Page 85 - 2020_11-Haematologica-web

TRPV4 mediates marrow adipocyte remodeling in AML
opment.8 As the breast cancer progresses, adipocytes de- differentiate to fibroblast-like cells.8 In mouse models of bone metastasis following prostate cancer, Herroon et al. showed that remodeled BM adipocytes support tumor growth by fatty acid-binding protein 4 (FABP4) trans- portation of fatty acids.9 These studies identified a func- tional role of remodeled adipocytes in supporting solid tumor metabolism. Thus, the adipocytes remodeled by cancer cells are also known as cancer-associated adipocytes.10 In the context of leukemia, there is a grow- ing consensus that reduction in BM adipocyte number, once believed to be merely due to mechanical squeezing by the rapid proliferation of leukemia cells in the limited BM cavity, is also actively regulated by leukemia cells.5,11 Indeed, we previously reported that growth differentia- tion factor 15 (GDF15) derived from leukemia cells regu- lates BM adipocyte remodeling by enhancing lipolysis.12 However, how extracellular GDF15 induces lipolysis within BM adipocytes remains elusive.
It has been reported that GDF15 enhances intracellular Ca2+ by increasing calcium voltage-gated channel subunit alpha1 C (Cav1.3) expression in rat cerebellar granule neurons, which induces the expression of genes essential for synaptic plasticity.13 As an important cellular signal for lipid metabolism, intracellular Ca2+ is involved in lipid synthesis and lipolysis in adipocytes.14,15 When the calci- um channels in the adipocytes are activated or upregulat- ed, accumulation of lipids is enhanced through increased [Ca2+]i.16,17 Conversely, when calcium channels are inhibit- ed or downregulated, decreased calcium influx may accel- erate fat breakdown.18,19 Thus, we hypothesized that cal- cium channels are involved in GDF15-induced BM adipocyte remodeling.
In this study, we examined a possible role of transient receptor potential vanilloid 4 (TRPV4) calcium channels in GDF15-driven remodeling of BM adipocytes. We unravel a novel function of transforming growth factor-β type II receptor (TGFβRII) that, in responding to GDF15 in BM adipocytes, activates the phosphatidylinositol 3- kinase (PI3K)/AKT transduction pathway, which in turn reduces the transcript factor Forkhead box C1 (FOXC1) level and subsequently downregulates TRPV4. We also provide evidence that inhibition of BM adipocyte remod- eling increases survival in the AML mouse model, imply- ing a novel therapeutic target for AML.
Methods
Patients’ samples
BM aspirates were collected from 16 patients diagnosed as having lymphoma without BM invasion, using procedures approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Hospital. Mesenchymal stem cells were derived from the BM of lymphoma patients without BM inva- sion, because marrow mesenchymal stem cells in this type of patients can be considered normal. The adipogenic induction of mesenchymal stem cells is described in the Online Supplementary Methods.
Chromatin immunoprecipitation-quantitative polymerase chain reaction
Adipocytes were collected from different groups and crosslinked with 1% formaldehyde for 10 min at 37°C. Cross- linking was blocked, then the cells were washed and lysed in
sodium dodecylsulfate lysis buffer (50 mM HEPE NaOH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% Na-deoxycholate, 1% Triton X100). The lysates were sonicated to shear DNA to a length between 200 and 500 base pairs with 10-second pulses using son- ication. The antibody against FOXC1 (5 mL, Abcam5079, USA) was then added to the supernatant, incubated overnight at 4°C with rotation and incubated with 100 mL Salmon Sperm DNA/Protein A agarose beads for 2 h at 4°C. The immunoprecip- itated complex was then washed and eluted. The histone DNA crosslinks were reversed and DNA was purified for real-time poly- merase chain reaction (PCR). Quantitative real-time PCR (RT- qPCR) was performed on bound and input DNA with the follow- ing primers for TRPV4: forward: 5-CTTTGCACTGGGGAGCA- GAGT-3, reverse: 5-ATTAACCG TGGGCTTCAGGCA-3.
Cell cultures and reagents
The cell cultures and reagents, as well as the co-culture assays are described in detail in the Online Supplementary Methods.
Animal experiments
All animal experiments were performed according to proce- dures approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Hospital. Five-week old C57BL/6 mice were fed with 60% high-fat diet (Research Diets, Inc. New Brunswick, NJ, USA) for 3 months to create an obese mouse model. Mice injected with FBL-3 cells (5×105) and mice injected with both FBL-3 cells (5×105) and 4a-phorbol 12,13-didecanoate (4aPDD) (200 mg/Kg according to the instructions for reagents) were used as experimental groups. The untreated obese mice were used as a control group. The volume of all solutions inject- ed was 200 mL. Mice were sacrificed and femora were removed after 3 weeks of treatment. Femora were fixed for 24 h with 4% paraformaldehyde and were decalcified for 2 days. BM sections from the mice were dewaxed by conventional methods and incubated with anti-perilipin1 monoclonal antibody (1:50, CST, USA) at 4°C overnight.
Other experimental details
Full descriptions of free fatty acid detection, lentiviral knock- down, RNA sequencing, western blot analysis and enzyme- linked immunosorbent assays, RT-qPCR (primers shown in Table 1), the cell counting and apoptosis assays, oil red O stain- ing, immunofluorescence studies, and adipocyte measurements are provided in the Online Supplementary Methods.
Statistical analysis
All statistical tests were performed with GraphPad Primer5. The data are presented as the mean ± standard deviation. A Student t-test was used for comparisons between two groups. A P value of less than 0.05 was considered statistically significant.
Results
Downregulated TRPV4 contributes to increased bone marrow adipocyte lipolysis
As an important channel for calcium ions, TRPV plays a critical role in the energy balance of adipocytes. RT- qPCR analysis showed that TRPV4 mRNA in BM adipocytes had the highest expression among TRPV fam- ily members (Figure 1A). Moreover, western blot analysis showed that BM adipocytes expressed TRPV4 protein highly (Figure 1B). To investigate whether TRPV4 plays an important role in BM adipocytes, we used TRPV4 inhibitor (RN1734) and agonist (4aPDD) to verify the
haematologica | 2020; 105(11)
2573