Overexpression of the Ubiquilin-4 (UBQLN4) is Associated with Cell Cycle Arrest and Apoptosis in Human Normal Gastric Epithelial Cell Lines GES-1 Cells by Activation of the ERK Signaling Pathway

Abstract

BACKGROUND Ubiquilin-4 (UBQLN4) is a component of the ubiquitin-proteasome system and regulates the degradation of many proteins implicated in pathological conditions. The aim of this study was to determine the role of UBQLN4 in regulating the proliferation and survival of the normal gastric epithelial cell line GES-1. MATERIAL AND METHODS We constructed GES-1 lines stably overexpressing UBQLN4 by lentiviral infection. Cell proliferation, apoptosis, and the cell cycle were analyzed using the MTT assay and flow cytometric assays. Phosphorylation of ERK, JNK, p38, and expression of cyclin D1 were detected by western blot analysis. RESULTS Overexpression of UBQLN4 significantly reduced proliferation and induced G2/M phase arrest and apoptosis in GES-1 cells. Moreover, upregulation of UBQLN4 increased the expression of cyclin D1 and phosphorylated ERK, but not JNK or p38. CONCLUSIONS These data suggest that UBQLN4 may induce cell cycle arrest and apoptosis via activation of the ERK pathway and upregulation of cyclin D1 in GES-1 cells.

Figure 1

Overexpression of UBQLN4 induced apoptosis in GES-1 cells. (A) Western blot analysis of UBQLN4 protein expression in uninfected (control) GES-1 cells or cells infected with empty (pLVX) or UBQLN4-encoding (pLVX-UBQLN4) lentiviruses. GAPDH was probed as a loading control. (B) Light microscopic images showing the morphology of GES-1 cells as described in (A). Scale bar, 30 μm. (C) Fluorescence microscopy of GES-1 cells expressing an EGFP–UBQLN4 fusion protein. Nuclei were stained with DAPI. Scale bar, 30 μm. (D) MTT proliferation assay of pLVX- or pLVX-UBQLN4-infected GES-1, MKN45, and BGC-823 cells. Data are shown as the mean ±SD of 8 experiments. * P<0.05 for comparison with the control.

Figure 2

UNQLN4 induced cell cycle arrest and apoptosis in GES-1 cells. (A) Flow cytometry dot plots and quantification of early apoptosis of Annexin V-PE/7-AAD-labeled control or UBQLN4-expressing GES-1 cells. (B) Flow cytometry histograms and quantification of PI-labeled GES-1 cells in each phase of the cell cycle at 48 hours (upper) and 72 hours (lower) after infection with pLVX or pLVX-UBQLN4. Data are shown as the mean ±SD of 3 experiments. * P<0.05 for comparison with the control.

Figure 3

UBQLN4 overexpression activated ERK signaling and increased cyclin D1 expression. (A) Western blot analysis of cyclin D1 in GES-1-pLVX-UBQLN4 and GES-1-pLVX cells. (B) Western blot analysis of total and phosphorylated ERK in GES-1, MKN45, and BGC-823 cells infected with pLVX-UBQLN4 or pLVX. (C) Western blot analysis of total and phosphorylated ERK and AKT in GES-1-pLVX and GES-1-pLVX-UBQLN4 cells after serum starvation for 12 hours followed by the addition of 10% FBS for the indicated times. (D) Western blot analysis of total and phosphorylated JNK and p38 in GES-1-pLVX and GES-1-pLVX-UBQLN4 cells. GAPDH served as a loading control for all blots.