Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2011) 35, 811–817 (Printed in Great Britain)
βig-h3 regulates store-operated Ca2+ entry and promotes the invasion of human hepatocellular carcinoma cells
Yun‑Shan Guo1, Juan Tang1, Bo Chen, Wan Huang, Yong Li, Hong‑Yong Cui, Xin Zhang, Shi‑Jie Wang, Zhi‑Nan Chen and Jian‑Li Jiang2
Cell Engineering Research Centre and Department of Cell Biology, State Key Laboratory of Cancer Biology, State Key Discipline of Cell Biology, Fourth Military Medical University, Xian 710032, Peoples Republic of China


βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.


Key words: βig-h3, Ca2+ mobilization, matrix metalloproteinase, metastasis

Abbreviations: [Ca2+]i, intracellular free calcium ion concentration, 8-Br-cGMP, 8-bromo-cGMP, ECM, extracellular matrix, ER, endoplasmic reticulum, FAK, focal adhesion kinase, FAS, fasciclin, FBS, fetal bovine serum, Fura-2/AM, Fura-2 acetoxymethyl ester, HCC, hepatocellular carcinoma, IP3, inositol trisphosphate, MMPs, matrix metalloproteinases, NO, nitric oxide, NPBS, normal PBS, OG, octyl glucoside, PI3K, phosphatidylinositide 3-kinase, PKG, protein kinase G, siRNAs, small interfering RNAs, SNAP, S-nitroso-N-acetylpenicillamine, snc-RNA, silencer negative control siRNA, SOC, store-operated Ca2+, TGF-β, transforming growth factor β

1Yun-Shan Guo and Juan Tang contributed equally to this work.

2To whom correspondence should be addressed (email jiangjl@fmmu.edu.cn).


1. Introduction

βig-h3 [also known as TGFBI (transforming growth factor, β-induced), MP78/70, RGD-CAP (Arg-Gly-Asp-containing collagen-associated protein) and keratoepithelin] is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein. It is a 68-kDa protein with an N-terminal signal peptide and four internal FAS (fasciclin)-1 domain repeats, which are homologous to FAS-1 in Drosophila (Kawamoto et al., 1998). Βig-h3 functions in cell growth and migration, apoptosis, wound healing and tumourigenesis (Bae, et al., 2002; Kim et al., 2003; Park et al., 2004). βig-h3 promotes human astrocytoma cell adhesion through α6β4 integrins (Kima et al., 2003). Further, it mediates the adhesion and inhibits the differentiation of osteoblasts via αvβ3/αvβ5 integrins (Thapa et al., 2005). βig-h3 enhances the expansion of chondrocytes and fibroblasts via α1β1 integrins (Shigeru et al., 1999). Our previous studies suggest that βig-h3 promotes the adhesion and increases the invasive potential of HCC (human hepatocellular carcinoma) cells (Tang et al., 2007). By interacting with α3β1 integrins, βig-h3 increases the phosphorylation and expression levels of FAK (focal adhesion kinase) and paxillin, and by FAK–paxillin signalling linkage, βig-h3 causes cytoskeleton reorganization (Tang et al., 2009). These observations suggested that βig-h3 may increase the adhesion potential of human HCC cells by focal adhesion formation and cytoskeleton reorganization, but the mechanism of βig-h3-mediated increase in the invasive potential of human HCC cells is still unknown.

Ca2+ is an important second messenger involved in the control of cell metastasis, proliferation, apoptosis, differentiation and metabolism (Roderick and Cook, 2008; Clapham, 2007). A previous study showed that the release of MMPs (matrix metalloproteinases), which are implicated in several aspects of tumour progression, is regulated by SOC (store-operated Ca2+) entry in human HCC cells (Jiang et al., 2004a). In most non-excitable cells, the depletion of ER (endoplasmic reticulum) stores elicits sustained Ca2+ influx by SOC entry, thus defining the major Ca2+ influx pathway (Feng et al., 2010). SOC influx is essential for maintaining the Ca2+ content in the ER at a precise level (Parekh and Putney, 2005; Brandman et al., 2007).

