Levamisole suppresses adipogenesis of aplastic anaemia‐derived bone marrow mesenchymal stem cells through ZFP36L1‐PPARGC1B axis

Abstract Aplastic anaemia (AA) is a life‐threatening hematopoietic disorder characterized by hypoplasia and pancytopenia with increasing fat cells in the bone marrow (BM). The BM‐derived mesenchymal stem cells (MSCs) from AA are more susceptible to be induced into adipogenic differentiation compared with that from control, which may be causatively associated with the fatty BM and defective hematopoiesis of AA. Here in this study, we first demonstrated that levamisole displayed a significant suppressive effect on the in vitro adipogenic differentiation of AA BM‐MSCs. Mechanistic investigation revealed that levamisole could increase the expression of ZFP36L1 which was subsequently demonstrated to function as a negative regulator of adipogenic differentiation of AA BM‐MSCs through lentivirus‐mediated ZFP36L1 knock‐down and overexpression assay. Peroxisome proliferator‐activated receptor gamma coactivator 1 beta (PPARGC1B) whose 3′‐untranslated region bears adenine‐uridine‐rich elements was verified as a direct downstream target of ZFP36L1, and knock‐down of PPARGC1B impaired the adipogenesis of AA BM‐MSCs. Collectively, our work demonstrated that ZFP36L1‐mediated post‐transcriptional control of PPARGC1B expression underlies the suppressive effect of levamisole on the adipogenic differentiation of AA BM‐MSCs, which not only provides novel therapeutic targets for alleviating the BM fatty phenomenon of AA patients, but also lays the theoretical and experimental foundation for the clinical application of levamisole in AA therapy.


| INTRODUCTION
Aplastic anaemia (AA) is a rare and life-threatening hematopoietic disorder characterized by hypoplasia and pancytopenia with fatty bone marrow (BM). 1 The pathogenic factors causally associated with AA may include immune abnormality, quantitative and qualitative defects in hematopoietic stem/progenitors cells and altered marrow microenvironment. 2,3 Bone marrow mesenchymal stem cells (MSCs) can differentiate into osteoblast, adipocytes and chondrocytes, which together constitute the major cellular components of BM microenvironment providing critical support for hematopoiesis. 4 There is increasing evidences that BM-MSCs derived from AA patients display decreased proliferation, aberrant morphology, altered transcriptome profile and impaired differentiation. [5][6][7] Aplastic anaemiaderived bone marrow mesenchymal stem cells are more susceptible to be induced to differentiate into adipocyte at the expense of osteoblast, 8

| Cell culture
Mesenchymal stem cells were isolated from the BM of AA patients and healthy controls and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco). Seven AA patients (4 males and 3 females aged 16-68) and 6 controls (3 male and 3 female aged 27-56) were enrolled in this study. The AA patients were diagnosed according to the guidelines previously reported. 14 All the human samples were obtained from haematology department of the affiliated hospital of Jining Medical University and informed consent to perform the biological studies was obtained from the examined subjects and the related study was approved by the Ethics Committees of the hospitals and the Institutional Review Board of Jining Medical University. 293TN cells were cultured in DMEM with 10% FBS.
Levamisole (Sigma) treatment was performed by adding levamisole into the AM at final concentration of 150 μg/mL.

| Oil red O staining
The cells were rinsed with PBS twice in the plates after discarding the supernatant and fixed with 4% paraformaldehyde at room temperature for 20 minutes. The cells were then washed with PBS and stained with oil red O (Solarbio) for 20 minutes followed by washing with PBS. Lipid droplets were observed and photographed using an IX71 Olympus microscope (Olympus, Tokyo, Japan). Quantification of the staining was performed using Image-Pro Plus 6.0 software.

| RNA extraction and qRT-PCR analysis
Total RNA was extracted from cell samples using TRIzol Reagent (Invitrogen) and quantified using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Bremen, Germany). The first strand of cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Oligo (dT) was used as the primer for reverse transcription of mRNAs. qRT-PCR was performed in a Bio-Rad CFX-96 System (Bio-Rad, Foster City, CA, USA) using the SYBR Premix (CWBio). The primers used for reverse transcription and qRT-PCR were listed in Table S1.
The shRNA sequences for PPARGC1B were synthesized, annealed and inserted into pSIH-H1 (System Biosciences, SBI). The primers LIU ET AL.

