Involvement of Hypoxia-inducing Factor-1α-dependent Plasminogen Activator Inhibitor-1 Up-regulation in Cyr61/CCN1-induced Gastric Cancer Cell Invasion*

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1α (HIF-1α) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1α protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1α protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl2 treatment, both well known for induction of HIF-1α. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1α up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1α protein accumulation. Blockage of HIF-1α activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1α strongly inhibited their invasion ability, suggesting that elevation in HIF-1α protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1α-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1α to block HIF-1α activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1α-dependent up-regulation of PAI-1.

In this study, we investigated the possible involvement of HIF-1␣ in Cyr61-enhanced invasion ability in gastric cancer cells. We found that HIF-1␣ protein was elevated and constitutively activated in Cyr61-overexpressing gastric cancer cells. Dominantnegative (DN)-HIF-1␣ mutant could abolish the invasive ability of Cyr61-overexpressing cells, suggesting a critical role of HIF-1␣ in such an invasion mechanism. Plasminogen activator inhibitor-1 (PAI-1), a HIF-1␣-dependent invasion gene, was significantly upregulated in Cyr61-overexpressing cells. Our results further delineate a novel mechanism for HIF-1␣-dependent up-regulation of PAI-1, which contributes to the invasion activity of Cyr61.
Cell Cultures-Human gastric carcinoma cells (AGS, N87, and SNU16) were obtained from the American Type Culture Collection (Manassas, VA and Rockville, MD), and TSGH was obtained from Food Industry Research and Development Institute (Hsinchu, Taiwan). All gastric carcinoma cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 2 mM L-glutamine, 100 g/ml streptomycin, and 100 units/ml penicillin (all from Invitrogen). Cell cultures were maintained at 37°C in a humidified 5% CO 2 atmosphere. Adherent cells were detached from the culture dishes with trypsin/EDTA (Sigma).
Transient and Stable Transfections-Details are available as supplemental information.
RT-PCR and Western Blot Analysis-Details are available as supplemental information.
Nuclear and Cytosolic Protein Extraction-For nuclear and cytosolic protein extraction, the protein extracts were prepared from rCyr61-treated AGS cells or Cyr61-stably transfected-AGS cells using a modified procedure, as described previously (22).
HIF-1␣ Reporter Activity Assay-Details are available as supplemental information.
Immunofluorescence Staining-Details are available as supplemental information.
Boyden Chamber Assay-The details of the Boyden chamber assay are available as supplemental information.
PAI-1 Antisense Oligonucleotides-The sequence of the PAI-1-specific antisense oligonucleotides was 5Ј-AGACATCTGC-ATCCTGAAGTT-3Ј, and that of control oligonucleotides was 5Ј-AACTTCAGGATGCAGATGTCT-3Ј. For PAI-1 blocking studies, gastric cancer cells were cultured to 70% confluence, control and PAI-1 antisense oligonucleotides (25-100 nM) were transfected into tumor cells. After 48 h of transfection, the cells were used for invasion assay or lysed for Western blot analysis.
Chromatin Immunoprecipitation Assay-Briefly, cells were grown to a confluency of 85-90% in complete media and treated with or without 40 ng/ml rCyr61 for 16 h. Cross-linking, lysate preparation, immunoprecipitations, and DNA purification were performed according to the experimental procedures described in Ref. 21. A 278-bp region (HRE3-5) of the PAI-1 promoter was amplified by using the following primers: 5Ј-TCTGGACACGTGGGGGAGTCA-3Ј and 5Ј-AGG-TCACTGTGGAGTTATCA-3Ј. PCR products were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide.

RESULTS
We first examined whether Cyr61 expression was associated with HIF-1␣ expression in a series of human gastric adenocarcinoma cell lines. Among these four cell lines tested, we found a strong association between the expression levels of Cyr61 and HIF-1␣ protein (Fig. 1A, bottom) but not mRNA levels (Fig. 1A,  top). Elevated expression of Cyr61 by transiently transfected with different doses of Cyr61 sense-oriented expression vector significantly induced HIF-1␣ protein level in AGS (Fig. 1B, upper) and N87 cells (Fig. 1B, middle) with a dose-dependent manner. In addition, TSGH cells were transiently transfected with Cyr61 antisense-oriented expression vector resulted in significantly decreased HIF-1␣ protein level (Fig. 1B, lower).