Our previous study showed that βig-h3, which is regulated by HAb18G/CD147, is involved in the HAb18G/CD147 signal transduction pathway and mediates HAb18G/CD147-induced invasion and metastasis of human HCC cells (Tang et al., 2007). CD147 is a transmembrane glycoprotein of the immunoglobulin superfamily and is broadly expressed in many cell types, especially in human tumour cells at high levels (Li et al., 2009). HAb18G/CD147 enhances the invasive potential of human HCC cells by disrupting the regulation of SOC entry by NO (nitric oxide)/cGMP, although the detailed mechanism of this process is unknown (Jiang et al., 2001). In co-operation with HAb18G/CD147 in the same signal transduction pathway, βig-h3 may increase the invasive potential of human HCC cells by regulating SOC entry.

In this study, we showed that βig-h3 promotes MMP-2 and -9 secretion by attenuating the negative regulation of NO/cGMP-sensitive SOC entry and, thus, enhances the invasive potential of human HCC cells.

2. Materials and methods

2.1. Cell culture

Human SMMC-7721 cells (obtained from the Institute of Cell Biology, Academic Sinica) were cultured with RPMI 1640 medium supplemented with 10% FBS (fetal bovine serum), 1% penicillin/streptomycin and 2% l-glutamine at 37°C in a humidified atmosphere of 5% CO2.

2.2. Gene silencing

The sense and antisense sequences for βig-h3 siRNA (small interfering RNA) were 5′-CCUUUACGAGACCCUGGGATT-3′ and 5′-UCCCAGGGUCUCGUAAAGGTT-3′ (Ambion), respectively. SMMC-7721 cells that were 60–70% confluent were transfected with 50 pM siRNA that specifically targets the βig-h3 gene or 50 pM snc-RNA (silencer negative control siRNA) by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The transfected cells were cultured for 24–48 h and used for further functional analysis.

2.3. Western blot analysis

Cells were lysed in 1% OG (octyl glucoside) buffer [20 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1% OG, 1 mM EDTA, 10 μg/ml leupeptin, 2 μg/ml aprotinin and 1 mM PMSF]. The BCA (bicinchoninic acid) Protein Assay Kit (Pierce Biotechnology) was employed to determine the total protein concentration; equal amounts of proteins were separated by 10% SDS/PAGE and then electrophoretically transferred to PVDF microporous membranes (Millipore). The membranes were blocked with 5% non-fat milk and incubated for 2 h at room temperature with anti-βig-h3 antibody (Santa Cruz Biotechnology). The membranes were extensively washed and incubated with a secondary horseradish peroxidase-conjugated rabbit anti-goat antibody (Pierce). Immunodetection was performed using the Western-Light chemiluminescence detection system (Applied Biosystems).

2.4. Reverse transcription PCR

Total RNA was extracted from the cells with TRIzol reagents (Invitrogen) and reverse transcribed with the ReverTra Ace kit (Toyobo). All primers and probes were synthesized by Shanghai Sangon Co., and their sequences were as follows: βig-h3: forward primer, 5′-CATTGAGAACAGCTGCATCG-3′ and reverse primer, 5′-AGTCTGCTCCGTTCTCTTGG-3′; β-actin (control): forward primer, 5′-CCCAGCCATGTACGTTGCTA-3′ and reverse primer, 5′-TCACCGGAGTCCATCACGAT-3′. The PCR conditions for βig-h3 were 1 cycle at 94°C for 4 min; 32 cycles at 94°C for 20 s, 54°C for 30 s and 72°C for 20 s and, finally, 1 cycle at 72°C for 10 min. The PCR products were electrophoresed on 1% agarose gels.

2.5. Immunofluorescence

Cells were allowed to attach to sterile coverslips for 6 h. The cells were then fixed in 3.7% formaldehyde in PBS, permeabilized with 0.5% Triton X-100 and blocked with 1% BSA (Fraction V) in PBS for 1 h. The coverslips were incubated with βig-h3 antibodies (Santa Cruz Biotechnology) at a dilution of 1:50 in PBS for 40 min. The antibody-treated cells were washed in PBS and incubated with Texas Red donkey anti-goat secondary antibodies (Pierce Biotechnology) at a dilution of 1:500 in PBS for 1 h. Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; Biotium). Finally, the cells probed with rhodamine–phalloidin were washed and immediately mounted using glycerol and observed using the FV1000 laser scanning confocal microscope (Olympus).

