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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Tumor cells can move as individual cells in two interconvertible modes: mesenchymal mode and amoeboid mode. Cytoskeleton rearrangement plays an important role in the interconversion. Previously, we reported that HAb18G/CD147 and annexin II are interacting proteins involved in cytoskeleton rearrangement, yet the role of their interaction is unclear. In this study we found that the depletion of HAb18G/CD147 produced a rounded morphology, which is associated with amoeboid movement, whereas the depletion of annexin II resulted in an elongated morphology, which is associated with mesenchymal movement. The extracellular portion of HAb18G/CD147 can interact with a phosphorylation-inactive mutant of annexin II and inhibit its phosphorylation. HAb18G/CD147 inhibits Rho signaling pathways and amoeboid movement by inhibiting annexin II phosphorylation, promotes membrane localization of WAVE2 and Rac1 activation by way of the integrin-FAK-PI3K/PIP3 signaling pathway, and promotes the formation of lamellipodia and mesenchymal movement. Conclusion: These results suggest that the interaction of HAb18G/CD147 with annexin II is involved in the interconversion between mesenchymal and amoeboid movement of hepatocellular carcinoma cells. (HEPATOLOGY 2011)

Abnormal migration and invasion are characteristics of malignant tumor cells and are important components of metastasis, which is the leading cause of tumor-related death. Cell movement is critical for tumor migration, invasion, and metastasis. Recent work has demonstrated that tumor cells can move as collective groups or as individual cells.1 Individual tumor cells exhibit two different interconvertible modes of cell movement.2, 3 Amoeboid movement is characterized by a rounded, blebbing morphology and independence from extracellular proteases. It requires high levels of actomyosin contractility downstream of RhoA-ROCK (Rho-kinase) signaling to deform the extracellular matrix and propel cell movement.4-7 In contrast, mesenchymal movement is characterized by an elongated cellular morphology, resulting from Rac-dependent actin assembly at the leading edge, and requires extracellular proteolysis.8 Plasticity permits tumor cells to undergo amoeboid-mesenchymal and mesenchymal-amoeboid transitions in different environments. Although mesenchymal and amoeboid movements employ different driving forces and signaling pathways, both types of movement are based on cytoskeletal rearrangement, and cytoskeletal rearrangement plays a critical role in the movement mode transition.1

Table 1. Overview of primers used in this study
  1. aRestriction Enzyme cutting sites introduced in the oligonucleotides is underlined.

Primer 1Forward5′-ACTGCGAATTCATGTCTACTGTTCACGA
Primer 2Reverse5′-TACGAGTCTAGATCAGTCATCTCCACCACAC
Primer 3Forward5′-AATAACTCGAGATGGCGGCTGCGCTGTTCG
Primer 4Reverse5′-ATAATGGATCCTCAGGAAGAGTTCCTCTGGCGGACGT
Primer 5Forward5′-AAGTAGCGAGCTCGGCTGCCGGCACAGTC
Primer 6Reverse5′-TAATAGCGGCCGCTTAGTGGCTGCGCACGCG
Primer 7Forward5′-ACATATGAGCTCGAAGCGCCGGAAG
Primer 8Reverse5′-TAATAGCGGCCGCGAGGTGAGAAC
Primer 9Forward5′-ACGGACTCGAGATGGCTGCCGGCACAGTCTT
Primer 10Reverse5′-ATAATGGATCCAGGTGGCTGCGCACGCGG
Primer 11Forward5′-CTCTACACCCCCAAGTGCAGAAGGGTCTGTCAAAGCC
Primer 12Reverse5′-GGCTTTGACAGACCCTTCTGCACTTGGGGGTGTAGAG
Primer 13Forward5′-CTCTACACCCCCAAGTGCATTTGGGTCTGTCAA
Primer 14Reverse5′-TTGACAGACCCAAATGCACTTGGGGGTGTAGAG
Primer 15Forward5′-ACGGACTCGAGATGTCTACTGTTCACGAAAT
Primer 16Reverse5′-ATAATGGATCCAAGTCATCTCCACCACACAG

CD147 is a highly glycosylated transmembrane protein that belongs to the immunoglobulin superfamily.9 It is also expressed in the endomembrane system (EMS). HAb18G, a CD147 family member, was identified by screening a hepatocellular carcinoma (HCC) complementary DNA (cDNA) expression library and was named HAb18G/CD147 in our laboratory.10, 11 Studies have demonstrated that HAb18G/CD147 plays important roles in cellular processes of HCC progression, including adhesion, migration, invasion, and extracellular matrix degradation.12-14 Our recent studies have indicated that HAb18G/CD147 increases integrin α3β1 and α6β1 activity, enhances expression and phosphorylation of FAK and paxillin, and subsequently leads to cytoskeletal rearrangement and a change of cell morphology.15, 16 However, the more in-depth mechanism responsible for the involvement of HAb18G/CD147 in cytoskeletal rearrangement and cell motility remains unclear.

