Wnt/β-catenin up-regulates Midkine expression in glioma cells

Abstract

Midkine, also known as neurite growth-promoting factor 2 (NEGF2), plays an important role in cell proliferation, apoptosis and differentiation. Recent studies have shown that Midkine is up-regulated in several types of human cancers. However, the molecular mechanism for its up-regulation remains poorly understood. Activation of Wnt/β-catenin signaling is viewed as crucial for multiple tumor growth and metastasis, including glioma. In the present study, we found that Wnt3a administration or transfection of a constitutively activated β-catenin promoted Midkine expression in glioma cells. We further identified a TCF/LEF binding site, with which beta-catenin interacts, on the proximal promoter region of Midkine gene, by luciferase reporter and chromatin immunoprecipitation assays. Thus, our results suggest a previously unknown Wnt/β-catenin/Midkine molecular network controlling glioma development.

Introduction

Midkine belongs to a type of neurite growth promoting factors [1]. The proteins of this family usually play a pivotal role in the regulation of neuritis outgrowth and neuronal survival [2]. Indeed, Midkine is abundantly expressed during early embryogenesis and markedly down-regulated in adult tissues [3,4]. Recent studies have demonstrated that Midkine is significantly up-regulated in a number of malignant human cancers, including breast, bladder, colon cancer, hepatocellular carcinoma and glioma [5-9]. For instance, it has been shown that Midkine is associated with neuroendocrine differentiation in castration-resistant prostate cancer [10]. Besides, Midkine over-expression is significant correlated to poor survival outcome in glioma [11], suggesting that Midkine might be an important therapeutic target. However, until now, the molecular mechanism for its up-regulation in human cancers remains poorly understood.

Aberrant activation of Wnt/β-catenin signaling has emerged as a major mechanism for the tumorigenesis, including glioma [12,13]. At the molecular level, The interaction of Wnt with Fzd/LRP (Frizzled/lipoprotein-receptor related protein) leads to the stabilization and nuclear-translocation of β-catenin, which in turn accumulates inside the nucleus and serves as a transcriptional coactivator to Tcf/Lef, to induce cell-cycle progression and cell metastasis [14,15].

In the present study, we aim to investigate whether Wnt/β-catenin could up-regulate Midkine expression in glioma. The interpretation of this question might put forward the possibility of understanding the molecular pathogenesis of glioma.

Materials and methods

Cell culture, transfection and luciferase assays

Glioma cell lines (SHG44 and U251 cells) were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (CAS, Shanghai). Cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Invitrogen) and maintained at 37°C in a humidified atmosphere with 5% CO₂. RNAi-mediated depletion of β-catenin was achieved by infecting cells with small interfering RNA (siRNA) oligos targeting β-catenin or negative controls (GE Dharmacon, Pittsburgh PA, United States). All transfections were performed using Lipofectamine 2000 reagents (Invitrogen) according to the manufacturer’s instructions. Human Midkine promoter was amplified by PCR and inserted into the KpnI and XhoI sites of PGL3 empty vector (Promega, Madison, WI, USA). For luciferase assays, cells were seeded in 24-well plates and transfection efficiency was normalized by co-transfecting Simian virus 40 (SV40) plasmids (Promega). Luciferase values were measured using the Dual-Luciferase Reporter Assay System (Promega).

BrdU incorporation and cell invasion assays

A cell proliferation enzyme-linked immunosorbent assay (BrdU kit; Beyotime) was used to analyze the incorporation of BrdU during DNA synthesis following the manufacturer’s protocols. Absorbance was measured at 450 nm in the Spectra Max 190 ELISA reader (Molecular Devices, Sunnyvale, CA). Invasion assays were conducted using a specialized Chemicon invasion chamber which included a 24-well tissue culture plate with 12 cell culture inserts (Millipore, Bedford, MA, USA).

Real-time PCR analysis

Total RNA from tissues and cells was extracted using the TRIzol Kit (Invitrogen) according to the manufacturer’s instructions. cDNA was transcribed from 1 g of total RNA following the manufacturer’s instructions (Promega). Quantitative real-time PCR was performed using SYBR Premix Ex Taq reagents (Takara, Shiga, Japan). Relative transcript quantities were calculated using the 2-ΔΔCt method with 36B4 as the endogenous reference gene.