Cyr61 contains a signal peptide that is believed to be a secreted protein. To test this hypothesis, we examined the Cyr61 protein amount in conditioned medium from the cultured gastric adenocarcinoma cells. Western blot analysis demonstrated a higher Cyr61 protein content in conditioned medium from TSGH cells. Significant amounts of secreted Cyr61 proteins were also obtained from Cyr61 stably transfected AGS cells (Fig. 1C, top). To further determine whether the secreted Cyr61 has an effect on HIF-1␣ expression, we depleted Cyr61 protein by adding neutralizing antibody (7, 23, FIGURE 1. Cyr61 stimulates HIF-1␣ accumulation and activation in human gastric carcinoma cells. A, the correlation between Cyr61 and HIF-1␣ protein levels in human gastric adenocarcinoma cell lines. Cells were cultured in the same condition and analyzed by RT-PCR and Western blot. B, AGS and N87 cells were transfected with different doses of empty vector or sense-oriented Cyr61 (S) plasmids followed by incubation for 48 h. Total proteins were isolated, and expression of Cyr61 and HIF-1␣ was analyzed by Western blot. TSGH cells were transfected with different doses of empty vector or antisense-oriented Cyr61 (AS) plasmids and used the same methods to analyze protein expression. C, upper: Cyr61 protein contents in conditioned medium from the gastric adenocarcinoma cells and Cyr61 stably transfected AGS cells by Western blot analysis. "50x": 1-ml samples from condition medium were typically concentrated into 20 l, using centrifugal filter devices (Amicon Inc., Beverly, MA). Lower: HIF-1␣ protein expression in TSGH and Cyr61 stably transfected AGS cells. Cyr61neutralizing antibody (10 g/ml) or control IgG was added in culture medium to deplete Cyr61 protein existence. D, duplicate plates of AGS and N87 cells were cultured in the absence of serum for 24 h, exposed to vehicle (lane 1), 10 -100 ng/ml recombinant Cyr61 (lanes 2-6) for 16 h. Western blot analyses of HIF-1␣ expression. Kinetics of HIF-1␣ mRNA (E) and protein (F) induction. Serum-starved cells were exposed to vehicle (lane 1), 40 ng/ml recombinant Cyr61, or 100 M CoCl 2 , or 100 ng/ml IGF-1 (lanes 2-7) prior to analysis of HIF-1␣ mRNA and protein. Data were quantified by Edit EZ-1D Software from EZLab Technology Co., Ltd. G, cytosolic and nuclear fractions were prepared from Cyr61 stably transfected AGS cells and AGS cells treated with rCyr61 or not. Cytosolic and nuclear fractions were subjected to Western blot analysis by using the indicated antibodies for HIF-1␣. ␣-Tubulin and SP-1 acted as the internal loading control for cytosolic and nuclear fractions, respectively. H, nuclear localization of HIF-1␣ was then observed by fluorescence microscopy. Cyr61 stably transfected AGS cells (upper panel) or AGS cells, which were incubated with rCyr61 for 8 and 16 h (lower panel), were fixed and immunostained with a mouse monoclonal anti-HIF-1␣ antibody, detected by fluorescein isothiocyanate-conjugated antibody. I, HIF-1␣ transcriptional activity was measured by the application of a luciferase assay using the reporter constructs containing tandem copies of hypoxia response element (HRE). pGL2 was used as vector control. Upper: Cyr61 stably transfected AGS cells were transiently transfected with pCEP4, DN-HIF-1␣, and then analyzed for luciferase activity. Lower: AGS cells transfected with various plasmids were treated with different doses of rCyr61 (20 -80 ng/ml) for 16 h, after which samples were analyzed for luciferase activity. All data are the average of three independent experiments. Columns, mean of three experiments; bars, Ϯ S.D. Statistical significance was determined with Student's t test (p Ͻ 0.05). 24) (kindly provided by Dr. Lester F. Lau (University of Illinois, Chicago, IL)) to the culture medium from TSGH and Cyr61 stably transfected AGS cells, and both cells secreted significant amounts of Cyr61 protein. As expected, decreasing Cyr61 protein concentration in culture medium significantly attenuated the endogenous HIF-1␣ protein level (Fig. 1C, bottom).