We recently found that annexin II interacts with HAb18G/CD147 in HCC cells. Effects of the interaction of these two molecules on tumor progression include MMP-2 production, migration, invasion, and cytoskeletal rearrangement.17

Annexin II, a 36-kDa Ca2+- and phospholipid-binding protein, is implicated in the organization of membrane domains and membrane-cytoskeleton contacts.18 In addition to interacting with Ca2+ and membrane lipids, annexin II can bind to G-actin and F-actin, inhibit filament elongation at the barbed ends, and be recruited to the sites of actin assembly at cellular membranes.19, 20 Hence, annexin II was reported to be closely linked to actin remodeling processes in cellular membranes. Membrane-bound annexin II presenting at the inner surface of the plasma membrane may serve as a platform for actin assembly and function in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton due to the direct interaction of annexin II with polymerized and monomeric actin, which plays an important role in cytoskeletal rearrangement and cell motility.21, 22 Cell motility requires a dynamic remodeling of the membrane-associated actin cytoskeleton in response to extracellular stimuli that are primarily transmitted through receptor tyrosine kinases.23 It was reported that annexin II is tyrosine-phosphorylated upon insulin receptor activation, and tyrosine (Tyr) phosphorylation of annexin II is involved in Rho/ROCK-mediated actin rearrangement and cell adhesion.23 It is not known whether the interaction of HAb18G/CD147 with annexin II is involved in cell motility.

In this study we investigated the role of HAb18G/CD147 and annexin II in cytoskeletal rearrangement and cell movement of HCC cells. The results showed that the two molecules caused diametrically different morphological changes in HCC cells. The extracellular portion of HAb18G/CD147 was confirmed to interact with annexin II and to inhibit Rho/ROCK signaling pathways and amoeboid movement in HCC cells by attenuating annexin II phosphorylation. HAb18G/CD147 can also promote the membrane localization of WAVE2 and Rac1 activation in HCC cells by way of the integrin-FAK-PI3K/PIP3 signaling pathway, and it can promote the formation of lamellipodia and mesenchymal movement.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Cell Lines.

Four human HCC cell lines were used: SMMC-7721, 7721-si18G, T7721, Huh-7, and MHCC97-H. SMMC-7721 cells were obtained from the Institute of Cell Biology, Academic Sinica (Shanghai, China). T7721 cells (HAb18G/CD147 is stably overexpressed) and 7721-si18G cells (HAb18G/CD147 is stably knocked down) were developed and preserved in our laboratory. Huh-7 cells were obtained from Cell Bank of the JCRB (Japanese Collection of Research Bioresources). MHCC97-H cells were supplied by the Liver Cancer Institute of Fudan University (Shanghai, China).

Cell Culture on a Thin Layer of Matrigel.

Matrigel was prepared at a concentration of 0.25 mg/mL in RPMI 1640 medium according to the manufacturer's protocol (BD), and 500 μL were placed in each well of the 24-well plates with or without a glass coverslip. Cells were seeded on top of the Matrigel in RPMI 1640 containing 10% serum and cultured for 16 hours. All cell imaging and immunoblotting were performed with cells cultured on a thin layer of Matrigel.

RNA Interference.

Chemically synthesized, double-stranded small interfering RNAs (siRNAs) with 19-nt duplex RNA and 2-nucleotide 3′dTdT overhangs were purchased from Shanghai GenePharma (Shanghai, China). Sequences for si-HAb18G were as described.12 Annexin II siRNA (sc-29199) was purchased from Santa Cruz Biotechnology. Lipofectamine 2000 reagent was employed according to the manufacturer's instruction (Invitrogen). Silencer negative control siRNA (snc-RNA) was used as a negative control under similar conditions.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Expression of HAb18G/CD147 and Annexin II Causes Morphological Changes in HCC Cells.

