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Experimental Biology and Medicine 232:344-352 (2007)
© 2007 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

ßig-h3 Is Involved in the HAb18G/CD147-Mediated Metastasis Process in Human Hepatoma Cells

Juan Tang*,1, Hong-wei Zhou{dagger},1, Jian-li Jiang*,1, Xiang-min Yang*, Yu Li*, Hong-Xin Zhang{dagger}, Zhi-nan Chen*,2 and Wei-ping Guo{dagger},2

* Cell Engineering Research Centre and Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi’an 710032, China; and {dagger} Department of Interventional Radiation, Tang-Du Hospital, Fourth Military Medical University, Xi’an 710032, China

To whom requests for reprints should be addressed at 2 Cell Engineering Research Centre and Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, 17 Changlexi Street, Xi’an 710032, China. E-mail: znchen{at}fmmu.edu.cn or Department of Interventional Radiation, Tang-Du Hospital, Fourth Military Medical University, 17 Changlexi Street, Xi’an 710032, China. E-mail: guoweip{at}fmmu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HAb18G/CD147, a new hepatoma-associated antigen cloned and screened from human hepatocellular carcinoma cDNA library, is closely correlated with metastasis process in human hepatoma cells. In the present study we aimed to identify the pivotal molecules of the HAb18G/CD147 signal transduction pathway. The investigation showed that ßig-h3, a secretory extracellular matrix (ECM) protein, was upregulated in HAb18G/CD147-expressing human hepatoma T7721 cells and was downregulated by depressing HAb18G/CD147 expression. The expression of ßig-h3, upregulated in human hepatoma cells, was positively relative to the expression of HAb18G/CD147 in different human hepatoma cell lines. By overexpressing ßig-h3 in human SMMC-7721 hepatoma cells, we discovered that ßig-h3 promoted cell adhesion, invasion, and matrix metalloproteinase (MMP) secretion potential. HAb18G/CD147-induced invasion and metastasis potential of human hepatoma cells can be attenuated by antibodies specific for ßig-h3, and no significant differences on inhibitory effects were observed among T7721 cells incubated with antibodies for ßig-h3 or HAb18G/CD147 or both types together. Taken together, our study suggests that ßig-h3, regulated by the expression of HAb18G/CD147, is involved in the HAb18G/CD147 signal transduction pathway and mediates the HAb18G/CD147-induced invasion and metastasis process of human hepatoma cells.

Key Words: HAb18G • CD147 • ßig-h3 • hepatoma cell • metastasis


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD147, a cell surface transmembrane glycoprotein widely expressed on many cell types, appears at especially high levels on human tumor cells (1–8). Elevated CD147 expression is correlated with tumor progression and invasion potential (9, 10). HAb18G, a new hepatoma-associated antigen, has an identical nucleic acid and amino acid sequence as CD147 (11). The transmembrane domain of HAb18G/CD147 contains a glutamic acid residue and a leucine zipper motif that is implicated in the protein association with the plasma membrane and the dimerization of DNA-binding proteins (12, 13). These structural characteristics may enable HAb18G/CD147 to interact with other membrane proteins, intracellular enzymes/factors as well as extracellular factors that are involved in the signaling pathways pertinent to the process of metastasis.

Our previous studies have demonstrated that HAb18G/ CD147 promotes the invasion activity of human hepatoma cells by stimulating fibroblast cells to produce elevated levels of several matrix metalloproteinases (MMPs; Ref. 7). By transfecting HAb18G/CD147 cDNA into human SMMC-7721 hepatoma cells to obtain a cell line, T7721, that stably expressed HAb18G/CD147, we have confirmed that the overexpression of HAb18G/CD147 enhances the metastatic potential of human hepatoma cells by disrupting the regulation of Ca2+entry by nitric oxide (NO)/cGMP (11, 14).