Next, to investigate whether the induction of HIF-1␣ directly depends on Cyr61, we added recombinant Cyr61 (rCyr61) into the cultures to examine the downstream signaling. Exposure of serum-starved AGS human gastric adenocarcinoma cells to rCyr61 for 16 h resulted in a concentration-dependent induction of HIF-1␣ protein expression and reached its maximal effect at 40 ng/ml rCyr61 (Fig. 1D, top). Similar results were obtained with N87 gastric adenocarcinoma cell lines (Fig. 1D, bottom). Moreover, we used CoCl 2 , a hypoxia mimetic, and IGF-1 as a comparison, because they are well known inducers of HIF-1␣ expression. We observed that exposure of serumstarved AGS cells to rCyr61, CoCl 2 , or IGF-1 did not significantly affect HIF-1␣ mRNA levels ( Fig. 1E). However, the rCyr61 treatment to serum-starved AGS cells resulted in HIF-1␣ protein expression after 8 h (Fig. 1F, top). The effect of CoCl 2 was extremely rapid (Fig. 1F, middle). We also found that CoCl 2 is a more potent inducer of HIF-1␣ expression compared with Cyr61. These observations suggest that Cyr61 and CoCl 2 play a distinct role in signaling pathways to increase the HIF-1␣ protein expression. Additionally, in the presence of IGF-1 (100 ng/ml), HIF-1␣ protein level increased at 2 h, suggesting the differences between Cyr61 and classic growth factors in HIF-1␣ induction mechanism (Fig. 1F, bottom). Taken together, Cyr61 stimulates HIF-1␣ protein accumulation in human gastric adenocarcinoma cells with a different induction kinetic as compared with CoCl 2 and IGF-1.
HIF-1␣ subunit is a pivotal transcription factor that requires nuclear translocation for activity. We therefore examined the nuclear translocation of HIF-1␣ protein in AGS cells after treatment with and without rCyr61. Western blot analysis revealed significant levels of HIF-1␣ in nuclear fractions of AGS cells treated with rCyr61 but not in control cells (Fig. 1G, right panel). Furthermore, immunofluorescent analysis also consistently revealed strong nuclear staining for HIF-1␣ in rCyr61treated cells but not in the control cells (Fig. 1H, lower panel). Similar results were obtained with the Cyr61 stably transfected AGS cells (Fig. 1, G (left panel) and H (upper panel)). Our results implicated that Cyr61 stimulates expression and nuclear translocation of HIF-1␣.
Moreover, we determined whether Cyr61-induced HIF-1␣ accumulation is correlated with the transcription activity of HIF-1. AGS cells were transfected with a luciferase reporter containing three hypoxia-response element sequences (pGL2-HRE). Treatment with 40 and 80 ng/ml rCyr61 resulted in 3.1and 3.7-fold increases, respectively, in HRE luciferase activity compared with untreated cells (Fig. 1I, lower panel). Additionally, the induction was also observed in Cyr61 stably transfected AGS cells (a 2.9-fold induction compared with that of AGS/Neo cell; Fig. 1I, upper panel). Furthermore, we transfected with a construct expressing a dominant-negative mutant version of HIF-1␣ (DN-HIF-1␣) that effectively competes with endogenous HIF-1␣ for dimerization with HIF-1␤ that resulting in blockage of the binding of HIF1␣⅐HIF-1␤ complex with target HRE-responsive element sequence and the subsequent transcriptional activation (22,25,26). The results showed that the Cyr61-induced HRE luciferase activity was significantly attenuated by the DN-HIF-1␣ in AGS cells. Our data thus suggest that Cyr61 not only stimulates the expression of functional HIF-1␣ protein but also induces the HIF-1␣-mediated HREdependent transcription activation.
Quantification of HIF-1␣ mRNA revealed that there was no significant increase in HIF-1␣ mRNA expression by rCyr61 in AGS human gastric carcinoma cells (Fig. 1E), indicating that Cyr61 was unable to regulate HIF-1␣ mRNA transcription. These results raised the question of whether increased amounts of HIF-1␣ protein were the result of reduced degradation of the protein in response to recombinant Cyr61 treatment or an increase in translation of the mRNA.