2.6. Measurement of [Ca2+]i (intracellular free calcium ion concentration)

[Ca2+]i was measured using Fura-2/AM (Fura-2 acetoxymethyl ester) (Molecular Probes). Cells were stained by incubating them with 5 μM Fura-2/AM for 45 min in the dark at 37°C in NPBS (normal PBS) containing 2 mM CaCl2 (pH 7.4). The cells were then washed and resuspended in NPBS. To start the experiment, cells were pretreated with 4 μM thapsigargin (Calbiochem) for 20 min. Subsequently, they were washed with and maintained briefly in PBS containing no Ca2+ and 2 mM EGTA. Unless stated otherwise, the cells were pretreated with or without chemicals [i.e. 8-Br-cGMP (8-bromo-cGMP) or SNAP (S-nitroso-N-acetylpenicillamine)] for 5 min. The fluorescence signal was monitored and recorded using the FV1000 laser scanning confocal microscope (Olympus).

2.7. Zymography experiments

To detect the expression and activation of MMPs, cells preincubated with antibodies or inhibitors were cultured in serum-free medium and incubated at 37°C for 5–20 h. The conditioned media were collected, and samples were separated by 8% acrylamide gels containing 0.1% gelatin. The gels were incubated in 2.5% Triton X-100 solution at room temperature with gentle agitation and were then soaked in the reaction buffer [0.05 mol/l Tris/HCl (pH 7.5), 0.2 mol/l NaCl and 0.01 mol/l CaCl2] at 37°C for 20 h. After the reaction occurred, the gels were stained with Coomassie Blue for 6 h and destained for 2 h. The zones of gelatinolytic activity were observed by negative staining.

2.8. Invasion assay

The chemotactic cell invasion assay was performed using 24-well Transwell units with an 8-μm pore size polycarbonate filter (Millipore). Each lower compartment of the Transwell contained 600 ml of 0.5% FBS as the chemoattractant or 0.5% BSA in RPMI 1640 medium as the negative control. The upper side of the filter was coated with Matrigel (Becton Dickinson Labware) to form a continuous thin layer. Before adding the cell suspension, the dried layer of the Matrigel matrix was rehydrated with the medium (without FBS) for 2 h at room temperature. Cells (1×105) were suspended in 0.1 ml RPMI 1640 medium containing 0.1% BSA, added into the upper compartment of the Transwell unit and incubated for 36 h at 37°C in a humidified atmosphere containing 5% CO2. After the incubation period, the cells remaining in the upper compartment were completely removed by gentle swabbing. The number of cells that invaded through the filter into the lower compartment was determined using a colorimetric Crystal Violet assay.

2.9. Statistical analysis

Results are expressed as mean values±S.D. and representative for at least three independently performed experiments. Statistical significance between the different values was analysed by one-way ANOVA (analysis of variance) for unpaired data with a threshold of P<0.05 (SPSS 13.0 statistical software).

3. Results

3.1. Ca2+ is involved in βig-h3-mediated increases in MMP secretion and invasive potential of human HCC cells

We have shown that βig-h3 is highly expressed in human HCC cells and may enhance the metastatic potential of these cells. To examine the mechanism underlying this process, we transfected siRNA targeted against βig-h3 into human SMMC-7721 HCC cells to silence βig-h3 expression. Western blot (Figure 1A) and RT (reverse transcription)-PCR (Figure 1B) were performed 24 h after transfection, and these analyses suggested that βig-h3 was significantly down-regulated in βig-h3 siRNA-transfected SMMC-7721 cells compared with that in snc-RNA-transfected SMMC-7721 cells. Further, immunofluorescence confirmed that a decrease in βig-h3 fluorescence occurred in βig-h3 siRNA-transfected SMMC-7721 cells (Figure 1C).

Elevated intracellular Ca2+ levels promote the release and activation of MMPs (Jiang et al., 2004b). To detect whether Ca2+ is involved in βig-h3-mediated increase in the invasive potential of human HCC cells, Ni2+, a potent blocker of Ca2+ entry that competes for Ca2+-binding sites, was used as previously reported (Jiang et al., 2001). As shown, the levels of MMP-2 and -9 in βig-h3 siRNA-transfected SMMC-7721 cells were markedly lower than those in snc-RNA-transfected SMMC-7721 cells (P<0.05, Figure 1D). However, the addition of 3 mM NiCl2 reduced the MMP-2 and -9 levels in snc-RNA-transfected SMMC-7721 cells to levels comparable with those in βig-h3 siRNA-transfected SMMC-7721 cells (P>0.05, Figure 1D).