Western blot

Cells and tissues were harvested and lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM 2-Mercaptoethanol, 2% w/v SDS, 10% glycerol). After centrifugation at 10,000× g for 10 min at 4°C, proteins in the supernatants were quantified and separated by 10% SDS PAGE. Immunoblots were performed using anti-β-catenin and Midkine antibody (Abcam, Cambridge, Massachusetts, USA). Protein levels were normalized to total GAPDH, using a rabbit anti-GAPDH antibody (Abcam). The proteins were visualized by an ECL chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK).

Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM). Analysis was conducted with GraphPad Prism version 6.01 (GraphPad Software). Significance between two groups was analyzed using the unpaired 2-tailed t test. Statistical significance is displayed as *(P < 0.05), **(P < 0.01) or ***(P < 0.001).

Results

Up-regulation of Midkine by Wnt/β-catenin signaling

First, SHG44 cells were treated with TNF-α, IL-6 and Wnt3a, agonists of NF-κB, Stat3 and Wnt/β-catenin signaling, respectively. Quantitative real-time PCR and western blot analysis were employed to examine the mRNA and protein levels of Midkine in SHG44 cells. As shown in the (Figure 1A-D), only Wnt3a significantly promoted Midkine expression in a time-dependent manner (Figure 1A-D). The up-regulation of Midkine by Wnt3a was also observed in U251 cells (Figure 1E, 1F). Besides, overexpression of a constitutively activated β-catenin also induced the expression levels of Midkine in both cells (Figure 2A-D).

Figure 1.

Up-regulation of Midkine by Wn3a in glioma cells. (A-C) Relative expression levels of Midkine were determined by quantitative real-time PCR analysis in SHG44 cells. Cells were treated with TNFa (10 ng/ml) (A), IL-6 (20 ng/ml) (B), Wnt3a (20 ng/ml) (C) or vehicle controls (Ctrl) for the indicated time. (D) Representative protein levels of Midkine in SHG44 cells treated with TNFa, IL-6, Wnt3a or vehicle controls (Ctrl) for 24 hr. (E, F) Relative mRNA and protein levels of Midkine in U251 cells treated with Wnt3a or vehicle controls (Ctrl) for 10 or 24 hr, respectively.

Figure 2.

Activated β-catenin promotes Midkine expression. (A-D) Relative mRNA (A, C) and protein (B, D) levels of Midkine in SHG44 and U251 cells transfected with constitutively activated β-catenin or empty vector (EV) for 24 or 36 hr, respectively.

Knockdown of β-catenin reduces Midkine expression

Next, to further establish the relationship between Wnt/β-catenin signaling and Midkine expression, small interfering RNA (siRNA) oligos were administrated into SHG44 cells to knockdown endogenous β-catenin expression (Figure 3A). As expected, ablation of β-catenin largely abolished the up-regulation of Midkine by Wnt3a (Figure 3B, 3C), further suggesting that Midkine expression was positively regulated by Wnt/β-catenin signaling.

Figure 3.

Knockdown of β-catenin reduces Midkine expression. A. Representative protein levels of Midkine in SHG44 cells transfected with siRNA oligos targeting β-catenin or negative controls (NC) for 36 hr. B, C. Relative mRNA and representative protein levels of Midkine in SHG44 cells. Cells were transfected with siRNA oligos targeting β-catenin or negative controls (NC) for 24 hr, and then treated with Wnt3a or vehicle controls (Ctrl) for another 12 hr.

β-catenin binding to the promoter region of Midkine

We further determined whether Wnt/β-catenin could be a transcriptional regulator of the Midkine gene. Therefore, the promoter region (from -1000 bp to +100 bp, relative to the transcription start site) was cloned into luciferase reporter vectors. In agreement, administration of Wnt3a induced the Midkine promoter activity, which was abrogated by mutation of the Tcf/Lef DNA-binding site in the Midkine promoter (Figure 4A). We then performed chromatin immunoprecipitation (ChIP) assays to determine whether β-catenin could bind the Midkine promoter. As shown in the (Figure 4B), β-catenin protein bound the Midkine promoter was significantly increased in SHG44 cells treated with Wnt3a (Figure 4B), indicating that Midkine was a direct transcriptional target of β-catenin.