We detected the expression of HAb18G/CD147 and annexin II in human SMMC-7721 and MHCC97-H HCC cells by western blotting. As shown in Fig. 1A, HAb18G/CD147 and annexin II were highly expressed in the two HCC cell lines. To investigate the role of HAb18G/CD147 and annexin II in cytoskeleton rearrangement and cell movement, the HCC cells were transfected with siRNA targeting these two molecules and plated on a thin layer of Matrigel. As shown in Fig. 1B and Supporting Fig. S1, depletion of HAb18G/CD147 produced a more rounded morphology with prominent cortical F-actin, whereas depletion of annexin II resulted in a more elongated morphology. The former is similar to the morphology observed during amoeboid movement, whereas the latter is similar to the morphology observed during mesenchymal movement. 3D tissue culture was also used to show the morphological changes of HAb18G/CD147 or annexin II knockdown cells (Fig. 1C), and similar results were obtained compared to that of thin layer Matrigel culture. These results demonstrate that HAb18G/CD147 and annexin II in HCC cells are important for two opposing types of cell morphology changes and may affect cell motility in HCC cells.

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Figure 1. HAb18G/CD147 and annexin II cause morphological changes in HCC cells. (A) Expression of HAb18G/CD147 and annexin II in SMMC-7721 and MHCC97-H cells were determined by western blotting. Total lysates were blotted for HAb18G/CD147, annexin II, and tubulin. (B) Confocal microscopy images of the effect of HAb18G/CD147 siRNA (si-18G)- or annexin II siRNA (si-AII)-mediated morphological changes in SMMC-7721 cells and MHCC97-H cells. Cells were transfected with si-18G, si-AII, or control siRNA (sncRNA) and were allowed to attach for 24 hours to Matrigel-precoated coverslips. Cells were stained with Alex 488-phalloidin solution (green). Cell nuclei were dyed with DAPI (blue). (C) Images of HAb18G/CD147 or annexin II siRNA-transfected SMMC-7721 cells were plated on a thick layer of Matrigel for 24 hours. (D) Expression of HAb18G/CD147 in SMMC-7721 (7721), 7721-si18G, and T7721 cells were determined by western blotting. Total lysates were blotted for HAb18G/CD147 and tubulin. (E) Morphology observation of SMMC-7721, 7721-si18G, and T7721 cells.

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We have demonstrated that annexin II is involved in the process of cytoskeletal rearrangement of HCC cells and that HAb18G/CD147 interacts with annexin II during tumor progression.17 To further explore the effects of HAb18G/CD147 on cytoskeletal rearrangement and cell morphology, three HCC cell lines, i.e., SMMC-7721, 7721-si18G, and T7721, were employed. The expression of HAb18G/CD147 in the three homologous HCC cell lines was detected (Fig. 1D; Fig. S3). In all, 38.82% ± 4.53% of SMMC-7721 cells showed an elongated morphology (Fig. 1E; Fig. S4). A smaller proportion of cells with elongated morphology was observed in the 7721-si18G cells as compared to in SMMC-7721 cells. On the contrary, more cells with elongated morphology were observed among the T7721 cells (Fig. 1E; Fig. S4), which strongly suggests that the expression level of HAb18G/CD147 was closely related to mesenchymal morphological changes of HCC cells.

These results demonstrate that both HAb18G/CD147 and annexin II play important roles in cytoskeletal rearrangement and may play some role in the interconversion between amoeboid movement and mesenchymal movement of HCC cells.

HAb18G/CD147 Regulates the Morphology of HCC Cells by Inhibiting the Phosphorylation of Annexin II.

We previously reported that HAb18G/CD147 and annexin II interact with each other and may work as a functional complex that plays an important role in cellular processes of HCC progression.17 In the present study we employed a mammalian protein-protein interaction trap (MAPPIT) system to analyze the interaction site of the two molecules. As shown in Fig. 2A, the extracellular portion (18GEP) but not the intracellular portion (18GIP) of HAb18G/CD147 interacted with annexin II. Consistently, HAb18G/CD147 and annexin II colocalized in the cytoplasm (Fig. 2B) of both SMMC-7721 cells and Huh-7 cells.