The process of proliferation, invasion, and metastasis is a complex one, involving both the autonomy of the malignant cells and their interaction with the cellular and extracellular environments (14). The way for tumor cells to respond to cellular and extracellular stimuli is regulated through transduction of those signals and translation into cellular activities. Although many researchers are focused on the function of CD147 in tumor process, the mechanism is still not well understood. It is not clear whether CD147 is involved in signal transduction and cell adhesion or whether it directly serves as a signal-transmitting adhesion molecule or as a regulator of adhesion.

In this study we identified that the expression of ßig-h3 is in positive correlation with HAb18G/CD147, and ßig-h3 promotes hepatoma cell adhesion, invasion, and MMP secretion. Additionally, the potential effect of HAb18G/ CD147 on the invasion and metastasis process in hepatoma cells was found to be mediated by its direct or indirect actions on expression of ßig-h3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture.
The human SMMC-7721 hepatoma cells, FHCC-98 hepatoma cells, human QSG normal hepatic cells (all obtained from the Institute of Cell Biology, Academic Sinica, Shanghai, China), and T7721 cells stably overexpressing HAb18G/CD147 (14) were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 2% L-glutamine at 37°C in a humidified atmosphere of 5% CO2. G418 was added into the medium for T7721 cells at a final concentration of 400 µg/ml.

Differential Analysis of Microarray.
Dual-color competitive microarray hybridization analysis was performed from digestive gland mRNA extracts using the mytox chip v1.1 as described previously (15). In brief, total RNA was isolated from 7721 and T7721 cells using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Cy3-labeled dCTP and Cy5-labeled dCTP (Amersham Biosciences, Freiburg, Germany) were directly incorporated into cDNA probes in a reverse transcription reaction, and were then analyzed using signal transduction–related microarray (Capital Biochip Corp., Beijing, China).

Transient Small Interfering RNA to T7721 Cells.
The sense sequence for HAb18G/CD147 small interfering RNAs (siRNAs) was 5'-GATCCGTCGTCAGAACACATCAACTTCAAGAGAGTTGATGTGTTCTGACGACTTTTTTGGAAA-3', and the antisense sequence was 5'-AGCTTTTCCAAAAAAGTCGTCAGAACACATCAACTCTCTTGAAGTTGATGTGTTCTGACGACG-3'. HAb18G/CD147 siRNA and the control siRNA (Ambion, Austin, TX) were cloned into the EcoRI and HindIII sites of pSilence r3h1 vector (Ambion). The T7721 cells, when at 70% to 80% confluence, were transfected with siRNA at a final concentration of 100 nM using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions.

Western Blot Analysis.
The expression of ßig-h3 and HAb18G/CD147 were analyzed by Western blotting as described previously (17). In brief, cells were suspended in serum-free medium at a density of 5 x 105/ml, and 2 ml of the cell suspension was seeded into a six-well plate. After culture for 48 hrs, the conditioned medium was collected. BCA protein assay kit (Pierce Biotechnology, Rockford, IL) was employed to determine the total protein density, and equal amounts of proteins were separated on 10% sodium dodecyl sulfide–polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA). After blocking with 5% nonfat milk, the membrane was incubated for 2 hrs at room temperature with the designated antibody. Immunodetection was performed by using the Western-Light chemiluminescent detection system (Applied Biosystems, Foster City, CA).

Quantitative Real-Time Polymerase Chain Reaction (PCR).
For quantitative analysis of ßig-h3 mRNA in human hepatoma cells 7721, T7721, and FHCC-98, and human novel hepatic cell QSG, the standard curve for quantifying mRNA copy number was established by amplifying nine aliquots of templates with known copy numbers (serial dilution from 108 to 1010 copies). The specific ßig-h3 cDNA was synthesized as follows: total RNA extracted as previously described was reverse transcribed into the first-strand cDNA using first-strand cDNA synthesis kit ReverTra Ace (Toyobo, Osaka, Japan). Forward and reverse primer sequences for ßig-h3 gene are 5 '-CATTGAGAACAGCTGCATCG-3' and 5'-AGTCTGCTCCGTTCTCTTGG-3', respectively. Amplification of cDNA templates was performed in a 50-µl reaction volume consisting of 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 200 µM deoxynucleotide triphosphates; 0.2 lM forward and reverse primers, 1 ll cDNA template from the reverse-transcribed product, and 1 unit Taq DNA polymerase (TaKaRa, Shiga, Japan) with the following cycling parameters: initial denaturation at 94°C for 3 mins, followed by 32 cycles at 94°C for 45 secs, 57°C for 45 secs, and 72°C for 45 secs, finally extended at 72°C for 10 mins.

