The Novel Secreted Adipokine WNT1-inducible Signaling Pathway Protein 2 (WISP2) Is a Mesenchymal Cell Activator of Canonical WNT

Abstract

WNT1-inducible-signaling pathway protein 2 (WISP2) is primarily expressed in mesenchymal stem cells, fibroblasts, and adipogenic precursor cells. It is both a secreted and cytosolic protein, the latter regulating precursor cell adipogenic commitment and PPARγ induction by BMP4. To examine the effect of the secreted protein, we expressed a full-length and a truncated, non-secreted WISP2 in NIH3T3 fibroblasts. Secreted, but not truncated WISP2 activated the canonical WNT pathway with increased β-catenin levels, its nuclear targeting phosphorylation, and LRP5/6 phosphorylation. It also inhibited Pparg activation and the effect of secreted WISP2 was reversed by the WNT antagonist DICKKOPF-1. Differentiated 3T3-L1 adipose cells were also target cells where extracellular WISP2 activated the canonical WNT pathway, inhibited Pparg and associated adipose genes and, similar to WNT3a, promoted partial dedifferentiation of the cells and the induction of a myofibroblast phenotype with activation of markers of fibrosis. Thus, WISP2 exerts dual actions in mesenchymal precursor cells; secreted WISP2 activates canonical WNT and maintains the cells in an undifferentiated state, whereas cytosolic WISP2 regulates adipogenic commitment.

Keywords: Adipose Tissue; Beta-catenin; Bone Morphogenetic Protein (BMP); Cell Biology; Obesity; Peroxisome Proliferator-activated receptor (PPAR); Wnt Pathway.

FIGURE 1.

WNT activation by WISP2 requires secretion of the ligand. A, full-length, but not the truncated WISP2 is secreted to the medium. Conditional medium from NIH3T3 cells transfected with wild-type Wisp2 (WT-Wisp2.myc) or mutated Wisp2 plasmid (MUT-Wisp2.myc), were collected followed by immunoprecipitation with anti-Myc antibody. B, full-length, but not the truncated WISP2, increases β-catenin and its nuclear targeting (Ser(P)-552) phosphorylation. Full-length WISP2 also increases the (Ser(P)-1490) phosphorylation of LRP6. NIH3T3 cells were transfected with wild-type Wisp2 (WT-Wisp2.myc) or mutated Wisp2- plasmid (MUT-Wisp2.myc) and/or Wisp2 siRNA. Cells were then incubated with or without WNT3A as shown for 48 h (n = 3). C, transcriptional activation of Tcf/Lef following addition of recombinant WISP2 (rec. WISP2), transfected Wisp2 (Wisp2.myc), or constitutively active β-catenin (β-catenin S33Y) in NIH-3T3 cells. Luciferase activity is normalized to that of the control samples (n ≥ 4). D, immunofluorescence staining of β-catenin (green) in the absence (left panel) or presence of recombinant WISP2 in NIH-3T3 cells for 48 h (right panel). DAPI staining (blue) for visualization of nuclear localization. E, the WNT antagonist DICKKOPF-1 (DKK1) reverses the inhibitory effect of recombinant WISP2 and WNT3A on activation of Pparg. F, Fabp4 in NIH3T3 cells incubated for 72 h with BMP4 and Wisp2 siRNA and DKK1 as shown (n = 3). G, Wisp2 siRNA, similar to adding DKK1, induces down-regulation of β-catenin protein, its nuclear targeting (Ser(P)-552) phosphorylation as well as (Ser(P)-1490) LRP6 phosphorylation. NIH3T3 cells were transfected with WT-Wisp2.myc or MUT-Wisp2.myc or Wisp2 siRNA. Cells were then incubated with or without DKK1 (200 ng/ml) as shown for 48 h (n = 2). ERK1/2 protein was used as a loading control. H, FLAG-tagged Wisp2-transfected NIH3T3 cells were incubated with the acylation inhibitor IWP2 (2 μm) for 24h. Medium was collected and immunoprecipitated with anti-FLAG antibody (n = 2). All data are means ± S.E. *, p < 0.05 and **, p < 0.01.

FIGURE 2.

WISP2 activates the canonical WNT pathway in 3T3-L1 adipose cells. A, WISP2 and WNT3A induce stabilization of β-catenin and its (Ser(P)-552) phosphorylation. WISP2 and WNT3A also activate and phosphorylate LRP6. ERK1/2 protein was used as a loading control. Quantification of β-catenin phosphorylation versus total β-catenin protein ratio and LRP6 phosphorylation versus total LRP6 protein ratio are shown. All proteins were normalized to ERK1/2 protein. B, WISP2 and WNT3A increase Axin2 mRNA level. Differentiated 3T3-L1 adipocytes were incubated with WISP2 or WNT3A as shown (n ≥ 6). Data are means ± S.E. *, p < 0.05 and **, p < 0.01. 1d, 1 day.

FIGURE 3.

WISP2 induces partial dedifferentiation of mature adipocytes. A, micro photos (10× magnifications) from Oil Red O-stained 3T3-L1 adipocytes incubated with/without recombinant WISP2 or WNT3A for 6 days. Both WISP2 and WNT3A significantly decreased the lipid accumulation (n = 7). Right, quantification of Oil Red O. Incubations with WISP2 and WNT3A as shown also reduced mRNA levels of Pparg, Cebpa, and Zfp423 (B) as well as Glut4, adiponectin, Fabp4, and Lpl (C) (n = 7). D, WISP2 increases the phosphorylation of ERK1/2 and p38 MAPK. Immunoblotting was performed on lysates from 3T3-L1 adipocytes incubated with WISP2 or WNT3A as shown (n ≥ 6). Data are means ± S.E. *, p < 0.05. 1d, 1 day.

FIGURE 4.

WISP2 induces a myofibroblast phenotype in 3T3-L1 adipose cells. A, α-SMA protein was increased by both WISP2 and WNT3A in the medium. ERK1/2 protein was used as a loading control and for normalization. B, Pdgfa, Syndecan-4, and Ctgf mRNA levels were increased following incubations with recombinant WISP2 or WNT3a as shown (n = 6). Data are means ± S.E. *, p < 0.05.

FIGURE 5.

Schematic illustration of the autocrine and paracrine effects of WISP2. Adipogenic differentiation of mesenchymal precursor cells involves both commitments to the adipose lineage with Pparg induction as well as adipose cell differentiation following PPARγ activation. WISP2 is both an intracellular and a secreted protein by mesenchymal precursor cells. Intracellular WISP2 retains ZFP423, the transcriptional activator of Pparg, from entering the nucleus and initiate adipogenic commitment of the precursor cells. Secreted WISP2, in an autocrine manner, activates the canonical WNT pathway through an unknown cellular signaling pathway involving LRP5/6. This prevents PPARγ activation and maintains the precursor cells in an undifferentiated state. This effect of WISP2 is antagonized by the canonical WNT inhibitor DICKKOPF-1 (DKK1). Thus, WISP2 exerts dual effects in the regulation of adipogenesis. As a secreted protein, WISP2 can also target differentiated 3T3-L1 adipocytes, inhibit PPARγ activation, and promote a myofibroblast phenotype. WISP2 may also target other peripheral cells, but this remains to be demonstrated.