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Figure 2. HAb18G/CD147 regulates morphology of HCC cells by inhibiting the phosphorylation of annexin II. (A) Interaction site analysis of HAb18G/CD147 and annexin II by using the mammalian protein-protein interaction trap (MAPPIT) system. HEK293T cells were transiently cotransfected with plasmids encoding an extracellular portion of HAb18G/CD147 (18GEP) bait or intracellular portion of HAb18G/CD147 (18GIP) bait together with the pMG1-annexin II (AII prey) constructs or mock vector, combined with the pXP2d2-rPAP1-luci. The transfected cells were stimulated for 24 hours with leptin (LS, leptin stimulated) or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (LS/NS) ± SD (n = 3, error bars indicate ± SD), and positive interactions were considered significant if LS/NS >5 (50). Upper panel: fold of induction (n = 3, error bars indicate ± SD). Lower panel: HAb18G/CD147 and annexin II structure. (B) Intracellular colocalization of annexin II with HAb18G/CD147 in SMMC-7721 cells and Huh-7 cells. Cells were stained with anti-CD147 (green) and anti-annexin II (red) antibodies, cell nuclei were dyed with DAPI (blue). Scale bar = 10 μm. (C) Tyr phosphorylation of annexin II was negatively related to the expression of HAb18G/CD147. Cells were starved 24 hours, then stimulated with 10 μg/mL insulin for 10 minutes. Do Ip with annexin II mAb, and detected annexin II (AII) and phosphor-annexin II (P-AII) by using western blot. Upper panel: a representative image. Lower panel: quantification (n = 3, error bars indicate ± SD; *P < 0.05, **P < 0.01). (D) 18GEP interacted with nonphosphorylated annexin II. HEK293T cells were transiently cotransfected with plasmids encoding 18GEP bait and pMG1-annexin II (AII prey), pMG1-AII-Y23E (AII-Y23E prey), or pMG1-AII-Y23F (AII-Y23F prey) constructs, or with mock vector, combined with the pXP2d2-rPAP1-luci. The transfected cells were stimulated for 24 hours with leptin or were left untreated. Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (LS/NS) ± SD (n = 3, error bars indicate ± SD).

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The Tyr23 phosphorylation switch in annexin II is an important event that triggers Rho/ROCK-dependent and actin-mediated cell morphology changes.23 We found that Tyr phosphorylation of annexin II was negatively related to the expression of HAb18G/CD147 (Fig. 2C), which suggests that HAb18G/CD147 may inhibit the Tyr phosphorylation of annexin II by way of 18GEP binding with annexin II. The MAPPIT system was employed again to further analyze which type of annexin II, phosphorylated or nonphosphorylated, could interact with 18GEP. A phospho-mimicking mutant (AII-Y23E) and a phosphorylation-inactive mutant (AII-Y23F) were generated. As shown in Fig. 2D, 18GEP bait could interact with AII-Y23F but not with AII-Y23E prey. The same results were detected by coimmunoprecipitation (Fig. 3A) and immunofluorescence (Fig. 3B). The phosphorylation of annexin II decreased 5-fold when the transmembrane and extracellular portion (18GTEP, 0-229 amino acids) were overexpressed in SMMC-7721 cells, and it decreased 2-fold when 18GEP was overexpressed in SMMC-7721 cells (Fig. 3C). These results suggest that HAb18G/CD147 inhibited the phosphorylation of annexin II in HCC cells by way of the combination of 18GEP with nonphosphorylated annexin II. Therefore, HAb18G/CD147 may regulate cytoskeletal rearrangement and cell movement by inhibiting the phosphorylation of annexin II.

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Figure 3. HAb18G/CD147 inhibited the phosphorylation of annexin II in HCC cells. (A) Coimmunoprecipitation assay. HEK293T cells were cotransfected with pMG1-18GEP and AII-Y23FEGFP or AII-Y23E-EGFP vector, and then IP done with GFP mAb or Flag mAb. (B) HAb18G/CD147 colocalized with AII-Y23F-EGFP in T7721 cells. (C) Overexpression of 18GEP and 18GTEP decreased the phosphorylation of annexin II in SMMC-7721 cells. Left panel: a representative image. Right panel: quantification (n = 3, error bars indicate ± SD; *P < 0.05, **P < 0.01).

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HAb18G/CD147 Inhibits the Rho/ROCK Signaling Pathway and Amoeboid Movement of HCC Cells by Inhibiting the Phosphorylation of Annexin II.

Mesenchymal and amoeboid modes of individual tumor cell movement are driven by different signaling transduction pathways, i.e., the Rac1/WAVE2 or Rho/ROCK signaling transduction pathway. It has been reported that the phosphorylation of annexin II is critical for the Rho/ROCK signaling pathway and actin-mediated cell morphology changes.23 Our present results indicate that HAb18G/CD147 inhibits the phosphorylation of annexin II in HCC cells (Fig. 2C). We hypothesized that HAb18G/CD147 may be involved in RhoA-mediated amoeboid movement by way of the regulation of annexin II phosphorylation. RhoA activity (GTP RhoA / Total RhoA) was detected in the SMMC-7721, 7721-si18G, and T7721 cells. As shown in Fig. 4A, RhoA activity was up-regulated in 7721-si18G cells (117.51% ± 12.17%) and down-regulated in T7721 cells (49.65% ± 13.42%) as compared to SMMC-7721 cells (upper panel). We obtained similar results in the Huh-7 cell line (Fig. 4B). We also detected MLC2 phosphorylation and showed that the expression of HAb18G/CD147 in the two HCC cell lines was negatively related to the Thr18 and Ser19 phosphorylation of MLC2 (Fig. 4A,B) and therefore to actomyosin contractility.