As shown, the invasive potential of βig-h3 siRNA-transfected SMMC-7721 cells was significantly lower than that of snc-RNA-transfected SMMC-7721 cells (P<0.05, Figure 1E). However, the addition of 3 mM NiCl2 reduced the invasive potential of snc-RNA-transfected SMMC-7721 cells to a level comparable with that of βig-h3 siRNA-transfected SMMC-7721 cells (P>0.05). These results indicate that extracellular Ca2+ entry is involved in βig-h3-mediated increase in the invasive potential of human HCC cells.

3.2. βig-h3 increases MMP secretion and the invasive potential of human HCC cells by increasing the sensitivity of these cells to Ca2+ entry

To further investigate the role of βig-h3-mediated regulation of extracellular Ca2+ entry in the invasion process of human HCC cells, βig-h3 siRNA- and snc-RNA-transfected SMMC-7721 cells were cultured in media containing serial doses of CaCl2 and incubated for 16–18 h. Zymographic analysis showed that an increase in CaCl2 concentration (0–1.2 mM) also increased MMP-2 and -9 secretion in a dose-dependent manner in both types of cells. The MMP-2 and -9 secretion reached a plateau in media containing more than 1.2 mM CaCl2. Moreover, at the same CaCl2 concentration, the levels of MMP-2 and -9 in βig-h3 siRNA-transfected SMMC-7721 cells were lower than those in snc-RNA-transfected SMMC-7721 cells (Figure 2A, P<0.05).

An invasion assay was performed to further test the effect of the addition of 1.20 mM CaCl2 on the invasive potential of human HCC cells. As shown, the invasive potential increased in both βig-h3 siRNA- and snc-RNA-transfected SMMC-7721 cells (Figure 2B). Moreover, the increased rate in βig-h3 siRNA-transfected SMMC-7721 cells was significantly less than that in snc-RNA-transfected SMMC-7721 cells (6.9±1.8% compared with 22.7±2.4%, respectively; P<0.05; Figure 2B). These results suggest that βig-h3 is involved in the increase in invasive potential mediated by the augmentation of extracellular Ca2+ in SMMC-7721 cells.

3.3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive SOC entry

We have previously shown the presence of a NO–cGMP-regulated SOC entry pathway in human SMMC-7721 cells (Jiang et al., 2001). cGMP, an activator of PKG (protein kinase G), negatively regulates SOC entry by the inhibition of phosphoinositide hydrolysis, mobilization of intracellular calcium and voltage-dependent activation of L-type Ca2+ channels (Lucas et al., 2000; Zucchi et al., 2001; Piper et al., 2004). NO is an important intracellular signalling molecule that activates soluble guanyate cyclase to synthesize cGMP (Denninger and Marletta, 1999). The present study investigated the involvement of cGMP and NO in the regulation of SOC entry. Thapsigargin, a potent inhibitor of the ER Ca2+–ATPase pump, was used to deplete intracellular Ca2+ stores and induce Ca2+ entry from the extracellular space. After the cells were treated with 4 μM thapsigargin in Ca2+-free medium for 20 min, the addition of 2 mM CaCl2 induced an increase in [Ca2+]i because of Ca2+ entry (Figures 3A, 3B); this finding suggested that thapsigargin induces SOC entry in both βig-h3 siRNA- and snc-RNA-transfected SMMC-7721 cells. Both 1 μM 8-Br-cGMP (a cGMP agonist) and 200 μM SNAP (an NO donor) reduced the thapsigargin-induced increase in [Ca2+]i in both βig-h3 siRNA- and snc-RNA-transfected SMMC-7721 cells. The inhibition rate of Ca2+ entry by 1 μM 8-Br-cGMP in βig-h3 siRNA-transfected SMMC-7721 cells was 76.5±2.3% and in snc-RNA-transfected SMMC-7721 cells was 41.2±4.8% (Figure 3A). The inhibition rate by 200 μM SNAP in βig-h3 siRNA-transfected SMMC-7721 cells (65.1±3.9%) was significantly greater than that in snc-RNA-transfected SMMC-7721 cells (22.7±5.4%; P<0.05; Figure 3B). These findings suggest that the inhibitory effect of cGMP and SNAP could be significantly enhanced by the down-regulation of βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive SOC entry.

