MID1 and MID2 regulate cell migration and epithelial-mesenchymal transition via modulating Wnt/β-catenin signaling

Abstract

Background: The ubiquitin E3 ligase activity has been ascribed to MID1, the causative gene of X-linked OS, and its homologue, MID2. Both alpha4, the common MID protein partner, and PP2Ac in MID-alpha4-PP2Ac complexes can be ubiquitylated. Ubiquitylation of alpha4 converted its function toward PP2Ac from protective to destructive, while PP2A also affected MID protein phosphorylation and their subsequent trafficking on microtubules. It was believed that disruption of the function of MID1-alpha4-PP2A complex was vital to the pathogenesis of craniofacial malformation, the most prominent clinical manifestation of OS, although the detailed molecular mechanisms was not unravelled.

Methods: The cellular level of PP2A and phosphor-PP2A in cells overexpressing MID1/MID2 or in cells with siRNA mediated MID1/MID2 gene silencing was analyzed using Western blot. The Wnt signaling in these cells was further monitored using TCF/LEF luciferase reporter assay and the cellular level of β-catenin was also verified using western blot. Given the crosstalk of E-cadherin and Wnt via the common effector β-catenin, the potential influences of MID1/MID2 on the cell migration and epithelial-mesenchymal transition (EMT) were investigated using wound healing assay and immunofluorescence for E-cadherin and vimentin, respectively.

Results: Here, we presented the increased phosphorylation of PP2Ac in cells overexpressing MID1/MID2, and vice versa, in vitro, while the cellular level of total PP2Ac was unaffected. In addition, β-catenin, the effector of canonical Wnt signaling, was downregulated in cells overexpressing MID1/MID2 and upregulated in cells with siRNA mediated MID1/MID2 gene silencing. Down-regulated Wnt/β-catenin signaling by Okadaic acid, a specific inhibitor of PP2A, was partially rescued by siRNA mediated MID1/MID2 gene silencing. In consistent, an activated EMT and accelerated cell migration in cells with MID1/MID2 gene silencing were observed, and vice versa.

Conclusions: The results in this study indicated roles for MID1 and MID2 in regulating cell migration/EMT via modulating Wnt/β-catenin signaling, which might help to understand the molecular etiology of the facial abnormalities that are usually the consequences of defective neural crest cells migration and EMT at the early stage of craniofacial development.

Keywords: MID1; MID2; Wnt signaling; craniofacial development.

Figure 1

The phosphorylation of PP2Ac, not the total level of PP2Ac, was affected by the expression of MID1/MID2. The total cellular PP2Ac and phosphor-PP2Ac 307 were determined by immunoblotting in cells overexpressing MID1/MID2 or in cells with siRNA mediated knock-down of MID1/MID2 (A and C). The relative expression of PP2Ac and p-PP2Ac were quantified by scanning densitometry and normalized with respect to total β-catenin (B and D). Graphs represent the average of 3 independent experiments (mean ± SEM; student t-test; *, P<0.05, **, P<0.01, ****, P<0.0001). It was observed that the phosphorylated PP2Ac was significantly increased in cells overexpressing MID1/MID2 and decreased in cells with MID1/MID2 gene silencing, while the change of the cellular level of total PP2A was unnoticed.

Figure 2

MID1/MID2 regulate Wnt signaling via their influence on PP2A’s enzyme activity. The expression of β-catenin were examined by immunoblotting in cells overexpressing MID1/MID2 or in cells with siRNA mediated knock-down of MID1/MID2, followed by quantification using scanning densitometry and shown as the average of 3 independent experiments (mean ± SEM; student t-test; *, P<0.05, **, P<0.01, ****, P<0.0001). In addition, the Wnt signaling monitored using Tcf/Lef luciferase reporter assay. The cells were transfected with pTOP/FOP-FLASH and the constitutively active vector encoding for Renilla luciferase, with MID1/MID2 expressing vector or MID1/MID2 siRNA. After 24 h, cells were homogenize and prepared for luciferase reporter assay. Data were obtained as the ratio luciferase/renilla activity, presented as relative values with respect to control cells transfected with empty vector or nonsense siRNA. Results were recorded as the means of triplicates and averaged from 3 independent experiments. (A) β-catenin levels were analyzed by Western blotting with specific antibody in cells transfected with siMID1/siMID2. (B) Knock-down of MID1/MID2 expression significantly increased β-catenin. (C) β-catenin levels were analyzed by Western blotting in cells transfected with pcDNA-MID1/pcDNA-MID2. (D) Overexpressing MID1/MID2 significantly decreased β-catenin. (E) The Wnt signaling was attenuated in cells overexpressing MID1/MID2, while was enhanced in cells with MID1/MID2 gene silencing. The compromised Wnt signaling in cells treated with Okadaic acid, a specific PP2A inhibitor, and was partially recovered by MID1/MID2 gene silencing.

Figure 3

The influence of altered MID1/MID2 expression on EMT. The cultured 293T cells were transfected with either pEGFP-MID1/pEGFP-MID2 or MID1/MID2 specific siRNA. The protein level of two EMT markers, E-Cadherin and Vimentin, was determined by western blots and quantified using scanning densitometry. Results were obtained from three independent experiments and were represented by mean ± SEM (student t-test; *, P<0.05, ***, P<0.001, ****, P<0.0001). Endogenous β-catenin was used as internal control. The significantly increased E-Cadherin and decreased vimentin in cells overexpressing GFP-MID1/GFP-MID2 was observed (A, B, and C). The opposite change was noticed in cells with siRNA mediated knock-down of MID1/MID2 expression, indicating a promoted EMT (D, E and F).

Figure 4

The immunofluorescence showing inhibited EMT by MID1/MID2 overexpression. The expression of E-cadherin and vimentin was monitored in 293T cells overexpressing GFP-MID1/GFP-MID2 using immunofluorescence. The fluorescence intensities of nine randomly chosen areas were quantified using ImageJ and normalized to the untransfected cells. The results was presented as mean ± SEM (student t-test; *, P<0.05, **, P<0.01, ***, P<0.001). The increased level of E-cadherin and decreased level of β-catenin were observed in cells overexpressing GFP-MID1 (A) or GFP-MID2 (B).

Figure 5

MID1/MID2 gene silencing promoted cell migration. The potential influence of MID1/MID2 on cell migration was analyzed using wound healing assay. The 293T cells were cultured in 6-well plates. The cells were transfected with indicated plasmids or siRNA at ~60% confluence and incubated overnight to allow to reach 90% confluence. A wound in each well was made by scratching and allowed to recover up to 24 h. The images were taken at indicated time intervals post transfection for cells overexpressing MID1/MID2 (A), as well as for cells with siRNA mediated knock-down of MID1/MID2 (B). Experiments were carried out in triplicate and repeated three times and the migration index were presented as mean ± SEM (student t-test; **, P<0.01). The cell migration was significantly accelerated in cells with siRNA mediated knock-down of MID1/MID2, while significantly slowed down in cells overexpressing GFP-MID1/GFP/MID2 at 24 h post transfection (C). Cells transfected with the empty GFP vector or nonsense siRNA were used as controls.