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TRPV4 mediates marrow adipocyte remodeling in AML
nificantly after BM adipocytes were treated with rhGDF15 for 1 h (Online Supplementary Figure S2F, G), sug- gesting that the role of GDF15 in promoting lipolysis may be different from the rapid action of β-adrenaline.25 Oil red O staining and quantitative analysis showed that the number and area of adipocytes did not change significant- ly on the fourth day, but decreased significantly from the sixth day (Online Supplementary Figure S2H, I). These results indicate that TRPV4 regulates the remodeling of BM adipocytes.
To further explore the potential role of TRPV4 in GDF15-induced BM adipocyte remodeling, oil red O staining and quantitative analysis was conducted and showed that the number and area of BM adipocytes were decreased in BM adipocytes treated with rhGDF15, whereas 4aPDD partly reversed the effect of rhGDF15 (Figure 2D, E). Optical density value measurements of lipid droplets showed similar results (Figure 2F). Accordingly, rhGDF15 could induce increased expression of lipolysis genes (ATGL and HSL) and increased release of free fatty acids from BM adipocytes, but activation of TRPV4 by 4aPDD partly reversed the effect of rhGDF15 (Figure 2G, H). Furthermore, rhGDF15 can inhibit Ca2+ influx in BM adipocytes (Online Supplementary Figure S3A). These findings strongly suggest that TRPV4 con- tributes to GDF15-induced remodeling of BM adipocytes. Although GDF15 has been reported to act on Cav1.3, Cav3.1, Cav3.2, Cav3.3,13,26 the expression of these chan- nels in BM adipocytes is much lower than that of TRPV4 (Online Supplementary Figure S3B). Moreover, when BM adipocytes were co-cultured with leukemia cell lines (THP-1, K562, HL-60), the expression of TRPV4 changed significantly (Online Supplementary Figure S3C). These results further suggest that TRPV4 may play an important role in GDF15-induced remodeling of BM adipocytes.
GDF15 activates the downstream genes PI3K and pAKT in bone marrow adipocyte remodeling
reduce the number and area of BM adipocytes. Accordingly, rhGDF15 did not significantly reduce the TRPV4 protein (Figure 3G). These results further con- firmed that GDF15 regulates BM adipocyte remodeling by binding to TGFβRII.
As an important signaling downstream pathway of the TGFβ family, GDF15 could not cause significant changes in Smad2 and Smad4 proteins in BM adipocytes (Figure 3H and Online Supplementary Figure S4C). Notably, when PI3K was blocked by PI3K-IN-1 (a PI3K inhibitor), rhGDF15 could not regulate AKT phosphorylation (Figure 3I), sug- gesting that GDF15 is involved in the remodeling of BM adipocytes by activating the PI3K/AKT pathway.
The PI3K/AKT pathway inhibits the TRPV4 promoter FOXC1
PI3K/AKT acts as a signaling pathway downstream of GDF15, which may affect the transcription or translation of TRPV4. We compared the expression of different tran- scription factors with or without rhGDF15 treatment by RNA-sequencing analysis. The results showed that the expression of several transcription factors decreased, including FOXC1, Spalt-like gene-2 (SALL2), and MYC associated factor X (MAX) (Figure 4A). Based on the cri- teria of a fold-change >2.0 and P-value <0.05, FOXC1 was identified as a transcription factor that was signifi- cantly changed in BM adipocytes after rhGDF15 treat- ment (Figure 4B). To investigate whether FOXC1 is responsible for GDF15 regulating TRPV4, we knocked down FOXC1 in BM adipocytes (Online Supplementary Figure S4D, E). The results showed that the expression of TRPV4 at both the mRNA and protein levels was inhibit- ed in FOXC1 knockdown adipocytes (Figure 4C, D), sug- gesting that GDF15 reduced the expression of TRPV4 by negatively regulating the transcription factor FOXC1.
To further demonstrate the link between FOXC1 and TRPV4, we used a FOXC1 antibody to pull DNA frag- ments containing FOXC1 and used RT-qPCR to detect the TRPV4 gene in the fragment. The results showed that the control group had the sequence of the TRPV4 gene and the amount of TRPV4 gene was correspondingly decreased after knocking down FOXC1 (Figure 4E). These data suggest that FOXC1 combines directly with TRPV4. As shown in Figure 4F, rhGDF15 downregulated the expression of FOXC1 and TRPV4 protein, but PI3K-IN-1 can block this process. Taken together, these results again demonstrate that GDF15 regulates TRPV4 channels through the PI3K/AKT pathway.
TRPV4 plays an important role in bone marrow adipocyte remodeling in acute myeloid leukemia mice
To better understand the role of TRPV4 in leukemia cell-induced BM adipocyte remodeling, we investigated the changes in number and size of BM adipocytes with 4aPDD in mice with leukemia. FBL-3 is a mouse-derived AML cell line, which can spontaneously induce leukemia. We first confirmed that FBL-3 cells secrete GDF15 (Online Supplementary Figure S5A). In vitro, FBL-3 cells co-cultured with BM adipocytes inhibited the expression of TRPV4 protein (Online Supplementary Figure S5B). Online Supplementary Figure S5C, D shows that FBL-3 cells reduced the number and area of BM adipocytes, while 4aPDD could partly reverse this effect. Correspondingly, FBL-3 cells significantly promoted the expression of ATGL and HSL mRNA in BM adipocytes, but 4aPDD
Extracellular GDF15 must bind to a receptor on the membrane surface to cause intracellular changes in BM adipocytes. We screened all of the reported GDF15 recep- tors by RT-qPCR and found that BM adipocytes mainly express TGFβRI and TGFβRII (Figure 3A). In order to determine whether GDF15 acts through binding to TGFβRI or TGFβRII on BM adipocytes, we conducted inhibitor experiments in vitro.27,28 Western blot results showed that rhGDF15 could downregulate TRPV4 expression in BM adipocytes treated with RepSox (a TGFβRI inhibitor) rather than ITD1 (a TGFβRII inhibitor) (Figure 3B). Moreover, we found that rhGDF15 could reduce the number and area of BM adipocytes treated with RepSox as compared to those treated with ITD1 (Figure 3C, D). In accordance with data from RT-qPCR experiments, the levels of expression of lipolysis genes (ATGL and HSL) were significantly elevated in BM adipocytes treated with RepSox compared with the levels in BM adipocytes treated with ITD1 (Online Supplementary Figure S3D). These results suggest that TGFβRII is the major receptor that mediates GDF15 action on BM adipocytes.
To further verify the function of TGFβRII on BM adipocytes, we knocked down TGFβRII expression by shTGFβRII lentivirus (Online Supplementary Figure S4A, B). As shown in Figure 3E and F, when TGFβRII was knocked down in BM adipocytes, rhGDF15 did not significantly
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