To analyze the possible effect of Cyr61 on HIF-1␣ protein synthesis, we performed a time-course analysis of HIF-1␣ turnover in the presence of the protein translation inhibitor, CHX. According to the Western blot analyses conducted and the results of quantitative data elicited ( Fig. 2A; quantification by Edit EZ-1D Software from EZLab Technology Co., Ltd., Taipei, Taiwan), the half-life of HIF-1␣ was much longer than 60 min in CoCl 2 -treated cells but Ͻ30 min in recombinant Cyr61-treated cells. As expected, observation is consistent with previous studies showing that CoCl 2 had no effect on HIF-1␣ synthesis but blocked its degradation. We also monitored that there was no significant change in HIF-1␣ protein half-life upon CHX treatment in the presence or absence of recombinant Cyr61 (both Ͻ30 min). Collectively, rCyr61 treatment seems to increase HIF-1␣ protein levels by increasing translation rather than by inhibiting degradation.
To corroborate these results, exposure of rCyr61 for 14 h in AGS cells and further treatment with or without the proteasome inhibitor MG132 (10 M) for 2 h, HIF-1␣ protein was highly elevated in rCyr61-treated cells than in control cells (Fig.  2B, lane 2 and 6). HIF-1␣ protein expression, promoted by MG132, was significantly inhibited by treatment with both MG132 and CHX (Fig. 2B, lane 4 and 8). If Cyr61 induces HIF-1␣ expression by stimulating synthesis of the protein, then it can be expected to have an additive effect with CoCl 2 , which can act by increasing the stability of the protein. Exposure of AGS cells to rCyr61 combined with CoCl 2 resulted in a greater increment of HIF-1␣ protein (Fig. 2C). The above results indi-cated that HIF-1␣ protein accumulation induced by Cyr61 is mainly through enhanced de novo protein synthesis.
To determine the signal transduction pathways mediating the effects of Cyr61 on HIF-1␣ protein expression, AGS cells were pretreated with PD98059, LY294002, and rapamycin, which are selective inhibitors of MEK, PI3K, and FRAP/ mTOR kinase activity, respectively. All these three agents inhibited the induction of HIF-1␣ protein expression in rCyr61-treated cells (Fig. 3A,  top). To examine whether the MAPK and PI3K pathways were activated serially or independently in rCyr61-treated cells, the phosphorylation of ERK and AKT were also analyzed. The increased phosphorylation of ERK that was induced by rCyr61 was blocked by PD98059 but not by LY294002 or rapamycin (Fig. 3A, middle). The increased phosphorylation of AKT, which was induced by the rCyr61 treatment, was blocked by LY294002 but not by PD98059 or rapamycin (Fig. 3A, bottom). Moreover, the induction of HIF-1␣ by rCyr61 was inhibited in a dose-dependent manner by PD98059 (Fig. 3B, upper) and LY294002 (Fig. 3B, lower). Thus, both MAPK and PI3K activities are required for induction of Cyr61-enhanced HIF-1␣ protein expression. In contrast, PD98059 and LY294002 had no significant inhibitory effect on the expression of HIF-1␣ in CoCl 2 -treated AGS cells (Fig. 3C). These results confer further evidence that Cyr61 and CoCl 2 act as a distinct molecular mechanisms.
The signal transduction pathway involving PI3K, AKT, and FRAP/mTOR has been shown to regulate protein translation via phosphorylation of p70 s6k and 4E-BP1 (27). Therefore we asked whether Cyr61 stimulates HIF-1␣ expression through a translation-dependent mechanism, and we also evaluated the phosphorylation status of p70 s6k and 4E-BP1 in response of rCyr61. In AGS cells, HIF-1␣ protein and the phosphorylation of both p70 s6k and 4E-BP1, which were induced by rCyr61 stimulation, could be blocked by rapamycin in a dose-dependent manner (Fig. 3D). Our results demonstrate that the Cyr61 induces HIF-1␣ through PI3K/mTOR and the MAPK-dependent pathway.
We previously found that Cyr61 was highly expressed in more advanced gastric adenocarcinoma, and overexpression of Cyr61 in human gastric cancer cell lines significantly increased their invasion abilities (11). To better understand the molecular mechanism, we examined whether HIF-1␣ participated in Cells were harvested after 16 h and subjected to immunoblot assay using antibodies specific for HIF-1␣, phosphorylated or total ERK, and phosphorylated or total AKT, respectively. B, AGS cells were exposed to vehicle or 40 ng/ml rCyr61 in the presence of 0 -40 M PD98059 (upper) or LY294002 (lower) for 16 h, and HIF-1␣ protein expression was determined by immunoblot assay. C, cells were exposed to 100 M CoCl 2 or 40 ng/ml rCyr61 without kinase inhibitor, or with 40 M PD98059, or 40 M LY294002. D, cells were exposed to vehicle or 40 ng/ml rCyr61 in the presence of 0 -100 nM rapamycin. Specific antibodies for phosphorylated or total 4E-BP and phosphorylated or total p70 S6K were used.