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Figure 4. HAb18G/CD147 inhibits the Rho/ROCK signaling transduction pathway and amoeboid mode of movement of HCC cells by way of inhibition of annexin II phosphorylation. (A,B) RhoA activation and phospho-MLC2 (p-T18/S19-MLC2) in SMMC-7721, T7721, 7721-si18G, Huh-7, Huh-7-si18G (HAb18G/CD147 knockdown), and T Huh-7 (HAb18G/CD147 overexpression) cells. Upper panel: a representative image. Lower panel: quantification (n = 3, error bars indicate ± SD). (C) Confocal microscopy images of the rescue effect of ROCK (Y27632 and H1152) or Rac1 (NSC23766) inhibitors in SMMC-7721 cells with silencing or overexpression of HAb18G/CD147 (7721-si18G cells or T7721 cells) on cell morphology. Cells were allowed to attach for 24 hours to Matrigel-precoated coverslips and were treated with 10 μM Y27632, 5 μM H1152, or 50 μM NSC23766. Cells were stained with Alex 488-phalloidin solution (green). Cell nuclei were dyed with DAPI (blue). Upper panel: representative image. Lower panel: relatively roundness (n = 100, error bars indicate ± SD, **P < 0.01, * P < 0.05).

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We further investigated the involvement of HAb18G/CD147 in RhoA/ROCK signaling pathway-mediated morphological changes and the motility of HCC cells. When treated with Rho/ROCK pathway inhibitors (H-1152 and Y27632), SMMC-7721 cells and 7721-si18G cells converted from having a rounded morphology to having an elongated morphology (Fig. 4C). In the T7721 cells, H-1152 and Y27632 had no effect on cell morphology. These results suggest that Rho/ROCK pathway inhibitors can convert the cell morphology from being rounded to elongated in SMMC-7721 cells and 7721-si18G cells with higher RhoA activity and lower HAb18G/CD147 expression. In contrast, in T7721 cells with lower RhoA activity and higher HAb18G/CD147 expression the morphological changes caused by HAb18G/CD147 overexpression were not affected by a Rho/ROCK pathway inhibitor. This indicates that HAb18G/CD147 inhibits the amoeboid mode of movement of HCC cells by inhibiting the Rho/ROCK signaling transduction pathway.

Migration ability assays (Fig. 5A) revealed that the migration ability increased when the SMMC-7721 cells were treated with H1152 and Y27632 but significantly decreased when the cells were treated with NSC23766 (Rac1 inhibitor). These results indicate that the Rac1 signaling pathway but not RhoA plays a dominant role in the migration ability of SMMC-7721 cells.

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Figure 5. Phosphor-annexin II suppress morphology change caused by HAb18G/CD147 overexpression. (A) Effects of ROCK inhibitor (Y27632 and H1152) or Rac1 inhibitor (NSC23766) in SMMC-7721 cells on cell motility. Cells were cultured until confluent. After scratching, cells were cultured with or without 10 μM Y27632, 5 μM H1152, or 50 μM NSC23766. Photomicrographs at 10× objective magnification were taken at various timepoints (0 and 24 hours). Upper panel: representative image. Lower panel: quantification (n = 3, error bars indicate ± SD, **P < 0.01, * P < 0.05). (B) Effects of ROCK inhibitor (Y27632 and H1152) or Rac1 inhibitor (NSC23766) in 7721-si18G and T7721 cells on cell motility. (C) Confocal microscopy images of the rescue effect of annexin II Tyr23 phospho-mimicking mutant (AII-Y23E-EGFP) in T7721 cells. Transfected cells were allowed to attach for 24 hours to Matrigel-precoated coverslips and were stained with rhodaminephalloidin solution (red). Cell nuclei were dyed with DAPI (blue). (D) RhoA activation of AII-Y23EEGFP vector-transfected T7721 cells. Upper panel: a representative pulldown image. Lower panel: quantification (n = 3, error bars indicate ± SD, **P < 0.01).

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We also detected the motility of 7721-si18G and T7721 cells by using a wound healing assay. As shown in Fig. 5B, when RhoA activity was inhibited by H-1152 and Y27632 in 7721-si18G, instead of decreasing the motility of 7721-si18G cell was increased. When we inhibited RhoA activity in T7721 cells the motility did not change much. The motility of both cell lines was decreased when Rac1 activity was inhibited in 7721-si18G and T7721 cells. But T7721 cells still moved faster than 7721-si18G cells.