3.4. NO/cGMP-sensitive Ca2+ entry is involved in βig-h3-mediated increases in MMP secretion and invasive potential

From the above results, we speculated that βig-h3 may promote the release and activation of MMPs by attenuating the negative regulation of NO/cGMP-sensitive Ca2+ entry to increase [Ca2+]i in HCC cells. To examine this hypothesis, invasion experiments by using Transwell and zymography experiments were performed. As shown, the addition of 1 μM 8-Br-cGMP reduced the secretion of MMP-2 and -9 by 38.8±5.2% in βig-h3 siRNA-transfected SMMC-7721 cells and by 24.0±2.4% in snc-RNA-transfected SMMC-7721 cells (Figure 4A). The inhibitory effect of 200 μM SNAP in βig-h3 siRNA-transfected SMMC-7721 cells (inhibition rate, 35.1±6.4%) was significantly greater than that in snc-RNA-transfected SMMC-7721 cells (inhibition rate, 18.2±3.2%).

As depicted, the addition of 1 μM 8-Br-cGMP or 200 μM SNAP reduced the invasive potential of both βig-h3 siRNA- and snc-RNA-transfected SMMC-7721 cells (Figure 4B). The inhibitory response of 8-Br-cGMP or SNAP in βig-h3 siRNA-transfected SMMC-7721 cells was significantly greater than that in snc-RNA-transfected SMMC-7721 cells. All the results indicate that βig-h3 reverses the inhibitory effect of NO/cGMP on MMP secretion and invasion in human HCC cells.

4. Discussion

βig-h3 was first identified in the human adenocarcinoma cell line A549 that had been treated with TGF-β (Skonier et al., 1992; Gibson et al., 1997; Hashimoto et al., 1997; Munier et al., 1997). βig-h3 is secreted into the ECM and might function as an extracellular attachment protein, which is involved in cell adhesion and metastasis. Previous studies suggest that βig-h3 is up-regulated in different human tumour cells (Tang et al., 2007, 2009) and might be involved in tumour formation and acquisition of the metastatic phenotype in human cancer cells (Kim et al., 2002; Nam et al., 2005). Our previous study shows that compared with that in normal cells, the expression of βig-h3 is up-regulated in human HCC cells. Overexpression of βig-h3 in human SMMC-7721 cells revealed that βig-h3 increased cell adhesion, invasion and MMP secretion potential. However, the underlying molecular mechanisms remain largely unknown.

Members of the MMP family play key roles in tumour cell invasion and metastasis; the degradation of ECM components by MMPs is critical for these processes (Jiang et al., 2004a). Our previous study showed that the release of MMPs in human HCC cells is regulated by Ca2+ entry (Jiang et al., 2004b). Our present data, obtained from gelatin zymography and invasion assay using Ni2+ (a potent blocker of Ca2+ entry), further confirmed this finding; we noted that extracellular Ca2+ entry plays a major role in βig-h3-mediated MMP secretion and tumour cell invasion. Furthermore, we showed that down-regulation of βig-h3 decreased the sensitivity of Ca2+ entry to NO/cGMP in SMMC-7721 cells.

Ca2+ is an essential and ubiquitous second messenger. Changes in the cytosolic Ca2+ concentrations trigger events that are critical for tumorigenesis, such as cellular motility, invasion, proliferation and apoptosis (Matsuo et al., 2004). The regulation of intracellular Ca2+ levels is a complicated process, which is associated with several intracellular signalling cascades (Kwan et al., 2000). Several studies have shown that the NO/cGMP/PKG signalling pathway can have an inhibitory effect on the elevation of intracellular Ca2+ concentrations by the inhibition of phosphoinositide hydrolysis, mobilization of intracellular calcium and voltage-dependent activation of L-type Ca2+ channels (Zucchi et al., 2001; Piper et al., 2004). The present results suggest that βig-h3 may attenuate the negative regulation of Ca2+ entry into the cells by NO/cGMP, thereby resulting in greater Ca2+ influx into the cells and increased secretion of MMPs, which would, therefore, increase the invasive potential of human HCC cells. However, how βig-h3 disrupts the NO/cGMP-sensitive SOC entry is still unknown. Βig-h3 is a 68-kDa secretory protein that contains four internal repeat domains, an RGD motif and a recognition sequence for some integrins on various cell types to influence cell attachment and migration in various tumour cells (Park et al., 2004). Our previous study revealed that α3β1 integrin is the principal integrin that interacts with βig-h3 in human HCC cells (Tang et al., 2009). The highly conserved amino acids (aspartic acid and isoleucine) in the second and fourth FAS-1 domains of βig-h3 are the binding motifs for α3β1 integrins in human corneal epithelial cells (Kim et al., 2002). Our previous study showed that HAb18G/CD147 co-immunoprecipitates and co-localizes with α3β1 integrins in human HCC cells. Further, HAb18G/CD147 reverses the inhibitory effect of NO/cGMP on SOC entry by activating the FAK–PI3K (phosphatidylinositide 3-kinase) pathway (Tang et al., 2008). These results indicate that α3β1 integrins and HAb18G/CD147 are in proximity to, if not directly associated with, human HCC cells. Even though the interaction of the α3β1 integrin and HAb18G/CD147 has been largely described in many other cell lines (Berditchevski et al., 1997; Engbring and Kleinman, 2003; Curtin et al., 2005), the exact mechanisms or the molecules that link CD147 to the α3β1 integrin have not yet been reported. The present study suggested that βig-h3 might act as a bridge between HAb18G/CD147 and the α3β1 integrin and then regulate HAb18G/CD147-induced metastasis of human HCC cells.