Cyr61-induced gastric cancer cell invasion. Co-transfection of DN-HIF-1␣ and sense-Cyr61 expression plasmids to AGS cells significantly abrogated the Cyr61-enhanced invasion ability (Fig. 4A, upper). Similar results were also observed in N87 cells (Fig. 4A, lower). In contrast, TSGH cells transiently transfected with Cyr61 antisense-oriented expression vector significantly abrogated cell invasion abilities and can be restored when cotransfected with WT-HIF-1␣ expression vector (Fig. 4B). Collectively, the Cyr61-induced invasion ability in gastric caner cells is largely due to the increase in HIF-1␣ expression.
The question remains as to which gene is the possible downstream effector that contributes to the Cyr61-mediated HIF-1␣-dependent cell invasion. It has been known that several invasion/metastasis-related genes such as PAI-1, VIM, c-MET, and ADM are the transcriptional targets of HIF-1␣ (27). To address this question, we analyzed the expression of these invasion/metastasis genes in rCyr61-treated cells by using RT-PCR. It appears that only the mRNA of PAI-1 was substantially increased in a dose-dependent manner in rCyr61-treated cells compared with control cells (Fig. 5A). Similar results of PAI-1 protein expression were also observed in Cyr61-overexpressing AGS cells (Fig. 5B).
In the previous investigation (11), we have found that Cyr61 is directly involved in invasion in human gastric cancer cell lines. In this study, we have demonstrated that Cyr61 up-regulates HIF-1␣-dependent invasion protein PAI-1. Therefore, to examine whether PAI-1 is involved in Cyr61-mediated gastric cancer cell invasion, three monolayer gastric cancer cell lines, including AGS, N87, and TSGH cells, were examined for their basal expression levels of PAI-1 protein and in vitro invasion capacity. Our results show that the level of PAI-1 protein was highly elevated in TSGH cells but was moderately expressed in N87 cells. AGS cells displayed an extremely low level of PAI-1 protein compared with other gastric cancer cells (Fig.  5C, top). In a modified Boyden chamber assay, TSGH cells exhibited the strongest invasive ability among these gastric cancer cell lines. In contrast, AGS cells, which expressed trace amounts of PAI-1, had very weak invasive potency (Fig.  5C, bottom). These results led us to hypothesize that PAI-1 could be directly involved in the malignant progression of human gastric cancer cells.
To ascertain the role of PAI-1 in Cyr61-mediated cell invasive effect, specific inhibition of PAI-1 expression was accomplished with antisense oligonucleotides. As anticipated, the PAI-1-specific antisense oligonucleotides used at 0.25 M significantly blocked the invasion ability by 60% in TSGH cells compared with the sense oligonucleotide group (Fig. 5D, top). Western blot analysis was used to confirm the PAI-1-specific antisense oligonucleotides activity (Fig. 5D, bottom). Furthermore, treatment with PAI-1-specific antisense oligonucleotides 0 -1 M in Cyr61-overexpressing AGS cells also abolished Cyr61-enhanced cell invasion in a dose-dependent manner (Fig. 5E). Supportively, treatment of Cyr61-overexpressing AGS cells with PAI-1-neutralizing antibody effectively inhibited Cyr61-induced gastric cancer cell invasion (Fig. 5F). The above studies using genetic inhibition and pharmacological treatments all vividly demonstrated a critical role of PAI-1 in Cyr61-mediated up-regulation of the gastric cancer cell invasion.
