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Molecular Cancer Research 3:227-236 (2005)
© 2005 American Association for Cancer Research

Signaling and Regulation

Akt Activation, but not Extracellular Signal–Regulated Kinase Activation, Is Required for SDF-1{alpha}/CXCR4–Mediated Migration of Epitheloid Carcinoma Cells

Sheng-Bin Peng, Victoria Peek, Yan Zhai, Donald C. Paul, Qinyuan Lou, Xiaoling Xia, Thomas Eessalu, Wayne Kohn and Shaoqing Tang

Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana

Requests for reprints: Sheng-Bin Peng, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285. Phone: 317-433-4549; Fax: 317-276-1414. E-mail: Peng_Sheng-Bin{at}Lilly.com


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Emerging evidence shows that the stromal cell–derived factor 1 (SDF-1)/CXCR4 interaction regulates multiple cell signaling pathways and a variety of cellular functions such as cell migration, proliferation, and survival. There is little information linking the cellular functions and individual signaling pathways mediated by SDF-1 and CXCR4 in human cancer cells. In this study, we have shown that human epitheloid carcinoma HeLa cells express functional CXCR4 by reverse transcription-PCR, immunofluorescent staining, and 125I-SDF-1{alpha} ligand binding analyses. The treatment of HeLa cells with recombinant SDF-1{alpha} results in time-dependent Akt and extracellular signal–regulated kinase 1/2 (ERK1/2) activations. The SDF-1{alpha}–induced Akt and ERK1/2 activations are CXCR4 dependent as confirmed by their total inhibition by T134, a CXCR4-specific peptide antagonist. Cell signaling analysis with pathway-specific inhibitors reveals that SDF-1{alpha}–induced Akt activation is not required for ERK1/2 activation and vice versa, indicating that activations of Akt and ERK1/2 occur independently. Functional analysis shows that SDF-1{alpha} induces a CXCR4-dependent migration of HeLa cells. The migration can be totally blocked by phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, whereas mitogen-activated protein/ERK kinase inhibitors, PD98059 and U0126, have no significant effect on SDF-1{alpha}–induced migration, suggesting that Akt activation, but not ERK1/2 activation, is required for SDF-1{alpha}–induced migration of epitheloid carcinoma cells.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Chemokines and their receptors play an important role in immune and inflammatory responses by mediating the directional migration and activation of leukocytes (1-3). These molecules have also been implicated in hematopoiesis, angiogenesis, and embryonic development (4-6). Much of what is currently known about chemokines and their receptors has been learned from studies of hematopoietic cells. Stromal cell–derived factor 1 (SDF-1/CXCL12) is a highly efficient chemotactic factor for T cells, monocytes, pre–B-cells, dendritic cells, and hematopoietic progenitor cells (1-6). It binds only to one receptor, CXCR4, for which SDF-1 is the only known ligand. Like other chemokine receptors, CXCR4 is a seven-transmembrane-domain G-protein–coupled receptor (7-9). The chemotactic effect of SDF-1 on hematopoietic progenitor cells has been shown to be mediated via the CXCR4 receptor. Targeted disruption in mice of either the SDF-1 or CXCR4 gene results in a very similar phenotype, is lethal, and accompanied by many severe developmental defects, including the absence of both lymphoid and myeloid hematopoiesis in the fetal bone marrow (10, 11). In addition, it was found that SDF-1 and CXCR4 play a critical role in the engraftment of hematopoietic stem cells to bone marrow (12). Collectively, these results suggest that SDF-1 and CXCR4 regulate hematopoiesis by modulation of migration and homing of hemotopoietic stem and progenitor cells.

In addition to the prominent role in regulating leukocytes and hematopoietic progenitor cells, recent research suggests that SDF-1 and CXCR4 also play an important role in tumorigenesis. CXCR4 was found to be expressed or overexpressed in a variety of cancer cell lines and tissues including breast cancer (13), prostate cancer (14), lung cancer (15), ovarian cancer (16), colon (17), pancreatic cancer (18), kidney cancer (19), and brain cancer (20-22), as well as non-Hodgkin's lymphoma (23) and chronic lymphocytic leukemia (24). The emerging evidence suggests that CXCR4 plays important roles in multiple phases of tumor progression, including tumor growth, invasion, and metastasis. In a preclinical mouse model of human high-grade non-Hodgkin's lymphoma, CXCR4 neutralization of Namalwa cells significantly delayed tumor growth (23). In vitro data showed that CXCR4 neutralization enhanced apoptosis of tumor cells, decreased cell proliferation, and inhibited cell migration and pseudopodia formation (23). In human glioblastoma and medulloblastoma xenograft models, the CXCR4-specific antagonist AMD3100 inhibited tumor growth by inhibiting cell proliferation and promoting apoptosis of the tumor cells (25). The role of CXCR4 in promoting brain tumor cell proliferation and survival was also observed under in vitro conditions (20-22).