FAK is a direct downstream molecule of integrin activation that interacts with the C-terminal domain of integrins. FAK activity is regulated by integrin-mediated cell metastasis as well as by the activation of growth factors and G-protein-linked receptors (Choi et al., 2004). Our previous study showed that βig-h3 increases FAK phosphorylation levels by interacting with the α3β1 integrin. One of the critical downstream signal molecules of FAK pathways is PI3K (Murillo et al., 2004). IP3 (inositol trisphosphate) is the initial trigger for the release of intracellular Ca2+ stores (Engbring and Kleinman, 2003), and the levels of intracellular IP3 mainly depend on the activities of PI3K. High levels of IP3, stimulated by PI3K, activate the Ca2+ channels, thus allowing greater Ca2+ influx into the cells. The greater Ca2+ influx in turn reduces the effectiveness of NO/cGMP on Ca2+ entry, thus attenuating the negative regulation of Ca2+ entry. We speculated that βig-h3 may up-regulate the activity of cellular factors such as PI3K. The investigation of the precise mechanism is currently underway in our laboratory.

In conclusion, our present study establishes that βig-h3 may promote MMP secretion by disrupting NO/cGMP-mediated negative regulation of SOC entry, thus enhancing the invasive potential of human HCC cells. These findings add new insights to the βig-h3-mediated invasion mechanism in human HCC cells and further highlight the importance of this molecule in tumour invasion.

Author contribution

The major contribution to the study was carried out by Yun-Shan Guo and Juan Tang under the guidance of Zhi-Nan Chen and Jian-Li Jiang. Bo Chen and Wan Huang were in charge of the provision of study materials and materials, Yong Li and Hong-Yong Cui were responsible for the data analysis. Xin Zhang and Shi-Jie Wang were involved in cell culture.

Funding

This work was supported by grants from the National Basic Research Program of China [grant number 2009CB521704]; the National S&T Major Project [grant number 2008ZX10002-0260]; and the National Natural Science Foundation of China [grant numbers 81071691 and 30900234].

REFERENCES

Bae, JS, Lee, SH, Kim, JE, Choi, JY, Park, RW and Yong, PJ (2002) Betaigh3 supports keratinocyte adhesion, migration, and proliferation through alpha3beta1 integrin. Biochem Biophys Res Commun 294, 940-8
Crossref   Medline   1st Citation  

Berditchevski, F, Chang, S, Bodorova, J and Hemler, ME (1997) Generation of monoclonal antibodies to integrin-associated proteins. J Biol Chem 272, 29174-80
Crossref   Medline   1st Citation  

Brandman, O, Liou, J, Park, WS and Meyer, T (2007) STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. Cell 131, 1327-39
Crossref   Medline   1st Citation  

Choi, YA, Lim, HK, Kim, JR, Lee, CH, Kim, YJ and Kang, SS (2004) Group IB secretory phospholipase A2 promotes matrix metalloproteinase-2-mediated cell migration via the phosphatidylinositol 3-kinase and Akt pathway. J Biol Chem 279, 36579-85
Crossref   Medline   1st Citation  

Clapham, DE (2007) Calcium signaling. Cell 131, 1047-1058
Crossref   Medline   1st Citation  

