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J. Teramachi et al.
of TNF-α signaling in BMSC by TAK1 inhibition. VLA-4, α4 b1 integrin, is constitutively overexpressed in MM cells. We found that the TAK1 inhibition with LLZ was able to reduce the expression of b1 integrin in MM cells (Online Supplementary Figure S3), indicating the contribu- tion of TAK1 to b1 integrin expression in MM cells. Consistently, treatment with LLZ suppressed MM cell adhesion to BMSC (Figures 3E; Online Supplementary Figure S4), although LLZ did not impair the viability of BMSC (Online Supplementary Figure S5).
BMSC are regarded as a major source of IL-6, a growth and survival factor for MM cells, and the critical osteoclas- togenic factor RANKL. Consistent with the previous observations,30,31 cocultures with MM cells potently aug- mented IL-6 (Figures 3F) and RANKL (Figures 3G) mRNA expression in BMSC. However, treatment with LLZ sup- pressed the upregulation of these factors in BMSC in cocultures with MM cells. These data demonstrate that TAK1 inhibition is able to efficaciously suppress MM cell adhesion to BMSC and thereby abolish the upregulation of IL-6 and RANKL in BMSC, which may alleviate MM tumor progression in the bone marrow and bone destruc- tion.
TAK1 inhibition suppresses osteoclastogenesis enhanced by RANKL and multiple myeloma cells
Consistent with previous observations,20,34 RANKL induced the phosphorylation of TAK1 in parallel with the degradation of IκBα and phosphorylation of p38MAPK and ERK (Figure 4A), and nuclear localization of the NF-κB subunit p65 (Figure 4B) in RAW264.7 preosteoclastic cells. However, treatment with LLZ abolished all of these RANKL-mediated changes, indicating critical involvement of TAK1 in RANKL-induced activation of the NF-κB and MAPK pathways. RANKL induced the expression of NFATc1 and c-fos, critical transcription factors for osteo- clastogenesis (Figure 4C), and the formation of TRAP-pos- itive multinucleated cells, namely OC, in RAW264.7 cells (Figures 4D); however, treatment with LLZ dose-depen- dently suppressed the RANKL-induced expression of NFATc1 and c-fos, and OC formation. TAK1 knockdown by siRNA also abolished the induction of NFATc1 and c-fos expression (Figure 4E) and osteoclastogenesis (Figure 4F) by RANKL. Furthermore, MM cells potently induced TRAP-positive multinucleated OC formation from bone marrow cells; however, treatment with LLZ suppressed OC formation (Figure 4G). These results demonstrate that TAK1 inhibition is able to suppress osteoclastogenesis enhanced by MM cells.
TAK1 inhibition restores osteoblastogenesis suppressed by multiple myeloma cells as well as major inhibitors for osteoblastogenesis in multiple myeloma
In contrast to the enhanced osteoclastogenesis, osteoblastogenesis or bone formation is suppressed in MM. Conditioned media (CM) from MM cell lines as well as inhibitory factors for osteoblastogenesis overproduced in MM, including IL-3, IL-7, TNF-α, TGF-b, and activin A,8-12 induced the phosphorylation of TAK1 (Figure 5A) and suppressed mineralized nodule formation (Figure 5B) in MC3T3-E1 preosteoblastic cells. However, treatment with the TAK1 inhibitor LLZ restored mineralized nodule formation (Figure 5B). Osterix is an essential transcription factor for osteoblastogenesis, known as a downstream tar- get of BMP-2. The upregulation of Osterix by BMP-2 was
reduced in MC3T3-E1 cells in the presence of CM from MM cell lines or TNF-α; however, treatment with LLZ restored the upregulation of Osterix by BMP-2 (Figure 5C).
TGF-b inhibits the terminal stage of OB differentiation or bone mineralization, whereas BMP-2 is a stimulator for osteoblastogenesis.11,35-38 We and others demonstrated that TGF-b plays a significant role in bone destruction in MM, and that the inhibition of the TGF-b signaling restored bone formation in MM animal models.11,39-41 Treatment with TGF-b induced the phosphorylation of Smad2 and Smad3 in MC3T3-E1 cells (Figure 5D). However, TAK1 inhibition with LLZ as well as TAK1 knockdown by siRNA abolished the phosphorylation of these factors. TGF-b has been shown to counteract the BMP-2 signaling to suppress the terminal differentiation of OB in part through the upregulation of Smad6, an inhibitory regula- tor for BMP-2 signaling.42 Treatment with LLZ for 24 hours dose-dependently reduced Smad6 protein levels in MC3T3-E1 cells (Figure 5E, upper). Moreover, LLZ inhib- ited TGF-b-induced upregulation of Smad6 in MC3T3-E1 cells (Figure 5E, lower). In contrast, treatment with LLZ as well as TAK1 knockdown by siRNA enhanced the phos- phorylation of Smad1/5 in MC3T3-E1 cells by BMP-2 (Figure 5F). These results collectively suggest that TAK1 inhibition may resume osteoblastogenesis suppressed in MM.
TAK1 inhibition suppresses vascular endothelial growth factor secretion by multiple myeloma cells
Angiogenesis also plays an important role in the patho- genesis and progression of MM. Vascular endothelial growth factor (VEGF) appears to be the most critical angiogenic factor in MM.43,44 VEGF has been demonstrated to be overproduced downstream of the signaling mediator ERK in MM cells.45 As expected, TAK1 inhibition with LLZ as well as TAK1 knockdown with siRNA substantial- ly reduced VEGF production by MM cells (Online Supplementary Figure S6). These results suggested that TAK1 inhibition can impair angiogenesis in MM to retard MM progression.
TAK1 inhibition suppresses multiple myeloma tumor progression and prevents bone destruction in vivo
We next examined the in vivo effects of the TAK1 inhibitor LLZ using MM mouse models by intratibial inoc- ulation of mouse 5TGM1 MM cells. Mice were treated with LLZ every other day for 2 weeks from day 6, the day on which 5TGM1 MM cell-derived IgG2b levels started to increase in mouse sera. Vehicle-treated mice showed at day 21 large tumor masses around the tibiae where MM cells were inoculated (Figure 6A), and a progressive increase in serum IgG2b levels over time (Figure 6B). Bone destruction in the tibiae was observed at day 21 in plain X-ray as well as m-computed tomography (m-CT) images (Figure 6C). Treatment with LLZ substantially suppressed tumor sizes (Figure 6A) with almost no increase in serum IgG2b (Figure 6B), and prevented bone destruction of the tibiae (Figure 6C). Cathepsin K-expressing OC increased in number on the surface of bone in 5TGM1-inoculated tibiae; however, treatment with LLZ reduced the OC numbers (Figure 6D). These results demonstrate that TAK1 inhibition is able to suppress MM tumor growth while preventing bone destruction in vivo.
In order to further clarify the effects of TAK1 inhibition
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