TaqMan probes for ßig-h3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-CGCTTTAGCATGCTGGTAGCTGCCA-3' and 5'-CCTCAACTACATGGTTTAC-3'. GAPDH was used as internal reference housekeeping gene to assess the status of sample mRNA. Forward and reverse primers used for amplification of human ßig-h3 were 5'-TGGATGTCCTGAAGGGAGACA-3' and 5'-TCTCCGTCAGTCCTGCAGACT-3', respectively. Forward and reverse primers used for amplification of GAPDH are 5'-CCATCAATGACCCCTTCATT-3' and 5'-CATGGGTGGAATCATAATGGAAC-3', respectively. The probes were labeled with the reporter fluorochrome 6-carboxyfluorescein (FAM) at the 5' end and the quencher fluorochrome 6-carboxy-tetramethyl-rhodamine (TAMRA) at the 3' end. Cycling and quantification was performed in DNA Engine Opticon (MJ Research, Hercules, CA) following the cycling conditions: initial denaturation at 94°C for 3 mins, followed by 30 cycles at 94°C for 45 secs, 60°C for 45 secs, and 72°C for 45 secs, finally extended at 72°C for 10 mins.

Cell Adhesion Assay.
The wells of the 96-well culture plate were coated with Matrigel (BD Bioscience, Franklin Lakes, NJ) at a concentration of 5 µg/ml and incubated at 4°C overnight. The coated wells were blocked with PBS containing 2% bovine serum albumin (BSA) for 30 mins and then washed with PBS. Cells in serum-free medium containing 0.1% BSA were added to the wells (2 x 104/well) and incubated at 37°C, 5% CO2 for 30 to 60 mins with or without antibodies specific for ßig-h3 and CD147. After the medium and nonattached cells were removed, 0.2% crystal violet was added for 10 min. The plate was gently washed with tap water and dried in air for 24 hrs. Then 0.1 ml of 5% SDS with 50% ethanol was added for 20 mins, and then the plate was read at 540 nm.

Cell Invasion Assay.
The chemotactic cell invasion assay was performed using 24-well Transwell units (Millipore) with an 8-µm pore size polycarbonate filter according to the method described by Mensing et al. (15). Each lower compartment of the Transwell contained 600 ml of 0.5% FBS as a chemoattractant in RPMI 1640. The upper side of a polycarbonate filter was coated with Matrigel (5 mg/ml in cold medium) to form a continuous thin layer. Prior to addition of the cell suspension, the dried layer of Matrigel matrix was rehydrated with medium without FBS for 2 hrs at room temperature. Cells (1 x 105) in 0.1 ml RPMI 1640 containing 0.1% BSA were added into the upper compartment of the Transwell unit and incubated for 24 hrs at 37°C with or without antibodies specific for ßig-h3 and CD147 in a humidified atmosphere containing 5% CO2. Cells remaining in the upper compartment were completely removed with gentle swabbing. The number of cells that had invaded through the filter into the lower compartment was determined by hematoxylin-eosin stain (HE stain).

Zymography Experiments.
To check the expression and activation of MMPs, cells incubated with or without antibodies specific for ßig-h3 and HAb18G/CD147 were cultured in serum-free medium and incubated at 37°C for 5 to 20 hrs. The conditioned media were collected by centrifugation, concentrated, and dialyzed. The dialyzed samples containing an equal amount of total proteins were mixed with sample buffer, incubated in a water bath (~55°C) for about 3 to 5 mins, and loaded onto a zymographic SDS gel containing gelatin (1 mg/ml) as previously described (16). The gels were washed in 2.5% Triton X-100 for 15 mins twice and incubated in incubation buffer (50 mM Tris-HCl [pH 8.8] containing 5 mM CaCl2 and 0.02% [w/v] NaN3) for 16 hrs. The gels were stained with Coomassie brilliant blue and destained. The zones of gelatinolytic activity were shown by negative staining.

