Abstract
Background: Receptor-interacting serine/threonine protein kinase-2 (RIPK2) has been reported to be an important regulator of tumor proliferation, differentiation and wound repair. We investigated the effects of RIPK2 knockdown in human hepatoma cells on epithelial-to-mesenchymal transition (EMT)-associated gene expression. Materials and Methods: HepG2 cells stably expressing RIPK2-shRNA (HepG2-shRIPK2) were generated after puromycin selection. Total RNAs from HepG2-shRIPK2 and from HepG2-shcontrol cells were isolated and PCR-based arrays were performed to compare the 84 EMT-associated gene expressions. Results: We observed that knockdown of RIPK2 down-regulated mRNA expression of jagged 1 (JAG1); plasminogen activator inhibitor-1 (PAI1); regulator of G-protein signalling 2, 24 kDa (RGS2); E-cadherin (CDH1); fibroblast growth factor binding protein 1 (FGFBP1); snail homolog 2 (SNAI2); protein tyrosine phosphatase type IVA, member 1 (PTP4A1); keratin 19 (KRT19); vimentin (VIM); and survival of motor neuron protein-interacting protein 1 (SIP1). Conclusion: We found that knockdown of RIPK2 down-regulated nuclear factor kappa B (NF-κB)-dependent PAI1 and VIM gene expressions. RIPK2 might play an important role in hepatic cell migration. These findings could shed new light on carcinogenesis and on liver regeneration.
Receptor-interacting protein (RIP) family kinases have emerged as essential sensors of cellular stress (1). RIP kinases (RIPKs) are closely related to members of the interleukin-1-receptor-associated kinase (IRAK) family. To date, five RIPKs are known (2). They play important roles in situations of cellular stress caused by different factors, such as pathogen infection, inflammation, cellular differentiation programs and DNA damage, and eventually lead to the activation of transcription factors, such as nuclear factor kappa B (NF-κB), the induction of apoptotic processes, or activation of mitogen-activated protein kinase (MAPK) (1, 2).
Our group as well as others showed that receptor-interacting serine/threonine protein kinase-2 (RIPK2, also known as RIP2, RICK or CARDIAK), a caspase-recruitment domain-containing kinase, plays an important role in cell migration and wound healing in keratinocytes as well as hepatocytes (2, 3). RIPK2 is also involved in the Toll-like receptor (TLR)-signalling pathway and plays an important role in the production of inflammatory cytokines through NF-κB activation (4, 5). RIPK2 also plays an important role in nucleotide-binding oligomerization domain containing 1 (NOD1) ligand-induced NF-κB activation in hepatocytes (3).
The extracellular matrix (ECM), which consists of collagens, glycoproteins, proteoglycans and glycosaminoglycans, provides cells with positional information and a mechanical scaffold for adhesion and migration. Chronic fibrogenesis can be regarded as a continuous wound-healing process that results in scar formation (6). Dynamic interactions between growth factors and the ECM are integral to wound healing (7, 8). Wound healing, and inflammatory processes, as well as changes in the tumor microenvironment through remodeling of the ECM, are important for cancer metastasis (8). The epithelial-to-mesenchymal transition (EMT) now takes center stage as the convergence point between inflammation and the progression of degenerative fibrotic diseases and cancer (9). EMT includes many processes associated with differentiation and development, morphogenesis, cell growth and proliferation, migration and motility, cytoskeleton formation, ECM and cell adhesion, and related signalling pathways, as well as transcription factors. The NF-κB family of transcription factors plays pivotal roles in both promoting and maintaining the cell phenotype (9, 10). Inflammation is necessary for EMT (11), and NF-κB plays an important role in the induction of inflammation (12).
In the present study, we uncovered a novel function of endogeneous RIPK2, which is located upstream of NF-κB. Knockdown of RIPK2 in human hepatoma cells affected EMT-associated gene expression.