| Lentivirus production and cell infection
The recombination lentiviruses for over-expression and knockdown were produced using the pmiRNA1-and pll3.7/pSIH-H1-based constructs. Lentivirus packaging was performed using the pPACKH1 ™ Lentiviral Vector Packaging Kit (LV500A-1; System Biosciences, SBI) according to the manufacturer's instructions. The culture medium supernatant containing the virus particles was directly used to infect the BM-MSCs in 12-well plates with 5 μg/mL polybrene (Sigma Aldrich). After 24-hour infection, the cells were replaced with fresh complete medium and induced towards adipogenic differentiation.

| GFP reporter assay
Two hundred and ninety-three TN cells were cotransfected with pcDNA6-GFP-PPARGC1B (or pcDNA6-GFP) and pcDNA6-ZFP36L1 (or pcDNA6) using UltraFection 2.0 (Beijing 4A Biotech) in 6-well plates. The transfection medium was replaced with complete medium after 5-6 hours. The cells were cultured at 37°C in 5% CO 2 for an additional 24-48 hours. Then, the GFP expression pictures were observed and captured under Fluorescence microscope Olympus IX71 (Olympus). The transfected cells were also collected, rinsed twice with PBS, resuspended in 20-μL PBS and analysed immediately using a FACSCalibur flow cytometer (BD Biosciences, USA).

| Statistical analysis
Student's t test (2-tailed) was performed to analyse the data. Statistical significance was set at P < .05, as indicated by an asterisk (*P < .05; **P < .01). The result obtained is also consistent with the previous literatures, 8 and to some extent, explained the fatty phenomenon of AA marrow.

| Levamisole inhibits the adipogenic differentiation of AA BM-MSCs
To seek a promising drug candidate which may be used to inhibit adipogenesis and improve BM microenvironment of AA, we tested a series of small molecules using the in vitro adipogenic differentiation model of AA BM-MSCs and finally focused on levamisole whose chemical structure was shown in Figure  F I G U R E 1 BM-MSCs derived from AA patients have increased adipogenic capacity relative to that from health controls. The 3rd passage BM-MSCs derived from AA patients and controls were incubated with adipogenic medium for 14 d. A, The cells were stained with oil red O and presented in the left, and the relative quantification of the staining was analysed and shown in the right. B, Adipogenic markers including PPARγ, PLIN1, LPL and FABP4 were detected using qRT-PCR. Actin was used as a loading control. *P < .05 and **P < .01, Student's t test. AA, aplastic anaemia; BM, bone marrow; MSCs, mesenchymal stem cells

| ZFP36L1 shows increased expression upon levamisole treatment and acts as a negative regulator of adipogenic differentiation
To reveal the underlying mechanism of levamisole in suppressing adipogenic differentiation of AA BM-MSCs, we first consulted the gene expression profiles of adipogenic differentiation and AA patients published before [16][17][18]

| PPARGC1B mRNA is identified as a direct target of ZFP36L1
ZFP36L1 is a RNA binding protein that mainly binds to the adenineuridine-rich elements (AREs) in the 3′-UTRs and promotes the decay of the target RNAs. 19 To search for downstream targets of ZFP36L1, we first consulted the AREsite (http://nibiru.tbi.univie.ac.at/AREsite 2/welcome) 20 and downloaded the mRNAs bearing UUAUUUAUU motif in their 3′UTRs, which is implicated by previous work that ZFP36L2, a ZFP36L1 homolog, binds to UUAUUUAUU motif with priority. 21 There are 1393 mRNAs containing UUAUUUAUU motif.
We first focused LPL and PPARGC1B that have been reported to be associated with adipogenesis. 22 binding motif were cloned into pcDNA6-GFP for GFP reporter assay, as depicted in Figure 5D. The GFP expression was presented as fluorescence pictures and flow cytometry analysis ( Figure 5E,F), which demonstrated that ZFP36L1 negatively regulates GFP expression in PPARGC1B-3′UTR-dependant manner. Similar results were not observed in LPL-3′UTR-GFP reporter assay ( Figure S1), implying that LPL may not be a direct target of ZFP36L1.