The SDF-1/CXCR4 interaction also plays a critical role in cancer cell metastasis. Muller and colleagues found that CXCR4 was highly expressed in malignant but not normal breast tissues, and its ligand SDF-1 was expressed in those organs where breast cancer metastases were frequently found, such as lymph node, lung, and bone marrow. SDF-1 stimulated breast cancer cells to carry out the basics of invasion [i.e., cells sent out extensions (pseudopodia), migrated in a directed manner, and penetrated barriers imposed by extracellular matrix (13)]. In a metastatic model of human breast cancer, CXCR4 neutralization significantly suppressed lymph node and lung metastases of tumor cells (13). The involvement of CXCR4 in cancer metastasis is not unique to breast cancer. In CXCR4-expressing prostate cancer cells, SDF-1 was shown to promote tumor cell transendothelial chemotaxis and adhesion to osteoclastic cells (14). Myeloma cells express CXCR4 and show a chemotactic response to SDF-1 associated with a rapid, transient up-regulation of very late antigen-4, promoting tumor cell adhesion to endothelial and marrow stromal cells (26). All B-cell lymphomas express CXCR4 and migrate towards lymph node stromal cells or SDF-1 (27). Chronic lymphocytic leukemic cells also typically express functional CXCR4, and SDF-1 promotes their adhesion to, and migration beneath, SDF-1 producing marrow stromal cells (24). CXCR4/SDF-1 may also play a role in the development of bone metastases in many neuroblastomas (28). Collectively, the emerging data convincingly suggest that CXCR4 is directly involved in promoting cancer metastases.

The role of SDF-1 and CXCR4 in angiogenesis is well documented. It was shown that SDF-1 acts as a direct chemoattractant for endothelial cells in vitro, and as an angiogenic factor in vivo (29-31). SDF-1 induces the expression of vascular endothelial growth factor by endothelial cells. Vascular endothelial growth factor can, in turn, up-regulate CXCR4 levels on endothelial cell surfaces. These observations indicate that SDF-1 and vascular endothelial growth factor act additively or synergistically to amplify angiogenic processes. In addition, SDF-1 alone can induce neovascularization in vivo and formation of sprouting vessels in an ex vivo rat aortic ring sprouting assay (30). Interestingly, knockout experiments revealed that SDF-1 and CXCR4 play a role in blood vessel development (32, 33). Human astrogliomas express elevated CXCR4 and respond to SDF-1 by secretion of chemokines expressed during angiogenesis and inflammation (21). In human glioblastoma, SDF-1 and CXCR4 expression increased with increasing tumor grade, and CXCR4 was also expressed in neovascular endothelial cells, again indicating a role in angiogenesis (22). In a prostate xenograft model expressing high levels of CXCR4, the blood vessel density in the tumor was 4.5-fold higher than in the control model (34).

Despite the apparently important role of CXCR4/SDF-1 in tumor cell growth, invasion, metastasis, and angiogenesis, relatively little is known about the signaling pathways that mediate these effects in cancer cells. In this study, we show that CXCR4 is functionally expressed in epitheloid carcinoma HeLa cells. SDF-1{alpha} treatment of HeLa cells results in the activation of Akt and mitogen-activated protein (MAP) kinases extracellular signal–regulated kinase (ERK) 1/2. SDF-1{alpha} also induces a CXCR4-dependent HeLa cell migration. Analysis with pathway-specific inhibitors reveals that Akt activation, but not ERK activation, is required for SDF-1{alpha}–induced HeLa cell migration.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
CXCR4 Expression in HeLa Cells
Human epitheloid carcinoma HeLa cells are adherent cells widely used for a variety of biological studies. We are interested in using HeLa cells to study CXCR4/SDF-1–mediated functions in tumor cells. To ensure adequate CXCR4 expression in HeLa cells, we first did a reverse transcription-PCR (RT-PCR) analysis, and the RNA prepared from cultured HeLa cells was analyzed using human CXCR4, SDF-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene–specific primers. As shown in Fig. 1A, CXCR4 was well expressed in HeLa cells, whereas SDF-1 expression is not detectable by RT-PCR in the given conditions. However, both genes were expressed in a prostate tumor tissue, a control sample used for this experiment. GAPDH was used as an internal control to monitor if RNA isolation and RT-PCR were reliable. As shown in Fig. 1A, GAPDH was detected in both samples as expected.



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  		FIGURE 1. Expression of CXCR4 in human epitheloid carcinoma HeLa cells. CXCR4 expression was analyzed by RT-PCR (A) and immunofluorescent staining with a CXCR4 monoclonal antibody (B) or a control immunoglobulin G (C). An RNA sample prepared from a prostate cancer sample (lane 1) was used as a control for RT-PCR. GAPDH was used as an internal control.

 
To investigate the cell surface expression of CXCR4 in HeLa cells, we did an immunofluorescent staining utilizing a monoclonal antibody that specifically recognizes CXCR4 on the cell surface. Confocal microscopic analysis revealed that CXCR4-specific immunofluorescent staining was observed when HeLa cells were incubated with CXCR4-specific antibody (Fig. 1B), whereas no significant fluorescence staining was observed when these cells were incubated with a control immunoglobulin G in the same conditions (Fig. 1C), suggesting that HeLa cells indeed express CXCR4 protein.