Materials and Methods
Cell culture. Human hepatoblastoma HepG2 cells were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum under 5% CO2, at 37°C.
Transfection of cells. HepG2 cells were transfected with plasmid control-shRNA or RIPK2-shRNA (Santa Cruz Biotechnology, CA, USA). After 48 h of transfection, cells were split and treated with puromycin for selection of antibiotic-resistant colonies. Individual colonies were picked-up and examined for expression of endogenous RIPK2 by western blotting with specific antibodies against RIPK2 (3), and clones HepG2-shC and HepG2-shRIPK2 [HepG2-shRIPK2-3 (3)], in which RIPK2 expression was knocked-down, were selected for subsequent studies.
RNA extraction. Cells were seeded into 6-well plates, and total cellular RNA was extracted 48 h later, using the RNeasy Mini kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. RNA samples were then stored at −80°C until use. RNA quality was examined using the A280/A260 ratio (Pharmacia Biotech, Bedford, MA, USA).
cDNA synthesis and real-time polymerase chain reaction (PCR). cDNA synthesis was performed using RT2 First Strand Kit (SABiosciences, Frederick, MD, USA). Each 1 μg of RNA was subjected to one reaction. The cDNA synthesis reaction was performed as follows: incubation at 42°C for 15 min and then reaction stoppage by heating at 95°C for 15 min. RNA quantification was conducted by real-time PCR with SyBr Green I, as described previously (12, 13). Gene quantification was determined using an ABI Prism 7300 instrument from Applied Biosystems (Foster City, CA, USA). Thermal cycling conditions were 95°C for 10 min, followed by 40 cycles at 95°C, 15 s for denaturation, and 1 min at 60°C for annealing and extension. All primers for examining human EMT-associated gene expression were purchased from SABiosciences. EMT-associated genes examined in the present study are listed in Table I. Gene expression was normalized to that of house-keeping genes (beta-2-microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L 13a, glyceraldehyde-3-phosphate dehydrogenase and beta-actin) to determine the fold-change in gene expression between test (HepG2-shRIPK2) and control (HepG2-shC) samples by the 2−ddCT (comparative cycle threshold) method (13). We performed each of these experiments in triplicate. Genes were annotated by Entrez Gene (NCBI, Bethesda, MD, USA).
Statistical analysis. Data were analyzed with RT2 prolifer PCR array data analysis software (http://www.superarry.com/pcrarraydataanalysis.php).
Results
Down-regulated genes among EMT-associated genes in RIPK2-knockdown HepG2 cells. RIPK2 functions as a signal transducer for both the innate and adaptive immune activation pathways (14). In our previous study, we observed that RIPK2 plays an important role in cell migration and wound healing in hepatocytes (3). We examined EMT-associated gene expression profiles using real-time PCR-based focused microarrays. A comparison of EMT-associated genes between HepG2-shC and HepG2-shRIPK2 is shown in Tables II and III. Among the 84 EMT-associated genes examined, collagen, type 1, alpha 2 gene (COL1A2) was undetected in both types of samples and the average threshold cycle of 12 genes was relatively high (>30), meaning that their relative expression level was low. We thus excluded these 13 genes from further analysis. Out of the remaining 71 genes, 10 (14.0%) were significantly down-regulated by the knockdown of RIPK2 (p<0.05; Table II). Among these 10 genes, six [jagged 1 (JAG1); plasminogen activator inhibitor-1 (PAI1); regulator of G-protein signalling 2, 24 kDa (RGS2); E-cadherin (CDH1); fibroblast growth factor binding protein 1 (FGFBP1); and snail homolog 2 (SNAI2)] were down-regulated by 1.5-fold or more in HepG2-shRIPK2 cells.
Up-regulated genes among EMT-associated genes in RIPK2-knockdown HepG2 cells. Out of the remaining 71 genes, six (8.4%) were significantly up-regulated by the knock-down of RIPK2 (p<0.05; Table III). Out of these six genes, four [collagen, type V, alpha 2 (COL5A2); matrix metalloproteinase 2 (MMP2); MMP3; and goosecoid homeobox (GSC)] were up-regulated by 1.5-fold or more in HepG2-shRIPK2 cells.
