Role of Raf-1 and FAK in cell density-dependent regulation of integrin-dependent activation of MAP kinase

Lianfeng Zhang, Mary Bewick and Robert M. Lafrenie,1

Division of Tumor Biology, Northeastern Ontario Regional Cancer Center, Sudbury, Ontario P3E 5J1, Canada


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MAP kinase can be activated by integrin-dependent adhesion in a FAK-dependent manner. Cell–cell contact inhibition is continuously active in controlling cell growth and the loss of cell–cell contact inhibition is correlated with the malignant characteristics of cancer cells. In this study we showed that cell adhesion to fibronectin for 1 h activated MAP kinase phosphorylation. However, when non-tumorigenic HSG cells, MCF-10A cells, or 293 cells were plated on fibronectin-coated substrates for 1 h at high cell density (which favors cell–cell contact), MAP kinase phosphorylation was not enhanced. Tumorigenic breast cancer cells, BT474, Cama, MCF-7, MDA-MB-231 and SKBR3, did not show inhibition of MAP kinase phosphorylation but rather enhanced MAP kinase phosphorylation when cultured at high density on fibronectin-coated substrates. Adhesion of HSG cells to fibronectin also increased FAK phosphorylation and this FAK phosphorylation was partially inhibited when cells were cultured at high density. Expression of Raf-1 catalytic domain-GFP in HSG cells could overcome the cell density-dependent inhibition of MAP kinase phosphorylation and FAK phosphorylation. The expression of Raf-1-catalytic domain-GFP also upregulated the expression of {alpha}v integrin and promoted cell–cell adhesion in HSG cells. These results suggest that the active form of Raf-1 may interrupt cell–cell contact inhibition by promoting {alpha}v integrin expression, which has been implicated in cell aggregation.

Abbreviations: DMEM, Dulbecco's modified essential medium; FCS, fetal calf serum


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Integrins comprise a large family of heterodimeric cell surface receptors that mediate cell adhesion to extracellular matrix proteins, such as fibronectin, collagens and laminins (1). Integrin-dependent adhesion can also induce signal transduction pathways that regulate cell spreading, migration, proliferation, and apoptosis (2–8). Changes in the expression of several different integrins have been implicated in the transition of normal breast epithelia to malignant cells (9–11).

Integrin-dependent adhesion promotes the formation of a large multicomponent complex of proteins including cytoskeletal components and multiple signaling molecules at the site of adhesion (2,5,6,8,12). Integrin-dependent adhesion also results in co-clustering and autophosphorylation of the cytoplasmic kinase, FAK (13–15). Phosphorylated FAK results in activation of several signaling pathways (3). The Raf-1 serine/threonine kinase is a central component of the MAP kinase cascade and is a key regulator of many growth and developmental pathways (16,17). The constitutively active form (the isolated catalytic domain) of the Raf-1 protein can transform normal cells (18,19). The N-terminal Raf-1 regulatory region (Reg/Raf-1) can autoregulate the kinase activity of the C-terminal kinase domain (Cat/Raf-1) and normal Raf-1 activation requires changes in conformation and phosphorylation within the regulatory region (20). The activation of Raf-1 following integrin-dependent cell adhesion is thought to involve recruitment and activation of Ras within the multicomponent complex formed around phosphorylated FAK. Activated GTP-Ras binds Raf-1 and directs Raf-1 to the plasma membrane where it is phosphorylated and activated (21–24). Raf-1 activates MEK, which in turn phosphorylates MAP kinase (23). Activated MAP kinase is translocated to the nucleus (25) where it directly, or indirectly, phosphorylates and activates a variety of transcription factors including AP-1, ELK and CREB-1 (26,27). MAP kinase activation has also been implicated in regulating cyclin expression and cell cycle progression through mid–late G1 phase (28,29).

Cell–cell adhesion or cell adhesion to the surrounding extracellular matrix are critical in the dynamic processes necessary for tissue morphogenesis in embryos and in tissue maintenance of adult organisms (30,31). A poorly understood process called cell–cell contact inhibition is thought to be continuously active in the control of cell growth (32). In vitro, this process is manifested by the ability of normal cells to grow only as a monolayer in contact with the culture substrate; cells in intimate contact with others stop growing. Although the mechanisms underlying cell–cell contact inhibition are largely unknown, it has been suggested that molecules that mediate cell–cell adhesion, such as cadherins, may initiate signals that mediate cell–cell contact inhibition of cell growth (33,34). Functional interactions between cadherins and integrins have been reported in various normal cell types and this cross talk has been shown to affect tumor cell differentiation, migration and invasiveness (35,36).