To further characterize the CXCR4 expression and function in HeLa cells, we established a whole cell–based 125I-SDF-1{alpha} ligand binding assay. As shown in Fig. 2, HeLa cells bind 125I-SDF-1{alpha} in a dose-dependent manner with a Kd of 0.2566 nmol/L. This 125I-SDF-1{alpha} binding can be specifically replaced by cold SDF-1{alpha}, confirming the specificity of SDF-1{alpha} binding. This ligand binding result further confirmed the CXCR4 expression and function in HeLa cells. Collectively, the results from RT-PCR, immunofluorescent staining, and ligand binding assays suggest that HeLa cells express endogenous CXCR4, and this endogenous CXCR4 expression avoids the potential conformational change of the receptor that could occur with overexpression by artificial transfection. Therefore, we believe that HeLa cell is a highly valid tumor cell line for CXCR4 functional studies.



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  		FIGURE 2. 125I-SDF-1{alpha} ligand binding by HeLa cells. 125I-SDF-1{alpha} at 0-1.6 nmol/L was used for determination of the 125I-SDF-1{alpha} binding affinity (A). The total binding (red line) and the nonspecific binding in the presence of 200-fold excess of cold SDF-1{alpha} (black line) were determined by filter binding assay as described under Materials and Methods. The net 125I-SDF-1{alpha} binding (blue line) was calculated by subtracting nonspecific binding from the total binding. A Kd of 0.2566 nmol/L was calculated by nonlinear progression analysis. The specificity of 125I-SDF-1{alpha} binding was further confirmed by a replacement experiment with cold SDF-1 (B). In B, 0.1 nmol/L 125I-SDF-1{alpha} and 1-64 nmol/L cold SDF-1{alpha} were used as indicated.

 
SDF-1{alpha} Treatment Activates Phosphoinositide 3-Kinase/Akt Pathway
Akt is a known downstream effector of the phosphoinositide 3-kinase (PI3K)–dependent signaling cascade. It has recently been shown that some chemokines stimulate PI3K, leading to the activation of protein Akt in some cell lines. The PI3K/Akt pathway is an important mediator of chemotaxis in many cell types (35-38). We were interested in investigating whether SDF-1{alpha} treatment could activate PI3K/Akt pathway in epitheloid carcinoma HeLa cells. For this purpose, we measured the phosphorylation of Akt by Western blot using a phospho-Akt–specific antibody. As revealed in Fig. 3A, SDF-1{alpha} treatment of HeLa cells stimulated a time-dependent and prolonged Akt phosphorylation. The phospho-Akt level reached a maximum 5 to 10 minutes after SDF-1{alpha} treatment, and the significantly enhanced Akt phosphorylation could still be observed 60 minutes after SDF-1{alpha} treatment, whereas no significant change in total Akt expression was observed over the course of the experiment. To address whether the SDF-1{alpha}–induced Akt activation was specifically via CXCR4, we synthesized T134, a peptide antagonist that has been characterized to block the CXCR4/SDF-1 interaction specifically (39). As revealed in Fig. 3B, the SDF-1{alpha}–activated Akt phosphorylation was inhibited by T134 in a dose-dependent manner. Complete inhibition was observed when 0.5 µmol/L T134 was included in the reaction. These results suggest that SDF-1{alpha}–induced Akt activation is specifically through its monogamous receptor, CXCR4.



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  		FIGURE 3. SDF-1{alpha}–induced Akt activation and its inhibition by T134. HeLa cells were treated with 12.5 nmol/L SDF-1{alpha} for 0, 1, 2, 5, 10, 20, 40, or 60 minutes. The cell lysates were prepared and used for Western blot analysis with phospho-Akt, total Akt, or actin antibody to investigate Akt activation (A). HeLa cells were preincubated with 0, 0.125, 0.25, 0.5, or 1 µmol/L of T134 for 3 hours, and then treated with 12.5 nmol/L SDF-1{alpha} for 5 minutes. The cell lysates were used for Western blot analysis to investigate inhibition of SDF-1{alpha}–induced Akt activation by T134 (B).

