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TRPV4 mediates marrow adipocyte remodeling in AML
were taken 21 days after tail vein injection of FBL-3 cells. The shape of BM adipocytes did not change significantly, but the number and area decreased (Figure 5A). Further quantitative analysis showed that the number of BM adipocytes in AML mice (219±37.7/mm2) was lower than that in the controls (505±49.7/mm2) and AML mice treat- ed with 4aPDD (334.4±39.6/mm2), but the number of BM adipocytes in AML mice treated with 4aPDD was still lower than that in the controls (the t-test for any two groups: P<0.05) (Figure 5B). Similarly, the BM adipocyte area in mice with leukemia was 860.0±142.5 mm2, which was smaller than that in the controls (1686.4±106.7 mm2, P<0.001). Meanwhile, the area of BM adipocytes in AML mice treated with 4aPDD was 1111.8±201.5 mm2, which was larger than that of the BM adipocytes in AML mice (P<0.01), and did not return to normal (P<0.001) (Figure 5C).
We further found that there were more CD117 (a pro- genitor cell expression marker, red fluorescence)-positive cells in AML mice than in the AML mice treated with 4aPDD (Figure 5D). This may be due to the fact that GDF15 secreted by AML cells promotes lipolysis and is beneficial to the proliferation of leukemia cells, while 4aPDD partly prevents lipolysis. In fact, the content of GDF15 in the BM supernatant was higher in AML mice than in the controls (Figure 5E), suggesting that GDF15 secreted by leukemia cells can promote lipolysis of BM adipocytes. Furthermore, we observed that treatment with 4aPDD significantly extended overall survival of AML mice (Figure 5F). In brief, these results suggest that targeting TRPV4 in BM adipocytes can delay the progres- sion of leukemia in mice.
Discussion
We have demonstrated a possible mechanism whereby TRPV4 mediates BM adipocyte responses to extracellular GDF15. Our data show that AML cells drive this remod- eling process, at least in part, through TRPV4-dependent lipolysis in the adipocytes. Our previous reports linked increased levels of small adipocytes in BM to poor prog- nosis in AML patients, and revealed that GDF15 derived from leukemia cells remodels mature BM adipocytes into small adipocytes.5,12 Here, we found that GDF15 binds to its receptor TGFβRII on BM adipocytes, which in turn activates downstream target genes, including PI3K and AKT. Subsequently, TRPV4 is inhibited via downregula- tion of its transcription factor FOXC1. These results sug- gest that GDF15 regulates TRPV4 through the above pathway, thereby promoting BM adipocyte remodeling (Online Supplementary Figure S6). This finding is consistent with several reports that TRPV4 acts as a volume receptor rather than an osmotic receptor.30-32
There has been a report that TRPV4 is located on the cell membrane and acts as a calcium channel.33 Therefore, TRPV4 can regulate energy metabolism of peripheral white adipocytes by facilitating Ca2+ influx, which in turn stimulates the ERK1/2-dependent pathway.34 GDF15 inhibits the expression of TRPV4 in BM adipocytes, resulting in a decrease of Ca2+ influx (Online Supplementary Figure S3A) and an increase in pHSL protein (Online Supplementary Figure S2E) after BM adipocytes were treat- ed with rhGDF15 for 4 days. It has been reported that reduced Ca2+ influx causes an increase in the expression of
pHSL, leading to lipolysis of adipocytes.17 Notably, the TRPV4 channel is a tetrameric complex formed by the same or similar monomeric subunits.35 Interestingly, cytosolic N- and C-terminal domains are involved in channel gating and mediating intracellular signaling,35,36 indicating that it is impossible for TRPV4 to interact directly with exogenous chemical factors. Hence, it would be interesting to examine how TRPV4 communi- cates with extracellular GDF15.
Given our findings that extracellular GDF15 inhibited the expression of TRPV4 in BM adipocytes, we speculat- ed that GDF15 acts on BM adipocytes through TGFβ receptors. As a member of the TGFβ superfamily, GDF15 is known to interact with receptors of TGFβ members, such as TGFβRI, TGFβRII, ALK4 and ACVR2.37-39 In addi- tion, GDF15 has unique cognate receptors, such as GFRAL, which is mainly expressed in the central nervous system and, at low levels, in testicular tissue.39 Our data show that BM adipocytes express TGFβ receptors, but not the known unique GDF15 receptors. Moreover, TGFβRII was shown to be associated with GDF15 activ- ity on BM adipocytes (Figure 3B-G). These experiments inform the first step of GDF15 acting on the adipocytes.
Our study further revealed that PI3K/AKT activation plays an essential role in driving GDF15 regulation of tar- get genes in BM adipocytes. In fact, GDF15 induced the activation of Smad, a component of the classic anti-apop- tosis pathway of cardiomyocytes which promotes the progression of lung cancer.40,41 However, we did not focus on the Smad pathway in this study because the activated Smad protein type is known to be determined by the TGFβRI present in the ligand-bound signal complex.42 In fact, GDF15 did not affect the Smad signaling pathway in BM adipocytes, which is consistent with our results (Figure 3H and Online Supplementary Figure S4C).Taken together, our data, when interpreted in the context of pre- vious reports, suggest that the PI3K/AKT pathway may be important for GDF15-induced remodeling of BM adipocytes.
Interestingly, we observed that PI3K/AKT activation downregulated the TRPV4-associated transcription factor FOXC1. FOXC1 is also a transcription factor of ITGA7 and FGFR4 in colorectal cancer, CXCR4 in endothelial cells, and FGF19 in ciliary body-derived cells.43-45 Moreover, the transcriptional function of FOXC1 has not been described previously for some pivotal adipogenic genes (FABP4, CEBPA and PPARG) and lipolytic genes (ATGL and HSL). Based on the knockdown of FOXC1 gene, the lipolytic gene in BM adipocytes increases (Online Supplementary Figure S4F), which confirmed that FOXC1 is important for regulating the metabolism of BM.
In addition, TRPV4 can be activated or inhibited by physical and chemical factors. When it comes to the mat- ter of the size of cells, TRPV4 acts as a volume receptor rather than an osmotic receptor,30 suggesting that TRPV4 is involved in the regulation of cell volume. Previous stud- ies have suggested that TRPV4 is an important inflamma- tory factor because TRPV4 levels are increased in inflamed tissues and activation of TRPV4 causes inflam- mation.46 This protein is also closely related to inflamma- tion of white adipose tissue.33,47 However, the decreased expression of TRPV4 in leukemia-associated BM adipocytes implies that TRPV4 is not a major pro-inflam- matory factor of leukemia-associated BM adipocytes. We conclude that downregulated TRPV4 preferentially pro-
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