To further explore whether HAb18G/CD147 inhibits RhoA activity and thus causes cell morphological changes by inhibiting the phosphorylation of annexin II, we constructed the AII-Y23E-EGFP vector. Lamellipodia retraction was observed when T7721 cells were transfected with the AII-Y23E-EGFP vector (Fig. 5C). RhoA activity was increased (by about 1.60-fold) in the T7721 cells when they were transfected with the AII-Y23E-EGFP vector (Fig. 5D). These results indicate that HAb18G/CD147 inhibits the Rho/ROCK signaling pathway in HCC cells by way of the inhibition of the phosphorylation of annexin II.

HAb18G/CD147 Promotes Membrane Localization of WAVE2 and Activation of Rac1 in HCC Cells by Way of the Integrin-FAK-PI3K/PIP3 Signaling Pathway, Thus Contributing to the Formation of Lamellipodia and Mesenchymal Movement.

Amoeboid and mesenchymal movement are interconvertible.2, 3 Previous studies have indicated that mesenchymal movement is supported by the Rac1/WAVE2 signaling pathway,8 which plays a dominant role in HCC cell migration ability (Fig. 5A). Therefore, we investigated whether HAb18G/CD147 regulates cell mesenchymal movement by way of the Rac1/WAVE2 signaling pathway. As shown in Fig. 6A, Rac1 activity and WAVE2 expression were positively related to the expression of HAb18G/CD147 in HCC cells. When Rac1 activity was blocked by a NSC23766, T7721 cells partially demonstrated a conversion from an elongated morphology to a small and rounded morphology (Fig. 4C). Previously, we demonstrated that HAb18G/CD147 can activate the integrin-FAK-PI3K pathway,15 thus causing the accumulation of PtdIns3-5 P3 (PIP3) in the cell membrane. It has been reported that membrane localization of WAVE2 caused by PIP3 accumulation is critical for lamellipodia formation.24 Therefore, we speculated that HAb18G/CD147 may regulate the membrane localization of WAVE2 and lamellipodia formation by way of the integrin-FAK-PI3K/PIP3 pathway. When the SMMC-7721 cells were transfected with CD147-pEGFP-N1, the lamellipodia formation was obvious (Fig. S2). The morphological changes caused by HAb18G/CD147 overexpression were inhibited by FAK (PF573,228) or PI3K (wortmannin) inhibitors (Fig. S2). The colocalization of HAb18G/CD147 and WAVE2 was observed in the SMMC-7721 cells when they were transfected with CD147-pEGFP-N1, especially on the plasma membrane (Fig. 6C). The colocalization was attenuated when PF573,228 was administered (Fig. 6C). We also detected lamellipodia marker (cortactin) distribution in the three cell lines and annexin Y23F and Y23E overexpression cell lines (Fig. 7A). Cortactin is localized to the leading edge of the CD147 overexpression cell line, especially when annexin II Y23F was overexpressed in cells. To further explore the mechanism outlined above, Rac1 activity was detected in the T7721 cells. As shown in Fig. 7B, Rac1 activity, in accordance with the expression of HAb18G/CD147, was significantly down-regulated by PF573,228 and wortmannin. When Rac1 activation was blocked by NSC23766, the expression of HAb18G/CD147 was also decreased in T7721 cells (Fig. 7C). These results strongly suggest that HAb18G/CD147 promotes membrane localization of WAVE2 and Rac1 activation in HCC cells by way of the integrin-FAK-PI3K/PIP3 signaling pathway, thus contributing to the formation of lamellipodia and mesenchymal movement.

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Figure 6. HAb18G/CD147 promotes membrane localization of WAVE2 and activation of Rac1 by way of the integrin-FAK-PI3K/PIP3 signaling pathway. (A,B) Rac1 activation and WAVE2 expression in SMMC-7721, T7721, 7721-si18G, Huh-7, Huh-7-si18G (HAb18G/CD147 knockdown), and T Huh-7 (HAb18G/CD147 overexpression) cells. Left panel: a representative image. Right panel: quantification (n = 3, error bars indicate ± SD). (C) Membrane colocalization of HAb18G/CD147 and WAVE2 in CD147-pEGFP-N1-transfected SMMC-7721 cells was blocked by FAK inhibitor (PF 573,228). Transfected cells were allowed to attach for 24 hours to Matrigel-precoated coverslips and were treated with 100 nM PF 573,228. Cells were stained with anti-WAVE2 (red) antibody. Cell nuclei were dyed with DAPI (blue). Scale bar = 20 μm.

