RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

Abstract

Multifunctional RNA helicase DDX3 participates in HCV infection, one of the major causes of hepatic steatosis. Here, we investigated the role of DDX3 in hepatic lipid metabolism. We found that HCV infection severely reduced DDX3 expression. Analysis of intracellular triglyceride and secreted ApoB indicated that lipid accumulations were increased while ApoB secretion were decreased in DDX3 knockdown HuH7 and HepG2 cell lines. Down-regulation of DDX3 significantly decreased protein and transcript expression of microsomal triglyceride transfer protein (MTP), a key regulator of liver lipid homeostasis. Moreover, DDX3 interacted with hepatocyte nuclear factor 4 (HNF4) and small heterodimer partner (SHP), and synergistically up-regulated HNF4-mediated transactivation of MTP promoter via its ATPase activity. Further investigation revealed that DDX3 interacted with CBP/p300 and increased the promoter binding affinity of HNF4 by enhancing HNF4 acetylation. Additionally, DDX3 partially relieved the SHP-mediated suppression on MTP promoter by competing with SHP for HNF4 binding which disrupted the inactive HNF4/SHP heterodimer while promoted the formation of the active HNF4 homodimer. Collectively, these results imply that DDX3 regulates MTP gene expression and lipid homeostasis through interplay with HNF4 and SHP, which may also reveal a novel mechanism of HCV-induced steatosis.

Figure 1. HCV infection suppresses DDX3, MTP expression and ApoB secretion.

(a) HCV infection reduces the expression of DDX3 and MTP. The total cell extracts of HCV-infected/uninfected Huh-7.5.1 (see Materials and Methods) were collected and subjected to immunoblotting using anti-MTP (Santa Cruz Biotechnology), anti-DDX3 and anti-core (Abcam) antibodies, and GAPDH was used as a loading control. Intracellular protein levels were quantified by ImageJ, normalized to GAPDH and represented as amount relative to that of uninfected Huh-7.5.1 cells. (b) HCV infection leads to reduced secretion of ApoB. Culture media harvested from HCV-infected/uninfected Huh-7.5.1 cells were subjected to ApoB detection by ELISA (see Materials and Methods). Results are represented as means ± S.D. for at least three independent experiments and ***indicates p < 0.001, is considered to be significant.

Figure 2. Knockdown of DDX3 inhibits the expression of MTP and leads to lipid accumulation.

(a) Lipid accumulation was visualized by Oil Red O staining. The parental HuH7 cells, vector control and two DDX3 knockdown cell lines (siDDX3-433-33 and siDDX3-433-52) were fixed and stained with Oil Red O. The images were observed by light microscopy. Scale bars, 25 μm. Absorbance of isopropanol-eluted Oil Red O was then detected by a spectrophotometer. Results shown in the right panel was represented as the relative fold of the absorbance compared with the vector control. (b and c) DDX3 knockdown leads to accumulation of triglycerides and reduced secretion of ApoB. The cell lysates and culture medium of parental HuH7 cells, vector control and two DDX3 knockdown cell lines were subjected to detection of triglycerides and ApoB as described in Materials and Methods, respectively. Relative ApoB secretion in panel c is compared to the ApoB level of vector control cells. (d) HuH7 and HepG2 cells were transfected with control siRNA (siCtrl) or DDX3 siRNA (siDDX3). After 72 hr, cells were fixed, stained and Oil Red O absorbance was measured as described in panel a. (e and f ) DDX3 silencing represses the expression of MTP. The total cell extracts of two DDX3 knockdown cell lines, vector control, parental HuH7 as well as siCtrl/siDDX3 HuH7 and HepG2 cells were collected then subjected to immunoblotting using anti-MTP and anti-DDX3 antibodies, and GAPDH was used as a loading control. The relative fold of MTP and DDX3 amount in panel e was normalized by GAPDH and compared to those in vector control cells. (g) Detection of DDX3 and MTP mRNA levels by quantitative RT-PCR assay. Total cellular RNA was isolated from two DDX3 knockdown cell lines and vector control, then real time RT-PCR was performed with primers specific to mRNA of DDX3 or MTP. The GAPDH mRNA served as an internal control. Results shown in panel a,b,c,d and g are represented as means ± S.D. for at least three independent experiments. **p < 0.01; ***p < 0.001.

Figure 3. DDX3 interacts with SHP and HNF4 in vitro and ex vivo.

(a) Coomassie brilliant blue staining of E. coli BL21 (DE3) expressed recombinant proteins, GST and GST-DDX3. (b) In vitro GST pull down assays. Purified GST and GST-DDX3 proteins prebound with glutathione-Sepharose 4B resins were incubated with nuclear extracts (200 μg) of HuH7 and HepG2 cells overexpressing HA-SHP or HA-HNF4 with or without RNase A treatment. Associated proteins were eluted and resolved by SDS-PAGE, then immunoblotted using anti-HA antibody (Roche). Input: nuclear extracts of HuH7 expressed HA-SHP (5%, 10 μg) or HuH7 expressed HA-HNF4 (2.5%, 5 μg). (c and d) In vivo co-immunoprecipitation assays. HuH7 cells were cotransfected with plasmids expressing Flag-DDX3 (10 μg) and HA-SHP (10 μg) (panel c) or with Flag-DDX3 (10 μg) and HA-HNF4 (10 μg) (panel d) expression constructs by calcium phosphate co-precipitation method. Nuclear extracts (200 μg) were isolated at 48 hr post-transfection and immunoprecipitated using anti-HA antibody- conjugated agarose beads (panel c) or anti-FLAG M2 affinity resins (panel d). The immunoprecipitates were analyzed by SDS-PAGE, then immunoblotting with anti-FLAG (Sigma) and anti-HA antibodies.

