The interaction of HAb18G/CD147 with integrin α6β1 and its implications for the invasion potential of human hepatoma cells

Background HAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin α3β1. However, it has never been investigated whether α3β1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells. Methods Human SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin α6β1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin α6β1. Invasion potential was evaluated with an invasion assay and gelatin zymography. Results We found that integrin α6β1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs) were partially blocked by integrin α6β1 antibodies (P < 0.01). Wortmannin, a specific phosphatidylinositol kinase (PI3K) inhibitor that reverses the effect of HAb18G/CD147 on the regulation of intracellular Ca2+ mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (P < 0.05). Importantly, no additive effect between Wortmannin and α6β1 antibodies was observed, indicating that α6β1 and PI3K transmit the signal in an upstream-downstream relationship. Conclusion These results suggest that α6β1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.

Integrins are cell surface adhesive receptors composed of αand β-chain heterocomplexes that mediate physical and functional interactions between cells and the extracellular matrix. Variant integrins can interact with different ligands and vice versa. Integrins thus serve as bidirectional transducers of extracellular and intracellular signals in the processes of cell adhesion, proliferation, differentiation, apoptosis, and tumor progression. Normal adult hepatocytes express low levels of only three integrins: α1β1 (a collagen and laminin receptor), α5β1 (a fibronectin receptor), and α9β1 (a tenascin receptor). In contrast, other integrins (such as α2β1, α3β1, α6β1, and α6β4) are not present in normal hepatocytes, but are expressed in hepatoma cells. However, the precise roles integrins play in liver carcinogenesis remain unclear.
In previous studies, CD147 was found to be associated with integrins α3β1 and α6β1, but not α2β1 and α5β1 [17]. In a recent study, we demonstrated that α3β1 plays a critical role in CD147-mediated liver carcinogenesis, indicating that the interaction between CD147 and various integrins is a necessary step for their tumor-promoting effects [18]. However, it is unknown whether α3β1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells. Given the fact that one of the most frequent alterations during liver carcinogenesis is de novo expression of the integrin α6β1 and that the induction of α6β1 expression is an early event in hepatocellular carcinogenesis [19][20][21][22][23], it is critical to clarify whether α6β1 interacts with CD147 and thus contributes to liver carcinogenesis.
In the present study, we demonstrated that HAb18G/ CD147 interacts with integrin α6β1, activates the PI3K signal pathway through phosphorylation, and thereby enhances the invasion potential of hepatoma cells.
Forty-eight hours after siRNA transfection, total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions for use in the analysis of CD147 mRNA levels. The isolation was followed by first-strand cDNA synthesis using True Script reverse transcriptase (BioRad, Hercules, CA, USA). The cDNA was amplified by PCR using a specific primer set for CD147 or glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal control. The sequences of the upstream and downstream primers were, respectively, as follows: Sense: 5'-ACA TCA ACG AGG GGG AGA CG-3'; Antisense: 5'-GGC TTC AGA CAG GCA GGA CA-3' for CD147 (492 bp); Sense: 5'-CTG AAC GGG AAG CTC ACT GG-3'; Antisense: 5'-TGA GGT CCA CCA CCC TGT TG-3' for GAPDH (313 bp). PCR analysis was performed under the following conditions: an initial denaturation at 94°C for 2 min followed by 25-30 cycles of a 30-sec denaturation step at 94°C, renaturation for 60 sec at 57°C, and extension for 90 sec at 72°C. The amplified products were analyzed by 1% agarose gel electrophoresis followed by GoldView™ staining. Forty-eight hours after siRNA transfection, FHCC98 and 7721 cells were harvested in a lysis buffer and equal amounts of cellular proteins were subjected to SDS-PAGE (10%). Proteins were transferred to polyvinylidene difluoride membranes and blots were probed with HAb18 mAb (prepared in our laboratory). GAPDH was chosen as an internal control and the blots were probed with mouse anti-GAPDH mAb (Chemicon International, Inc.).