Curtin, KD, Meinertzhagen, IA and Wyman, RJ (2005) Basigin (EMMPRIN/CD147) interacts with integrin to affect cellular architecture. J Cell Sci 118, 2649-60
Crossref   Medline   1st Citation  

Denninger, JW and Marletta, MA (1999) Guanylate cyclase and the NO/cGMP signaling pathway. Biochim Biophys Acta 1411, 334-50
Crossref   Medline   1st Citation  

Engbring, JA and Kleinman, HK (2003) The basement membrane matrix in malignancy. Pathol J 200, 465-70
Crossref   1st Citation   2nd  

Feng, M, Grice, DM, Faddy, HM, Nguyen, N, Leitch, S and Wang, Y (2010) Store-independent activation of Orai1 by SPCA2 in mammary tumors. Cell 143, 84-98
Crossref   Medline   1st Citation  

Gibson, MA, Kumaratilake, JS and Cleary, EG (1997) Immunohistochemical and ultrastructural localization of MP78/70 (βig-h3) in extracellular matrix of developing and mature bovine tissues. J Histochem Cytochem 45, 1683-96
Crossref   Medline   1st Citation  

Hashimoto, K, Noshiro, M, Ohno, S, Kawamoto, T, Satakeda, H and Akagawa, Y (1997) Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/βig-h3) that binds to collagen. Biochim Biophys Acta 1355, 303-14
Crossref   Medline   1st Citation  

Jiang, JL, Zhou, Q, Yu, M, Ho, LS, Chen, ZN and Chan, HC (2001) The involvement of HAb18G/CD147 in regulation of store-operated calcium entry and metastasis of human hepatoma cells. J Biol Chem 276, 46870-7
Crossref   Medline   1st Citation  

Jiang, JL, Chan, HC, Zhou, Q, Yu, MK, Yao, XY and Lam, SY (2004a) HAb18G/CD147-mediated calcium mobilization and hepatoma metastasis require both C-terminal and N-terminal domains. Cell Mol Life Sci 61, 2083-91
Medline   1st Citation   2nd  

Jiang, JL, Yao, XY, Zhou, J, Huang, Y and Chen, ZN (2004b) Effect of Ca2+ mobilization on release and activation of matrix metalloproteinases in hepatocellular carcinoma cells. Chin J Oncol 26, 9525-7
1st Citation   2nd   3rd   4th  

Kawamoto, T, Noshiro, M, Shen, M, Nakamasu, K, Hashimoto, K and Kawashima-Ohya, Y (1998) Structural and phylogenetic analysis of RGD-CAP/beta-ig-h3, a fasciclin like-adhesion protein expressed in chick chondrocytes. Biochim Biophys Acta 1395, 288-92
Medline   1st Citation  

Kim, JE, Jeong, HW, Nam, JO, Lee, BH, Choi, JY and Park, RW (2002) Identification of motifs in the fasciclin domains of the transforming growth factor-beta-induced matrix protein betaig-h3 that interact with the alphavbeta5 integrin. J Biol Chem 277, 46159-65
Crossref   Medline   1st Citation   2nd  

Kim, JE, Kim, SJ, Jeong, HW, Lee, BH, Choi, JY and Park, RW (2003) RDG peptides released from βig-h3, a TGF-β induced cell adhesive molecule, mediate apoptosis. Oncogene 22, 205317-45
1st Citation  

Kima, MO, Yuna, SJ, Kimb, IS, Sohnc, SY and Lee, EH (2003) Transforming growth factor-b-inducible gene-h3 (βig-h3) promotes cell adhesion of human astrocytoma cells in vitro: implication of α6β4 integrin. Neurosci Lett 336, 93-6
Crossref   Medline   1st Citation  

Kwan, HY, Huang, Y and Yao, X (2000) Store-operated calcium entry in vascular endothelial cells is inhibited by cGMP via a protein kinase G-dependent mechanism. J Biol Chem 275, 6758-63
Crossref   Medline   1st Citation  

Li, Y, Xu, J, Chen, L, Zhong, WD, Zhang, Z and Mi, L (2009) HAb18G (CD147), a cancer-associated biomarker and its role in cancer detection, Histopathology. 54, 677-87
Medline   1st Citation  

Lucas, KA, Pitari, GM, Kazerounian, S, Ruiz-Stewart, I, Park, J and Schulz, S (2000) Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev 52, 375-414
Medline   1st Citation  

