Pref-1 interacts with fibronectin to inhibit adipocyte differentiation

Abstract

Pref-1/Dlk1 is made as an epidermal growth factor (EGF) repeat-containing transmembrane protein but is cleaved by tumor necrosis factor alpha converting enzyme (TACE) to generate a biologically active soluble form. Soluble Pref-1 inhibits adipocyte differentiation through the activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and the subsequent upregulation of Sox9 expression. However, others have implicated Notch in Pref-1 signaling and function. Here, we show that Pref-1 does not interact with, or require, Notch for its function. Instead, we show a direct interaction of Pref-1 and fibronectin via the Pref-1 juxtamembrane domain and fibronectin C-terminal domain. We also show that fibronectin is required for the Pref-1-mediated inhibition of adipocyte differentiation, the activation of ERK/MAPK, and the upregulation of Sox9. Furthermore, disrupting fibronectin binding to integrin by the addition of RGD peptides or by the knockdown of alpha 5 integrin prevents the Pref-1 inhibition of adipocyte differentiation. Pref-1 activates the integrin downstream signaling molecules, FAK and Rac, and ERK activation by Pref-1 is blunted by the knockdown of Rac or by the forced expression of dominant-negative Rac. We conclude that, by interacting with fibronectin, Pref-1 activates integrin downstream signaling to activate MEK/ERK and to inhibit adipocyte differentiation.

FIG. 1.

Pref-1 does not interact with Notch or require Notch for its signaling. (A) COS-7 cells were cotransfected with Notch together with Dll-HA, desnutrin-HA, and Pref-1 EC-HA. Cell lysates were subjected to immunoprecipitation (IP) with control IgG or anti-HA agarose beads followed by Western blotting with anti-Notch antibody. (B) 3T3-L1 cells were treated with 1 μM γ-secretase inhibitor L-685458 (Tocris Bioscience) for 24 h, serum starved for 4 h, and then treated with 50 nM hFc or Pref-1-hFc for 15 min. Western blotting using anti-phosphorylated ERK1/2 and total ERK1/2 antibodies are shown. 3T3-L1 cells were transfected with either control or Notch siRNA. After 48 h of transfection, cells were treated with hFc or Pref-1-hFc. Western blotting was performed using anti-Notch, phosphorylated ERK1/2, and total ERK1/2 antibodies. (C) 3T3-L1 cells were subjected to adipocyte differentiation in the presence of 1 μM γ-secretase inhibitor L-685458 or after Notch siRNA transfection in the presence of Pref-1-hFc. Oil red O staining (upper), quantification of lipid staining (lower left), and adipocyte marker expression levels by RT-qPCR (lower right) are shown.

FIG. 2.

Pref-1 interacts with fibronectin. (A) Pref-1JM interacts with C-terminal fibronectin in yeast-two hybrid assay (left). Staining of β-galactosidase activity of filter replicas of yeast plates double transformed with indicated plasmids. 1106A, positive clone; Pas2, empty vector. Schematic illustration of fibronectin structure and fibronectin fragments used (right panel). (B) COS-7 cells were cotransfected with Pref-1-HA and 52Fn-Myc, and lysates were subjected to IP followed by Western blotting. (C) Pulldown of 52Fn-Myc by Pref-1-HA beads. Lysates from COS-7 cells transfected with 52Fn-Myc were used for pulldown using Pref-1-HA beads and were detected by Western blotting (IB). The asterisk represents the IgG band. (D) ³⁵S-labeled Pref-1JM-HA and 52Fn were incubated, followed by IP with HA-antibody before SDS-PAGE and autoradiography. (E) Purified Pref-1EC was incubated with purified fibronectin fragments before IP with anti-Pref-1 antibody or normal IgG followed by Western blotting using anti-fibronectin antibody. (F) Fibronectin fragments separated by SDS-PAGE were transferred to nitrocellulose before incubation with Pref-1EC or Pref-1JM-AP in far-Western analysis. (G) Myc-tagged FnI or FnII was transfected into COS-7 cells, and lysates were used for pull-down assay with Pref-1EC-HA beads. The asterisk represents the IgG band.

FIG. 3.