Since MAP kinase activation is an important regulator of cell growth, and since FAK and Raf-1 are components of the pathway that activates MAP kinase in response to extracellular stimuli, it seems possible that these kinases may be involved in the process of cell–cell contact inhibition. The goal of this work was to determine whether integrin-dependent activation of MAP kinase was regulated by changes in cell density that increase cell–cell contact and whether this effect was mediated by activation of FAK and Raf-1.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construction of Raf-1-GFP, Cat/Raf-1-GFP and Reg/Raf-1-GFP
The full length Raf-1 (residues 1–648), catalytic region (Cat/Raf-1, residues 325–648) and regulatory region (Reg/Raf-1, residues 1–303) fragments (17–20) were generated by PCR amplification using Raf-1 cDNA (Upstate Biotechnology, Lake Placid, NY) as the template. The PCR product was then inserted into the pEGFP-N1 vector (Clontech, Palo Alto, CA) upstream of sequences encoding GFP. The DNA fragments encoding Raf-1-GFP, Cat/Raf-1-GFP and Reg/Raf-1-GFP fusion proteins were then subcloned into the pIND ecdysone-inducible vector (Invitrogen, Carlsbad, CA) to generate pIND-Raf-1-GFP, pIND-Cat/Raf-1-GFP and pIND-GFP respectively. The catalytic region of Raf-1 fused with GFP was termed Cat/Raf-1-GFP. The regulatory region of Raf-1 fused with GFP was termed Reg/Raf-GFP and Raf-1 fused with GFP was termed Raf-1-GFP.

Cell culture
The HSG human salivary gland cell line (37) was maintained in Dulbecco's Modified Essential Medium (DMEM, Canadian Life Technologies, Burlington, ON) supplemented with 10% fetal calf serum (FCS) (Hyclone, PDI Biosciences, Aurora, ON), 100 µg/ml streptomycin and 100 U/ml penicillin (Canadian Life Technologies). Cells expressing Cat/Raf-1-GFP, Reg/Raf-1-GFP, Raf-1-GFP, and GFP (control) were produced by co-transfecting each pIND construct with the pVgRXR (ecdysone receptor expressing) plasmid into HSG cells using Lipofectamine (Canadian Life Technologies) followed by selection in G418 (Sigma, Chemical Co., St Louis, MO) and Zeocin (Invitrogen) selection media (700 µg/ml G418 and 100 µg/ml Zeocin in DMEM plus 10% FCS). Clonal lines were isolated and lines expressing similar amounts of Raf-1-GFP, Cat/Raf-1-GFP and Reg/Raf-1-GFP were pooled and maintained in G418 and Zeocin selection media. The four cell lines were treated with 10 µg/ml Ponasterone (Invitrogen) in culture media for 24 h to induce expression of the GFP, Cat/Raf-1-GFP, Reg/Raf-1-GFP, or Raf-1-GFP proteins. The BT474, Cama, MCF-7, MDA-MB-231 and SKBR3 breast cancer cell lines and 293 fibroblasts (American Type Culture Collection, Rockville, MD) were maintained in DMEM media supplemented with 10% FCS, 100 µg/ml streptomycin and 100 U/ml penicillin. The non-tumorigenic MCF-10A epithelial cell line (ATCC), was maintained in a 1:1 mixture of Ham's F12 and DMEM supplemented with 20 ng/ml epidermal growth factor (Calbiochem, San Diego, CA), 100 ng/ml cholera toxin (Sigma), 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5% horse serum (Hyclone).

Cell attachment to fibronectin-coated substrates
Cells were harvested with 10 mM EDTA in PBS, washed and resuspended to 105 cells/ml in DMEM supplemented with 1% BSA. Non-tissue culture 96-well plates (Costar, Cambridge, MA) were precoated with 50 µl of 10 µg/ml fibronectin or BSA. The cells were incubated in the presence or absence of 50 µg/ml of anti-ß1 (mAb13, K.M. Yamada, National Institutes of Health, Bethesda, MD), anti-{alpha}5 (mAB16), or anti-{alpha}2 (P1E6, Life Technologies) integrin antibodies and then 50 µl of the cell suspension (5000 cells) were added to each well. The cells were incubated with the substrate for 1 h. The wells were washed with PBS and the number of adherent cells per high power microscope field were counted. All experiments were conducted in quadruplicate and data analyzed using a Student's= t-test.

Preparation of lysates from cells adherent to fibronectin-coated substrates
The indicated cell lines were harvested by incubation with 10 mM EDTA in PBS. The cells were washed twice and resuspended in serum-free DMEM and incubated at 37°C for 1 h. The cells were then cultured on non-coated or fibronectin-coated 6-well plates at the indicated cell densities for 1 h and harvested in RIPA buffer (1% Triton X-100, 0.5% SDS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, and 5 mM EDTA) containing 1 mM sodium orthovanadate and protease inhibitors (Roche, Laval, QB). The plates were coated by incubation in 10 µg/ml fibronectin (Roche) at 4°C for 16 h.