Matsuo, Y, Hashimoto, S, Koga, T, Yonemitsu, Y, Yoshino, I, Sugimachi, K, Honda, H, Masuda, K and Sueishi, K (2004) Growth pattern correlates with the distribution of basement membrane and prognosis in lung adenocarcinoma. Pathol Res Pract 200, 517-29
Crossref   Medline   1st Citation  

Munier, FL, Korvatska, E, Djemai, A, Le Paslier, D, Zografos, L and Pescia, G (1997) Kerato-epithelin mutations in four 5q31-linked corneal dystrophies. Nat Genet 15, 247-51
Crossref   Medline   1st Citation  

Murillo, CA, Rychahou, PG and Evers, BM (2004) Inhibition of alpha5 integrin decreases PI3K activation and cell adhesion of human colon cancers. Surgery 136, 143-9
Crossref   Medline   1st Citation  

Nam, JO, Jeong, HW, Lee, BH, Park, RW and Kim, IS (2005) Regulation of tumor angiogenesis by fastatin, the fourth FAS1 domain of betaig-h3, via alphavbeta3 integrin. Cancer Res 65, 4153-61
Crossref   Medline   1st Citation  

Parekh, AB and Putney, JW Jr (2005) Store-operated calcium channels. Physiol Rev 85, 757-810
Crossref   Medline   1st Citation  

Park, SW, Bae, JS, Kim, KS, Park, SH, Lee, BH and Choi, JY (2004) Betaig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with alpha3beta1 integrin. Exp Mol Med 36, 211-9
Medline   1st Citation   2nd  

Piper, HM, Abdallah, Y and Schafer, C (2004) The first minutes of reperfusion: a window of opportunity for cardioprotection. Cardiovasc Res 61, 365-71
Crossref   Medline   1st Citation   2nd  

Roderick, HL and Cook, SJ (2008) Ca2+ signalling checkpoints in cancer: remodelling Ca2+ for cancer cell proliferation and survival. Nat Rev Cancer 8, 361-375
Crossref   Medline   1st Citation  

Shigeru, O, Mitsuhide, N, Seicho, M, Takeshi, K, Ming, S and Yan, WQ (1999) RGD-CAP (βig-h3) enhances the spreading of chondrocytes and fibroblasts via integrinα1β1. Biochim Biophys Acta 1451, 196-205
Crossref   Medline   1st Citation  

Skonier, J, Neubauer, M, Madisen, L, Bennett, K, Plowman, GD and Purchio, AF (1992) cDNA cloning and sequence analysis of βig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-β. DNA Cell Biol 11, 511-22
Crossref   Medline   1st Citation  

Tang, J, Zhou, HW, Jiang, JL, Yang, XM, Li, Y and Zhang, HX (2007) βig-h3 is involved in HAb18G/CD147 mediated metastasis process in human hepatoma cells. Exp Biol Med 232, 344-52
1st Citation   2nd   3rd  

Tang, J, Wu, YM, Zhao, P, Yang, XM, Jiang, JL and Chen, ZN (2008) Overexpression of HAb18G/CD147 promotes invasion and metastasis via alpha3beta1 integrin mediated FAK–paxillin and FAK–PI3K–Ca2+ pathways. Cell Mol Life Sci 65, 2933-42
Crossref   Medline   1st Citation  

Tang, J, Wu, YM, Zhao, P, Jiang, JL and Chen, ZN (2009) {beta}ig-h3 interacts with {alpha}3{beta}1 integrin to promote adhesion and migration of human hepatoma cells. Exp Biol Med 234, 35-9
Crossref   1st Citation   2nd   3rd  

Thapa, N, Kang, KB and Kim, IS (2005) βig-h3 mediates osteoblast adhesion and inhibits differentiation. Bone 36, 232-42
Crossref   Medline   1st Citation  

Zucchi, R, Ronca, F and Ronca-Testoni, S (2001) Modulation of sarcoplasmic reticulum function: a new strategy in cardioprotection. Pharmacol Ther 89, 47-65
Crossref   Medline   1st Citation   2nd  


Received 28 December 2010/13 February 2011; accepted 11 March 2011

Published as Cell Biology International Immediate Publication 11 March 2011, doi:10.1042/CBI20100916


© The Author(s) Journal compilation © 2011 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)