 
SDF-1{alpha}/CXCR4 Interaction Activates Mitogen-Activated Protein Kinases Extracellular Signal–Regulated Kinase 1/2 and Their Downstream Effectors
To further study cell signaling mediated by the CXCR4/SDF-1 interaction, we also investigated whether SDF-1{alpha} could activate MAP kinases ERK1/2, p38, or c-jun-NH2-kinase. Western blot analysis revealed that SDF-1{alpha} treatment of HeLa cells resulted in a dramatic and rapid activation of both ERK1 and ERK2. The maximum phospho-ERK1/2 level was observed 5 minutes after SDF-1{alpha} treatment, with phospho-ERK1/2 level returning to near the basal level 20 to 40 minutes after SDF-1{alpha} treatment. No significant change was noticed in total ERK1/2 protein expression over the course of the investigation (Fig. 4A). In contrast to ERK1/2, the phosphorylation of other MAP kinases, p38 or c-jun-NH2-kinase, was not significantly affected by SDF-1{alpha} in these assay conditions (data not shown). To further confirm the ERK pathway activation, Western blot analysis was done for phospho-Elk-1 and phospho-90rsk, the two immediate downstream effectors of ERK1/2. As shown in Fig. 4A, both phospho-Elk-1 and phospho-90rsk levels were increased in response to SDF-1{alpha} treatment in a time-dependent manner similar to ERK1/2 activation, indicating that ERK1/2 pathway was indeed activated. Again, T134, a peptide antagonist specific for CXCR4, blocked SDF-1{alpha}–induced ERK1/2 activation in a dose-dependent manner, and 0.5 µmol/L T134 completely abolished the activation (Fig. 4B). This observation suggests that SDF-1{alpha}–induced ERK1/2 activation in HeLa cells was via its receptor, CXCR4.



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  		FIGURE 4. SDF-1{alpha}–induced activation of ERK1/2 and their downstream effectors Elk-1 and 90rsk. HeLa cells were treated with 12.5 nmol/L SDF-1{alpha} for 0, 1, 2, 5, 10, 20, 40, or 60 minutes. The cell lysates were prepared and used for Western blot analysis with phospho-ERK, total ERK, phospho-Elk-1, phospho-90rsk, or actin antibody (A). HeLa cells were preincubated with 0, 0.125, 0.25, 0.5, or 1 µmol/L of T134 for 3 hours, and then treated with 12.5 nmol/L SDF-1{alpha} for 5 minutes. The cell lysates were used for Western blot analysis to investigate inhibition of SDF-1{alpha}–induced ERK1/2 activation by T134 (B).

 
SDF-1{alpha}/CXCR4–Mediated Akt and ERK Activations Are Independent from Each Other in HeLa Cells
Intracellular signaling involves a complex network of often interacting pathways. We are interested in investigating the relationship between SDF-1{alpha}–induced activation of Akt and ERK, and in defining whether activation of the PI3K/Akt cascade is required for SDF-1{alpha}–induced ERK activation and vice versa. For this purpose, we did some pathway-specific inhibitor studies. When HeLa cells were treated with PI3K inhibitors, wortmannin or LY294002, SDF-1{alpha}–induced Akt activation was inhibited in a dose-dependent manner, as expected. However, no significant difference in SDF-1{alpha}–induced ERK activation was observed with or without a PI3K inhibitor (Fig. 5A and B), suggesting that Akt activation is not required for ERK activation. Similarly, when HeLa cells were treated with MAP/ERK kinase (MEK) inhibitors, PD98059 or U0126, SDF-1{alpha}–induced ERK activation was blocked in a dose-dependent manner, whereas no significant effect of PD98059 or U0126 on SDF-1{alpha}–induced Akt activation was observed (Fig. 5C and D). Therefore, Akt activation is independent of ERK activation. Overall, these results clearly show that SDF-1–induced activations of Akt and ERK in HeLa cells are not linear and arise from different signaling pathways.



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  		FIGURE 5. A pathway-specific inhibitor study showing that SDF-1{alpha}–induced Akt and ERK activations are independent of each other. HeLa cells, 2.5 x 105, were preincubated for 3 hours with different concentrations of PI3K inhibitor, LY294002 (A) or wortmannin (B), or MEK inhibitors, PD98059 (C) or U0126 (D), followed by treatment with or without 12.5 nmol/L SDF-1{alpha} for 5 minutes. The preparation of cell lysates and Western blot analysis were done as described under Materials and Methods.

 
SDF-1{alpha} Induces CXCR4-Dependent Epitheloid Carcinoma Cell Migration
SDF-1{alpha} and other chemokines regulate leukocyte trafficking by mediating the adhesion of leukocytes to endothelial cells, the initiation of transendothelial migration, and tissue invasion (40). These are also processes used by cancer cells during invasion and metastasis. Chemotaxis is the central function of SDF-1–regulated leukocyte trafficking and tumor cell metastasis. Due to the important role of the CXCR4/SDF-1 interaction in cancer metastasis, we were interested in investigating whether SDF-1{alpha} induces HeLa cell migration. For this purpose, we developed a cell migration assay in which HeLa cells were evaluated for their ability to migrate through 3-µm pores of bare filters as described under Materials and Methods. As shown in Fig. 6A, SDF-1{alpha} induced a dose-dependent HeLa cell migration, and the maximum migration was observed when 89 nmol/L SDF-1{alpha} was applied in the assay conditions. The migration was inhibited by T134 in a dose-dependent manner by incubation of the HeLa cells with T134, indicating that SDF-1{alpha}–induced HeLa cell migration was specifically via its receptor CXCR4 (Fig. 6B).



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  		FIGURE 6. SDF-1{alpha}/CXCR4–mediated epitheloid carcinoma HeLa cell migration. In A, 0-200 nmol/L concentrations of SDF-1{alpha} as indicated were used for this experiment, and SDF-1{alpha} induced a dose-dependent HeLa cell migration. In B, 60 nmol/L SDF-1{alpha} was used, and SDF-1{alpha}–induced HeLa cell migration was inhibited by T134 in a dose-dependent manner.