Phosphorylation of FAK and MAP kinase in the presence of a MEK inhibitor
HSG cells expressing Cat-GFP/Raf-1 and GFP were harvested with 10 mM EDTA in PBS, washed, resuspended in serum-free DMEM supplemented with 5 ng/ml PD98059 (Calbiochem, La Jolla, CA), and incubated for 2 h at 37°C to inhibit MEK and block MAP kinase activation. The treated cells were then cultured at a density of 7 x 105 cells/well on non-coated or fibronectin-coated plates for 1 h at 37°C and harvested in RIPA buffer containing 1 mM sodium orthovanadate and protease inhibitors.

Immunoprecipitation and immunoblot analysis
HSG cells were lysed in RIPA buffer containing 1 mM sodium orthovanadate and protease inhibitors. For immunoprecipitation analysis, the lysates from HSG cell lines expressing Raf-1-GFP, Cat/Raf-1-GFP, Reg/Raf-1-GFP, or GFP were mixed with 1 µl of polyclonal rabbit anti-GFP antibodies (Invitrogen) and 30 µl of 10% protein A/G-linked agarose beads (Santa Cruz, Santa Cruz, CA) and incubated at 4°C for 16 h. The protein A/G agarose beads were washed twice with RIPA buffer. The beads were resuspended in loading buffer and subjected to electrophoresis on polyacrylamide gels and the proteins electrophoretically transferred to nitrocellulose sheets for immunoblot analysis. The different Raf-1-GFP proteins were detected by incubation with a monoclonal mouse anti-Raf-1 that recognizes the N-terminal region of Raf-1 (Transduction Laboratories, Lexington, KY) or a polyclonal rabbit anti-Raf-1 antibody that recognizes the C-terminal end of Raf-1 (Santa Cruz). The levels of MAP kinase, phosphorylated MAP kinase, FAK, phosphorylated FAK, {alpha}v integrin, and ß1 integrin were determined in the cell lysates (normalized for 40 µg total proteins) by immunoblot analysis with polyclonal rabbit anti-phospho-p42/44 MAP kinase (New England Biolab), polyclonal rabbit anti-phospho-FAK (pTyr576, Biosource, Camarillo, CA), monoclonal mouse anti-{alpha}v (Canadian Life Technology), or monoclonal rat anti-ß1 integrin. The primary antibodies were detected by incubation with polyclonal goat anti-rabbit IgG coupled to HRP or polyclonal goat anti-mouse IgG-HRP (Santa Cruz). HRP was detected by chemiluminescence using the Supersignal reagent (Pierce Chemical Co., Rockford, IL) and ECL Hyperfilm (Amersham, Oakville, ON). The nitrocellulose sheets were then stripped by incubation in stripping buffer (1% SDS, 100 mM ß-mercaptoethanol in TBS), washed in TBS, and reblotted with polyclonal goat anti-MAP kinase (a 1:1 mixture of anti-ERK-1 and anti-ERK-2 antibodies, Santa Cruz) or monoclonal mouse anti-FAK antibodies (Transduction Laboratories).

Aggregation assays
HSG cells expressing Cat/Raf-1-GFP or GFP were harvested with 10 mM EDTA in PBS, washed twice, and then resuspended in serum-free DMEM medium at a density of 106 cells/ml. The cells were kept at room temperature for 30 min to allow the cells to form multicellular aggregates and then dispersed by vortexing for 10 s. The cells were seeded onto 6-well plates, cultured at 37°C for 3 h and then visualized using a Zeiss Axiophot phase-contrast microscope fitted with an electronic camera. The aggregation was quantitatively analyzed by counting multicellular aggregates that contained at least five cells.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell adhesion to fibronectin is mediated by ß1 integrins
The integrin adhesion molecules that mediate adhesion to fibronectin-coated substrates was determined by measuring cell adhesion in the presence or absence of anti-integrin antibodies. The adhesion of HSG cells to fibronectin was almost completely inhibited by inclusion of the anti-ß1 (91 ± 11%) or anti-{alpha}5 (82 ± 12%) integrin antibodies but not by anti-{alpha}2 antibodies. Similarly, adhesion of MCF-10A, 293, BT474, MCF-7, MDA-MB-231, and SKBR3 cells to fibronectin was almost completely inhibited by anti-ß1 (inhibition was >88% for each of the cell lines) or anti-{alpha}5 antibodies (inhibition was >84% for each of the cell lines) (not shown).