Clone ßig-h3 Gene and Transient Transfected to 7721 Cells.
To clone ßig-h3 cDNA, poly(A)+ RNA was isolated from FHCC-98 cells, which express high levels of ßig-h3, and then reverse transcribed into the first-strand cDNA using superscript II reverse transcriptase and oligo(dT) primer (NEB, Ipswich, MA). Human ßig-h3 cDNA PCR amplified using high-fidelity MasterAmpm DNA polymerase (New England Biotechnology, Beverly, MA) and synthetic primers (5'-TAACTCGAGGCTTGCCCGTCGGTCGCTAGCT-3', 5'-ATCCGCGGGCCTCCAAGCCACGTGTAGATGT-3') was then cloned into the XhoI and SacI sites of pEGFP-C2 expression vector (Invitrogen), which express green fluorescent protein (GFP). The recombinant plasmid was transfected into 7721 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The expression of the enhanced green fluorescent protein (EGFP) was detected under a fluorescent microscope (Olympus, Nagoya, Japan). Schizolysis transfected 7721 cells 48 hrs after transfection. Western blot was adopted using lysate of transfected cells, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ßig-h3 Is Upregulated in T7721 Cells, Which Stably Overexpress HAb18G/CD147.
In our previous study, the overexpression of HAb18G/CD147 was shown to be able to enhance metastasis potential and elevate MMP secretion when it was tranfected into human 7721 hepatoma cells, hence renamed T7721 cells. To identify pivotal molecules in the HAb18G/CD147 signal transduction pathway in human hepatoma cells, we screened the differentially expressed genes by cDNA microarray between 7721 cells and T7721 cells. According to the final data from Capital Biochip Corp., the screening standard for a differentially expressed gene was that the fluorescence intensity ratio of T7721 to 7721 should be either >2.0 or <0.5. The final data showed that there were 13 differentially expressed genes, in which the expression of 7 genes was upregulated in T7721 cells compared with 7721 cells. We found that the expression of the transforming growth factor-ß (TGF-ß)–induced molecule ßig-h3 in T7721 cells was upregulated significantly—4.4-fold over that in 7721 cells—whereas the expression of TGF-ß1 was not obviously changed in both types of cells (Fig. 1Go), indicating that overexpression of HAb18G/CD147 may, at least in part, result in the upregulation of the ßig-h3 gene, which encodes a secretory extracellular matrix (ECM) protein involved in cell growth, tumorigenesis, wound healing, apoptosis, and migration (16–21).


Figure 1
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  		Figure 1. The overlay image of signal transduction-related microarray. (A) RNA of 7721 cells was directly labeled with Cy3 by reverse transcription, and RNA of T7721 cells was labeled with Cy5. The fluorescein labeled to RNA of 7721 and T7721 was opposite (B). The plots arrows point to present ßig-h3 (row 24, column 1) and TGF-b1 (row 3, column 21).

 
Downregulation of HAb18G/CD147 Depresses the Expression of ßig-h3.
To further investigate whether HAb18G/CD147 regulates the expression of ßig-h3, HAb18G/CD147-specialized siRNA was used to suppress the expression of HAb18G/CD147. T7721 cells transfected with HAb18G/CD147-specialized siRNA reduced the expression of HAb18G/CD147 protein by 80% (Fig. 2Go). This reduction of HAb18G/CD147 was accompanied by concomitant decreases of ßig-h3 expression in T7721 cells. These results suggest that HAb18G/CD147 may be involved in mediating the expression of ßig-h3.