 
Akt Activation Is Required for SDF-1{alpha}–Induced Cell Migration
Activation of Akt and ERK1/2 after SDF-1{alpha} treatment indicates that SDF-1/CXCR4 signaling involves multiple pathways. To determine the functional role of PI3K/Akt activation in SDF-1{alpha}–induced migration of epitheloid carcinoma cells, HeLa cells were treated with different concentrations of PI3K inhibitors, wortmannin or LY294002. Cell migration in response to SDF-1{alpha} was examined by the migration assay described under Materials and Methods. As shown in Fig. 7A, the treatment of HeLa cells with wortmannin inhibited SDF-1{alpha}–induced cell migration in a dose-dependent manner, and a nearly complete inhibition was observed with 200 nmol/L wortmannin. In addition, we also tested the effect of LY294002, an ATP-competitive and specific inhibitor of PI3K, because the specificity of wortmannin at low doses was questioned previously (41). Similar to the result with wortmannin, treatment of HeLa cells with LY294002 also inhibited SDF-1{alpha}–induced cell migration in a dose-dependent manner (Fig. 7B). Collectively, we have shown that PI3K inhibitors, wortmannin and LY294002, could completely inhibit SDF-1–induced HeLa cell migration, suggesting that PI3K/Akt activation is absolutely required for SDF-1{alpha}–induced migration of epitheloid carcinoma cells.



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  		FIGURE 7. Akt activation is required for SDF-1{alpha}/CXCR4–mediated HeLa cell migration. SDF-1{alpha}–induced HeLa cell migration was dramatically inhibited by PI3K inhibitors, wortmannin (A) and LY294002 (B), in a dose-dependent manner. SDF-1{alpha} at 60 nmol/L was used in all the cell migration reactions.

 
ERK Activation Is Not Required for SDF-1{alpha}–Induced HeLa Cell Migration
We have shown that ERK1/2 and its downstream effectors, Elk-1 and 90rsk, were activated by SDF-1{alpha} treatment. To evaluate the functional role of the MEK/ERK pathway in SDF-1{alpha}–induced migration of epitheloid carcinoma cells, we next examined the effects of MEK inhibitors, PD98059 and U0126, on the migration of HeLa cells in response to SDF-1{alpha} treatment. As shown in Fig. 8A, the incubation of HeLa cells with PD98059 over a concentration range from 2 to 50 µmol/L did not significantly alter cell migration. As shown in Fig. 5, ERK activation was significantly inhibited by 2 to 50 µmol/L of PD98059. Similarly, incubation of HeLa cells with another MEK inhibitor, U0126, over a concentration range from 0.4 to 10 µmol/L, resulted in no significant effect on SDF-1{alpha}–induced migration (Fig. 8B), although nearly complete inhibition of ERK activation was achieved by 0.6 to 10 µmol/L U0126 (Fig. 5B). Thus, it seems that ERK activation is not required for SDF-1{alpha}–induced migration of epitheloid carcinoma cells, although ERK1/2 are dramatically activated after SDF-1{alpha} treatment.



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  		FIGURE 8. ERK activation is not required for SDF-1{alpha}–induced HeLa cell migration. When HeLa cells were preincubated with MEK inhibitors, PD98059 (A) and U0126 (B), no significant inhibition of SDF-1{alpha}–induced HeLa cell migration was observed. SDF-1{alpha} at 60 nmol/L was used in all the cell migration reactions.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
The chemokine SDF-1 and its cognate receptor CXCR4 have recently sparked substantial interest because of their role in tumorigenesis including tumor growth, invasion, and metastasis (13, 23, 25). These molecules mediate multiple signal transduction pathways and a variety of cellular functions such as cell migration, proliferation, and survival. However, there is little information linking the cellular functions and individual signaling pathways mediated by SDF-1/CXCR4 interaction within the cells, particularly cancer cells. In this study, we have shown that SDF-1{alpha} treatment of epitheloid carcinoma HeLa cells results in CXCR4-dependent activation of Akt and ERK1/2. Cell signaling analysis using pathway-specific inhibitors revealed that Akt and ERK activations seem to be independent of each other. Functionally, SDF-1{alpha} induces a CXCR4-dependent cell migration, and Akt activation, but not ERK activation, is required for SDF-1{alpha}–induced migration of epitheloid carcinoma cells.