Cell density differently regulates activation of MAP kinase in non-tumorigenic and tumorigenic cells adherent to fibronectin
Integrin-dependent adhesion of cells to fibronectin can activate the MAP kinase signal transduction pathway in a FAK-dependent way (23,38,39). To determine the effects of cell density on the regulation of MAP kinase in response to adhesion to fibronectin, HSG cells were seeded onto fibronectin at different cell densities. The activation of MAP kinase was detected by measuring phosphorylated MAP kinase using an anti-phospho-MAP kinase antibody in an immunoblot assay and then normalized for expression of total MAP kinase (Figure 1AGo). When HSG cells were seeded at a density of 7 x 105 cells/well in a 6-well plate, the plated cells were completely separated and there was very little cell–cell contact. Under these conditions of low cell density, HSG cells cultured on fibronectin-coated substrates expressed much higher levels of phosphorylated MAP kinase than on non-precoated substrates. When HSG cells were seeded onto 6-well-plates at densities of 1.4 x106 cells/well or 2.8 x 106 cells/well, the plated cells showed extensive cell–cell contact. Under these conditions of high cell density, HSG cells cultured on both fibronectin-coated and non-precoated substrates expressed very low levels of phosphorylated MAP kinase similar to background. Therefore, it appeared that the activation of MAP kinase in response to integrin-dependent adhesion of HSG cells to fibronectin was inhibited in cells cultured at high density under conditions that favor cell–cell contact.



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Fig. 1. MAP kinase phosphorylation in HSG, MCF-10A, 293, BT474, Cama, MCF-7, MDA-MB-231, and SKBR3 cells following adhesion to fibronectin was differently regulated by cell–cell contact. HSG (A); MCF-10A (B); 293 (C); BT474 (D); Cama (E); MCF-7 (F); MDA-MB-231 (G); and SKBR3 (H) cells were harvested using 10 mM EDTA in PBS and washed with serum-free DMEM culture medium. The cells were seeded onto fibronectin-coated or non-coated 6-well plates at the indicated cell densities and cultured at 37°C for 1 h. The cells were harvested and subjected to immunoblot analysis with anti-phospho-p42/44 MAP kinase (left panels) and reblotted with the polyclonal goat anti-MAP kinase antibodies (a 1:1 mixture of anti-ERK-1 and anti- ERK-2 antibodies) (right panels). The relative intensity of phosphorylated MAP kinase was determined by densitometric analysis of autoradiographs and normalized for the intensity of total MAP kinase (I): the non-tumorigenic cells (HSG, MCF-10A, and 293 cells) are shown with solid lines and the tumorigenic cells (BT474, Cama, MCF-7, MDA-MB-231, and SKBR3 cells) are shown with broken lines.

 
In order to determine if cultured non-tumorigenic cells and cultured cancer cells differ in the ability of cell density to regulate MAP kinase activation, we compared MAP kinase activation in MCF-10A, 293, BT474, Cama, MCF-7, MDA-MB-231, and SKBR3 cells cultured on fibronectin at different cell densities (Figure 1B–IGo). Cell density regulated activation of MAP kinase in MCF-10A and 293 cells adherent to fibronectin in a similar way to HSG cells. This suggests that the integrin-dependent activation of MAP kinase was inhibited in HSG, MCF-10A, and 293 cells cultured at high cell densities. However, when the BT474, Cama, MCF-7, MDA-MB-231 and SKBR3 breast cancer cells were cultured at high cell densities on fibronectin-coated substrates, they all showed a strong upregulation of MAP kinase phosphorylation. Further, MDA-MB-231 and SKBR3 cells cultured at the highest density on non-precoated substrates also upregulated MAP kinase phosphorylation. These results suggest that the cancer cell lines have lost the ability of high cell density, which favors cell–cell contact, to inhibit integrin-dependent activation of the MAP kinase.

The regulatory and catalytic domain of Raf-1 regulate MAP kinase phosphorylation
Stable HSG cell clones expressing similar amounts of Cat/Raf-1-GFP, Reg/Raf-1-GFP and Raf-1-GFP were isolated and characterized by immunoblot analysis (Figure 2AGo). The inducer, Ponasterone, enhanced expression of the transgene in all of the cell lines. HSG cells expressing Cat/Raf-1-GFP expressed greater levels of phosphorylated MAP kinase and HSG cells expressing Reg/Raf-1-GFP expressed lower levels of phosphorylated MAP kinase than did the control cells expressing GFP (Figure 2BGo). However, the low level of Reg/Raf-1-GFP expressed in the transfected HSG cells was not sufficient to completely block MAP kinase phosphorylation although induction of Reg/Raf-1-GFP with Ponasterone did dramatically decrease MAP kinase phosphorylation.