Figure 2
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  		Figure 2. Downregulation of HAb18G/CD147 depresses the expression of ßig-h3 protein in T7721 cells. T7721 cells were transfected with HAb18G/CD147-specific siRNA (HAb18G-siRNA) or control siRNA (CTL-siRNA) by Lipofectamine 2000 (Invitrogen). The ßig-h3 protein expressions were determined by using Western blot analysis as described in Materials and Methods. Results are representative of triplicate experiments with similar results.

 
Expression of ßig-h3 Is in Positive Correlation with HAb18G/CD147 in Different Human Hepatoma Cells and Normal Hepatic Cells.
It has been reported that HAb18G/CD147 is highly expressed in hepatoma cells, especially in hepatoma cells with high metastasis potential, whereas it is lowly expressed in normal hepatic cells (11). Our results above suggest that ßig-h3 expression may be in positive correlation with HAb18G/CD147 expression in 7721 cells. To further confirm this conclusion, we tested the expression of HAb18G/CD147 in different human hepatoma cells and normal hepatic cells. Quantitative real-time PCR analysis results showed that the expression level of ßig-h3 mRNA in hepatoma cells 7721, T7721, and FHCC-98 was higher than that in normal hepatic cells QSG. We also found that the ßig-h3 mRNA level in FHCC-98 cells with high metastasis potential was much higher than that in 7721 cells with low metastasis potential (Fig. 3AGo). The results of Western blot showed that the expression level of ßig-h3 protein was in accordance with the level of mRNA (Fig. 3BGo). These results further confirm that the expression of ßig-h3 is in positive correlation with HAb18G/CD147 in hepatoma cells and suggest that ßig-h3 may be related to invasion and metastasis process in human hepatoma cells.


Figure 3
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  		Figure 3. The mRNA (A) and protein (B) expressions of ßig-h3 in different hepatoma cells and normal hepatic cells. The mRNA and protein expressions were tested using quantitative real-time PCR and Western blot analyses, respectively. The expression of ßig-h3 mRNA was normalized by the expression of GAPDH mRNA, and ßig-h3 protein was normalized by human ß-actin expression. Data are shown as mean ± SE from three independent experiments. *P < 0.01 vs. QSG cells; #P < 0.01 vs. 7721 cells (Student’s t test).

 
ßig-h3 Enhances Hepatoma Cell Invasion and Metastasis Potential.
Given our finding that ßig-h3 is highly expressed in human hepatoma cells, we overexpressed ßig-h3 in 7721 cells by transiently transfecting recombinant plasmid pEGFP-C2/ßig-h3. After 48 hrs, ßig-h3 was highly expressed in transfected 7721 cells (Fig. 4Go).


Figure 4
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  		Figure 4. Intracellular expressions of the GFP (A) and protein expression of ßig-h3 (B) in 7721 cells transfected with ßig-h3. Cells were transfected with ßig-h3 using a pEGFP-C2 expression vector (Invitrogen) as described in Materials and Methods. The expression of GFP was detected under a fluorescent microscope (Olympus). ßig-h3 protein expressions were determined using Western blot analysis as described in Materials and Methods. ßig-h3 protein was normalized by human ß-actin expression and is shown as mean ± SE from three independent experiments. Cells transfected with the pEGFP-C2 vector alone served as control. *P < 0.01 vs. pEGFP-C2 vector–transfected 7721 cells (Student’s t test).

 
As shown in Figure 5Go, the amount of attached cells observed in 7721 cells transfected with ßig-h3, expressed as a percentage of total seeded cells, was 63.2% ± 2.8%, compared with 44.3% ± 1.6% in pEGFP-C2 vector–transfected 7721 cells (P < 0.01; Fig. 5AGo). Antibodies are useful tools for blocking or activation assay via binding with extracellular and transmembrane antigen or internalization. In the present study, the commercial polyclonal antibody for ßig-h3 was introduced in the functional assay systems. The amount of attached cells was significantly decreased after treatment with antibodies specific for ßig-h3 (29.1% ± 4.4%, P < 0.01). The ability of ßig-h3–transfected 7721 cells to invade through Transwell chambers was greater than that of pEGFP-C2 vector–transfected 7721 cells: 42.9% ± 4.0% compared with 21.4% ± 3.1%, respectively (P < 0.01). Similarly, treatment with antibodies specific for ßig-h3 decreased the invasion ability of ßig-h3–transfected 7721 cells (15.2% ± 1.3%, P < 0.01; Fig. 5BGo).