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Figure 7. HAb18G/CD147 affect lamellipodia formation. (A) HAb18G/CD147 and annexin II Y23FEGFP overexpression promote lamellipodia marker cortactin localized to the leading edge of cells. (B) Rac activation of T7721 cells after treatment with 100 nM PF 573,228 or 100 nM wortmannin for 15 hours. Left panel: representative pulldown. Right panel: quantification (n = 3, error bars indicate ± SD; Student's t test was used to generate P values, **P < 0.01). (C) HAb18G/CD147 expression inhibited by Rac1 inhibitor (NSC23766) in T7721 cells. Left panel: a representative image. Right panel: quantification (n = 3, error bars indicate ± SD).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Interconversion of amoeboid and mesenchymal movement of tumor cells is mediated by the dynamic polymerization of actin filaments (F-actin).25 Several molecules have been implicated in this process, including Rac1, RhoA, PIP3, ROCK, WAVE2, MLC2, and the actin-related protein 2/3 (Arp2/3) complex. Our present study suggests that HAb18G/CD147 is involved in the interconversion of HCC cellular movement. Localizing to both the cell membrane and EMS in HCC cells (Fig. 2B), HAb18G/CD147 inhibits the RhoA/ROCK signaling pathway and amoeboid movement by way of depression of annexin II phosphorylation. On the contrary, HAb18G/CD147 enhances the Rac1/WAVE2 signaling pathway and mesenchymal movement by way of activation of the integrin-FAK-PI3K/PIP3 signaling pathway (Fig. 8).

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Figure 8. Schematic representation of the major molecular mechanisms of HAb18G/CD147 in the interconversion between amoeboid and mesenchymal movement. EMS: endomembrane system; Arp2/3: actin-related protein 2/3; ROCK: Rho-kinase; MLC2: myosin light chain 2; FAK: focal adhesion kinase; MLCK: myosin light chain kinase.

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RhoA is a member of the Rho subfamily GTPases, which regulate the stability of the actin cytoskeleton, and is also known to play important roles in cell migration, cell polarity, endocytosis, vesicular trafficking, oncogenesis, and gene transcription.26 ROCK is one of the RhoA downstream effectors, elevating MLC2 phosphorylation by inhibiting MLC2 phosphatase to thereby enhance myosin activation.27 A previous study showed that tyrosine phosphorylation of annexin II is involved in Rho/ROCK-mediated actin rearrangement and cell adhesion.23 Our present study indicated that CD147 inhibits the phosphorylation of annexin II by the extracellular portion of HAb18G/CD147 (18GEP) and thus indirectly decreases the activity of RhoA and phosphorylation of MLC2. Although HAb18G/CD147 is a cell-surface transmembrane protein localized in both the cell membrane and EMS, few studies have focused on the functions of HAb18G/CD147 in the EMS. We report in this study that HAb18G/CD147 inhibits annexin II phosphorylation by way of an interaction between 18GEP and annexin II, which suggests that HAb18G/CD147 localized in the EMS can also mediate changes in the internal cell architecture. Other studies also reported that CD147 is coexpressed with monocarboxylate transporter 1 (MCT1) and MCT4 on the plasma and mitochondrial membranes.28-30 The mature, glycosylated form of CD147 works as a chaperone of MCT1, MCT3, and MCT4, regulating the expression and function of these MCTs on plasma or mitochondrial membranes.31, 32 We also reported that HAb18G/CD147 was located on the membrane of the endoplasmic reticulum and interacted with Cyclophilin A in the process of invasion of host cells by SARS-CoV.33 These studies provided another clue for the intracellular function of CD147.