CXCR4 is a Gi-coupled receptor, and studies have shown that SDF-1, after binding to CXCR4, causes mobilization of calcium, decrease of cyclic AMP within the cells, and activation of multiple signaling pathways, including PI3K, phospholipase C-{gamma}/protein kinase C, and MAP kinases ERK1/2 (42-45). However, almost all of these studies were done with immune cells or stem cells. Due to the important roles of CXCR4/SDF-1 in tumorigenesis, we are interested in investigating CXCR4/SDF-1{alpha}–regulated cell signaling in cancer cells. For this purpose, we treated HeLa, a widely used human epitheloid carcinoma cell line, with SDF-1{alpha} and analyzed the regulation of multiple signal transduction components including MAP kinases ERK1/2, p38, and c-jun-NH2-kinase, Akt, IkB{alpha}, Stat, and GSK. Among the components analyzed, significant and time-dependent activation of Akt, ERK1/2, and their downstream effectors, Elk-1 and 90rsk, was observed. However, no significant regulation of p38, c-jun-NH2-kinase, NF{kappa}B, Stat, or GSK was observed by Western blot analysis with the antibodies used in this study (data not shown). Due to the important role of Akt and ERK1/2 in cancer biology, this observation triggered us to explore further the relationship between Akt and ERK1/2 activations, and their respective roles in cellular function.

Cell signaling is a very complex network, and in many cases it is cell type dependent. Although SDF-1/CXCR4–mediated Akt and ERK activations were observed in multiple cell types, including lymphocytes and stem cells, their relationship in cell signaling was not well characterized. In this study, we clearly show that SDF-1{alpha}–induced Akt and ERK activations are independent of each other in HeLa cells. PI3K inhibitors, wortmannin and LY294002, at concentrations totally blocking SDF-1{alpha}–induced Akt activation, have no effect on ERK activation. Similarly, MEK inhibitors, PD98059 and U0126, at concentrations totally blocking ERK activation, have no significant effect on Akt activation. These results suggest that SDF-1{alpha}–induced Akt and ERK activations in HeLa cells are independent and not linear in signal transduction. It is in accordance with other results obtained with human glioblastoma cells where ERK activation does not require PI3K/Akt activation (46). However, it was reported that SDF-1–stimulated ERK activation was inhibited by PI3K inhibitors in T lymphocytes, ERK1/2 activation is at least partly dependent on PI3K activation, and both biochemical events seem to be involved in the regulation of SDF-1–stimulated chemotaxis (43). Therefore, it seems that the relationship between SDF-1–induced Akt and ERK activations is cell type dependent.

From a functional standpoint, we found that SDF-1{alpha} treatment induced a dose-dependent HeLa cell migration, an important function related to cancer cell metastasis. Recent studies show that the SDF-1/CXCR4 interaction plays a critical role in cancer cell metastasis (13, 14, 26-28). Therefore, it is important to define the molecular mechanisms involved in SDF-1/CXCR4–mediated tumor cell migration. Accumulating data have implicated that multiple signaling mechanisms exist to regulate cell migration. Both ERK (47, 48) and PI3K/Akt (46, 49, 50) signaling pathways have been shown to mediate the cell migration induced by chemokines or cytokines in different cell types. However, the molecular mechanisms of SDF-1/CXCR4–regulated cell migration in tumor cells are poorly understood to date. To investigate the functional roles of PI3K/Akt and MEK/ERK signaling cascades in SDF-1–induced cell migration of epitheloid carcinoma cells, we did studies with PI3K-specific inhibitors (wortmannin and LY294002) or MEK-specific inhibitors (PD98095 and U0126). The results showed that PI3K inhibitors wortmannin and LY294002 totally blocked SDF-1{alpha}–induced HeLa cell migration. In contrast, MEK inhibitors PD98059 at 50 µmol/L and U0126 at 10 µmol/L, both of which totally inhibited SDF-1{alpha}–induced ERK activation, had no significant effect on SDF-1{alpha}–induced cell migration, indicating that PI3K/Akt activation, but not ERK activation, is required for SDF-1{alpha}–induced HeLa cell migration. Although activation of the ERK signaling pathway has been shown to promote cell mobility either by regulating gene expression in carcinoma cells (47) or directly activating myosin light chain kinase in COS-7 cells (48), it seems that it is not the case in SDF-1{alpha}–induced migration of epitheloid carcinoma cells. However, our results are similar to the results observed in T lymphocytes, where the SDF-1/CXCR4 interaction led to activation of multiple signal transduction components, and the MAP kinase inhibitor PD98059 had no effect on SDF-1–induced chemotaxis (51). Our findings are also consistent with results obtained with hematopoietic progenitor cells and primary marrow CD34+ cells where PI3K/Akt seemed to be required for SDF-1–mediated cell migration, but ERK1/2 were not (52).

