DDX5/p68 RNA helicase expression is essential for initiating adipogenesis
© Ramanathan et al. 2015
Received: 29 July 2015
Accepted: 1 December 2015
Published: 4 December 2015
DDX5/p68 RNA helicase is a member of the DEAD (Asp-Glu-Ala-Asp) box proteins. Apart from RNA unwinding, DDX5 is an important transcriptional factor and co-activator in cell proliferation and differentiation.
Here, we have characterised the role of DDX5 in adipogenesis in 3T3-L1 cells using siRNA mediated suppression. Transient inhibition of Ddx5 mRNA expression at the start of adipogenesis impairs the differentiation programme even when DDX5 expression is restored later in adipogenesis. However transient suppression of Ddx5 at the later stages of adipogenesis do not impair adipogenesis or triglyceride accumulation suggesting Ddx5 expression is dispensable in a mature adipocyte.
These results implicate DDX5 as a crucial factor involved in the complex transcriptional cascade of events that regulate adipogenesis and essential to the initiation of adipogenesis.
DEAD (Asp-Glu-Ala-Asp) Box Protein 5 (DDX5), or p68 RNA helicase is a multifunctional nuclear factor, first identified by its immunological cross-reactivity to a monoclonal antibody to the large T antigen of simian virus 40 . DDX5 is a prototypical member of the DEAD/H-box protein family. The DEAD box family is characterized by a region of nine conserved amino acid motifs including Asp-Glu-Ala-Asp (DEAD) which are required in cellular functions such as pre-mRNA processing and ribosome biogenesis . Co-purification of DDX5 with spliceosomes suggested a role in RNA splicing and this was subsequently confirmed when DDX5 was shown to be an essential splicing protein acting at the U1 snRNA-5′ splice site . More recently, DDX5 has been reported to co-activate several transcription factors in proliferation and differentiation that are themselves highly regulated, including the estrogen receptor α (ERα), tumour suppressor p53, androgen receptor (AR), myogenic regulator MyoD, and the osteoblast differentiation factor Runx2 . DDX5 has been hinted at to be involved in adipogenesis in one report , although with rudimentary characterisation. Here we sought to clarify the requirement of DDX5 in adipogenesis using a transient knockdown strategy.
Materials and methods
Cell culture and adipogenesis
3T3-L1 murine preadipocytes and C3H10T1/2 murine mesenchymal stem cells were cultured in high glucose (4500 mg/L) Dulbecco’s modified Eagle medium (DMEM) supplemented by 10 % heat-inactivated Newborn Calf Serum (NCS) (Sigma-Aldrich N4637) as described . Two days after reaching 100 % confluence, cells were induced to differentiate into adipocytes in high glucose DMEM supplemented with 10 % Fetal Calf Serum (FCS) as described . Oil Red O staining of lipid accumulation was performed as described  and triglyceride levels were quantified using a triglyceride quantification kit (Abcam, ab65336) according to the manufacturer’s protocol.
RNA isolation and quantitative real-time PCR (qRT-PCR)
SYBR green primers used for quantitative real-time PCR
Western blot analysis
Cell lysis and western blotting was performed as described . Antibodies used for western blotting were against PPARγ (E8)(Santa Cruz, sc-7273, 1:1000), DDX5 (PAb204, kind gift from Frances-Fuller Pace, University of Dundee, 1:500) and GAPDH (FL-335, sc-25778, 1:2000).
siRNA mediated suppression of gene expression
The SMARTpool siRNA ON-TARGETplus mouse siRNA (Thermo Scientific) designed to specifically target mouse Ddx5, Pparg and Cebpb. The SMARTpool siRNA consists of four individual duplexes designed to obtain a high level of gene silencing. The non-targeting siRNA is a negative control siRNA with at least four mismatches to any human mouse or rat gene
Non-targeting siRNA #2
Cell viability assay
Cells were treated with WST-1 reagent using the WST-1 Cell Viability Assay Kit (Cayman Chemicals, #10008883) according to the manufacturer’s protocol. Briefly, preadipocytes were seeded at a density of 104 cells/cm2 in a 96-well plate in triplicates. Cells were normalized to untreated cells. Bleomycin (Sigma-Aldrich, B8416) was used as a negative control (cytotoxic agent) at 200 μg/ml. Both siRNA treatment and bleomycin treatment were administered for 48 h.
Here we demonstrate that DDX5/p68 RNA helicase peaks in expression on day 2 of adipogenesis in two different murine cell lines (Fig. 1a–d). These observations led us to speculate that this transient expression is required at the early stage of adipogenesis. The transcriptional pathways controlling adipocyte differentiation are relatively well characterized . Adipogenesis is regulated by a cascade in the expression of a variety of transcription factors where an early wave of factors is induced immediately at the start of differentiation. These factors then activate a second set of transcription factors that are responsible in maintaining the mature and functional adipocyte. Utilising a transient knockdown strategy, we observed that DDX5 is essential during the initial stages of adipogenesis (Fig. 3) but not at later stages (Fig. 4). This suggests a role for DDX5, potentially as a transcriptional co-activator, at the early stages of adipocytic differentiation, placing it in the same category as other well established transcriptional factors such as C/EBPβ/δ and the Kruppel-like factors (KLFs) . In future studies it will be of interest to examine how the loss of Ddx5 affects lipid droplet biology given the increasing evidence of genes expressing lipid droplet associated proteins being implicated in metabolic diseases such as lipodystrophy . This unappreciated role of DDX5 in adipogenesis provides further insight into adipogenic molecular pathways, providing an additional potential target to address metabolic diseases including obesity and type 2 diabetes that are of increasing importance to global public health.
NR and CLS are supported in part by the Singapore Biomedical Research Council and the Singapore Agency for Science, Technology and Research (A*STAR). NL is supported in part by Nanyang Technological University (NTU), Singapore and the Singapore Biomedical Research Council.
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