Maternal embryonic leucine zipper kinase inhibits epithelial-mesenchymal transition by regulating transforming growth factor-β signaling

Abstract

Maternal embryonic leucine zipper kinase (MELK) performs an important role in self-renewal and proliferation of progenitor cells or tumor stem cells, and is expressed in aggressive cancers, contributing to tumorigenesis. However, the function of MELK in metastasis is unknown. In the present study, the lung cancer A549 cell line was utilized in order to study the role of MELK in epithelial-mesenchymal transitions (EMTs), the initial step of tumor metastasis. It was identified that transforming growth factor-β (TGF-β) could downregulate the expression of MELK, and that MELK could inhibit EMT by regulating TGF-β signaling. MELK can interact with Smad proteins, which represses TGF-β/Smad-mediated signaling activity. The findings of the present study identified the effect of MELK in TGF-β signaling and the EMT process.

Keywords: A549; Smad; epithelial-mesenchymal transition; maternal embryonic leucine zipper kinase; transforming growth factor-β.

Figure 1.

Knockdown of MELK promotes the EMT. (A) MELK expression was detected by western blot analysis, showing that MELK was decreased in shMELK (sh1 and sh2) cells compared with that in shMock cells. The star symbol shows the MELK band. (B) Proliferation rate was measured using MTS solution. Proliferation was decreased in shMELK1 and shMELK2 cells compared with shMock cells. (C) E-cadherin level was detected prior to or following TGF-β stimulation. Western blot analysis shows that E-cadherin was dramatically decreased in shMELK (sh1 and sh2) cells. (D) Wound healing assay. Confluent A549 cells were scratched and then stimulated with or without TGF-β for 24 h. Images were captured at 0 or 24 h, showing that shMELK (sh1 and sh2) cells migrate faster than shMock cells following TGF-β stimulation. The quantification of distance is presented. The experiments were performed in triplicate. *P<0.05. TGF-β, transforming growth factor-β; MELK, murine embryonic leucine zipper kinase; sh, short hairpin.

Figure 2.

(A) TGF-β inhibits MELK protein expression. The A549 cells were stimulated with TGF-β1 for 0, 1, 3, 6, 12 and 24 h. MELK expression was detected by western blot analysis. The star symbol shows the MELK band. (B) TGF-β inhibits the expression of MELK mRNA. MELK mRNA expression was detected with reverse transcription polymerase chain reaction. E-cadherin mRNA expression was detected as a positive control, demonstrating the effect of TGF-β stimulation. The experiments were performed in triplicate. *P<0.05. TGF-β, transforming growth factor-β; MELK, murine embryonic leucine zipper kinase.

Figure 3.

Knockdown of MELK enhances TGF-β signaling activity. (A) The p3TP reporter gene was used to detect TGF-β signaling activity in shMock cells and shMELK (sh1 and sh2) cells. The reporter gene activity was increased in shMELK cells with or without TGF-β stimulation (*P<0.05). (B) shMock and shMELK1 cells were stimulated by TGF-β for 15, 30 or 60 min, and western blot analysis was then performed using different antibodies (p-Smad2, Smad2, MELK and β-actin). The phosphorylation level of Smad2 did not change following MELK knockdown compared with shMock cells. TGF-β, transforming growth factor-β; MELK, murine embryonic leucine zipper kinase; sh, short hairpin; p-Smad2, phosphorylated Smad2.

Figure 4.

MELK interacts with the Smad protein and represses TGF-β signaling activity. (A) MELK interacts with Smad2, Smad3, or Smad4 protein. The Flag-tagged MELK was co-transfected with HA-tagged Smad2, Smad3, or Smad4 plasmids. The HA antibodies were used to immunoprecipitate the immunocomplex, and the Flag antibodies were used to detect the MELK signals using western blot analysis. (B) MELK represses Smad2- or Smad3-induced activity of TGF-β signaling. The p3TP reporter gene was transfected with different combinations of plasmids. The experiments were performed in triplicate (*P<0.05). HA, hemagglutinin; TGF-β, transforming growth factor-β; MELK, murine embryonic leucine zipper kinase; IP, immunoprecipitation; IB, immnuoblot.