A characteristic of migrating cells is the formation of ruffles.34, 35 Membrane ruffles are closely related to lamellipodia, a characteristic of mesenchymal movement. Frequently, lamellipodia evolve into ruffles when membrane protrusions fail to adhere and are swept backward onto the dorsal surface. In Drosophila melanogaster, CD147 promotes cytoskeletal rearrangements and the formation of lamellipodia.36 Consistent with this result, our present study indicates that overexpression of HAb18G/CD147 induces lamellipodia formation in HCC cells and subsequently promotes mesenchymal movement. These processes induced by HAb18G/CD147 can be inhibited by FAK and PI3K inhibitors. Rac1, a member of the Rac-like subfamily, stimulates the formation of lamellipodia and membrane ruffles,37 playing an essential role in cell motility and chemotaxis.38 In migrating cells, activated Rac1 was concentrated in the leading lamellipodia or in membrane ruffles.39 We observed that Rac1 activity (GTP Rac1/Total Rac1) is positively related to the expression of HAb18G/CD147 (Fig. 6A,B). Rac1 activation induced by overexpression of HAb18G/CD147 can be inhibited by FAK and PI3K inhibitors (Fig. 7B). WAVE2, a downstream effector of Rac1,40, 41 plays a central role in cell motility, cell polarity, and morphological changes of cells. Membrane localization of WAVE2 is critical for lamellipodia formation. PIP3 can promote the membrane localization and activation of WAVE2 by way of activation of Rac1, thus promoting Arp2/3 complex activation and actin polymerization. The present study showed that expression of WAVE2 is promoted by overexpression of HAb18G/CD147 in HCC cells (Fig. 6A,B). Membrane localization of WAVE2 is increased in HAb18G/CD147-overexpressing cells, and this effect of HAb18G/CD147 can be blocked by an FAK inhibitor. FAK is the upstream effector of PI3K-PIP3, and its activation has been linked to cell spreading and migration.42 We speculate that HAb18G/CD147 enhances actin polymerization and lamellipodia formation by promoting Rac1 activation and WAVE2 expression.

It is known that Rac1 is activated by guanine nucleotide exchange factors (GEFs) and is hydrolyzed by GTPase-activating proteins (GAPs). ARHGAP22 is a member of the GAPs whose activity is promoted by ROCK signaling. The Rho/ROCK signaling pathway prevents Rac activation by promoting ARHGAP22 activity.8 In the present study, blockade of RhoA signaling pathway by ROCK inhibitors (H1152 and Y27632) enhanced lamellipodia formation in HCC cells (7721-si18G) with higher RhoA activity and lower HAb18G/CD147 expression. However, the inhibitors could not further increase lamellipodia formation of T7721 cells with lower RhoA activity and higher HAb18G/CD147 expression, which indicates that HAb18G/CD147 promotes lamellipodia formation and mesenchymal movement in HCC cells through two signaling pathways: inhibition of the RhoA/ROCK signaling pathway and enhancement of the Rac1/WAVE2 signaling pathway.

Many cell adhesion molecules have been shown to have effects on Rho and Rac activity, including integrins,43 cadherins44 and Ig superfamily members.45 Integrins are heterodimers composed of α- and β-chain subunits that mediate interactions between cells and the ECM, thus serving as bidirectional transducers of extracellular and intracellular signals in the processes of cell adhesion, proliferation, differentiation, apoptosis, and tumor progression. Integrin αvβ3 is likely to be important for many migrating cells where lamellipodia are stabilized by new adhesions.46 Our previous study showed that integrin and HAb18G/CD147 are interacting proteins.15, 16 The function of HAb18G/CD147 in HCC cells can be blocked by an arginine-glycine-aspartic acid (RGD) peptide, which inhibits the interaction between HAb18G/CD147 and integrins (Li et al., unpubl. data). Consistent with these results, our previous study also showed that HAb18G/CD147 enhances the invasion and metastatic potential of human hepatoma cells by way of integrin α3β1-mediated FAK-paxillin and FAK-PI3K-Ca2+ signal pathways.15 Many Rac GEFs are activated by PI3K/PIP3 signaling.47 Interestingly, when Rac1 activation was blocked by a Rac1 inhibitor (NSC23766), the expression of HAb18G/CD147 was also decreased in T7721 cells (Fig. 7C), indicating that a positive-feedback loop also exists between Rac1 activation and HAb18G/CD147 expression, which may play a role in the long-term reaction to extracellular signaling changes.

Consistent with our findings that HAb18G/CD147 plays an important role in the interconversion of amoeboid movement and mesenchymal movement in HCC cells, Wu et al. (unpubl. data from our lab) found that the epithelial-mesenchymal transition (EMT) of normal liver cells is regulated by HAb18G/CD147. This provides another piece of evidence for the function of HAb18G/CD147 in cytoskeleton rearrangement and cell mesenchymal movement as well as in the pathology of tumor development and metastatic dissemination.

In summary, we found that HAb18G/CD147 inhibits the RhoA/ROCK signaling pathway and amoeboid movement by inhibiting annexin II phosphorylation, and it enhances the Rac1/WAVE2 signaling pathway and mesenchymal movement through the integrin-FAK-PI3K signaling pathway. Therefore, HAb18G/CD147 was identified as a regulator of the interconversion between amoeboid and mesenchymal movement and is involved in the invasion and metastatic processes of HCC cells.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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