Immunofluorescence and confocal microscopy
Cells were allowed to attach to precoated glass coverslips overnight. They were fixed the following day in 4% paraformaldehyde and then blocked with 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 0.5 h. Coverslips were incubated with the primary antibodies (Santa Cruz) at a 1:200 dilution in PBS for 1 h. Primary antibody-treated cells were washed in PBS and then incubated with Allex594 goat anti-mouse (Invitrogen-Molecular Probe, USA) or fluorescein isothiocyanate (FITC)conjugated donkey anti-goat secondary antibodies (Santa Cruz, USA) at a 1:500 dilution in PBS for 1 h. Cell nuclei were dyed with 4,6-diamino-2-phenyl indole (DAPI) (Invitrogen-Molecular Probe) for 3 min. Finally, the cells were mounted using glycerol and observed by FV1000 laser scanning confocal microscope (Olympus).

Invasion assay
The assay was performed using chambers with polycarbonate filters with 8-μm nominal pore size (Millipore, USA) coated on the upper side with Matrigel (Becton Dickinson Labware, USA). The chambers were placed into a 24-well plate. Hepatoma cells were harvested after 24 h of siRNA transfection. Five groups of transfected cells (1×10 5 ) were harvested and placed in the upper chamber: transfected cells alone, cells treated with 10 μg/mL antiintegrin α6β1 mAb, cells treated with 1 μg/mL Wortmannin in 100 μL RPMI 1640 containing 0.1% BSA, sncRNAtransfected cells (negative control), and untreated cells (blank control). The lower chamber was filled with 600 μL RPMI 1640 containing 5% FBS and then incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO 2 . Cells remaining in the upper chamber were completely removed by gently swabbing. The number of cells that passed through the filter and invaded the lower chamber was determined using a colorimetric crystal violet assay.

Gelatin zymography
Twenty-four hours after siRNA transfection, cells were treated with antibodies or inhibitors in serum-free medium. Cells were incubated at 37°C for 20 h. The conditioned medium was collected and separated by 8% acrylamide gels containing 0.1% gelatin. The gels were incubated in a 2.5% Triton X-100 (Sigma, USA) solution at room temperature with gentle agitation and then were soaked in reaction buffer [50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 10 mM CaCl 2 ] at 37°C overnight. The gels were stained for 6 h and destained for 0.5 h. The zones of gelatinolytic activity were revealed by negative staining.

Western blot for Akt phosphorylation
FHCC98 and 7721 cells were harvested in a lysis buffer, and equal amounts of cellular proteins were subjected to SDS-PAGE (10%). Proteins were transferred to PVDF membranes, and blots were probed with phosphorylated and non-phosphorylated forms of Akt. Blots were washed 3-5 min with Tris-buffered saline/0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody (1:2000) for 1 h. The membranes were washed as described above, and the bands detected by chemilluminescence (Amersham, Freiburg, German).

Statistical analysis
Statistical significance was determined using a one-way ANOVA analysis or Student's t-test. GraphPad Prism software (Cricket Software, Philadelphia, PA) was used for the above analyses and P values less than 0.05 were considered significant.

HAb18G/CD147 co-localized with α6β1 integrin on the surface of human hepatoma cells
Immunofluorescent double-staining and confocal imaging analysis demonstrated that integrin α6β1 and HAb18G/CD147 were strongly expressed at the marginal areas of the two different hepatoma cell lines FHCC98 (Fig. 1) and 7721 (not shown). HAb18G/CD147 signals co-localized with integrin α6 and β1 on the surface of HCC cells. The co-localizations were distributed diffusely throughout the membrane.

HAb18G/CD147 immunoprecipitates with α6β1 integrin in human hepatoma cells
To verify whether HAb18G/CD147 interacts with integrin α6β1 in hepatoma cells, we performed co-immunoprecip-itation experiments in FHCC98 (Fig. 2) and 7721 (not shown) cells. As Fig. 2 demonstrates, integrin α6 and β1 subunits co-immunoprecipitate with endogenous HAb18G/CD147 in FHCC98 cells. The same finding occurred in 7721 cell lysates (results not shown). The results indicated that HAb18G/CD147 and α6β1 integrin interact in their native conformations.