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Fig. 2. Expression of Cat/Raf-1-GFP, Reg/Raf-1-GFP, Raf-1-GFP and GFP and the effect on phosphorylation MAP kinase in transfected HSG cell lines. HSG cell lines expressing Cat/Raf-1-GFP, Reg/Raf-1-GFP, Raf-1-GFP and GFP were induced with Ponasterone for 24 h and harvested in RIPA buffer. The cell lysates were subjected to immunoprecipitation or immunoblot analysis. The total cell lysates were immunoprecipitated with an anti-GFP antibody and subjected to immunoblot analysis with an anti-Raf-1 (C-terminal region) antibody (A, top) or an anti-Raf-1 (N-terminal region) antibody (A, bottom). The cell lysates were subjected to immunoblot analysis with anti-phospho-p42/44 MAP kinase (B, top) and reblotted with polyclonal goat anti-MAP kinase (anti-ERK-1 and ERK-2) antibodies (B, bottom).

 
The catalytic domain of Raf-1 abolishes the cell density-dependent inhibition of MAP kinase activation and increases phosphorylation of FAK at high cell density
To determine the effect of Raf-1 on cell density-dependent inhibition of integrin-dependent activation of MAP kinase, HSG cells transfected with the different Raf-1 constructs were cultured at different cell densities on fibronectin-coated substrates. MAP kinase phosphorylation in HSG cells expressing GFP, Cat/Raf-1-GFP, Reg/Raf-1-GFP, and Raf-1-GFP following adhesion to fibronectin- or non-precoated substrates was determined by immunoblot analysis (Figure 3AGo). All four HSG cell lines, cultured at low density on fibronectin-coated substrates showed upregulation of MAP kinase phosphorylation compared with cells cultured on non-precoated substrates. This suggests that integrin-dependent signaling can still augment MAP kinase activation in HSG cells expressing low levels of Reg/Raf-1-GFP, Cat1/Raf-1-GFP, or Raf-1-GFP. Similar to the parental HSG cells, HSG cells expressing GFP, Reg/Raf-1-GFP, or Raf-1-GFP expressed higher levels of phosphorylated MAP kinase when cultured on fibronectin-coated substrates at low density (7 x 105 cells/well) but when cultured at high density (2.8 x 106 cells/well) they expressed low levels of phosphorylated MAP kinase on both fibronectin- and non-precoated substrates. However, HSG cells expressing Cat/Raf-1-GFP expressed high levels of phosphorylated MAP kinase following adhesion to fibronectin at low cell densities and when cultured on both non-precoated and fibronectin-coated substrates at high cell densities.



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Fig. 3. Phosphorylation of MAP kinase and FAK in HSG cells transfected with Raf-1 constructs following adhesion to fibronectin-coated substrates at different cell densities. HSG cells expressing Cat/Raf-1-GFP, Reg/Raf-1-GFP, Raf-1-GFP and GFP were treated with 10 mM EDTA in PBS and washed with serum-free DMEM culture medium. The cells were then seeded onto fibronectin-coated or non-precoated 6-well plates at the indicated cell densities and cultured at 37°C for 1 h. The cells were harvested and subjected to immunoblot analysis with the anti-phospho- p42/44 MAP kinase antibody (A, top) and reblotted with the polyclonal goat anti-MAP kinase (anti-ERK-1 and ERK-2) antibodies (A, bottom). HSG cells or HSG cells expressing GFP or Cat/Raf-1-GFP were seeded onto fibronectin-coated or non-precoated 6-well plates at the indicated cell densities and cultured at 37°C for 1 h. The cells were harvested in RIPA buffer and subjected to immunoblot analysis using polyclonal rabbit anti-pTyr574-FAK (B, top) and reblotted with the monoclonal mouse anti-FAK antibody (B, bottom).

