulled down with streptavidin beads and were immunoblotted with a p65 antibody. Nucleophosmin in nuclear extracts served as an input control. (E) Similar to (A). (F) BMMs were cultured with 20 ng/ml M-CSF and 100 ng/ml RANKL with or without 10 μM ACCX. Cell lysates were collected at the indicated time periods and were subjected to immunoblotting with antibodies as indicated. Chemistry & Biology 2007 14, 860-869DOI: (10.1016/j.chembiol.2007.06.010) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 4 ACCX Blocks TNFα-Induced Osteoclastogenesis in Vitro and Alters TNFα- Elicited Signaling Events (A) BMMs were cultured with 50 ng/ml M-CSF and 50 ng/ml TNFα with or without 10 μM ACCX for 5 days. Cells were stained for TRAP. (B and C) BMMs were grown in serum-free medium with or without exposure to 10 μM ACCX for 4 hr. Cells were stimulated with 50 ng/ml TNFα for the indicated time periods. Cell lysates were analyzed by immunoblotting with indicated antibodies. (D) BMMs pretreated with or without 10 μM ACCX for 4 hr were stimulated with 200 ng/ml RANKL or 50 ng/ml TNFα for 30 min. Levels of TNFα, IκBα, and TLR2 mRNA were determined by RT-PCR. GAPDH mRNA served as loading controls. Chemistry & Biology 2007 14, 860-869DOI: (10.1016/j.chembiol.2007.06.010) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 5 In Vivo Osteoclastogenesis Induced by TNFα Is Reduced by ACCX Mice were injected daily with DMSO or ACCX with or without supracalvaria injection of TNFα for 5 days and were sacrificed the following day. (A) Histological sections of calvaria from these mice were stained for TRAP activity. (B) Histomorphometric quantitation of the percentage of bone surface covered by osteoclasts in calvaria of each group of mice (∗p < 0.005, ∗∗p < 0.02, versus DMSO; #p < 0.04, versus TNFα + DMSO; the error bars show standard deviations). Chemistry & Biology 2007 14, 860-869DOI: (10.1016/j.chembiol.2007.06.010) Copyright © 2007 Elsevier Ltd Terms and Conditions