However, in some cell types and under certain stimuli, MAP kinase signaling has been shown to regulate cell mobility (53). In T lymphocytes, SDF-1–induced chemotaxis is dependent on PI3K activation, and actin polymerization requires additional biochemical inputs. SDF-1–stimulated ERK activation was inhibited by a PI3K inhibitor, Wortmannin. In addition, MEK inhibitor PD98059 partially attenuated chemotaxis in response to SDF-1. Hence, it seems that ERK1/2 activation is dependent on PI3K activation, and both biochemical events are involved in the regulation of SDF-1 stimulated chemotaxis (43). These results are somewhat different from those reported by another group with the same cells (51). The differences observed by these two groups are likely due to the nonspecificity of PI3K inhibitor, wortmannin (41). In fact, the so-called PI3K-dependent ERK activation was only observed at high concentration (100 nmol/L) of wortmannin (43). In human neuroepithelioma CHP100 cells expressing functional CXCR4, inhibition of either the ERK or the PI3K pathways blocked the SDF-1–induced chemotaxis, suggesting that both Akt and ERK activations were involved in SDF-1–regulated cell migration (54). In human embryonic kidney 293/hCXCR4 transfected cells, ß-arrestin 2 amplified CXCR4-mediated activation of both p38 MAP kinase and ERK, and the suppression of ß-arrestin 2 expression blocked the activation of the two kinases (55). Interestingly, inhibition of p38 activity (but not ERK activity) by selective inhibitors or by expression of a dominant-negative mutant of p38 MAP kinase effectively blocked the chemotactic effect (55). Therefore, it seems that the molecular mechanisms involved in CXCR4-mediated cell migration are also cell type dependent.

Although ERK activation is not required for SDF-1{alpha}–induced migration of HeLa cells as revealed by this study, we believe that it may play an important role in promoting tumor cell proliferation and survival. Indeed, many studies with cultured tumor cells or cancer xenografts imply that CXCR4/SDF-1 interaction stimulates tumor cell growth and promotes cell survival (20, 23, 25). The functional roles of SDF-1–inducd ERK1/2 and Akt activations in cell proliferation and survival of HeLa cells are currently under investigation in our laboratory.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Reagents
Anti-ERK1/2, anti–phospho-ERK1/2, anti-Akt, anti–phospho-Akt, anti–phospho-Elk-1, and anti–phospho-90rsk polyclonal antibodies for Western blot analysis were purchased from Cell Signaling (Beverly, MA). Anti–ß-actin monoclonal antibody was from Sigma (St. Louis,MO). Anti-hCXCR4 monoclonal antibody for immunofluorescent staining was from R&D Systems (Minneapolis, MN). Recombinant human SDF-1{alpha} was purchased from PeproTech EC Ltd. (London, United Kingdom). PI3K inhibitors, wortmannin and LY294002, and MEK inhibitors, PD98059 and U0126, were from Calbiochem (San Diego, CA). T134, a CXCR4-specific peptide antagonist, was synthesized based on the published structure (35).

Cell Culture
Human epitheloid carcinoma HeLa Cells were purchased from the American Type Culture Collection (Rockville, MD), and grown in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (FBS). Adherent cultures were passaged twice each week at subconfluence after trypsinization. Cultures were maintained in an incubator at 37°C in an atmosphere of 5% CO2 and 95% air. The cells used for all experiments do not exceed 15 passages.

Reverse Transcription-PCR
Total cellular RNA was prepared using Absolutely RNA RT-PCR Miniprep Kit from Invitrogen (Carlsbad, CA). Briefly, cells were grown in flasks to 80% confluence, and 3 x 106 trypsinized cells were lysed with lysis buffer and digested with RNase-free DNase following the instruction of the manufacturer. For RT-PCR, cDNA was synthesized from DNase-treated RNA (200 ng) using Moloney murine leukemia virus reverse transcriptase. The gene-specific primers used for human CXCR4, SDF-1, and GAPDH amplification were designed as follows: CXCR4, 5'-CTTCTACCCCAATGACTTGTGG-3' (sense) and 5'-AATGTAGTAAGGCAGCCAACAG-3' (antisense); SDF-1, 5'-ATGAACGCCAAGGTCGTGGTC-3' (sense) and 5'-CTCACATCTTGAACCTCTTGTT-3' (antisense); and GAPDH, 5'-ATGTCGAAGCGCGACATCGTC-3 (sense) and 5'-CACGACCAGTTGTCCATTCCT-3' (antisense). For PCR reactions, 25 µL of sample contained synthesized cDNA, 1 unit of AmpliTaq DNA polymerase, PCR buffer, deoxynucleotide triphosphates, and 4 µmol/L of each primer. The following PCR reaction conditions were used in a DNA Engine PTC-200 (MJ Research): 94°C for 5 minutes, 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The PCR products were electrophoresed through 2.0% agarose gel and visualized by ethidium bromide.

Immunofluorescence Microscopy
Ten thousand HeLa cells in 800 µL medium (RPMI 1640 + 10% FBS) were seeded in a four-chambered cover glass (Lab-Tek II Chamber Slides System, Nalge Nunc International, Rochester, NY) and grown overnight at 37°C to reach about 80% confluence. The cells were fixed in 1% formaldehyde prepared in 1x PBS for 10 minutes at room temperature, washed thrice for 10 minutes each with 1x PBS/1% bovine serum albumin (BSA), and then permeabilized with 1x PBS/1% BSA/0.025% NP40 for 15 minutes at room temperature. After permeabilization, the cells were washed twice with 1x PBS/1% BSA, and blocked for 30 minutes at room temperature with protein blocker buffer (Dako, Carpinteria, CA), and then incubated with anti-CXCR4 monoclonal antibody in 1:200 dilution or a control immunoglobulin G in 1:100 dilution for 1 hour at room temperature. After incubation with primary antibody, the cells were washed and incubated with goat anti-mouse immunoglobulin G conjugated with Alexa 488 (Molecular Probes, Eugene, OR) in 1:500 dilution for 1 hour in a dark humid chamber. Finally, the cells were counterstained with propidium iodide. After careful washing with 1x PBS/1% BSA, the cells were visualized for cell surface expression of CXCR4 under a confocal microscope.