 
Integrin-dependent adhesion of cells to fibronectin can promote FAK phosphorylation on Tyrosine-576 (Tyr576). Since HSG cells expressing Cat/Raf-1-GFP no longer showed cell density-dependent inhibition of MAP kinase activation, the effect of Cat/Raf-1-GFP on cell density-dependent inhibition of FAK phosphorylation was also examined. HSG cells, HSG cells expressing GFP, or HSG cells expressing Cat/Raf-1-GFP cells were cultured on fibronectin-coated substrates at high and low cell densities and the levels of phosphorylated FAK were detected by immunoblot analysis with an anti-phospho-FAK (pTyr576) antibody (Figure 3BGo). Abundant phosphorylated FAK was detected in HSG cells on fibronectin-coated substrates at low cell density (7 x 105 cell/well) when compared with cells cultured on non-precoated substrates. However, at high cell density (2.8 x 106 cell/well), lower levels of phosphorylated FAK were detected in HSG cells cultured on fibronectin-coated substrates although these levels were still higher than in the cells cultured on non-precoated substrates. This suggests that the phosphorylation of FAK in response to cell adhesion to fibronectin was partially inhibited under high cell density conditions that favor cell–cell contact. HSG cells expressing GFP cultured on fibronectin-coated substrates at low cell density showed abundant FAK phosphorylation while FAK phosphorylation was partially inhibited in cells cultured on fibronectin-coated substrates at high cell density. However, at high cell density, HSG cells expressing Cat/Raf-1-GFP showed hyper-induced levels of phosphorylated FAK when cultured on fibronectin-coated substrates. These results suggest that the expression of active Raf-1 feeds back and results in activation of FAK especially in cells cultured at high cell density.

The effects of a MEK inhibitor on FAK and MAP kinase phosphorylation following adhesion to fibronectin
Activated Raf-1 is able to phosphorylate MEK, the next kinase in the cascade pathway (3,43), Therefore, MEK, activated by Cat/Raf-1-GFP (a constitutively active form of Raf-1), may be involved in the increased phosphorylation of FAK. To determine if Cat/Raf-1-GFP increased FAK phosphorylation in a MEK-dependent manner, the effect of a specific MEK inhibitor, PD98059, on FAK and MAP kinase phosphorylation was determined (Figure 4Go). HSG cells expressing GFP or Cat/Raf-1-GFP were pretreated in the presence or absence of PD98059 and then cultured on fibronectin-coated substrates. Incubation with PD98059 inhibited MEK and resulted in inhibition of MAP kinase phosphorylation in HSG cells adherent to fibronectin-coated substrates. However PD98059 had no effect on the phosphorylation of FAK by either GFP or Cat/Raf-1GFP expressing HSG cells. This result suggested that the effect of activated Raf-1 on FAK phosphorylation was not mediated through activation of MEK, the best characterized Raf-1 substrate.



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Fig. 4. Effect of MEK inhibition on FAK and MAP kinase phosphorylation following adhesion to fibronectin-coated substrates. HSG cells expressing GFP and Cat/Raf-1-GFP were seeded onto fibronectin-coated plates and cultured in the presence of PD98059 (0.2 mM) at 37°C for 2 h. The cells were harvested in RIPA buffer, normalized for protein concentration and subjected to immunoblot analysis with polyclonal rabbit anti-anti-phospho-p42/44 MAP kinase (A, top) and reblotted with anti-MAP kinase (anti-ERK-1 and ERK-2) antibodies. The lysates were also subjected to immunoblot analysis with a polyclonal rabbit anti-pTyr574-FAK antibody (B, top) and reblotted with an anti-FAK antibody (B, bottom).

 
The catalytic domain of Raf-1 increased the expression of {alpha}v integrin and enhanced the aggregation of HSG cells
The expression levels of various integrins were determined in order to investigate the mechanism by which Cat/Raf-1-GFP enhanced FAK phosphorylation in cells cultured on fibronectin at high density and enhanced MAP kinase phosphorylation on both non-precoated and fibronectin-coated substrates cultured under conditions which favor cell–cell contact (Figure 5A–EGo). Immunoblot analysis showed that the expression of the {alpha}v integrin was increased in HSG cells expressing Cat/Raf-1-GFP, but that the expression of the ß1integrin was not changed.



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Fig. 5. Effect of Cat/Raf-1-GFP on the expression of {alpha}v integrin and cell aggregation in HSG cells. HSG cells expressing GFP or Cat/Raf-1-GFP were harvested in RIPA buffer and subjected to immunoblot analysis with the anti-{alpha}v antibody (A) or anti-ß1 antibody (B). The cell–cell aggregation of HSG cells expressing GFP (C) or Cat/Raf-1-GFP (D) were determined. The effect of anti-{alpha}v antibody on the aggregation of cells expressing Cat/Raf-1-GFP was also determined (E).

 
HSG cells suspended in culture medium form multicellular aggregates. Comparative aggregation assays showed that HSG cells expressing Cat/Raf-1-GFP aggregated to a greater extent than HSG cells expressing only GFP. Further, the aggregation between cells expressing Cat/Raf-1-GFP was partially inhibited in the presence of an anti-{alpha}v integrin antibody.