Discussion
In the present study, we observed that 10 genes were significantly down-regulated in HepG2-shRIPK2 cells. Out of these 10 genes, PAI1 and vimentin (VIM) are NF-κB-dependent genes (15, 16). We also observed 6 genes were significantly up-regulated in HepG2-shRIPK2 cells. It was reported that NF-κB is involved in the expression of the wingless-type mouse mammary tumor virus (MMTV) integration site family, member 5B (WNT5B), MMP2 and MMP3 (17-19). Our previous study (3) showed that knockdown of RIPK2 has an effect on NOD1 ligand C12-iE-DAP-induced NF-κB activation in HepG2 cells, suggesting that RIPK2 plays an important role in NF-κB activation induced through NOD1 triggering in hepatocytes. We also observed that silencing of RIPK2 was associated with the reduction of interleukin-6 (IL6), IL8 and hepatic wound closure (3).
PAI1, a multifaceted proteolytic factor, plays an important role in the plasminogen/plasmin system as it is the main inhibitor of tissue-type and urokinase-type plasminogen activator (20). PAI1 also plays an important role in signal transduction, cell adherence and cell migration (21). It was reported that PAI1 is associated with poor prognosis in several types of cancers (21) and that it is associated with hepatocellular carcinoma (HCC) caused by hepatitis B and C (22). Recently, single-nucleotide polymorphism (SNP) of PAI1 was reported to be associated with treatment response in patients with chronic hepatitis C, treated with pegylated-interferon plus ribavirin (23).
Genes associated with epithelial-to-mesenchymal transition (EMT) assessed by real-time RT-PCR in the present study.
VIM is a member of the intermediate filament family of proteins. VIM provides cellular integrity under mechanical stress in vivo with a resilience not related to microtubule or actin filament networks (24). It was reported that VIM interacts with hepatitis B or C viral proteins (25, 26). Our previous study (3) showed that hepatitis B virus e antigen inhibits RIPK2 expression and interacts with RIPK2, which might represent two mechanisms through which hepatitis B virus e antigen blocks NOD1 ligand-induced NF-κB activation in HepG2 cells. We also observed that hepatitis B virus e antigen inhibits cell migration (3).
Our hypothesis is that RIPK2 plays an important role in hepatic cell migration and wound repair in the liver, possibly due to: i) activation of NF-κB through RIPK2; ii) production of inflammatory cytokines IL6 and IL8, which are also important for regeneration of the liver through activation of NF-κB; and iii) up-regulation of EMT-associated NF-κB-dependent genes, such as PAI1 and VIM. In the present study, we also observed up-regulated genes such as MMPs, especially important in ECM and EMT. Further studies are required to determine this.
In conclusion, we observed that knockdown of RIPK2 down-regulated the expression of the NF-κB-dependent genes PAI1 and VIM. RIPK2 might play an important role in hepatic cell migration. These findings could shed new light on carcinogenesis and regeneration of the liver.
Genes significantly down-regulated by knockdown of Receptor-Interacting Serine/Threonine Protein Kinase-2 (RIPK2) in HepG2 cells.
Genes significantly up-regulated by knockdown of Receptor-Interacting Serine/Threonine Protein Kinase-2 (RIPK2) in HepG2 cells.
Acknowledgements
This work was supported by the Japan Science and Technology Agency, Ministry of Education, Culture, Sports, Science and Technology, Japan (TK), the Chiba University Young Research-oriented Faculty Member Development Program in Bioscience Areas (TK) and a Research Grant-in-Aid from the Miyakawa Memorial Research Foundation (WS).
Footnotes
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Declaration of Interest
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
- Received May 10, 2012.
- Revision received July 18, 2012.
- Accepted July 19, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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