Relationship between Pref-1 and fibronectin in the regulation of adipocyte differentiation. (A) Semiquantitative RT-PCR and Western blotting for various genes and proteins, respectively, during 3T3-L1 adipocyte differentiation. (B) Immunofluorescence micrograph for localization of Pref-1 and fibronectin. 3T3-L1 cells were immunostained with goat anti-Pref-1 antibody as the primary antibody and labeled with anti-goat IgG-Alexa fluor 488 (green). The cells also were stained with mouse antifibronectin antibody and labeled with anti-mouse IgG-Alexa fluor 594 (red). DAPI staining (blue) also is shown. (C) Pref-1EC and 52Fn-Myc were transfected into COS-7 cells (left), and the collected conditioned media were added to 3T3-L1 cells during differentiation. Oil red O staining (right upper panel) and its quantification (right lower left panel) are shown. Results are means ± SEM. The intensity of control cells after differentiation was defined as 1. P < 0.05 (*) and P < 0.01 (**) compared to control cells. Semiquantitative RT-PCR for the expression of adipocyte genes is shown.

FIG. 4.

Fibronectin is required for Pref-1 inhibition of adipocyte differentiation. (A) The 3T3-L1 cells transfected with fibronectin or control siRNA were subjected to adipocyte differentiation in the presence of hFc or Pref-1-hFc. Microscopic morphology (upper), Oil red O staining, and the quantification of lipid accumulation (middle) are shown. RT-qPCR and semiquantitative RT-PCR are shown as well (bottom). The value of control siRNA-transfected cells treated with hFc after differentiation was defined as 1. P < 0.05 (*) and P < 0.01 (**) compared to control cells. (B) Pref-1 null MEFs were transfected with control or fibronectin siRNA and treated with Pref-1-hFc. Western blotting was performed with antifibronectin, phosphorylated ERK1/2, and total ERK1/2 antibodies (left). Control or fibronectin siRNA-transfected cells were treated with hFc or Pref-1-hFc for 8 h before being harvested for Sox9 protein levels via Western blotting (right). (C) 3T3-L1 cells were transfected with fibronectin siRNA along with 1.0 kb C/EBPβ promoter-luciferase construct (left) or 2.0 kb C/EBPδ-promoter-luciferase construct (right). After 48 h, cells were treated with control hFc or Pref-1-hFc for an additional 16 h. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega). pRL-SV40 was used as internal control. Results are means ± SEM. **, P < 0.01. n.s, no significant difference.

FIG. 5.

α5 integrin is required for Pref-1 inhibition of adipocyte differentiation. (A) 3T3-L1 cells were differentiated in the presence of control DGR or RGD peptides and in the presence of hFc or Pref-1-hFc. Morphology and adipocyte marker expression levels by RT-qPCR and RT-PCR are shown. The value from cells treated with DGR peptides and hFc after differentiation was defined as 1. P < 0.05 (*) and P < 0.01 (**) compared to control cells. (B) Knockdown of α5 integrin in 3T3-L1 cells was verified by RT-PCR (left). Cells transfected with control or α5 integrin siRNA were subjected to adipocyte differentiation in the presence of hFc or Pref-1-hFc. Microscopic morphology, Oil red O staining (middle), and expression levels by RT-qPCR (right) are shown. **, P < 0.01 compared to control cells.

FIG. 6.

Downstream signaling pathway involved in the activation of ERK by Pref-1. (A) 3T3-L1 cells were treated with hFc or Pref-1-hFc for 1 h. Western blotting is shown. (B) Cells stably expressing control GFP or dominant-negative Rac (RacDN-GFP) were treated with Pref-1-hFc. Western blotting was performed with anti-GFP, phosphorylated ERK1/2, and total ERK1/2 antibodies (left). Oil red O staining and adipocyte marker expression levels determined by RT-qPCR are shown. The values from cells that first were transfected with control GFP vector or cells treated with control hFc after differentiation was defined as 1. P < 0.05 (*) and P < 0.01 (**) compared to control cells. (C) Western blotting of lysates from 3T3-L1 cells transfected with either control or Rac siRNA, treated with hFc or Pref-1-hFc, using the indicated antibodies. Oil red O staining and adipocyte marker expression levels by RT-qPCR are shown. The values from cells transfected with control siRNA or treated with control hFc after differentiation was defined as 1. P < 0.05 (*) and P < 0.01 (**) compared to control cells.