Figure 5
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  		Figure 5. Adhesion potential (A), invasive potential (B), and MMP levels (C) of ßig-h3–expressing 7721 cells incubated with or without antibodies specific for ßig-h3. The invasion assay, invasive assay, and zymography experiment were adopted as described in Materials and Methods. Values are the means ± SE from six independent experiments. *P < 0.01 vs. pEGFP-C2 vector–transfected 7721 cells (Student’s t test).

 
As shown in Figure 5CGo, MMP-2 and MMP-9 levels were enhanced by 62.3% ± 3.2% in ßig-h3–transfected 7721 cells and were reduced by 40.8% ± 1.3% after treatment with antibodies specific for ßig-h3 compared with 7721 cells transfected with pEGFP-C2 vector (P < 0.01). This finding suggests that ßig-h3 may enhance invasion and metastasis potential of human hepatoma cells.

ßig-h3 Mediates HAb18G/CD147-Induced Metastasis of Human Hepatoma Cells.
To investigate the involvement of ßig-h3 in the HAb18G/CD147-induced invasion and metastasis process in human hepatoma cells, we incubated T7721 cells with monoclonal antibodies specific for HAb18G/CD147 (developed in our lab), multiclonal antibodies specific for ßig-h3 (Santa Cruz Biotechnology, Santa Cruz, CA), and both types of antibodies mixed to test the inhibitory effect on cell adhesion and invasion and MMP secretion.

Matrigel-coated plates and chambers were used to investigate the roles of ßig-h3 and HAb18G/CD147 in metastasis-related processes in human hepatoma cells. The percentage of attached T7721 cells incubated with antibodies specific for ßig-h3 and HAb18G/CD147 were significantly reduced (33.7% ± 8.7% and 28.3% ± 5.6%, respectively) compared with T7721 cells without either treatment (60.8% ± 11.3%; P < 0.01; Fig. 6AGo). Similar results also were observed in invasion tests. Compared with 29.7% ± 1.9% in T7721 cells without antibody treatment, the percentages of the invaded T7721 cells were 17.9% ± 2.4% in ßig-h3 antibody group and 15.7% ± 1.4% in HAb18G/CD147 antibody group (P < 0.01; Fig. 6BGo).


Figure 6
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  		Figure 6. Adhesion potential (A), invasive potential (B), and MMP levels (C) of T7721 cells incubated with antibodies specific for ßig-h3 (0.02 mg/ml), HAb18G/CD147 (0.1 mg/ml) and both types together. (A) T7721 + anti–ßig-h3; (B) T7721 + anti-HAb18; (C) T7721 + anti–ßig-h3 + anti-HAb18; (D) 7721; and (E) T7721. The invasion assay, invasive assay, and zymography experiment were adopted as described in Materials and Methods. Values are the means ± SE from six independent experiments. *P < 0.01 vs. T7721 cells (one-way ANOVA).

 
Since MMPs are well known for promoting cancer metastasis by degradation of the extracellular matrix, the effect of ßig-h3 and HAb18G/CD147 on metastatic potential could be mediated by MMPs. T7721 cells incubated with antibodies specific for ßig-h3 and HAb18G/CD147 reduced MMPs levels by 40.8% ± 2.1% and 42.0% ± 5.5%, respectively, compared with T7721 cells without antibody treatment (P < 0.01; Fig. 6CGo).