| Knockdown of PPARGC1B impairs adipogenic differentiation of AA BM-MSCs
PPARGC1B was originally identified by virtue of its function to stimulate the activity of several transcription factors and nuclear receptors. 24 To investigate the effect of PPARGC1B on adipogenic differentiation, we make use of the recombined lentivirus that express specific short hairpin RNA for PPARGC1B (lenti-shPPARGC1B) to infect AA BM-MSCs followed by adipogenic induction for 14 days. qRT-PCR analysis revealed that lenti-shPPARGC1B infection significantly decreased PPARGC1B mRNA expression ( Figure 6A), which resulted in significant down-regulation of the mRNA levels of the adipogenic differentiation markers (PPARγ, PLIN1, LPL and FABP4; Figure 6B) as compared with the lenti-ctrl F I G U R E 5 PPARGC1B mRNA is verified as a direct target of ZFP36L1. A, PPARGC1B expression was detected using qRT-PCR during the adipogenic differentiation with and without levamisole treatment. B-C, The mRNA level of ZFP36L1 and PPARGC1B was determined in AA BM-MSCs infected with lenti-shZFP36L1 (or lenti-ctrl) and lenti-ZFP36L1 (or lenti-ctrl), respectively. D, Schematic outline of PPARGC1B-GFP reporter construct. E-F, GFP reporter assay. 293TN cells were co-transfected with each pcDNA6-GFP-based constructs (pcDNA6-GFP and PPARGC1B) and pcDNA6-ZFP36L1 (or pcDNA6). The relative GFP expression was presented as fluorescence pictures (E) and also analysed by flow cytometry (F). **P < .01, Student's t test. AA, aplastic anaemia; BM, bone marrow; MSCs, mesenchymal stem cells; PPARGC1B, peroxisome proliferator-activated receptor gamma coactivator 1 beta LIU ET AL.  25 It is known that the adipogenic and osteogenic differentiation of MSCs is a dynamic process and well balanced in normal BM, the imbalance of which may lead to diseases. 26,27 Previous studies have suggested that BM adipocytes are predominantly negative regulators of the BM microenvironment and are less supportive to hematopoiesis than those of other cell types derived from MSCs, such as osteoblasts. 28,29 The BM of AA patients often exhibits increasing adipocytes and decreasing osteoblasts, 3  RNA binding proteins are a kind of post-transcriptional regulator and have been implicated to influence various aspects of RNA metabolism and participate in many physiological and pathological processes, including hematopoiesis 34 and adipogenesis. 35 To reveal the underlying mechanism of levamisole in inhibiting adipogenesis, we unexpectedly found that levamisole could increase the expression of ZFP36L1 which is an AREs binding protein. Adenine-uridine-rich elements are a group of loosely defined adenylateuridylate-rich instability determinants with sizes ranging from 50 to 150 nucleotides mainly found in the 3′UTRs of mRNAs. 36 ZFP36L1 belongs to the zinc finger protein family and has been implicated to bind AREs in the 3′UTRs of mRNAs through its RNA binding domain, resulting in destabilization of the target RNAs. 37 As a post-transcriptional regulator, ZFP36L1 has been reported to participate in erythroid differentiation and monocyte/macrophage differentiation by directly targeting stat5b and CDK6 expression, 38,39 respectively. ZFP36L1 can also act as a tumour suppressor to influence tumorigenesis through negatively regulating VEGFA and Bcl2 expression. 40,41 However, the role of ZFP36L1 in adipogenesis has only been implicated by the report that it could enhance the degradation of PPARγ2 mRNA transcript in 3T3-L1 preadipocytes. 42 In this study, through lentiviruses-mediated ZFP36L1 over-expression and knockdown assay, we demonstrated that ZFP36L1 functions in the downstream of levamisole and acts as a negative regulator in the adipogenic differentiation of the human AA BM-MSCs. Further mechanistic analysis revealed that PPARGC1B mRNA who contains AREs in its 3′UTRs is verified as a direct target of ZFP36L1. As a PPARγ coactivator, PPARGC1B has previously been reported to be correlated with fatty acid oxidation and mitochondrial biogenesis, 43

CONFLI CT OF INTERESTS
The authors declare that they have no competing interests.