Inhibition of HIF-1␣ by transfection with DN-HIF-1␣ significantly reduced the amount of PAI-1 mRNA expression in AGS/Cyr61 cells, suggesting that the HIF-1␣ pathway is required for the Cyr61-induced PAI-1 mRNA expression (Fig.  6A, upper). Western blot analysis further confirmed that the increase of PAI-1 protein present in AGS/Cyr61 cells was also effectively attenuated by transfection with DN-HIF-1␣ (Fig. 6A,  lower). Finally, we examined whether HIF-1␣ would directly bind to the HRE (hypoxia response element) within the PAI-1 gene promoter by chromatin immunoprecipitation assay (Fig. 6  B). As shown in Fig. 6B (upper panel), there were three HRE sites located between Ϫ687 and Ϫ411 bp of the PAI-1 pro- moter. Treatment of AGS cells with rCyr61 or using AGS/ Cyr61 cells, followed by chromatin immunoprecipitation assay, showed that HIF-1␣ was constitutively bound to this region of the PAI-1 promoter. In summary, the Cyr61 up-regulated PAI-1 expression is mediated by the HIF-1␣-dependent pathway.

DISCUSSION
Members of the CCN family are multifunctional growth factors, and the nature of their regulation seems to be dependent on the cellular circumstance. Recent clinical evidence shows that Cyr61, one of CCN family members, is substantially involved in the development and progression of human malignancies. The significant importance of Cyr61 in human malignant alterations can be explained by its active regulation of multifaceted biological functions. For tumor invasiveness, Cyr61 can enhance motility of fibroblasts and microvascular endothelial cells. Recently, Nguyen et al. (28) have demonstrated that Cyr61 expression causes tumor cell migration and invasion in breast cancer cells, and their preliminary report indicated that gene silencing of Cyr61 in breast cancer cells suppressed matrix metalloproteinase-1 induction in stromal fibroblasts. However, the detailed mechanism by which Cyr61 enhances tumor invasiveness has not yet been characterized.
In the present study, we demonstrate a novel function of Cyr61 in gastric cancer cell invasion by molecular dissection. This study demonstrated, for the first time, that Cyr61 is able to up-regulate HIF-1␣ through de novo protein synthesis. This up-regulation of HIF-1␣ by Cyr61 commonly occurs in a variety of gastric cancer cell lines, involving PI3K/AKT/mTOR and ERK1/2 signaling pathways. Furthermore, we decipher the molecular mechanism for Cyr61-enhanced cell invasion, which is mediated by HIF-1␣-dependent PAI-1 up-regulation.
The role of HIF-1␣ in tumor invasion/metastasis has been extensively exploited in different cell systems (29 -34). Here we show the first time that Cyr61 enhances HIF-1␣ expression through a translation-dependent mechanism rather than inhibition of protein degradation. The underlying process is distinct from that observed during hypoxia. The hypoxia inhibits HIF-1␣ proteasomal degradation, leading to its accumulation (35). Previous studies supported that heregulin was found to activate HER2 tyrosine kinase receptor and stimulate HIF-1␣ translation (36). The stimulatory action on HIF-1␣ translation seems to be a general mechanism used by tyrosine kinase receptors, such as insulin and IGF-I receptors, HER2, and by cytosolic tyrosine kinases such as Src (36,37). Our finding revealed that Cyr61 regulates HIF-1␣ expression through both the PI3K and the MAPK cascades. Moreover, Cyr61 induces phosphorylation of p70 S6K and 4E-BP1 through a PI3K/mTOR-dependent pathway. The phosphorylation of 4E-BP1 leads to the release of the eukaryotic initiator factor 4E (eIF4E), and the phosphorylation of p70 S6K results in the phosphorylation and activation of the 40 S ribosomal protein S6. The ultimate outcome of all these events is the stimulation of protein synthesis. We found that MAPK is not involved in the phosphorylation of 4E-BP1 and p70 S6K in response to Cyr61. This is in contrast to the previous study in which IGF-1 stimulates phosphorylation of p70 S6K and 4E-BP1 through both PI3K-and MAPK-dependent pathways in colon cancer cells (38). We propose that MAPK regulates downstream of 4E-BP1 in response to Cyr61 in gastric cancer cells. Indeed, it has been shown that MnK1 (MAPK signal integrating kinase-1) phosphorylates eIF4E leading to increased protein synthesis (39).