Figure 4.

β-catenin binding to the promoter region of Midkine. A. SHG44 cells were transfected with the indicated plasmids for 24 hr, and treated with Wnt3a or vehicle controls (Ctrl) for another 12 hr. Then, cells were harvested and the luciferase activity was measured. B. Two antibodies (anti-IgG and β-catenin) were used in the ChIP assays using SHG44 cells treated with Wnt3a or vehicle controls (Ctrl) for 4 hr.

Knockdown of Midkine attenuated the proliferative roles of Wnt/β-catenin signaling

Since the oncogenic role of Wnt/β-catenin signaling in the onset and progression of glioma [12,13], we speculate the biological function of Midkine in this process. As shown in the (Figure 5A-C), administration of Wnt3a significantly in-creased the cell proliferation and invasion abilities in SHG44 cells. However, knockdown of Midkine expression largely attenuated the proliferative roles of Wnt3a (Figure 5A-C). Therefore, our data suggest that activation of Wnt/β-catenin signaling could promote tumorigenesis, at least in part, through induction of Midkine.

Figure 5.

Knockdown of Midkine attenuated the proliferative roles of Wnt/β-catenin signaling. A. Representative protein levels of Midkine in SHG44 cells transfected with siRNA oligos targeting Midkine or negative controls (NC) for 36 hr. B, C. Cell proliferation and invasion abilities were determined in SHG44 cells. Cells were transfected with siRNA oligos targeting Midkine or negative controls (NC) for 24 hr, and then treated with Wnt3a or vehicle controls (Ctrl) for another 12 hr.

Discussion

In the current study, we identified for the first time the role of Wnt/β-catenin signaling in the regulation of Midkine in glioma. This is supported by multiple lines of evidence. Firstly, activation of Wnt/β-catenin by Wnt3a or transfection of a constitutively activated β-catenin increased mRNA and protein levels of Midkine. Secondly, knockdown of endogenous β-catenin abolished the up-regulation of Midkine by Wnt3a. More importantly, we identified a Tcf/Lef DNA-binding site in the Midkine promoter, which mediates the induction of β-catenin. However, further studies are still needed to further establish the precise relationship between Wnt/β-catenin signaling and Midkine expression in other types of human cancers. Besides, Luo J et al. revealed that transcriptional factor specificity protein 1 (SP1) promoted the expression of Midkine, by which SP1 enhanced proliferation of glioma cells [16]. In addition, a recent study found that TNFα-mediated NF-κB activation could induce midkine expression in prostate cancer cells, which was not observed in our experiments [17]. Although the inconsistence remains poorly understood now, the expression of Midkine and its molecular determinants might be cell-or tissue-specific.

Activation of Wnt/β-catenin signaling or (and) increased β-catenin expression has been consistently found in glioma tissues relative to normal brain regions [12,13]. Besides, it has been correlated with poor prognosis and short survival of glioma patients [18,19]. At the molecular level, Wnt/β-catenin signaling could up-regulate multiple cell-cycle regulators, including Cyclin D1 and Cyclin E, while down-regulate cell-cycle inhibitors, such as p21 and p27 [20]. Moreover, recent studies indicate that Wnt/β-catenin could form a cross-talk with epidermal growth factor receptor (EGFR) pathway [21], which is probably the most significant signaling pathway clinically implicated in glioma. Here, we found that knockdown of Midkine expression significantly blocks the oncogenic roles of Wnt/β-catenin, as shown by cell proliferation and invasion assays. Interestingly, Liedert A et al. reported that Midkine may exert a negative effect on Wnt signaling, through down-regulation of Wnt target genes [22]. Therefore, a cross-talk or negative feedback may exist between Wnt/β-catenin and Midkine, which needs to be determined in the future studies.

In summary, our data provide a novel insight into how Wnt/β-catenin activation could affect Midkine expression. These results may be helpful to understand the roles of Wnt signaling in the glioma progression and see efficient anti-cancer drugs for patients.

Disclosure of conflict of interest

None.