Effects of silencing HAb18G/CD147 on FHCC98 and 7721 cells
To investigate the role of HAb18G/CD147 in FHCC98 and 7721 cells, RNA interference was used to knock down the expression of HAb18G/CD147 in these two cell lines. The HAb18G/CD147-specific siRNA (siCD147) and sncRNA were tested for their ability to specifically suppress HAb18G/CD147. RT-PCR showed that the sncRNA was incapable of inhibiting HAb18G/CD147 gene expression, whereas siCD147 could effectively decrease the mRNA expression of HAb18G/CD147 (Fig. 3A). Similar results were obtained in 7721 cells (data not shown). These results were confirmed by Western blot. The protein expression of HAb18G/CD147 was obviously decreased in siCD147-transfected cells, but not in sncRNA-transfected cells 48 h after siRNA transfection (Fig. 3B). These data show that siCD147 treatment effectively decreased HAb18G/CD147 expression in FHCC98 and 7721 cells.
After siRNA transfection or treatment with blocking mAb against integrin α6β1, the supernatants of FHCC98 and 7721 cells were collected to test the levels of MMP secre-Expression and co-localization of HAb18G/CD147 with integrin α6 and β1 subunits in FHCC98 cells Figure 1 Expression and co-localization of HAb18G/CD147 with integrin α6 and β1 subunits in FHCC98 cells. FHCC98 cells were double-stained for HAb18G/CD147 (red) and integrin α6 or β1 (green).
Immunoprecipitation of HAb18G/CD147 and integrin α6 and β1 subunits in FHCC98 cells tion. Results from the gelatin zymography assay showed that secretion of both MMP-2 and MMP-9 was significantly reduced in FHCC98 and 7721 cells after treatment with siRNA or antibodies. As shown in Fig. 5, the density of MMPs in FHCC98 cell lines after treatment with sncRNA associated with GoH3, 3S3, and GoH3+3S3 was reduced by 32.55 ± 3.01%, 34.81 ± 6.25%, and 29.09 ± 5.33%, respectively (P < 0.01), as compared to the blank group. The MMPs levels in siHAb18G/CD147-transfected FHCC98 cells were also significantly reduced by 21.31 ± 9.98% (P < 0.01), but there was no significant difference in comparison to the sncRNA+GoH3, sncRNA+3S3, and sncRNA+GoH3+3S3 groups. The density of MMPs in the siHAb18G/CD147+GoH3, siHAb18G/CD147+3S3, and siHAb18G/CD147+GoH3+3S3 groups were also significantly reduced by 27.75 ± 5.03%, 32.78 ± 6.30%, and 32.63 ± 11.90% (P < 0.05), respectively, as compared to the blank group, but no significant difference was found in comparison to the siHAb18G/CD147-transfected cells alone. Similar results were obtained in 7721 cells (data not shown). The results of the invasion assay and gelatin zymography suggested that there were no additive effects between HAb18G/CD147 gene silencing and α6β1 antibodies.
The results of gelatin zymography also showed that MMP secretion was significantly reduced in FHCC98 and 7721 cells after treatment with Wortmannin and anti-α6 mAb. The density of MMPs in the anti-α6 mAb, Wortmannin, and Wortmannin-associated anti-α6 mAb was reduced by 18.82 ± 7.27%, 19.26 ± 5.21%, and 16.85 ± 4.13%, respectively, as compared to the blank control group in FHCC98 cells (P < 0.05) ( Fig. 6D and 6E). In 7721 cells, these same groups correspondingly manifested reduced MMP density by 22.82 ± 12.32%, 18.18 ± 4.97%, and 20.03 ± 3.74% (P < 0.05) (Fig. 6D and 6F). There were no significant differences among groups treated with anti-α6 mAb, Wortmannin, or anti-α6 mAb associated with Wortmannin (P > 0.05). To identify whether Akt phosphorylation is involved in the HAb18G/CD147-mediated and integrin-activated invasion processes of human hepatoma cells, the expression levels of Akt and P-Akt were tested by Western blot. The expression levels of P-Akt decreased 57.62 ± 3.61% and 68.06 ± 4.43%, respectively, in siCD147-transfected FHCC98 cells and siCD147-transfected 7721 cells when compared to expression levels in sncRNA-transfected cells (P < 0.01, Fig. 7). All these results suggest that PI3K, a key downstream signal molecule of integrin α6β1, is involved in HAb18G/CD147-induced invasion and metastatic processes of human hepatoma cells.
Effect of silencing HAb18G/CD147 in FHCC98 and 7721 cells