Figure 4. DDX3 up-regulates the HNF4-mediated transactivation of MTP promoter.

(a) Schematic representation of pGL2/MTP-Luc (−611/+87) reporter plasmid. (b) DDX3 potentiates transactivation activity of HNF4 on MTP promoter. MTP promoter (−611~+87) -driven reporter plasmid (0.25 μg) was transfected alone or together with HA-HNF4 and HA-DDX3 expressing constructs in HuH7 and HepG2 by lipofectamine 2000. The total amount of transfected plasmids was adjusted to 2.5 μg/35 mm dish by supplementing with control vector, pcDNA/HA. After 48 hr, cells were collected and subjected to luciferase activity assay. (c) Schematic representation of pMTP(mutant)-Luc reporter plasmid, with the mutated sequences specified (underlined). (d) Destruction of HNF4-responsive elements blocks transactivation activity of HNF4 on MTP promoter. pGL2/MTP-Luc (−611/+87) reporter plasmid (Wild-type; 0.25 μg) or pMTP(mutant)-Luc plasmid (mutant; 0.25 μg) was transfected alone or together with HA-HNF4 expressing construct (0.25 μg) as indicated in HuH7 by lipofectamine 2000. The total amount of transfected plasmids was adjusted to 2.5 μg/35 mm dish by supplementing with control vector, pcDNA/HA. Forty-eight hours later, cells were collected and subjected to luciferase activity assay. (e) Destruction of HNF4-responsive elements blocks synergistic transactivation activity of HNF4/DDX3 on MTP promoter. pMTP(mutant)-Luc reporter plasmid (0.25 μg) was transfected alone or together with HA-HNF4 and HA-DDX3 expressing constructs as indicated amount in HuH7 and HepG2 by lipofectamine 2000. The total amount of transfected plasmids was adjusted to 2.5 μg/35 mm dish by supplementing with control vector, pcDNA/HA. Cells were collected and subjected to luciferase activity assay 48 hr post-transfection. The relative luciferase activity of pGL2/MTP-Luc (−611/+87) reporter alone is arbitrarily taken as one. All data are represented as the average (mean ± S.D.) of at least three independent experiments. (f) ATPase activity of DDX3 is required for its transactivation activity of HNF4 on MTP promoter. HuH7 and HepG2 cells were transfected with pGL2/MTP-Luc (−611/+87) reporter plasmid (0.25 μg) alone or together with indicated amount of plasmids expressing HA-HNF4, HA-DDX3(WT), HA-DDX3(DQAD) and HA-DDX3(AAA). Luciferase activity was measured 48 hr post-transfection.

Figure 5. DDX3 enhances HNF4 binding to the MTP promoter.

(a) Western blot analysis of subcellular localization of DDX3. HuH7 cells were transfected with plasmid expressing HA-HNF4 alone or together with HA-DDX3 expression construct as indicated. After 48 hr, cytosolic fractions and nuclear extracts (20 μg of each) were prepared and subjected to SDS-PAGE then immunoblotting with anti-HA antibody. (b) HNF4 DNA binding ability is enhanced by DDX3. The nuclear extracts of transfected HuH7 cells were incubated with annealed biotinylated probe containing HNF4-responsive element and subjected to binding with streptavidin agarose. After extensive washing, the bound fractions were analyzed by SDS-PAGE, followed by immunoblotting with anti-HA antibody. (c) Western blot analysis of the ectopically expressed Flag-HNF4 and HA-DDX3. HuH7 cells were transfected with plasmids expressing HA-DDX3 and Flag-HNF4 as indicated. Western blot analysis was performed 48 hr posttransfection. The expression level of HA-DDX3 and Flag-HNF4 was detected using anti-HA and anti-Flag antibodies, respectively. (d) Overexpression of DDX3 enhances the DNA binding activity of Flag-HNF4 on MTP promoter. Chromatin immunoprecipitation assay was performed by isolation of soluble chromatin fragments prepared from transfected-HuH7 cells and then immunoprecipitated with anti-FLAG M2 agarose resins. Immunoprecipitates were analyzed by quantitative real-time PCR with specific primers for MTP promoter (as described in Materials and Methods). Results are expressed as means ± S.D. for at least three independent experiments and **p < 0.01, is considered to be significant.

Figure 6. DDX3 interacts with CBP/p300 and induces the acetylation status of HNF4.