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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The MAP kinase cascade is a critical signal transduction pathway in transmitting extracellular signals to the nucleus (25–28,40,41). Therefore, the MAP kinase cascade may mediate the cell–cell contact-dependent inhibition of cell growth that occurs when non-tumorigenic cells are grown to high cell density. Malignant cells have lost their ability to implement cell–cell contact-dependent inhibition of cell growth and can therefore form multilayer cultures. This characteristic has been shown to correlate with the invasive phenotype of tumor cells. The results of this study showed that several non-tumorigenic and malignant cell lines differed in their ability to phosphorylate MAP kinase in response to integrin-dependent stimuli under high cell density conditions that favor cell–cell contact. Further, cell density-dependent inhibition of both MAP kinase and FAK phosphorylation can be overcome by the expression of activated Raf-1 (Cat/Raf-1). In fact, the expression of activated Raf-1 can promote MAP kinase activation in cells cultured on non-precoated substrates at high cell density but not at low cell density.

The mechanisms underlying the effect of cell density on cellular signaling could be mediated by signals induced by cell–cell contact, changes in cell spreading or cell shape, or by density-dependent changes in culture conditions such as oxygen tension. In fact, several investigators have shown that hypoxic culture conditions can induce phosphorylation of FAK, p38 MAP kinase, and MAP kinase (42–44). However, the experiments in the current proposal were conducted over only 1 h in a normal air (20% oxygen) environment with cell densities which are expected to have small effects on oxygen tension (45). Further, others have shown that cell density can regulate phosphorylation and localization of several proteins including FAK, paxillin, Rap1 and MAP kinase in a manner which appears to require cell–cell contact (46–48).

Integrin-dependent adhesion of cells to fibronectin can activate MAP kinase in a FAK-dependent manner (25,38,39). We showed that both HSG, MCF-10A, or 293 cells cultured at low density on fibronectin-coated substrates, mediated by ß1 integrin, resulted in an increase in MAP kinase phosphorylation. When HSG, MCF-10A, or 293 cells were cultured at high density on fibronectin, which favors cell–cell contact, MAP kinase phosphorylation was not upregulated. The HSG, MCF-10A, and 293 cell lines are non-tumorigenic. (HSG cells do not form tumors in nude mice, are not immortal, and differentiate when cultured on extracellular matrices (49,50).) However, the BT474, Cama, MCF-7, MDA-MB-231, and SKBR3 breast cancer cell lines did not show cell density-dependent inhibition of MAP kinase activation but rather showed that at high density these cells activated MAP kinase on fibronectin-coated substrates. This correlation between tumorigenicity and density-dependent regulation of MAP kinase suggests a fundamental difference in the effects of cell density on adhesion-dependent activation of MAP kinase between malignant and non-tumorigenic cells. Further, this suggests that the cancer cell lines no longer recognize inhibitory signals initiated by increased cell density and perhaps by cell–cell contact. Since cell–cell contact inhibition is an important regulatory mechanism of cell growth (32), the loss of cell–cell contact-dependent inhibition of MAP kinase activation could contribute to uncontrolled tumor cell growth and might correlate with the invasive phenotype of tumors.

HSG cells that express an active form of Raf-1 (Cat/Raf-1-GFP) showed increased phosphorylation of MAP kinase. HSG cells expressing GFP, Reg/Raf-1-GFP and Raf-1-GFP showed upregulation of MAP kinase phosphorylation when cultured on fibronectin at low cell density. Therefore, the low level of Reg/Raf-1-GFP expressed in HSG cells was not sufficient to block adhesion-dependent activation of MAP kinase. Further, HSG cells expressing GFP, Reg/Raf-1-GFP, and Raf-1-GFP still showed cell density-dependent inhibition of MAP kinase phosphorylation. Interestingly, only the HSG cells expressing Cat/Raf-1-GFP lost the cell density-dependent inhibition of MAP kinase following adhesion to fibronectin-coated (and non-precoated) substrates at high cell density. Therefore, the expression of Cat/Raf-1-GFP was sufficient to overcome cell density-dependent inhibition of MAP kinase phosphorylation following adhesion to fibronectin. In addition, the expression of Cat/Raf-1-GFP was sufficient to upregulate MAP kinase phosphorylation in HSG cells cultured at high density on non-precoated substrates. Since cell density-dependent regulation of MAP kinase could occur upstream or downstream of Raf-1, we investigated the role of FAK and MEK.