Discussion
The invasion process is a complicated and fatal cascade process in the human hepatoma, but the underlying molecular events remain largely unknown. Our previous studies implicated a hepatoma-associated antigen, HAb18G/CD147, in metastatic processes [28][29][30]. However, ligands or receptors for HAb18G/CD147 remain obscure, and whether HAb18G/CD147 directly interacts with extra-or intracellular molecules to execute its function is still unknown. We report here that HAb18G/ CD147, by interacting with α6β1 integrin, enhances the metastatic potential of hepatoma cells.
In this study, we identified previously uncharacterized roles for HAb18G/CD147. The expression of integrin α6β1, a receptor of laminin, was positively related to the expression of HAb18G/CD147 as indicated the immunofluorescent double-staining and co-immunoprecipitation assay. When the specific antibodies for α 6 and β1 were added to the culture system, effective blocking of the interaction of HAb18G/CD147 and integrin α6β1 reduced cell invasion and MMP secretion. In siHAb18Gtransfected cells, the addition of specific α6 and β1 antibodies provides no significant additive inhibitory effect. These results suggest that the enhancing effect of HAb18G/CD147 on cell invasion and metastatic potential is mediated through α6β1 integrin.
Integrins serve as the major receptors mediating cellmatrix adhesion, which plays an important role in the invasive process of tumor cells. Therefore, it is not surprising that their interaction with laminin, the major compo-Invasion of hepatoma cells with or without integrin α6β1 mAbs and siHAb18G/CD147 treatment nent of the basement membrane, serves as a key event in the tumor invasion and metastatic process [31]. Through the interaction with integrins, CD147 may regulate the integrin-laminin association and then influence diverse processes such as basement membrane formation. The interaction of CD147 with integrin is conserved in Drosophila and is proposed to play a role in dorsal closure and extraembryonic membrane apposition [32] as well as in the maintenance of cellular architecture through cytoskeletal rearrangement [18]. Within cultured insect cells, the CD147-integrin interaction is essential for lamellipodia formation. Within retinal cells, disruption of the CD147-integrin interaction results in aberrant organelle distribution, including mitochondria, nuclei, and rough endoplasmic reticulum [18]. The elevated expression levels of both CD147 and integrin α6β1 have been observed in most metastatic and primary cancers [33,34]. Compar-ison of normal prostate cells to cancerous cells showed a preferential pairing of α6 with β4 transitioning to α6 with β1 in cancer cells [35]. The significance of this transition, while still speculative, is most likely related to alterations in downstream signaling events. But there are no reports about the signal mechanism of the CD147-integrin/α6β1 interaction.
The signaling pathways of the integrins consist of many cytoskeleton proteins and enzymes, of which focal adhesion kinase (FAK), paxillin, and PI3K are crucial components. We previously discovered that FAK, paxillin, and their phosphorylation levels closely correlate with HAb18G/CD147 expression in human hepatoma cells [30]. In the present study, a specific inhibitor of PI3K, a key protein kinase downstream of integrin α6β1, significantly blocked HAb18G/CD147-induced invasion and MMP secretion of hepatoma cells with or without integrin α6β1 mAbs and siHAb18G/CD147 treatment MMP release. Our previous data have already demonstrated that elevated HAb18G/CD147 expression leads to attenuation of the store-operated Ca 2+ entry response to NO/cGMP and enhanced metastatic potential [26], but the precise mechanism is still unknown. As a strong inducer for intracellular Ca 2+ store release, IP3 is stimulated by PI3K and activates the Ca 2+ channels, allowing greater Ca 2+ influx into the cells. We may speculate that HAb18G/CD147 enhances the invasion and metastatic potential of human hepatoma cells via integrin α6β1-PI3K-Ca 2+ signaling pathways.

Conclusion
In the present study, we have identified the interaction of HAb18G/CD147 with integrin α6β1 in human hepatoma cells. We demonstrated that HAb18G/CD147 promotes invasion potential of hepatoma cells by interacting with integrin α6β1 and further activating its downstream PI3K-Akt signaling pathway. These findings shed new light onto the mechanisms underlying HAb18G/CD147-induced invasion and human hepatoma cell metastatic processes. The CD147-integrin α6β1 interaction might be a novel potential target for tumor metastasis therapy.