The adhesion potential, invasion potential, and MMP levels of T7721 cells incubated with the two types of antibodies mixed were reduced compared with T7721 cells without antibody treatment, with the percentages of 27.0% ± 3.1% (adhesion potential), 14.6% ± 2.7% (invasion potential), and 51.5% ± 4.1% (MMP levels) for each (P < 0.01). When comparing the groups treated with either antibody or both antibodies, we did not observe any significant difference in adhesion potential, invasion potential, or MMP seceration (P > 0.05). The results above indicate that ßig-h3 might be involved in the progress of HAb18G/CD147-induced invasion and metastasis activity of human hepatoma cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tumor growth and metastasis are multistep processes involving cell adhesion, proteolytic enzyme degradation of ECM, and motility factors that influence cell migration, in which the interaction between cells and ECM plays an essential role. The interaction of cells with ECM is mediated by contacts between cellular surface proteins and components of ECM, including laminin, fibronectin, and collagen (22). Invasion and metastasis are complicated and fatal cascades in human hepatoma, but the underlying molecular events remain largely unknown. Our previous studies implicated a hepatoma-associated antigen, HAb18G/ CD147, in metastasis processes (11, 23, 24), and they demonstrated the involvement of HAb18G/CD147 in the modulation of NO/cGMP-sensitive, store-operated Ca2+ entry and the metastasis potential of human hepatoma cells (14, 25). However, ligands or receptors for HAb18G/CD147 remain obscure, and whether HAb18G/CD147 directly interacts with extracellular molecules to execute its function is still unknown.

The present study demonstrated that expression levels of ßig-h3, a secretory ECM protein, were positively relative to the expression of HAb18G/CD147 by cDNA microarray, quantitative real-time PCR, Western blot, and siRNA assay. The results also showed that the expression level of ßig-h3 in hepatoma cells was much higher than that in normal hepatic cells, and in hepatoma cells with high metastasis potential it was much higher than that in hepatoma cells with low metastasis potential, which was coincident with the expression of HAb18G/CD147. This suggested that the expression of ßig-h3 was mediated by HAb18G/CD147, and ßig-h3 might be in involved in mediating the metastasis processes of hepatoma cells.

The regulatory mechanisms of the expression of ßig-h3 are still obscure. ßig-h3, first identified from the A549 lung adenocarcinoma cell line after long-term treatment by TGF-ß1, could be similarly induced in H2981 cells (lung adenocarcinoma cell line), HEPM cells, human melanoma cells, mammary epithelial cells, keratinocytes, and fibro-blasts. However, it has been reported that the same induced results were not observed in 293S cells (embryo kidney cell lines) and MCF-7, MDA453, and MDA468 cells (breast carcinoma cell lines; Refs. 16, 26). In the present study, the results of cDNA microarray indicated that ßig-h3 was upregulated in T7721 cells compared with 7721 cells, but the expression of TGF-ß1 had no obvious changes in the above two cell lines, suggesting that the upregulation of ßig-h3 is not a universal result of TGF-ß1 treatment.

It has been reported that CD147 stimulates the secretion of MMP-1 by activating a p38-dependent signaling pathway. This is the first available information regarding the mechanism by which tumor-associated molecules upregulate MMP synthesis in stromal fibroblasts. p38 is the only one of three major MAP kinases (ERK1/2, SAPK/JNK, and p38) that undergoes phosphorylation (an index of activation) in response to CD147 (27). So we supposed that HAb18G/CD147 regulates the expression of several proteins, such as MMP and ßig-h3, possibly via a MAPK-dependent signaling pathway. Expression, localization, and the intracellular signal mechanism of ßig-h3 were not yet well investigated. Studies to determine the signaling molecules in HAb18G/CD147 regulated ßig-h3 expression pathway are in progress.

