Elsevier

Experimental Cell Research

Volume 358, Issue 2, 15 September 2017, Pages 279-289
Experimental Cell Research

CD147-induced cell proliferation is associated with Smad4 signal inhibition

https://doi.org/10.1016/j.yexcr.2017.07.003Get rights and content

Highlights

  • Smad4 interacts with CD147 via the MH2 domain.

  • CD147 prevents Smad4 translocation into the nuclear to regulate p21 transcription.

  • CD147 inhibits p21 transcription and expression independent on p53.

  • pCD147-S252 is responsible for interaction with Smad4 and p21 down-regulation.

Abstract

CD147 is a multifunctional trans-membrane glycoprotein, which is highly expressed in many cancers. However, the mechanism by which CD147 modulates cell proliferation is not fully understood. The aim of this study is to investigate the role of CD147 in cell proliferation associated with the TGF-β/Smad4 signaling pathway. Here, we used cell viability and clone formation assays in LNCaP prostate cancer cells to demonstrate that CD147 promotes cell proliferation. The luciferase assay and western blotting show that silencing CD147 using shRNA enhances transcription and expression of p21WAF1. Using immunofluorescence and nuclear-cytoplasmic separation, we show that this is primarily attributed to transport of Smad4 from the cytoplasm to nucleus. Other assays (GST pull-down, co-immunoprecipitation and immunofluorescence) demonstrate that Smad4 is a new interaction partner of CD147, with the Smad4 MH2 domain and CD147 intracellular domain (CD147-ICD) being involved in the interaction. Furthermore, we report that a phosphoserine (pSer) in CD147 (pSer252) is responsible for this interaction and inhibition of the Smad4/p21WAF1 signal that promotes cell proliferation. Our results provide a novel molecular mechanism for CD147-induced cell proliferation associated with Smad4 signal inhibition.

Introduction

CD147 (or BSG) is a 65 kDa transmembrane glycoprotein that belongs to the immunoglobulin (Ig) superfamily [1]. CD147 plays a pivotal role in normal tissue development [2], [3], Alzheimer's disease (AD) [3], and various cancers [4], [5], [6], [7]. The broadly expressed CD147 is a two Ig-like domain isoform of CD147 and is composed of 269 residues composed of an extracellular domain (206 residues), a transmembrane domain (24 residues) [8], and an intracellular domain (ICD, 39 residues) [9], [10]. Although the CD147 gene encodes for a 29 kDa protein, it has a highly glycosylated mature form that migrates on SDS-PAGE like a 65 kDa species [11]. In addition, a three Ig-like domain isoform of CD147 has also been reported; however this isoform is rarely expressed [12]. Increasing evidence indicates that CD147 is involved in numerous processes related to tumor progression. For instance, CD147 directly regulates tumor cell adhesion [13], angiogenesis [14], chemoresistance [5], cell migration [15], invasion and metastasis [14], [15], [16]. However, its function in the regulation of cell proliferation is still poorly understood. Recently, CD147 has also been identified as a mediator of anti-apoptotic function and chemoresistance in several cancer cell lines [17], [18], once again with the underlying mechanism of action being poorly understood.

Transforming growth factor-β (TGF-β) plays a pivotal role in both homeostasis and cell differentiation. Studies have found that the TGF-β signaling pathway not only inhibits tumor proliferation, but also promotes tumor progression, a function that mainly depends on Smad4 activation and deactivation [19], [20]. Recently, crosstalk between TGF-β and CD147 has been reported. Here, activation of the TGF-β1-CD147 complex was shown to induce a positive feedback loop in the regulation of hepatic stellate cells that facilitate liver fibrosis [21]. However, the correlation between CD147 and Smad4 remains unclear.

The inhibition of cell proliferation induced by Smad4 is mediated by p21WAF1 that are inhibitors of cyclin-dependent kinase (CDK) [22]. p21WAF1 is one of the Smad4/DPC4-regulated downstream target genes, and over-expression of the Smad4 gene can bypass TGF-β receptor activation and re-establish one of the key regulatory controls of cell proliferation [23]. Following TGF-β and receptor binding, phosphorylated R-Smads form complexes with the common-mediator (Co-) Smad4 which is translocated to the nucleus to activate cell cycle inhibition genes, such as p21WAF1 [24].

Until now, direct evidence for the crosslink between CD147 and the Smad4/p21WAF1 pathway has been lacking. Therefore, we initiated the current study to understand the correlation between CD147 and the Smad4/p21WAF1 pathway in mediating the inhibition of cell proliferation inhibition and examining the roles of CD147 in p21WAF1 regulation, one of the downstream target genes of the Smad4 pathway. We also explored the CD147 phosphorylation site involved in Smad4/ p21WAF1 pathway regulation.

Section snippets

Plasmids construction

The Flag-tagged Smad4 was a gift from Dr. Hsiu-Ming Shih (Taipei, China) [25]. The MH1、Linker and MH2 domain of Smad4 were amplified by PCR and then cloned into pGEX4T–1 vector. The CD147/GFP expression plasmid was kindly provided by Bryan P. Toole [26]. CD147 intracellular domain was constructed to pGEX4T–1 vector (GST-CD147-ICD), and then used site-directed mutagenesis to construct GST-CD147-ICD/S246A and GST-CD147-ICD/S252A. The same strategy was used to construct CD147/S246A and

CD147 facilitates cancer cell proliferation

To investigate the role of CD147 in prostate cancer cell proliferation, we first determined the expression level of CD147 in BPH-1, LNCaP, and PC-3 cells and found that CD147 was expressed at higher levels in LNCaP cells than in BPH-1 or PC-3 cells (Fig. 1A). Then we used shRNAs to show that CD147 expression is significantly silenced in LNCaP cells (Fig. 1B), whereas its over-expression enhances protein production. Next, we tested viability of cells in each group. As shown in Fig. 1C,

Discussion

CD147 plays a significant role in tumor progression, because of its ability to stimulate cancer cells promoting cell invasion [39], metastasis [39], [40], and angiogenesis [41]. Here, we demonstrated that CD147 can facilitate prostate cancer cell proliferation and inhibit the transcription and expression of p21WAF1. The inhibitory effect of CD147 on p21WAF1 primarily occurs via the Smad4 pathway. We showed that CD147 and Smad4 directly interact with each other in both co-immunoprecipitation and

Disclosure of conflict of interest

None.

Acknowledgments

This study was supported by the Ministry of Science and Technology (2016YFE0128500), Jilin Provincial Science & Technology Department (20170204009YY, 20150101187JC, 20150414007GH), Jilin Province Development and Reform Commission (2016C047-3), Jilin Province Education Department (2015-526, 2015-551) the Fundamental Research Funds for the Central Universities (2412015ZH005, 2412016KJ037, 130017507, 130028633, 130014637). University S & T Innovation Platform of Jilin Province for Economic Fungi (

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