Ligand Binding Assay
Ligand binding assay was done in a 96-well U-bottomed microplate. One-hundred-microliter reactions containing 100,000 cells, 50 mmol/L HEPES (pH 7.5), 1 mmol/L CaC12, 5 mmol/L MgCl2, 0.5% BSA, and different concentrations of 125I-SDF-1{alpha} and cold SDF-1{alpha} were incubated for 2 hours at 4°C. The reaction mixtures were then transferred to a 96-well MultiScreen-FB filter plate, which was preequilibrated with 0.3% polyethyleneimine and 0.2% BSA for 30 minutes. The plate was then washed thrice by filtration with 300 µL of washing buffer containing 50 mmol/L HEPES (pH 7.5), 1 mmol/L CaC12, 5 mmol/L MgCl2, 0.5 mol/L NaCl, and 0.5% BSA with a vacuum filtration apparatus from Millipore. After washing, the filter plate was adapted to a MultiScreen Adaptor plate (Perkin-Elmer), 100 µL of Microscint 20 were added to each well, and the radioactivity was determined on a Microplate Scintillation Counter from Packard (Meriden, CT).

SDF-1{alpha} Treatment and Preparation of HeLa Cell Lysates
HeLa cells, 2 x 105, were seeded and grown in 1 mL of RPMI 1640 with 10% FBS in six-well plates for overnight, then starved for 3 hours by growing them in RPMI 1640 without FBS. After starvation, the cells were treated with human SDF-1{alpha} at 37°C for various periods as indicated in figure legends, and then washed with 1 mL of ice-cold PBS. Cells were then immediately lysed with 100 µL of lysis buffer consisting of 25 mmol/L Tris (pH 7.5), 2.5 mmol/L Na2H2P2O7, 150 mmol/L NaCl, 2 mmol/L TAMP, 15 mmol/L p-nitropenyl phosphate, 5 mmol/L benzamidine, 60 mmol/L ß Gly PO4, 1 mmol/L Na-vanadate, 10 mmol/L Na-fluoride, 1 mmol/L DTT, 15 mmol/L EDTA, 5 mmol/L EGTA, 1 µmol/L okadaic acid, 1 µmol/L microsystin, 1% Triton X-100, and 1x protease inhibitor cocktail from Roche (Indianapolis, IN). Total cell lysates were clarified by centrifugation at 10,000 rpm for 10 minutes using an Eppendorf mini-centrifuge. Protein concentrations were determined with Bio-Rad (Hercules, CA) protein assay agents using BSA as a standard.

Western Blot Analysis
A total of 10 µg of control or SDF-1{alpha}–treated cell lysate was separated on a 4% to 20% SDS-polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane (Invitrogen), and the membrane was blocked in blocking solution (5% nonfat dry milk/Tris-buffered solution/0.01% Tween 20) and then incubated overnight with a primary antibody in the appropriate dilution. The unbound primary antibody was removed by washing the membrane with Tris-buffered solution/0.01% Tween 20, followed by incubation with horseradish peroxidase–conjugated anti-rabbit or anti-mouse secondary antibody diluted 1:5,000 in 3% nonfat dry milk/Tris-buffered solution/0.01% Tween 20. Protein was then visualized using enhanced chemiluminescence solution from Amersham (Piscataway, NY) and X-ray film.

Cell Migration Assay
HeLa cells were grown in RPMI 1640 + 10% FBS to 80% confluence on the day of experiment. The cells were harvested and washed once with migration assay buffer [HBSS consisting of 20 mmol/L HEPES (pH 7.5) and 0.1% BSA]. After washing, the cells were pretreated with an inhibitor at certain concentration for 15 minutes at 37°C with 5% CO2. Then, 1 x 105 cells in 250 µL of migration assay buffer were added to each of FALCON HTS FluoroBlock insert with pore size of 3.0 µm (BD Bioscience, Palo Alto, CA). To each bottom well, 750 µL of 60 nmol/L SDF-1 or a mixture of 60 nmol/L SDF-1 and an inhibitor in certain concentration prepared in migration assay buffer were added. After assembly of inserts and bottom wells, the migration plate was incubated for 22 hours at 37°C with 5% CO2. Following the incubation, the top inserts were transferred into a second Falcon non-TC-treated 24-well plate containing 2 µg/well Calcein AM (Molecular Probes) prepared in 0.5 mL of HBSS. The plate was then incubated for 90 minutes at 37°C, and the total cell migration was obtained by measuring the fluorescence in the CytoFluor 400 microplate spectrofluorometer using excitation/emission wavelength of 485/530 nm.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/19/04; revised 2/11/05; accepted 2/22/05.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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