ßig-h3, involved in cell growth, migration, apoptosis, wound healing, and tumorigenesis (16, 28–33), might function as either a promoter or an inhibitor of carcinogenesis, depending on cells and tumor types. The gain or loss of expression of ßig-h3 might be involved in tumor formation and the acquisition of a metastatic phenotype in human cancer. Analysis of cultured human bronchial smooth muscle cells identified ßig-h3 in both the ECM and the cytoplasm, and also in the nucleus, which suggests that it may have other as yet unknown functions in addition to its role as a structural ECM component. One possible explanation for nuclear localization is that the protein is secreted and subsequently taken up by cells, perhaps by receptor-mediated endocytosis. Alternatively, the protein may have multiple isoforms that potentially could be targeted to specific compartments; some isoforms are secreted and deposited in the ECM, whereas others lacking the signal peptide are translocated to the nucleus, transferring signals or regulating gene transcription (34). To detect its functions in human hepatoma carcinoma cells, we cloned ßig-h3 without the signal sequence into a GFP vector. The present results obtained from assays for cell adhesion and invasion processes involved in metastasis indicated a greater metastatic potential of the ßig-h3–expressing 7721 cells compared with 7721 cells transfected with vector as control. The fact that cell attachment and invasion could be greatly inhibited by antibody for ßig-h3 further confirmed the results. MMPs have been implicated in several aspects of tumor progression, including invasion through basement membrane and interstitial matrices, angiogenesis, and tumor cell growth (35, 36). Our data obtained from a gelatin zymography assay showed that ßig-h3 promoted MMP secretion, and the ßig-h3 special antibody inhibited this stimulated effect. All of the results clearly demonstrated that ßig-h3 as a promoting factor possibly enhances hepatoma cell invasion and metastasis potential.

Previous studies have demonstrated that HAb18G/ CD147 promotes the invasive and metastatic ability of human hepatoma cells (7, 11), so what is the relationship between HAb18G/CD147 and ßig-h3 in promoting invasion and metastasis potential? The phenomenon that the antibody specialized for ßig-h3 inhibited HAb18G/CD147-induced invasion and metastasis potential can partly explain the question. The similar inhibitory effects on cell attachment and invasion and MMP secretion also were observed among T7721 cells incubated with antibodies for ßig-h3, HAb18G/ CD147, or both types together. These results indicated that ßig-h3 may be involved in HAb18G/CD147-induced cell invasion and metastatic progress; that is, they cooperate in one signal transduction pathway promoting hepatoma cell metastasis.

Integrins are cell surface adhesive receptors composed of {alpha}- and ß-chain heterocomplexes, which mediate the physical and functional interactions between a cell and its surrounding ECM, thus serving as bidirectional transducers of extracellular and intracellular signals in the processes of cell adhesion, proliferation, differentiation, apoptosis, and tumor progression (37). Berditchevski et al. documented that CD147 may form complexes with integrin {alpha}3ß1 (37). Conversely, caveolin-1 may associate with CD147 from CD147-{alpha}3 integrin complexes, thereby diminishing both CD147 clustering and CD147-dependent, MMP-1–inducing activities (24). Moreover, ßig-h3, which contains an amino terminal signal peptide, four internal FASI repeat domains, and an RGD (Arg-Gly-Asp) motif, may interact with {alpha}3ß1, {alpha}1ß1, {alpha}6ß4, {alpha}vß3, or {alpha}vß5 in various tumor cells (30, 31, 33, 38, 39). These findings shed new light on the opportunity for integrin (especially {alpha}3ß1) to act as a bridge between HAb18G/CD147 and ßig-h3 to form a trimer, and then regulate HAb18G/CD147-induced metastasis of human hepatoma cells. The investigation of the relationship of HAb18G/CD147, ßig-h3, and integrin is underway in our laboratory.

In summary, all the results suggest that ßig-h3, the expression of which is regulated by HAb18G/CD147, may be involved in the HAb18G/CD147 signal pathway, and it plays important roles in invasion and metastatic processes in human hepatoma cells, although the precise mechanism calls for further attention.


   Acknowledgments
 
We thank Xi-ying Yao for her help in our experiments and Fan Peng for her careful reading of the manuscript.


   Footnotes
 
Supported by National Natural Science Foundation of China grant 30200144.

1 Drs. Tang, Zhou, and Jiang contributed equally to this work. Back

Received for publication July 21, 2006. Accepted for publication October 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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