Studies to uncover the signaling pathways involved in nonhypoxic HIF-1␣ activation revealed accumulation and transactivation of not only MAPKs and PI3K/AKT kinases but also NF-B (40). Our previous studies suggest that the Cyr61 elevates functional COX-2 via an integrin ␣v␤3/NF-B-dependent pathway in gastric cancer cells. Under such a scenario, we suggested that Cyr61 and IGF-1 influence HIF-1␣ up-regulation through quite distinct pathways. Effect of IGF-1 on HIF-1␣ accumulation is mediated directly through MAPKs or PI3K/ AKT signaling pathways (37,38,41). However, the effect of Cyr61 on HIF-1␣ expression is very complex, and this regula-tion might involve not only MAPKs or PI3K/AKT pathways but also NF-B/COX-2-related pathways. To support this, our preliminary data showed that celecoxib, a COX-2-specific inhibitor, inhibits Cyr61-induced HIF-1␣ elevation, indicating that COX-2 may have an intermediate role in regulating Cyr61-induced HIF-1␣ expression. This observation could account for the different kinetic induction patterns of HIF-1␣ in response to IGF-1 and rCyr61 treatments in AGS cells (Fig. 1F).
PAI-1, a member of the serpin family, has shown that its high expression in various cancers correlates with unfavorable prognosis (42)(43)(44). It has been initially suggested that increased PAI-1 expression in tumors could reflect a general up-regulation of the plasminogen activator system in proliferating cancer cells and a stromal reaction aimed at limiting excessive degradation of the extracellular matrix in more aggressive and invasive cancer forms (45). This issue is further strengthened in our current study where oligonucleotide antisense PAI-1 treatment not only reduced Cyr61-induced elevated PAI-1 protein but also significantly suppressed invasion ability of Cyr61-expressing cells. Recently, PAI-1 has been reported to be elevated in many human gastric cancer cell lines and tumor specimens (46,47). Inhibition of PAI-1 activity by MAI-12, a PAI-1-specific blocking antibody, significantly reduced cell invasion in human fibrosarcoma cancer cells (48). Concomitantly, these results have provided strong evidence that PAI-1 may be a critical factor in modulating cell invasion in human gastric cancer cells, and its expression can be regulated by Cyr61. We also investigated HIF-1␣-regulated invasion/metastasis genes. Out of six genes screened, only PAI-1 was significantly up-regulated in Cyr61-treated AGS cells. This specific association between PAI-1 and Cyr61 expression in gastric cancer cells is particularly intriguing. Accumulating evidence shows that PAI-1 can be enhanced by intracellular calcium via HIF-1␣ (49,50) or up-regulated by Cyr61 expression in the wound-healing process of fibroblasts (51). Recent studies showed that IGF-1 activates human PAI-1 gene expression through activation of PI3K/AKT and ERK1/2 via HIF-1␣ in HepG2 cells (49). Our findings are consistent with the results of others that PAI-1 plays a critical role in Cyr61-enhanced gastric cancer invasion via HIF-1␣.
It appears to be contradictory that PAI-1 is required for invasion, because elevated PAI-1 should reduce plasmin generation, whereas invasion requires cooperation between both proteolytic and inhibitory activities, probably to control and confine the areas of invasive growth. It has been suggested that the role of PAI-1 may be merely to shield the tumors from ongoing urokinase-type plasminogen activator-mediated proteolysis (52). More recent studies support the role of PAI-1 in promoting invasion and metastasis (48,(53)(54)(55)(56). PAI-1 specifically binds to the somatomedin domain of vitronectin and competes with and inhibits urokinase-type plasminogen activator receptor-mediated cell attachment to vitronectin (15). Vitronectinbound PAI-1 is still able to bind urokinase-type plasminogen activator, and the resulting complex has a highly reduced affinity for vitronectin and allows the migratory process to occur.
In conclusion, we have shown that Cyr61 activates the transcription factor HIF-1␣ protein synthesis through PI3K/AKT/ mTOR and ERK1/2 signaling pathway in AGS cells. Aug-  ). B, coimmunoprecipitation of HIF-1␣ on the PAI-1 promoter. ChIP analysis was done in Cyr61 stably transfected AGS cells and AGS cells, which were treated with 40 ng/ml rCyr61 for 16 h. Immunoprecipitations were carried out using HIF-1␣-specific antibody. Primers for the Ϫ687 bp to Ϫ411 bp region of the human PAI-1 promoter were used for PCR. mented Cyr61 expression is correlated with up-regulated HIF-1␣ and its downstream gene PAI-1. Blockage of HIF-1␣ activation led to reduced PAI-1 levels, and in turn, attenuated cell invasion. We propose a detailed mechanism underlying the role of Cyr61 in gastric cancer cell-invasive ability via an HIF-1␣-dependent up-regulation of PAI-1.