FAK links integrins and the downstream components of integrin-dependent signaling, such as Src or Fyn and Ras to the activation of MAP kinase (14–16,38,39,51). As expected, FAK was phosphorylated at Tyr576 following adhesion to fibronectin-coated substrates at low cell density. However, when the HSG cells were cultured on fibronectin-coated substrates at high cell density FAK (Tyr576) phosphorylation was significantly, but not completely, inhibited. These results suggest that cell density altered signals that interfere with FAK phosphorylation. Previously, Batt and Roberts (46) showed that culture of Balb3T3 cells for >2 h at high density can enhance FAK phosphorylation while Xu and Zhao (47) showed that cell–cell contact for >2 h in NIH3T3 cells can inhibit FAK phosphorylation. In our current studies, we have shown that both the substratum and the expression of Cat/Raf-1 can affect density-dependent FAK phosphorylation. Culturing HSG cells at high density for 1 h on non-precoated substrates (mimicking the previous studies) resulted in a modest increase in FAK phosphorylation compared with cells cultured at low densities while HSG cells cultured on fibronectin at higher density expressed lower levels of phosphorylated FAK than HSG cells cultured on fibronectin at low density. Further, HSG cells expressing the Cat/Raf-1 (oncogenic) transgene expressed higher levels of phosphorylated FAK than in cells cultured at high density on fibronectin (see below). Therefore, the differences between the previous studies and with our own may be related to cell type-specific differences in the ability of the cell line to produce extracellular matrix or to differences in their degree of transformation. The expression of Cat/Raf-1-GFP, which can overcome the cell density-dependent inhibition of MAP kinase activation in cells adherent to fibronectin, increased FAK phosphorylation in an unknown way. When HSG cells expressing Cat/Raf-1-GFP were cultured on fibronectin-coated substrates at high cell density, the phosphorylation level of FAK was even higher than when cultured at low cell density. However, unlike MAP kinase phosphorylation, Cat/Raf-1-GFP did not induce FAK phosphorylation in cells cultured on non-precoated substrates at high cell density. Therefore, the activation of MAP kinase phosphorylation in Cat/Raf-1-GFP-transfected cells cultured on non-precoated substrates was not dependent on FAK.

Since it has been shown that FAK may be downstream of MAP kinase in some conditions (52) we tested whether the activation of FAK phosphorylation in Cat/Raf-1-GFP-transfected cells was dependent on MAP kinase. The results showed that PD98059, an inhibitor of MEK, inhibited MAP kinase phosphorylation but did not alter FAK phosphorylation. Thus, MEK or MAP kinase were not involved in the enhanced phosphorylation of FAK and Cat/Raf-1-GFP must act via some other mechanism. Expression levels of several integrins in HSG cells expressing GFP or Cat/Raf-GFP were analyzed by immunoblot analysis. The expression of {alpha}v integrin was increased in HSG cells expressing Cat/Raf-1-GFP, but the expression levels of ß1 integrin was not changed.

It has been reported that the {alpha}v integrin causes cell aggregation (53) and further that {alpha}v integrin-dependent adhesion can promote FAK phosphorylation (54). The expression of Cat/Raf-1-GFP in HSG cells correlated with increased {alpha}v integrin expression and with the increased aggregation of those cells in suspension. Further, the aggregation of HSG cells expressing Cat/Raf-1-GFP was partially inhibited by an anti-{alpha}v antibody. It has also been reported that the increased expression of the {alpha}v integrin suppresses E-cadherin-dependent morphological changes (55). Since lower levels of E-cadherin expression correlate with increased invasiveness of epithelial cancers, the disruption of E-cadherin mediated cell–cell adhesion may promote tumor progression (56–59). It has also been shown that expression of an oncogenic form of Raf-1 in rat salivary gland epithelial cells can disrupt cell–cell contact growth control and epithelial tight junctions by inhibiting expression of the tight junction proteins occludin and claudin-1 in a MAP kinase-dependent manner (60).

In conclusion, our results suggest that integrin-dependent MAP kinase activation is inhibited by increased cell density in HSG, MCF-10A, 293 cells. The breast cancer cell lines BT474, Cama, MCF-7, MDA-MB-231, and SKBR3 no longer inhibit integrin-dependent MAP kinase activation when cultured at high cell density and increased MDA-MB-231 and SKBR3 cell density can activate MAP kinase even on non-precoated substrates. Expression of the active form of Raf-1 in HSG cells can overcome the cell density-dependent inhibition of integrin-dependent MAP kinase activation and enhance phosphorylation of FAK. The effect of Cat/Raf-1-GFP on cell density-dependent regulation of MAP kinase may be mediated by changes in {alpha}v integrin which promote cell aggregation and which may interfere with E-cadherin-mediated or other mechanisms of cell–cell contact.


    Notes
 
1 To whom correspondence should be addressed Email: rlafrenie{at}neorcc.on.ca Back


    Acknowledgments
 
We would like to thank Dr K.M.Yamada of the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD for providing the anti-integrin antibodies mAb13 and mAb16. We also thank the Northeastern Ontario Regional Cancer Centre Foundation for funding this research.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 26, 2001; revised April 19, 2002; accepted April 29, 2002.