(a) DDX3 interacts with CBP and p300 in vivo. HuH7 cells were transfected with HA-DDX3 expressing plasmid or vector control. After 48 hr, the nuclear fractions (200 μg) were collected and incubated with antibodies against CBP or p300, and then the mixtures were bound to protein G sepharose beads. The precipitates were subjected to immunoblotting with anti-HA antibody. The IgG-conjugated protein G sepharose beads (mouse, lane 3 and lane 8) incubated with HA-DDX3 expressed nuclear extracts were used as negative control. Input; 10% (20 μg) of the nuclear extract. (b) Ectopic expression of DDX3 induces the acetylation status of HA-HNF4. HuH7 cells were transfected with plasmid expressing HA-HNF4 alone or together with HA-DDX3 expression construct as indicated. The whole cell extracts (1 mg) prepared from the transfected cells were subjected to immunoprecipitation of HA-HNF4 with anti-HA agarose beads. After extensive wash, the total amounts and acetylated forms of immunoprecipitated HA-HNF4 were detected by Western blot analysis with anti-HA and anti-acetylated lysine (Cell Signaling Technology) antibodies, respectively. (c) In situ proximity ligation assay (PLA) indicates that ectopic expression of DDX3 induces endogenous HNF4 acetylation. HuH7 cells transfected with either GFP or GFP-DDX3 expressing plasmids for 48 hr were fixed and subjected to PLA with anti-HNF4 (Abcam) and anti-acetylated lysine antibodies. Anti-NS5A antibody (Austral Biologicals) served as control. Nuclei were stained with DAPI (blue). The numbers of PLA signals per cell were quantified using MetaMorph software (Molecular Devices) and are shown as means ± S.D. relative to that of GFP-transfected cells. n = 50. ***p < 0.001.

Figure 7. DDX3 partially relieves the SHP-mediated repression effect on MTP promoter.

HuH7 cells were transfected with pGL2/MTP-Luc(−611/+87) reporter plasmid (0.5 μg) alone or together with a combination of indicated amount of HA-HNF4, HA-SHP and HA-DDX3 expression plasmids. After 48 hr, cells were harvested and lucifearse activity assay was performed. The total amount of transfected plasmids was kept constant by adding control vector, pcDNA/HA. The relative luciferase activity is represented as activation fold compared to basal expression level of the MTP reporter plasmid alone (lane 1). Results are represented as the average (mean ± S.D.) of at least three independent experiments.

Figure 8. HNF4 interacts with DDX3 through one of its SHP-interacting domains.

(a) Schematic representation of the GST fusion proteins containing N-terminal, middle and C-terminal regions of HNF4. The binding results of the GST pull down analysis shown in Fig. 6b are summarized. (b) Mapping the interaction domains of DDX3 and SHP with HNF4. The GST and GST-HNF4 truncated derivatives prebound with glutathione Sepharose beads were incubated with whole cell lysates (500 μg) from HuH7 cells expressing HA-DDX3 and HA-SHP, respectively. After extensive wash, the bound proteins were resolved by SDS-PAGE and analyzed by Western blot with anti-HA antibody. Lane 5, input: total cell lysate (20 μg) of HuH7 cells expressing HA-tagged DDX3 or HA-tagged SHP. Lane 6, total cell lysate (20 μg) of HuH7 cells. Coomassie brilliant blue staining of the GST-HNF4 truncated derivatives is shown in the lower panel.

Figure 9. DDX3 disrupts the formation of SHP/HNF4 heterodimer and promotes the formation of the active HNF4 homodimer.

(a and b) HuH7 cells were transfected with HA-SHP expression plasmid alone or together with Flag-HNF4 expression plasmid as indicated. (a) Immunoblotting was performed with antibodies against HA and Flag. (b) The total cell lysates (2 mg) of co-expressed Flag-HNF4 and HA-SHP were incubated with anti-Flag M2 agarose resins. After extensive washing, increasing amounts of purified GST-DDX3 proteins (as indicated on the top, lane 3–5) were added to the mixtures of the beads with bound fractions. The immunoprecipitated proteins were analyzed by immunoblotting with antibodies against Flag, HA and DDX3. The GST-DDX3 alone (lane 1) and cell lysates of expressed HA-tagged SHP (lane 2) incubated with anti-Flag M2 agarose resins were used as negative control. (c,d and e) Similar experiments were performed as shown in panel a and b, except HuH7 cells were transfected with HA-HNF4 expression plasmid alone or together with Flag-SHP expression plasmid (c and d), or purified GST protein was used instead of GST-DDX3 in (e). (f) Overexpression of DDX3 increases HNF4 homodimer formation. HuH7 cells were transfected with either GFP or GFP-DDX3 expressing plasmids together with HA and Flag vectors (−) or expressing plasmids for HA-HNF4 and Flag-HNF4 (+). Forty-eight hours later, cells were fixed and analyzed by PLA with anti-HA (Abcam) and anti-Flag antibodies. Nuclei were stained with DAPI (blue). In cells cotransfected with HA-HNF4 and Flag-HNF4, the numbers of PLA signals per cell were quantified using MetaMorph software and are shown as means ± S.D. relative to that of GFP-cotransfected cells. n = 50. ***p < 0.001.